Medicine, Faculty of
Medical Genetics, Department of
DSpace
UBCV
Mirjalili Mohanna, Seyedeh Zeinab
Doctor of Philosophy - PhD
University of British Columbia
Aniridia is a rare eye disorder with no cure and is caused by pathogenic variants in the paired box 6 (PAX6) gene. Patients have low vision at birth due to retinal foveal hypoplasia, which typically progresses to blindness by early adulthood due to corneal clouding and glaucoma. Since aniridia is caused by PAX6 haploinsufficiency, one potential approach to treating it is targeted CRISPR-based gene editing. Preclinical studies in an appropriate animal model are important first steps towards the translation of the research into a clinical trial. Thus, in Chapter 2, we inserted a 3xFLAG tag on the Pax6 small eye (Sey) allele, creating a novel mouse model for aniridia that is ideal for testing CRISPR strategies. Next, we developed a CRISPR strategy in vitro and subsequently evaluated its efficacy in vivo in the germline of our new mouse. We showed that our CRISPR strategy corrected the Sey variant in the mouse germline, resulting in the full rescue of the eye phenotype. Next, we worked towards developing a somatic therapy, the success of which relies on effective delivery of CRISPR components to therapeutically significant cells. In Chapter 3, we tested a novel lipid nanoparticle-based platform, and successfully edited cultured mouse cortical neurons. We then directly injected LNP-CRISPR into the mouse cornea to evaluate in vivo delivery. We demonstrated wide-spread gene editing of corneal stromal and endothelial cells but not the PAX6-expressing epithelial or limbal stem cells (LSCs). Hence, in Chapter 4, we shifted our focus to recombinant adeno-associated viruses (rAAVs), a more conventional delivery vector for CRISPR. We carried out a series of experiments to test viral capsid, dose, and route of administration. We also investigated if functional LSCs are present in the already cloudy aniridic mouse corneas and whether rAAV can transduce them. We reported unexpected ataxia and lethality in mice after intraocular rAAV-PHP.B injections. We also showed that intrastromal and intravitreal delivery of rAAV9 transduced functional LSCs, as well as all PAX6-expressing retinal cell types in aniridic eye, respectively, with no observed adverse events, making rAAV9 the capsid of choice for the future gene therapy development for aniridia.
Successful Gene Editing in a Novel Mouse Model of Aniridia Towards Prevention of Blindness by Seyedeh Zeinab Mirjalili Mohanna B.Sc., University of Malaya, 2014 M.Sc., The University of Sheffield, 2015 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Medical Genetics) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August 2023 © Seyedeh Zeinab Mirjalili Mohanna, 2023 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Successful Gene Editing in a Novel Mouse Model of Aniridia Towards Prevention of Blindness submitted by Seyedeh Zeinab Mirjalili Mohanna in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Medical Genetics Examining Committee: Dr. Elizabeth M. Simpson, Professor, Medical Genetics, UBC Supervisor Dr. Cheryl Gregory-Evans, Professor, Ophthalmology & Visual Sciences, UBC Supervisory Committee Member Dr. Perry Howard, Associate Professor, Biochemistry & Microbiology, University of Victoria External Examiner Dr. Deborah Giaschi, Professor, Ophthalmology & Visual Sciences, UBC University Examiner Dr. Robert Molday, Professor, Ophthalmology & Visual Sciences, UBC University Examiner Additional Supervisory Committee Members: Dr. Orson Moritz, Professor, Ophthalmology & Visual Sciences, UBC Supervisory Committee Member Dr. Colin Ross, Associate Professor, Medical Genetics, UBC Supervisory Committee Member iii Abstract Aniridia is a rare eye disorder with no cure and is caused by pathogenic variants in the paired box 6 (PAX6) gene. Patients have low vision at birth due to retinal foveal hypoplasia, which typically progresses to blindness by early adulthood due to corneal clouding and glaucoma. Since aniridia is caused by PAX6 haploinsufficiency, one potential approach to treating it is targeted CRISPR-based gene editing. Preclinical studies in an appropriate animal model are important first steps towards the translation of the research into a clinical trial. Thus, in Chapter 2, we inserted a 3xFLAG tag on the Pax6 small eye (Sey) allele, creating a novel mouse model for aniridia that is ideal for testing CRISPR strategies. Next, we developed a CRISPR strategy in vitro and subsequently evaluated its efficacy in vivo in the germline of our new mouse. We showed that our CRISPR strategy corrected the Sey variant in the mouse germline, resulting in the full rescue of the eye phenotype. Next, we worked towards developing a somatic therapy, the success of which relies on effective delivery of CRISPR components to therapeutically significant cells. In Chapter 3, we tested a novel lipid nanoparticle-based platform, and successfully edited cultured mouse cortical neurons. We then directly injected LNP-CRISPR into the mouse cornea to evaluate in vivo delivery. We demonstrated wide-spread gene editing of corneal stromal and endothelial cells but not the PAX6-expressing epithelial or limbal stem cells (LSCs). Hence, in Chapter 4, we shifted our focus to recombinant adeno-associated viruses (rAAVs), a more conventional delivery vector for CRISPR. We carried out a series of experiments to test viral capsid, dose, and route of administration. We also investigated if functional LSCs are present in the already cloudy aniridic mouse corneas and whether rAAV can transduce them. We reported unexpected ataxia and lethality in mice after intraocular rAAV-PHP.B injections. We also showed that intrastromal and iv intravitreal delivery of rAAV9 transduced functional LSCs, as well as all PAX6-expressing retinal cell types in aniridic eye, respectively, with no observed adverse events, making rAAV9 the capsid of choice for the future gene therapy development for aniridia. v Lay Summary Aniridia is a rare eye disorder that reduces vision from birth and causes a lifetime of visual impairment. Current treatments are unable to prevent the typical eventual blindness, and thus new treatment strategies are needed. Since, aniridia is caused by defects in the paired box 6 (PAX6) gene, one possible approach for treating it is gene editing. Effective delivery is a major challenge when it comes to developing a gene editing therapy. Thus, our broad goal was to identify an appropriate delivery system for transporting the gene editing components to the affected cells in the mouse eye. Towards this end, we tested two commonly-used delivery systems and showed that a non-pathogenic virus successfully targets the affected cells after direct-to-eye injections. We recommend that this virus can be used for developing a gene editing treatment for aniridia in the future. vi Preface Chapter 2 has been originally published as: Mirjalili Mohanna SZ, Hickmott JW, Lam SL, Chiu NY, Lengyell TC, Tam BM, Moritz OL, and Simpson EM. (2020). Germline CRISPR/Cas9-mediated gene editing prevents vision loss in a novel mouse model of aniridia. Mol Ther Methods Clin Dev. 17, 478–490. EMS designed the experiments, supervised the whole study, secured funding, reviewed and revised the manuscript. OLM supervised the Xenopus laevis experiments and secured funding. BMT performed all the Xenopus laevis experiments. TCL generated the mouse embryonic stem cell lines used for the in vitro testing of various CRISPR strategies and produced the transgenic mice by microinjecting the optimized CRISPR strategies into mouse zygotes. NYC performed visual inspection and slit lamp imaging of a subset of mouse eyes. SLL designed, tested, and optimized the CRISPR components for the insertion of the 3xFLAG tag. JWH cloned all plasmids for in vitro transcription, and made the figure that shows the results from the Xenopus laevis experiments. SZMM was in charge of all aspects of the mouse husbandry. SZMM designed, tested, and optimized the CRISPR components for the Sey correction strategy. SZMM analyzed all the Sanger sequencing results, prepped the CRISPR components for the microinjections, fully characterized all the transgenic strains, made the figures, and wrote the original draft of the manuscript. Chapter 3 has been originally published as: Mirjalili Mohanna SZ, Djaksigulova D, Hill AM, Wagner PK, Simpson EM, and Leavitt BR. LNP-mediated delivery of CRISPR RNP for wide-spread in vivo genome editing in mouse cornea. (2022). J Control Release. 350, 401–413. vii This study was conducted as a collaboration with the company Incisive Genetics and was co-supervised by EMS and BRL. The experiments in this study were conceptualized by EMS and BRL. EMS was also in charge of funding acquisition and project administration, as well as reviewing and revising the manuscript. Incisive Genetics designed, produced, and characterized the LNPs. AMH designed the CRISPR components for both the tdTomato expression and the luciferase non-targeting strategies, performed immunocytochemistry analysis of cortical neurons treated with the tdTomato strategy, generated images of tdTomato expression in the cortical neurons, and subsequently quantified the tdTomato signal in these cells. AMH also helped with project administration. PKW isolated and cultured the ex vivo mouse cortical neurons, encapsulated the LNPs with the RNP and DNA template prior to each experiment, and performed all the ex vivo gene editing experiments. DD generated and genotyped all the embryos and mice used in this study. SZMM performed immunocytochemistry analysis of cortical neurons treated with the Sey correction strategy, and quantified FLAG expression in these cells. SZMM analyzed the Sanger sequencing results, did all the statistical analysis in the study, conducted all aspects of the in vivo gene editing experiments, and subsequently analyzed the results. SZMM also made the figures and wrote the original draft of the manuscript. Chapter 4 has been originally published as: Mirjalili Mohanna SZ, Korecki AJ, and Simpson EM. rAAV-PHP.B escapes the mouse eye and causes lethality whereas rAAV9 can transduce aniridic corneal limbal stem cells without lethality. (2023). Gene Ther (online ahead of print). EMS designed the experiments, supervised the whole study, and secured funding, as well as reviewed and revised the manuscript. AJK generated and genotyped the mice, performed all the intravenous experiments including the post-injection monitoring of mice, and edited the viii manuscript. SZMM conducted the intrastromal and intravitreal injections of both viruses, monitored the mice, analyzed the results, performed the statistical analysis, made all the figures, and wrote the original draft of the manuscript. Ethics statement: All procedures involving mice in chapters 2-4 were in accordance with the guidelines set by the Canadian Council on Animal Care and approved by UBC Animal Care Committee (Protocols A17-0204, A17-0205, A21-0140, and A21-0184). ix Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ......................................................................................................................... ix List of Tables ................................................................................................................................xv List of Figures ............................................................................................................................. xvi List of Abbreviations ................................................................................................................. xix Acknowledgements ................................................................................................................... xxii Dedication ................................................................................................................................. xxiv Chapter 1: Introduction ................................................................................................................1 1.1 Aniridia, an eye disorder without a vision-saving therapy ............................................. 1 1.2 Extra-ocular phenotypes associated with PAX6 haploinsufficiency .............................. 4 1.3 Aniridia-associated microdeletion syndrome ................................................................. 4 1.4 Clinical diagnosis and management of aniridia .............................................................. 5 1.5 Therapeutic window for saving vision ............................................................................ 6 1.6 Clinical trials for aniridia ................................................................................................ 7 1.7 Recent advances in the preclinical treatment of aniridia ................................................ 8 1.8 CRISPR: a promising tool for treating aniridia .............................................................. 9 1.9 Viral and nonviral delivery vectors for CRISPR-based therapies ................................ 11 1.10 Sey mice mimic human aniridia .................................................................................... 13 1.11 Thesis objectives ........................................................................................................... 14 x Chapter 2: Germline CRISPR/Cas9-Mediated Gene Editing Prevents Vision Loss in a Novel Mouse Model of Aniridia ..................................................................................................16 2.1 Introduction ................................................................................................................... 16 2.2 Materials and Methods .................................................................................................. 19 2.2.1 Cloning ...................................................................................................................... 19 2.2.2 mRNA production and injection into Xenopus laevis............................................... 20 2.2.3 Cell Culture ............................................................................................................... 21 2.2.4 In vitro gene editing .................................................................................................. 22 2.2.5 Sanger and site-specific next generation sequencing ................................................ 23 2.2.6 Mouse husbandry ...................................................................................................... 23 2.2.7 In vivo gene editing ................................................................................................... 24 2.2.8 Slit lamp imaging ...................................................................................................... 25 2.2.9 Immunohistological analysis and image processing ................................................. 25 2.2.10 Embryonic day 18.5 fetus generation ................................................................... 26 2.2.11 Western blot analysis ............................................................................................ 27 2.3 Results ........................................................................................................................... 27 2.3.1 PAX6 and 3xFLAG/PAX6 constructs induced ectopic eyes in Xenopus laevis tadpoles ................................................................................................................................. 27 2.3.2 An optimized CRISPR strategy successfully inserted 3xFLAG tag in mouse embryonic stem cells............................................................................................................. 30 2.3.3 Endogenous Sey tagging created a novel mouse model for aniridia without compromising the protein function in the control mice ........................................................ 31 2.3.4 Sey mutation was efficiently corrected in mouse ESCs ............................................ 34 xi 2.3.5 In vivo gene editing corrected the Sey mutation with a 25% success rate ................ 37 2.3.6 Germline correction of the Sey mutation restored 3xFLAG-tagged PAX6 expression and normal eyes .................................................................................................................... 38 2.3.7 PAX6 level was increased in the brain of germline-corrected mice ......................... 40 2.4 Discussion ..................................................................................................................... 41 2.5 Acknowledgments......................................................................................................... 44 Chapter 3: LNP-Mediated Delivery of CRISPR RNP for Wide-Spread In Vivo Genome Editing in Mouse Cornea.............................................................................................................45 3.1 Introduction ................................................................................................................... 45 3.2 Materials and Methods .................................................................................................. 49 3.2.1 Mouse husbandry and breeding ................................................................................ 49 3.2.2 Isolation and culture of ex vivo mouse cortical neurons ........................................... 50 3.2.3 Preparation of Incisive DS LNP and encapsulation of RNP and DNA template ...... 51 3.2.4 Ex vivo genome editing ............................................................................................. 52 3.2.5 Immunocytochemistry (ICC) .................................................................................... 53 3.2.6 Stereological analysis................................................................................................ 54 3.2.7 Sanger sequencing and peak quantification .............................................................. 54 3.2.8 Statistical analysis ..................................................................................................... 55 3.2.9 In vivo genome editing .............................................................................................. 55 3.2.10 Immunohistochemistry (IHC) ............................................................................... 56 3.2.11 Microscopy and image processing ........................................................................ 57 3.2.12 Quantification of tdTomato expression in the cornea ........................................... 57 3.3 Results ........................................................................................................................... 57 xii 3.3.1 LNP delivery of an effective CRISPR RNP strategy turned on tdTomato expression in ex vivo cortical neurons..................................................................................................... 57 3.3.2 LNP delivery of the CRISPR RNP strategy corrected the Sey variant and restored FLAG-tagged PAX6 expression in 38% of ex vivo cortical neurons ................................... 59 3.3.3 LNP delivery of the CRISPR RNP strategy corrected the Sey variant in 17% of ex vivo cortical neurons ............................................................................................................. 62 3.3.4 Repeated treatment with LNP-RNP significantly increased the expression of both tdTomato (30%) and FLAG-tagged PAX6 (35%) proteins in ex vivo cortical neurons ....... 65 3.3.5 Repeated treatment with LNP-RNP increased, but not significantly, the correction of the Sey variant in ex vivo cortical neurons ............................................................................ 68 3.3.6 In vivo intrastromal delivery of the LNP-RNP CRISPR strategy turned on wide-spread tdTomato expression in the cornea ............................................................................ 70 3.3.7 In vivo intrastromal delivery of the LNP-RNP CRISPR strategy did not result in detectable correction of the Sey variant in the cornea........................................................... 73 3.4 Discussion ..................................................................................................................... 74 3.5 Acknowledgments......................................................................................................... 78 Chapter 4: rAAV-PHP.B Escapes the Mouse Eye and Causes Lethality Whereas rAAV9 can Transduce Aniridic Corneal Limbal Stem Cells Without Lethality ................................79 4.1 Introduction ................................................................................................................... 79 4.2 Materials and Methods .................................................................................................. 83 4.2.1 Mouse husbandry and breeding ................................................................................ 83 4.2.2 rAAV Production ...................................................................................................... 84 4.2.3 Intravenous injections ............................................................................................... 85 xiii 4.2.4 Intravitreal and intrastromal injections ..................................................................... 85 4.2.5 Slit lamp imaging ...................................................................................................... 87 4.2.6 Histology ................................................................................................................... 87 4.2.7 Microscopy and image processing ............................................................................ 88 4.2.8 Statistical analysis ..................................................................................................... 89 4.3 Results ........................................................................................................................... 89 4.3.1 PHP.B iCre injections caused lethality in mice, regardless of administration route 89 4.3.2 Intraocular injected rAAVs escaped from the eye .................................................... 94 4.3.3 In PHP.B ocular-injected mice tdTomato signal directly correlated with severity of ataxia ................................................................................................................................... 98 4.3.4 Intravitreal PHP.B and rAAV9 iCre injections resulted in transduction of a variety of retinal cells ...................................................................................................................... 100 4.3.5 Intrastromal PHP.B and rAAV9 iCre injections resulted in transduction of all three corneal cell layers ............................................................................................................... 102 4.3.6 Intrastromal rAAV9 iCre injections successfully transduced corneal LSCs in adult aniridic mouse eyes ............................................................................................................. 104 4.4 Discussion ................................................................................................................... 107 4.5 Acknowledgments....................................................................................................... 111 Chapter 5: Discussion ................................................................................................................112 5.1 Overview of major findings ........................................................................................ 112 5.2 Significance beyond the scope of the current thesis ................................................... 114 5.3 Limitations and caveats............................................................................................... 116 5.4 Future directions ......................................................................................................... 120 xiv 5.4.1 CRISPR/Cas9-mediated somatic gene editing in Fey mouse model of aniridia .... 120 5.4.2 Prospect of an in vivo CRISPR-based gene therapy for aniridia patients ............... 122 5.4.3 Prospect of an ex vivo CRISPR-based gene therapy for aniridia patients .............. 123 5.4.4 Finding a phenotype in the aniridic mouse retina ................................................... 124 5.4.5 Evaluating the potential of LNP delivery platform in the retina ............................ 125 5.4.6 Investigating the potential role of exogenous Apolipoprotein E in cellular uptake of LNPs in the cornea .............................................................................................................. 126 5.4.7 Improved administration routes for targeting the cornea ........................................ 127 5.4.7.1 Central vs. edge intrastromal injections .......................................................... 127 5.4.7.2 Dissolving microneedle patches ..................................................................... 128 5.4.7.3 Topical application.......................................................................................... 129 5.5 Concluding remarks .................................................................................................... 130 Bibliography ...............................................................................................................................131 Appendices ..................................................................................................................................173 Appendix A Supplementary Materials for Chapter 2 ............................................................. 173 Appendix B Supplementary Materials for Chapter 3 ............................................................. 177 Appendix C Supplementary Materials for Chapter 4 ............................................................. 180 xv List of Tables Table 2.1. The Sey phenotype was rescued in two mice by germline correction. ........................ 38 Table A.1. Western blot densitometry data generated for two technical replicates. .................. 175 Table A.2. CRISPR RNAs (crRNAs) and single-stranded oligodeoxynucleotide (ssODN) templates used for in vitro and in vivo gene editing. .................................................................. 176 Table B.1. CRISPR guide RNAs (gRNAs) and DNA templates used for ex vivo and in vivo genome editing. ........................................................................................................................... 178 Table B.2. Final mouse numbers and experimental groups for in vivo genome editing............. 179 xvi List of Figures Figure 1.1. Schematic of the human eye. ........................................................................................ 2 Figure 1.2. Schematic of key stages in embryologic development of the human eye. ................... 3 Figure 2.1. Generation of a new transgenic mouse model of aniridia for quantifying the impact of CRISPR therapy. ........................................................................................................................... 19 Figure 2.2. FLAG tag does not disrupt the ability of PAX6 to induce ectopic eyes in Xenopus laevis. ............................................................................................................................................ 29 Figure 2.3. Site-specific next generation sequencing revealed the correct insertion of 3xFLAG tag in 5.14% of sequences. ............................................................................................................ 31 Figure 2.4. Fey is a novel mouse model of aniridia and importantly the Fax control mice showed no phenotype. ................................................................................................................................ 33 Figure 2.5. In vitro CRISPR-mediated gene editing corrects the Sey mutation in Pax6Sey/Sey embryonic stem cells..................................................................................................................... 36 Figure 2.6. Germline correction of the Sey mutation rescued the mutant eye phenotype. ........... 39 Figure 2.7. Expression of 3xFLAG-PAX6 was confirmed in Fax and FeyCor mice. ................. 41 Figure 3.1. An optimized CRISPR RNP genome editing strategy resulted in the successful disruption of stop signals and turn on of tdTomato expression in ex vivo cortical neurons. ........ 59 Figure 3.2. A pre-established CRISPR RNP genome editing strategy resulted in FLAG-tagged PAX6 expression in 38% of ex vivo cortical neurons. .................................................................. 62 Figure 3.3. An optimized CRISPR RNP genome editing strategy corrected the Sey variant in 17% of ex vivo cortical neurons. ................................................................................................... 64 Figure 3.4. Repeated treatment significantly increased both tdTomato (30%) and FLAG-tagged PAX6 (35%) proteins in ex vivo cortical neurons. ........................................................................ 68 xvii Figure 3.5. Repeated treatment with LNP-RNP increased, but not significantly, the correction of the Sey variant in ex vivo cortical neurons. ................................................................................... 69 Figure 3.6. Intrastromally-delivered CRISPR genome editing resulted in wide-spread tdTomato expression in vivo in the cornea. ................................................................................................... 72 Figure 3.7. Intrastromally-delivered CRISPR genome editing strategy did not target PAX6-expressing corneal epithelial cells in vivo. .................................................................................... 74 Figure 4.1. Mice injected with PHP.B or rAAV9 iCre viruses had different survival curves. ..... 93 Figure 4.2. Intravitreally-delivered PHP.B and rAAV9 iCre viruses escaped the eye. ................ 95 Figure 4.3. Intrastromally-delivered PHP.B and rAAV9 iCre viruses escaped the eye. .............. 97 Figure 4.4. In PHP.B ocular-injected mice tdTomato signal was higher in ataxic versus healthy mice. ............................................................................................................................................ 100 Figure 4.5. Intravitreally-delivered iCre viruses successfully transduced a variety of retinal cells and some corneal cells. ............................................................................................................... 102 Figure 4.6. Intrastromally-delivered iCre viruses successfully transduced all three corneal cell layers. .......................................................................................................................................... 103 Figure 4.7. Corneal clouding was present in 3-month-old Sey corneas. ..................................... 105 Figure 4.8. Intrastromal injections of rAAV9 iCre successfully transduced corneal LSCs in Sey mice. ............................................................................................................................................ 107 Figure A.1. Eyes of adult homozygous Fax mice were not significantly different from WT. ... 173 Figure A.2. Eyes of heterozygous and homozygous Fax and FeyCor E18.5 fetuses were not significantly different from WT. ................................................................................................. 174 Figure B.1. The average diameter distribution of RNP loaded LNPs was 151.6 ± 0.8 nm. ....... 177 xviii Figure C.1. A variety of retinal and corneal cells were successfully transduced in WT and Sey mice after intravitreal delivery of PHP.B and rAAV9 iCre viruses. .......................................... 180 Figure C.2. All three corneal cell layers were successfully transduced in WT and Sey mice after intrastromal delivery of PHP.B and rAAV9 iCre viruses. .......................................................... 181 xix List of Abbreviations ApoE Apolipoprotein E B6 C57BL/6J BBB blood-brain barrier Cas CRISPR-associated protein cgEMS CRISPR guide EMS CNS central nervous system CRISPR clustered regularly interspaced short palindromic repeats crRNA CRISPR RNA DEV day ex vivo DPBS Dulbecco's phosphate-buffered saline DRG dorsal root ganglia DS delivery system DSB double stranded break E embryonic day EmGFP emerald green fluorescent protein EMS Elizabeth M. Simpson ESC embryonic stem cell Fax FLAG-tagged Pax6 Fey FLAG-tagged Sey FeyCor Fey corrected gRNA guide RNA HBSS Hank's balanced salt solution xx HDR homology-directed repair ICC immunocytochemistry iCre improved Cre iPSC induced pluripotent stem cell IHC immunohistochemistry ILM inner limiting membrane indels insertions and deletions LDL low-density lipoprotein LNP lipid nanoparticle LRP1 LDL receptor-related protein 1 LSC limbal stem cell mRNA messenger RNA NGS next generation sequencing NHEJ non-homologous end joining NHP non-human primate oEMS oligonucleotide EMS ORF open reading frame P postnatal day PAM protospacer adjacent motif PAX6 paired box 6 PB phosphate buffer PBS phosphate buffer saline PBS+P PBS plus 0.001% pluronic acid xxi PCR polymerase chain reaction PDI polydispersity index PFA paraformaldehyde PHP.B rAAV-PHP.B rAAV recombinant adeno-associated virus RNP ribonucleoprotein RPE retinal pigment epithelium Sey Small eye SD standard deviation smCBA small chicken beta actin promoter/CMV enhancer SpCas9 Streptococcus pyogenes Cas9 ssODN single-stranded oligodeoxynucleotide tdTomato tandem dimer Tomato THB tissue homogenization buffer tracRNA trans-activating crRNA vg viral genome WPRE woodchuck hepatitis virus post-transcriptional regulatory element WT wild type xxii Acknowledgements First and foremost, I would like to thank my supervisor, Dr. Elizabeth M. Simpson, without whom the completion of this work could not have been possible. Dr. Simpson, I would like to thank you for your open-door policy, never being too busy to answer my questions, and for patiently hearing out my ideas and giving me a chance to plan some of my experiments during my stay at your lab. Your commitment and kindness to your students, as well as your unmatched passion and dedication to aniridia research have continually inspired me and left a long-lasting, positive impact on me as a person and as a young scientist. Indeed, I could not have imagined having a better supervisor and mentor for my Ph.D. studies. Secondly, I would like to express my heartfelt gratitude to my Ph.D. supervisory committee members (Dr. Cheryl Gregory-Evans, Dr. Orson L. Moritz, and Dr. Colin Ross) for attending my progress meetings rain or shine, taking the time to read this thesis, and for providing me with invaluable feedback at various stages of my research that has greatly helped to improve the work presented here. Special thanks to the past and present members of the Simpson lab (Andrea Korecki, Diana Djaksigulova, Bethany Adair, Tess Lengyell, Siu Ling Lam and Nina Chiu) for their warm friendship, continuous support, and many stimulating discussions over the past seven years. I would also like to thank Dr. Jack Hickmott, an outstanding scientist and an incredibly kind human being, whom I had the pleasure of working with during my first two years in the Simpson lab. Jack, thank you for being an amazing mentor and for sharing your grad school experiences with me. Additionally, thanks for teaching me about the imposter syndrome and how not to fall victim to it. xxiii My highest praise to Allah, the almighty, for blessing me with the knowledge, creativity, perseverance, and skills needed to complete this work. I would like to particularly thank my parents, my sister, and my in-laws for their love, care, prayers, and encouragements that have always brought me joy and comfort and got me through challenging times over the course of this Ph.D. Thanks to my sweet daughter, Zoya, who was born during the final year of my Ph.D. studies and has filled my heart with love and my life with happiness ever since. Dokhtaram, thanks for being my biggest stress-reliever and my main source of entertainment during write up of this thesis. Last but not least, I am extremely grateful to my soulmate, Zamly, for always believing in me and constantly motivating me to be a better scientist. My love, thank you for all the rides to and from CMMT, satisfying my sweet tooth with my favorite desserts, lifting my spirit during difficult times of my Ph.D., and most importantly for giving up so much to make my studies a priority in our lives. You always cheered me and rushed to celebrate my academic achievements, big or small, and I adore you for that. I would like you to know that your unwavering support played a huge role in my academic performance and achievements. xxiv Dedication To the children with aniridia and their families who were the main motivation behind this work. 1 Chapter 1: Introduction 1.1 Aniridia, an eye disorder without a vision-saving therapy Although named for the lack of an iris, congenital aniridia is actually a panocular disorder. The ocular abnormalities seen in patients affect the iris, retinal fovea, optic nerve, lens, and cornea (Hingorani et al., 2012) (Figure 1.1). Foveal hypoplasia is one of the most frequently reported ocular abnormalities in aniridia and is responsible for low vision and nystagmus at birth. By adulthood, vision further deteriorates in patients due to the progressive corneal pannus and clouding (termed aniridia-associated keratopathy), cataracts, and glaucoma, leading to lifelong vision impairment and eventual blindness (Daruich et al., 2022; Yazdanpanah et al., 2020). Aniridia is a rare disorder with a worldwide prevalence of 1 in 40,000 to 1 in 100,000 live births, with no known influence of race or sex (Hingorani et al., 2012). In Canada, aniridia is estimated to affect 1 in 60,000 individuals [Ross M, retrieved from https://emedicine.medscape.com/article/1208379-overview, March 27, 2018]. Considering the current population of Canada, an estimated 644 individuals suffer from this debilitating eye disorder. Aniridia is a monogenic disorder caused by autosomal dominantly inherited, or less commonly, de novo variants in the paired box gene 6 (PAX6) gene (Hingorani et al., 2012). PAX6 encodes a highly conserved transcription factor that is crucial for normal development of the eye (Glaser et al., 1992). Moreover, PAX6 is regarded as the master regulator of the eye, since its overexpression is sufficient to result in the formation of ectopic eyes in Drosophila melanogaster (Halder et al., 1995) and Xenopus laevis (Chow et al., 1999). In the eye, during early embryonic development, PAX6 is expressed in both neuroectoderm and surface ectoderm. During late stages of embryogenesis, PAX6 is expressed in both neural and pigmented retinal 2 layers that are derived from neuroectoderm. PAX6 is also expressed in the lens vesicle and corneal epithelium that are derived from the surface ectoderm (Grindley et al., 1995; Nishina et al., 1999; Terzic and Saraga-Babic, 1999) (Figure 1.2). Postnatally, PAX6 expression is limited to the retina, ciliary body, iris, lens, conjunctiva, and cornea (Davis et al., 2009; de Melo et al., 2003; Hickmott et al., 2016; Koroma et al., 1997; Roesch et al., 2008; Shaham et al., 2009; Stanescu et al., 2007). Aniridia is a haploinsufficiency disorder, which means one functional PAX6 allele is unable to produce sufficient PAX6 protein necessary for the normal development of the eye (Azuma et al., 1998; Schedl et al., 1996; Ton et al., 1991). Figure 1.1. Schematic of the human eye. Light enters the eye through the cornea. The iris together with the pupil control the amount of light that reaches the lens. The lens refracts the light and focuses it onto the retina. The retina processes the light into neural signals, and subsequently transmits them to the brain through the optic nerve, where they can be interpreted. 3 Figure 1.2. Schematic of key stages in embryologic development of the human eye. A) In the fourth week of gestation, neural ectoderm grows towards the overlying surface ectoderm to form the optic vesicle. The area of surface ectoderm that comes into contact with optic vesicle thickens and forms the lens placode. B) In the fifth week of gestation, optic vesicle folds on itself to form a two-fold structure known as optic cup. The lens placode invaginates to form the lens pit. C) In the sixth week of gestation, the inner layer of optic cup becomes deeper due to the lens pit invagination. The lens pit completely closes and detaches from the surface ectoderm to form a hollow sphere known as the lens vesicle. Surface ectoderm adjacent to lens vesicle differentiate into presumptive cornea. D) In the seventh week of gestation, optic cup continuous to differentiate. The inner layer of optic cup forms the neural retina and the outer layer of it forms the retinal pigment epithelium. The lens vesicle is filled up to become the crystalline lens. Corneal cells are completely separate from the lens surface. 4 1.2 Extra-ocular phenotypes associated with PAX6 haploinsufficiency Besides the eye, PAX6 is also expressed in both the developing and adult brain, olfactory system, and endocrine pancreas (Hart et al., 2013; Simpson and Price, 2002; Yogarajah et al., 2016). PAX6 haploinsufficiency affects these extra-ocular tissues, resulting in a spectrum of medical conditions in aniridia patients, such as hearing difficulties, anosmia or hyposmia, challenges in information processing, sleep disorders, impaired glucose tolerance, increased average body mass index, and obesity (Abouzeid et al., 2009; Bamiou et al., 2007; Berntsson et al., 2020; Hanish et al., 2016; Hanish and Han, 2018; Netland et al., 2011; Yasuda et al., 2002; Yogarajah et al., 2016). There are other sites of PAX6 expression such as testis, appendix, small intestine, colon, rectum and stomach [The Human Protein Atlas, retrieved from https://www.proteinatlas.org/ENSG00000007372-PAX6/tissue#expression_summary, April 22, 2023] that do not cause a known phenotype, and thus are presumably not sensitive to PAX6 dosage. 1.3 Aniridia-associated microdeletion syndrome Aniridia can be part of WAGR syndrome, where the letter W stands for Wilms tumor, A for aniridia, G for genitourinary anomalies, and R for mental retardation. WAGR is a rare syndrome that occurs once in every 500,000 to 1,000,000 live births in the general population, regardless of sex, and involves contiguous deletion of PAX6 and WT1 genes on a region of Chromosome 11 (11p13) (Breslow et al., 2003). Deletion of PAX6 causes the classical aniridia, while deletion of WT1 predisposes the children to developing Wilms tumor, which is a malignant kidney tumor (van Heyningen et al., 2007). The clinical symptoms of WAGR vary from person to person depending on the extent of the deletion of chromosomal region 11p13 (Fischbach et al., 2005). Therefore, some patients with WAGR do not manifest all conditions associated with this 5 syndrome. A patient is formally diagnosed with WAGR syndrome when they have aniridia and at least one other main clinical symptom (Wilms tumor, genitourinary anomalies, or mental retardation). 1.4 Clinical diagnosis and management of aniridia Complete or partial lack of the iris (termed iris hypoplasia) is often detected soon after birth. However, some individuals are born with subtle abnormalities in the iris that might be harder to detect by untrained eyes (Hingorani et al., 2012). Therefore, a thorough examination by an ophthalmologist is needed to further evaluate the iris and other parts of the eye in these patients. Clinical testing is often accompanied by genetic testing to confirm the diagnosis (Daruich et al., 2022). Once a diagnosis has been established, patients with aniridia must undergo frequent eye examinations for early detection of secondary ocular complications that can further deteriorate the vision. Early detection is especially critical for glaucoma (defined as increased intraocular pressure leading to eventual death of retinal ganglion neurons and optic nerve damage) that happens in about 50% of aniridia patients (Netland et al., 2011; Vernazza et al., 2021). Some of the current interventions for aniridia include: 1) low vision aids to improve the visual function, especially in younger patients; 2) tinted or photochromic glasses to manage photophobia; 3) topical application of preservative-free lubricants for mild keratopathy, autologous serums, and amniotic membrane transplants for moderate keratopathy, and limbal stem cell (LSC) transplants or keratoprosthesis for treating severe keratopathy; 4) surgical extraction of cataracts; and 5) use of intraocular pressure–lowering drops, laser treatment or surgery for managing glaucoma (Lee et al., 2008a; Samant et al., 2016). Early detection and intervention for glaucoma has shown to noticeably delay the legal blindness or in some cases even prevent the legal blindness caused by glaucoma in aniridia (Jacobson et al., 2022; Netland, 6 2015). Nevertheless, new treatment strategies are urgently needed for aniridia, since in most cases, the current treatments for aniridia-associated keratopathy cannot prevent the typical eventual legal blindness in patients. 1.5 Therapeutic window for saving vision The fovea is a pit-like structure, which is located in the central part of the macula within the retina and it is responsible for the sharp central vision (Rehman et al., 2023). Foveal hypoplasia is present in as high as 90% of patients with aniridia and results in variably reduced visual acuity from birth (Chang et al., 2014; Landsend et al., 2021). The fovea begins to form during early gestation but its development does not complete prenatally. In fact, the fovea continues to develop after birth and only reaches morphological maturity when a child is four to five years old (Hendrickson et al., 2012; Hendrickson and Yuodelis, 1984), offering a therapeutic window for intervention in children with aniridia to improve vision by supporting the postnatal development of the fovea. Corneal clouding, which is secondary to LSC deficiency, is another clinical symptom that is reported in up to 90% of aniridia patients (Dyer et al., 2021). Although varying degrees of peripheral pannus can be present in aniridic corneas at birth, the majority of patients have a clear central cornea during early childhood. Usually the slowly progressing corneal clouding advances from the periphery, to the center of the eye, over the first two to three decades of life, leading to a gradual vision loss in these patients (Eden et al., 2010; Lee et al., 2008a; Schanilec and Biernacki, 2014). The progressive nature of vision-threatening corneal clouding in aniridia presents a great opportunity for early intervention to delay or prevent blindness. 7 1.6 Clinical trials for aniridia Nonsense suppression therapy is a viable approach for increasing the protein levels in haploinsufficiency disorders such as aniridia, where a substantial number of patients carry variants that introduce premature stop codons in the coding region of PAX6 (Kit et al., 2021; Tzoulaki et al., 2005), leading to the degradation of truncated mRNA transcripts via the process of nonsense mediated decay (Latta et al., 2017; Liu et al., 2015; Zhang et al., 2017). Topical application of Ataluren, a nonsense suppression drug, has shown to reverse the abnormal eye phenotype and subsequently prevent vision loss in a Pax6-deficient mouse model of aniridia (Gregory-Evans et al., 2013; Wang et al., 2017). This promising preclinical data paved the way for a double-masked, placebo-controlled, phase 1/2 clinical trial for aniridia (ClinicalTrials.gov identifier: NCT02647359), aiming to evaluate the safety and efficacy of orally-administered Ataluren for the treatment of nonsense-variant aniridia. Despite the preclinical success, unfortunately this clinical trial failed to meet its primary endpoint of improving vision, resulting in the cancellation of the planned phase 2 open label extension study (ClinicalTrials.gov Identifier: NCT04117880). The difference in the administration route of Ataluren between the preclinical studies (topical) and clinical trial (oral) could be a contributing factor to the unsatisfactory results achieved in the clinical trial. Perhaps the use of an alternative Ataluren eye drop with an improved formulation and stability (Djayet et al., 2020) can open up the possibility for further clinical investigation. Currently, there is an interventional study in clinical trial for aniridia that involves transplantation of both healthy limbal epithelial stem cells and a stromal support layer to the patients’ eyes (ClinicalTrials.gov: NCT05044598). However, if this therapy is successful, it will 8 be exclusive to patients who are diagnosed with advanced aniridia-associated keratopathy and will not help with managing the initial ocular problems and other aspects of this disorder. 1.7 Recent advances in the preclinical treatment of aniridia Gene augmentation therapy is an ideal approach for haploinsufficiency disorders, as it aims to deliver a functional copy of the gene to the affected cells to increase the protein levels and treat the condition. Luxturna® (Russell et al., 2017) and Zolgensma® (Day et al., 2021) are examples of FDA-approved gene augmentation therapies that have achieved stunning results for treating individuals with Leber congenital amaurosis and spinal muscular atrophy, respectively. The major obstacle facing effective PAX6 augmentation gene therapy is the extreme dosage sensitivity of this gene. Like haploinsufficiency, PAX6 overexpression in mice has been shown to negatively affect normal eye development (Davis and Piatigorsky, 2011b; Dora et al., 2008; Manuel et al., 2008; Ouyang et al., 2006; Schedl et al., 1996). Thus, a precise regulation of PAX6 in many different cell types will be necessary to achieve therapeutic success. Gene augmentation therapy for aniridia has been shown to reverse the corneal epithelial thinning after a single intrastromal injection of a PAX6 augmentation vector directly into the cornea of Pax6-deficient mice (Hickmott, 2018). However, the observed increase in the thickness of the epithelial layer was transient, which is likely due to the augmentation of PAX6 levels in the terminally differentiated cells of the corneal epithelium that are rapidly turned over and lost from the surface of the cornea (Douvaras et al., 2013). Therefore, more research must be done on optimizing a PAX6 augmentation therapy for aniridia to safely prolong the effect of the treatment. Other emerging therapies for aniridia take advantage of the existing functional copy of PAX6 to postnatally increase PAX6 levels in the target cells. One such therapy demonstrated that 9 topical and oral administration of PD0325901, a mitogen-activated protein kinase kinase inhibitor, to Pax6-deficient mouse pups was able to induce the expression of the wild-type Pax6 allele, which in turn resulted in the elevation of PAX6 levels in the cornea, reduced corneal clouding, and improved vision (Rabiee et al., 2020). Since there was no therapeutic impact on the retinal structure, authors attributed the observed improvement in vision to enhanced light passage into the eye as a result of a reduction in the cloudiness level in the cornea in treated mice. Interestingly, in a follow-up study authors evaluated the long-term effects of PD0325901 on maintaining the visual acuity, and reported that even about a year after stopping the drug application, the treated mice had reduced intraocular pressure and better vision, compared to untreated mice (Cole et al., 2022). As exciting as this approach appears, it has the potential pitfall that prolonged inhibition of mitogen-activated protein kinase kinase may induce ocular toxicity (LoRusso et al., 2010; Stjepanovic et al., 2016), leading to retinal optic neuropathy and retinal vein occlusion that can accelerate vision loss in aniridia patients. Hence, even though the initial studies did not report any adverse effects of PD0325901 in mice (Cole et al., 2022; Rabiee et al., 2020), the safety of this drug must be further assessed in future preclinical studies that ideally utilize nonhuman primates. 1.8 CRISPR: a promising tool for treating aniridia CRISPR/Cas was first discovered as part of the prokaryotic adaptive immune system that aids in protection from repeated viral infections (Ishino et al., 1987; Jansen et al., 2002; Mojica et al., 2005). Repurposing this system for gene editing of eukaryotic cells (Cong et al., 2013) has revolutionized the biomedical field and intrigued researchers worldwide to explore further the utility of this system for treating many genetic disorders (Chen et al., 2022a; Erkut and Yokota, 2022; Mohammadian Gol et al., 2022; Yu and Wu, 2020). 10 CRISPR-associated protein 9 (Cas9) is the most common and well-studied Cas nuclease for gene editing (Makarova and Koonin, 2015). CRISPR/Cas9-mediated gene editing is carried out in three steps. In the first step, a guide RNA (gRNA) directs the Cas9 nuclease to the correct region of the genome through complementary base pairing with the target DNA. In the next step, the Cas9 nuclease recognizes the appropriate protospacer adjacent motif (PAM) and subsequently introduces a double-stranded break (DSB) in the DNA. In the final step, the DSB is either repaired by the error-prone non-homologous end joining (NHEJ) pathway, resulting in the creation of insertions or deletions (indels), or in the presence of homologous template DNA, the DSB may be repaired through homology-directed repair (HDR), allowing for precise editing of the DNA (Asmamaw and Zawdie, 2021). The CRISPR-mediated HDR pathway was initially thought to only occur in actively dividing cells (Cox et al., 2015), subsequently it was discovered that HDR can even happen in non-dividing (post-mitotic) cells (Nishiyama et al., 2017). In the last 8 years, CRISPR gene editing has shown great potential for successfully treating various ocular disorders in vivo (Gumerson et al., 2022; Jain et al., 2017; Kim et al., 2017; Maeder et al., 2019; Shahin et al., 2022). The most exciting of them is EDIT-101 (the first in-human administration of a CRISPR-based therapy), which was developed for the treatment of Leber congenital amaurosis 10 (Maeder et al., 2019). In this approach, CRISPR components are directly delivered to the photoreceptors via a subretinal injection, in order to remove an aberrant splice donor, created by an intronic c.2991+1655A>G variant in the CEP290 gene, leading to the restoration of full-length and functional CEP290 proteins. EDIT-101 is currently being evaluated for safety, efficacy, and tolerability in a phase 1/2 clinical trial (ClinicalTrials.gov identifier: NCT03872479). CRISPR-mediated gene editing is a particularly appealing approach for treating aniridia, as it allows for correction of the PAX6 gene while it is expressed under its natural 11 promoter and other regulatory elements, thereby eliminating any concerns with inappropriate expression of this dosage-sensitive gene. 1.9 Viral and nonviral delivery vectors for CRISPR-based therapies A successful gene editing event relies on the efficient delivery of CRISPR components to the nucleus of the target cells. Components of the CRISPR gene editing machinery can be delivered in the format of: plasmid DNA, messenger RNA, or ribonucleoprotein (RNP) complex (Zhang et al., 2021). Approaches for the delivery of these CRISPR cargos fall into two main categories: viral and nonviral vectors (Wilbie et al., 2019). Each of these vectors have their own unique advantages and disadvantages that are briefly discussed here. Recombinant adeno-associated viruses (rAAVs) are the leading viral vector for the in vivo delivery of CRISPR/Cas9 components (Asmamaw Mengstie, 2022). rAAVs are non-pathogenic in humans, generally elicit a tolerable immune response (Verdera et al., 2020), and most importantly exhibit broad tissue tropism for achieving optimal transduction efficiency, which is required for many gene therapy applications (Castle et al., 2016; Srivastava, 2016). In addition, rAAVs have been approved by the FDA for clinical use in two gene augmentation therapies (Luxturna® and Zolgensma®) (Day et al., 2021; Russell et al., 2017). rAAV is also the vector used in the EDIT-101 clinical trial (Maeder et al., 2019). Some of the main disadvantages of rAAVs include: cargo size constraints (Dong et al., 1996), undesirable toxicity at high doses, immunogenicity of capsids upon re-administration (Ertl, 2022), and high manufacturing cost (Hitchcock, 2021). The limited cargo capacity of rAAV (up to 4.9 kb) is especially problematic, since it means packaging the widely-used Cas9 nuclease derived from Streptococcus pyogenes (SpCas9) (4.1 kb) only leaves room for a minimal promoter in the vector construct, which might result in reduced gene expression in comparison to a full-length promoter (Gray et al., 2011). 12 Furthermore, the gRNA and its promoter, as well as the template DNA (if required), must be packaged in a second rAAV vector and co-delivered with the SpCas9 rAAV. This “two-virus system” makes delivery of the gene editing machinery to the target cells more challenging, since successful gene editing depends on the transduction of target cells by both these rAAVs. For many applications such as gene augmentation therapy, sustained gene expression of rAAV in nondividing and slowly dividing target cells is an advantage (Herzog et al., 1999; Ramsingh et al., 2018; Wang et al., 2007). In contrast, CRISPR-based therapies often benefit from a hit-and-run strategy, and persistent expression of Cas9 and gRNA, can significantly increase the chance of off-target events (Hanlon et al., 2019). Due to the mentioned disadvantages of viral vectors, particularly in the context of CRISPR therapy, the use of nonviral vectors is becoming more popular. Lipid nanoparticles (LNPs) are the most clinically advanced nonviral vector (Yan et al., 2022). LNPs are capable of packaging large cargo, are easier to manufacture, and are scalable (Kenjo et al., 2021). Furthermore, the low immunogenicity of LNPs allows for re-administration of the therapy (Kenjo et al., 2021). Recently, LNPs have been successfully used in numerous preclinical studies (Gillmore et al., 2021), and a phase 1 clinical trial (ClinicalTrials.gov Identifier: NCT04601051) for the systemic delivery of CRISPR components to treat the life-threatening disease, transthyretin amyloidosis. The biggest advantage of LNPs for CRISPR delivery is the ability to directly deliver the RNP format of CRISPR components, bypassing the need for both the transcription and translation steps (Wei et al., 2020). This gives LNPs better control over the duration of Cas9 and gRNA expression, and thereby greatly reduces potential off-target events. Despite these advantages, the lower efficiency of LNP delivery of CRISPR components in comparison to rAAV, is currently the main barrier preventing broader use of this 13 vector in gene editing therapies (Duan et al., 2021). LNP composition optimization and/or chemical modification may aid in overcoming this obstacle. 1.10 Sey mice mimic human aniridia Mouse models are a valuable tool for therapy development for aniridia, because of the many similarities between mouse and human in terms of eye development, anatomy, and physiology (Chang, 2013). Furthermore, the mouse and human PAX6 proteins have identical amino acid sequences (Tyas et al., 2006). Short generation time, small size, and relatively low maintenance cost are some of the other advantages that make mice the animal model of choice for large experimental cohort studies (Bryda, 2013). To date, several Pax6 disease mouse models have been described in the literature. These mice either have a Pax6 pathogenic variant or whole gene deletion that has occurred spontaneously, induced by chemicals/radiation, or created by targeted mutation methods (Abdolkarimi et al., 2022). Among these models, the Pax6 Small eye (Sey) is a well-characterized and widely-acceptable mouse model for aniridia (Collinson et al., 2004; Gregory-Evans et al., 2013; Hickmott et al., 2018; Li et al., 2007; Ramaesh et al., 2003; Wang et al., 2017). Sey mice arose as a result of a spontaneous variant and were first described in the literature in 1967 (Roberts, 1967). Eventually, it was discovered that these mice carry a heterozygous nonsense variant in exon 8 of Pax6 (Hill et al., 1991), which has been also reported in an aniridia patient (Hever et al., 2006; Kit et al., 2021). The iris hypoplasia, as well as the lens and corneal abnormalities seen in these mice, closely resemble those of aniridia patients (Gregory-Evans et al., 2013; Hickmott et al., 2018; Ramaesh et al., 2003; Wang et al., 2017), making Sey an excellent model for testing new therapeutics for aniridia. Sey mice have been used in preclinical studies for testing new drugs for patients with aniridia (Gregory-Evans et al., 2013; Wang et al., 2017). 14 Despite significant contributions to the field, mouse models are not without limitations. The mouse retina lacks a fovea (Huberman and Niell, 2011) and this major anatomical difference makes it difficult to draw any direct conclusions regarding the impact of a therapy on foveal hypoplasia, which is one of the main features of aniridia. Additionally, the smaller corneal thickness (Matthaei et al., 2012) and lower level of vitreous body (a gel-like matrix that fills the space around the lens and between the lens and the retina) due to presence of a large lens (Rosch et al., 2014), make delivery by intrastromal and intravitreal injections technically challenging. Finally, it is known that the severity of phenotypes observed in different mouse strains can differ due to the epistasis between the Pax6 gene and genetic background (Hickmott et al., 2018). For example, inbred C57BL/6J Sey mice have been reported to have severe microphthalmia or even in some instances unopened eyes that are not part of the typical aniridia phenotype. Inbred mice are homozygous at all loci, hence, the observed phenotype in these mice might not represent human aniridia well, confounding therapy development for this disease (Hickmott et al., 2018). To overcome this hurdle, researchers can use hybrid mice in preclinical studies of aniridia. Hybrid mice genetically deviate from the two inbred parental strains, therefore, can relatively better capture the genetic variation seen in humans. Epistasis between Sey genotype and hybrid genetic background normalizes severe phenotypes observed in unnatural inbred strains and contributes to a more patient-like expression of aniridia phenotypes in these mice (Hickmott et al., 2018). 1.11 Thesis objectives Preclinical studies on small animal models are an essential first steps towards developing a gene therapy. The results from such studies indicate if there is any value in proceeding to safety and efficacy studies in available intermediate animal models (for instance dogs, pigs, or 15 nonhuman primates are often used as a secondary model between rodent and human studies), and the potential for future translation to clinical trials. Aniridia does not have an intermediate animal model at this time, and despite this, the FDA approved Ataluren for clinical trial (ClinicalTrials.gov identifier: NCT02647359). This suggests that the lack of an intermediate animal model for aniridia may similarly not hold back any CRISPR therapies from moving directly from pre-clinical mouse work into clinical trials. The broad goal of this thesis is to develop a CRISPR therapy for treating a novel mouse model of aniridia as a proof-of-principle. Since effective delivery is a pivotal step in ensuring the success of a therapy, the main focus of this thesis is to find an optimal method for delivering CRISPR components to the target cells of interest. We hypothesize that delivering CRISPR components with an optimized method can increase the expression of PAX6 in the target cells, improve the structure and function of the eye, and ultimately rescue the phenotype in the novel mouse model of aniridia. The specific objectives of each data chapter are: Chapter 2: to create a novel mouse model of aniridia that is optimized for CRISPR therapy development and subsequently develop an efficient CRISPR strategy to correct the Sey pathogenic variant in vitro in the mouse embryonic stem cells and in vivo in the germline of the mouse; Chapter 3: to evaluate the potential of LNPs for the ex vivo and in vivo delivery of Cas9 RNPs to the mouse cortical neurons and cornea, respectively; Chapter 4: To determine the best rAAV capsid and administration route for a future CRISPR gene editing therapy for aniridia. 16 Chapter 2: Germline CRISPR/Cas9-Mediated Gene Editing Prevents Vision Loss in a Novel Mouse Model of Aniridia1 2.1 Introduction The paired box 6 gene (PAX6) encodes a transcription factor that controls many aspects of early development of the central nervous system, eye, and some non-neuronal tissues such as the pituitary and pancreas (Simpson and Price, 2002). This transcription factor is conserved in evolution and has complex regulatory regions that span beyond 200 kilobases (Tyas et al., 2006). Heterozygous loss-of-function mutations of PAX6 result in a rare congenital eye disorder known as aniridia (Vincent et al., 2003). Patients with aniridia are typically born with low vision, mainly due to fovea hypoplasia, and become legally blind in young adulthood due to progressive sight-threatening complications including glaucoma, cataract, and corneal opacification (Calvao-Pires et al., 2014). Currently, there are no long-term vision-saving therapies or cures for aniridia, however some of the secondary ocular abnormalities can be partially managed through medical and surgical interventions with limited success, as surgeries on aniridic eyes can expedite aniridia-related keratopathy and worsen vision (Eden et al., 2010; Tsai et al., 2005). Therefore, new treatment strategies are needed for aniridia. One possible approach to treating aniridia is gene therapy, where genetic material is delivered into cells to make the required protein (augmentation) or correct the defective gene (gene editing) (Naldini, 2015). However, like haploinsufficiency, PAX6 overexpression has been 1 This chapter has been published in Molecular Therapy–Methods & Clinical Development as: Mirjalili Mohanna et al. (2020). Germline CRISPR/Cas9-Mediated Gene Editing Prevents Vision Loss in a Novel Mouse Model of Aniridia. Mol Ther Methods Clin Dev. 17:478-490. PMID: 32258211. See Preface for details of my contributions. 17 shown to negatively affect normal eye development (Davis and Piatigorsky, 2011a; Manuel et al., 2008; Ouyang et al., 2006; Schedl et al., 1996). Thus, very precise regulation of PAX6 would be necessary to achieve therapeutic success with augmentation gene therapy (Hickmott et al., 2016). In contrast, gene editing, for example by clustered regularly interspaced short palindromic repeats (CRISPR), can be used to revert a mutation to the correct sequence in the genome (Ran et al., 2013), thereby allowing for the normal regulation of expression levels and timing in appropriate cell types. Unfortunately, CRISPR-mediated gene editing can also create small insertions and deletions (indels), or even large deletions and complex rearrangements, which are important considerations when taking this approach (Kosicki et al., 2018). Studies on cellular and animal models are often the first steps towards developing a gene therapy. In the case or aniridia, this approach is supported by the fact that the human PAX6 gene is homologous to the mouse Pax6 gene, and the protein products from these two genes have identical amino acid sequences (Gehring, 1996). The small eye (Sey) mouse was first described in 1996 and is a widely accepted model for aniridia, which carries a spontaneous point mutation causing a pre-mature stop codon in exon 8 of the Pax6 gene (Hill et al., 1991; Hogan et al., 1986; Roberts, 1967). Mice that are homozygous for the Sey allele are anophthalmic and die shortly after birth, whereas mice that are heterozygous for Sey are viable and exhibit iris hypoplasia, lens abnormalities, and corneal clouding (Hill et al., 1991; Hogan et al., 1986; Ramaesh et al., 2003). In both cases, the phenotypes are comparable in humans, with the latter modeling aniridia (Ramaesh et al., 2003; Yokoi et al., 2016). Furthermore, the mutation present in Sey mice (G194X) has been reported in a patient with aniridia (Hever et al., 2006), making Sey an exciting model for proof-of-principle gene therapy studies. 18 Given the role of PAX6 in stem/progenitor cells (Sansom et al., 2009), and the overall plasticity of the eye (Gregory-Evans et al., 2013; Wang et al., 2017), we think a postnatal CRISPR-based gene therapy is a feasible option for treating aniridia. Supporting this is evidence of successful delivery of CRISPR/Cas9 components via viral vectors to rodent and non-human primate eyes (Giannelli et al., 2018; Hung et al., 2016; Jo et al., 2019; McCullough et al., 2019; Ruan et al., 2017; Yu et al., 2017), as well as the ability of the aniridic mouse eye to improve after augmenting PAX6 levels postnatally (Gregory-Evans et al., 2013; Wang et al., 2017). Therefore, we hypothesized that increasing the expression of PAX6 through a CRISPR-based gene editing strategy would improve the structure and function of the eye, and ultimately rescue the mutant phenotype in a mouse model of aniridia. For in vivo CRISPR strategy development, it would be advantageous to have a mouse model that allows us to distinguish both the gene and protein of wild-type (WT) PAX6 from CRISPR-corrected mutant PAX6, for quantification of the targeted genome editing efficacy (Figure 2.1). Thus, using CRISPR for endogenous-gene tagging we have created a new model for preclinical CRISPR-based therapy development for aniridia. Furthermore, since the Sey mutation was first identified over half a century ago, there was a possibility that the Sey locus might have acquired additional genetic mutations over time (Roberts, 1967). Hence, as an important step, we validated the causality of this mutation by germline correction. Lastly, we developed a CRISPR allele-distinguishing strategy that successfully corrected the Sey mutation both in vitro and in vivo in the mouse germline. 19 Figure 2.1. Generation of a new transgenic mouse model of aniridia for quantifying the impact of CRISPR therapy. Sey mice are not optimal for testing CRISPR-based therapeutic approaches for aniridia, as the CRISPR-corrected protein would be not different from the wild-type (WT) counterpart. Insertion of a 3xFLAG tag on the Sey allele will enable quantitative assessment of our Pax6 gene editing therapy by creating a corrected form that can be distinguished from WT by antibody labeling. Inserting the same tag on the WT allele will create an essential control mouse to ensure the addition of the 3xFLAG tag does not interfere with the PAX6 protein function. Ab, antibody; Fax, FLAG-tagged Pax6; Fey, FLAG tagged Sey; Sey, Pax6 small eye. 2.2 Materials and Methods 2.2.1 Cloning To generate plasmids for in vitro transcription, emerald GFP (EmGFP), PAX6, 3xFLAG/PAX6, and PAX6/3xFLAG open reading frames (ORFs) were cloned into pBluescript as previously described (de Leeuw et al., 2016; Hickmott et al., 2016). Briefly, EmGFP, PAX6, 3xFLAG/PAX6, and PAX6/3xFLAG ORFs, flanked by NotI restriction sites, were commercially synthesized (DNA2.0, Menlo Park, CA). pBluescript and DNA2.0 plasmids containing EmGFP, 20 PAX6, 3xFLAG/PAX6, and PAX6/3xFLAG were electroporated into 50 μL of One Shot™ TOP 10 electrocomp™ Escherichia coli (C404052; Thermo Fisher Scientific, Waltham, MA) as directed, and cultured on Luria-Bertani (LB) plates. Resulting colonies were picked, cultured, and the DNA was prepped (27104; Qiagen, Germantown, MD). The resulting plasmids were digested with NotI (R0189; New England Biolabs, Ipswich, MA), size separated, gel extracted, and the ORFs and ligated into pBluescript using T4 DNA ligase (M0202; New England Biolabs). Ligation products were electroporated into TOP 10 cells, cultured, and DNA was prepared as described above. Correct ligation was confirmed by restriction digest with NcoI (R0193; New England Biolabs) and XhoI (R0146; New England Biolabs), as well as Sanger sequencing that was executed at Centre for Molecular Medicine and Therapeutics (CMMT) DNA Sequencing Core Facility. 2.2.2 mRNA production and injection into Xenopus laevis To produce mRNA for injection into Xenopus laevis (X. laevis) embryos, EmGFP, PAX6, 3xFLAG/PAX6, and PAX6/3xFLAG ORFs were in vitro transcribed, polyadenylated, and 5′ capped. Plasmids were linearized and the T7 promoter was used to produce 5′ capped, polyadenylated, mRNA using the mMESSAGE mMACHINE T7 ultra kit (AM1345; Thermo Fisher Scientific) as directed. The integrity of the resulting mRNA was confirmed by gel electrophoresis, and mRNA concentration was determined using a nanodrop spectrophotometer. X. laevis females were injected with 700 units of human chorionic gonadotropin and housed in 18oC tadpole ringer overnight. Embryos were produced by in vitro fertilization and 50 ng of mRNA was injected into each embryo at the 16-cell stage. The resulting embryos were reared in 18°C tadpole ringer solution, in 4 L tanks, on a 12-hour light/dark cycle. Fourteen days post 21 conception, tadpoles were fixed in 4% paraformaldehyde and screened for the formation of ectopic eye structures using a white light dissecting microscope. 2.2.3 Cell Culture Pax6Sey/Sey and Pax6+/+ embryonic stem cells (ESCs) were produced by setting up timed pregnancies with B6-Pax6Sey/+ dam mice and 129-Pax6Sey/+ stud males, and B6-Pax6+/+ dam mice and 129-Pax6+/+ stud males, respectively. The resulting embryos were harvested and treated as previously described (Yang et al., 2009). The ESCs were cultured in high glucose Dulbecco's modified Eagle's medium (DMEM) (LS11960044; Thermo Fisher Scientific) supplemented with 1,000 units/ml ESGRO recombinant mouse leukemia inhibitory factor (ESG1107; Chemicon/MilliporeSigma, Burlington, MA), 16% fetal bovine serum (080-150; Wisent Bioproducts, Canada), 0.1 mM MEM non-essential amino acids solution (11140050; Thermo Fisher Scientific), 2 mM L-Glutamine (25030081; Thermo Fisher Scientific), and 0.01% 2-Mercaptoethanol (M7522; Sigma-Aldrich, St. Louis, MO). ESC lines were genotyped for Pax6 using WT specific polymerase chain reaction (PCR) assay with primer pair oEMS6076 (AACACCAACTCCATCAGTTCTAATG), and oEMS6075 (GCAAAATATAAGCATCCCAGTGCAT), as well as, Sey specific PCR assay with primer pair oEMS6071 (TCACTCTATTTTCCCAACACAGCC), and oEMS6073 (CTGAGCTTCATCCGAGTCTTCTTA). The selected ESC lines were expanded on mouse embryonic fibroblast cells until passage 8. Subsequently, the cells were maintained on tissue culture plates coated with 0.1% gelatin (G7-500; Thermo Fisher Scientific) in sterile distilled water for minimum of 5 passages prior to use in in vitro gene editing experiments. 22 2.2.4 In vitro gene editing The online tool Benchling (https://benchling.com/crispr) was used to design guide RNAs (gRNAs) for the Streptococcus pyogenes CRISPR associated protein 9 (Cas9) nuclease in the region of interest. The top three gRNAs were selected based on their predicted on-target and off-target effects, and their proximity to either the Pax6 start codon for 3xFLAG insertion, or the Sey mutation for the correction strategy. The commercially synthesized CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) (GenScript, Piscataway, NJ) were annealed at a 1:1 ratio by incubating at 95°C for 5 minutes to generate gRNA. Next, single-stranded oligodeoxynucleotide (ssODN) templates with 60-80 bp homology on each side of a Cas9 cleavage site were ordered (Integrated DNA Technologies, Coralville, IA). When required, a silent mutation was introduced in protospacer adjacent motif (PAM) to prevent Cas9 nuclease from re-cleaving the desired sequence after the initial editing step. A purified high concentrated Cas9 nuclease protein with nuclear localization signal (CP02; PNA Bio, Thousand Oaks, CA), which is suitable for electroporation was purchased. The Cas9 and gRNA stocks were 5 µg/µL, and 3.3 µg/µL, respectively. For 3xFLAG insertion, the ssODN concentration was 12 µg/µL, while the ssODN concentration for the Sey correction was 10 µg/µL. 10 µL Neon Transfection System tip was used according to the manufacturer’s protocol (Thermo Fisher Scientific) to deliver the CRISPR components into ESCs. Briefly, ribonucleoprotein (RNP) complex was formed by mixing 0.3 µL of Cas9 with 0.2 µL of annealed gRNA and incubated at room temperature for 20 minutes. Right before electroporation, 0.3 µL of ssODN was added to the RNP complex. 2x105 cells were used for each reaction with Neon optimization setting 14, which was previously determined to best suit our cells. Electroporated cells were plated as duplicates in a 24-well plate containing pre-warmed media. The plate was incubated in a 37°C incubator with 5% CO2. At 48 hours post-23 electroporation, cells were lysed in tissue homogenization buffer (THB) according to a previously described protocol (Yang et al., 2009). The sequences for the best performed crRNAs and ssODNs are listed in Table A.2. 2.2.5 Sanger and site-specific next generation sequencing The region around Sey mutation was PCR amplified using Q5 high-fidelity PCR kit (E0555S; New England Biolabs), with primer pair oEMS6299 (ATGCCGTTCCGATTTCTC), and oEMS6171 (GCTGTGGCATTTCCTTTG). Following gel electrophoresis, DNA bands were excised and purified using Qiaquick gel purification kit (29706; QIAGEN). Cleaned DNA samples were then sent to CMMT DNA Sequencing Core Facility for Sanger sequencing using the same primers as above. Whole cell lysates from electroporated cells were sent to Desktop Genetics (London, United Kingdom) to be assayed for gene-editing frequency by site-specific next generation sequencing (NGS) with self-designed primers. 2.2.6 Mouse husbandry All mice were bred and maintained in the pathogen-free mouse core facility of the CMMT at The University of British Columbia (UBC). Animal work was performed in accordance with the guidelines set by the Canadian Council on Animal Care and adhered to the Association for Research in Vision and Ophthalmology statement for the use of animals in ophthalmic and vision research. All work was done following protocols approved by the UBC Animal Care Committee (protocol A17-0204, and A17-0205). For characterizing the strains, WT C57BL/6J (B6; JAX strain code: 000664) dams were bred with heterozygous transgenic sires (backcross N2 or higher). Pups were weaned and ear notched at post-natal day 21. Collected ear notches were digested in THB with protein kinase, and genotyped by PCR using primer pair 24 oEMS6145 (GATTCTCCAGTTCAGGCACC) and oEMS6146 (CTCAGTGTTGCCCGGATAGA), and primer pair oEMS6077 (CGTATATCCCAGTTCTCAGGC) and oEMS6144 (CTTGTCATCGTCATCCTTGT); allowing for detection of 3xFLAG insertion based on size difference and sequence specificity, respectively. Primer pairs used for WT and Sey specific PCR assays were as mentioned earlier. For phenotyping the B6-Pax6em3(3xFLAG)Ems (herein called Fax) strain, WT and homozygous Fax mice litter mates (N2F2 or higher) obtained from intercrosses were used to set up WT or Fax incrosses. The progeny eyes were scored for abnormal phenotypes by visual inspection at 4 weeks, and representative eyes were imaged by slit lamp at a minimum of 3 months of age. 2.2.7 In vivo gene editing For the creation of the B6-Pax6Sey-em1(3xFLAG)Ems (herein called Fey) and Fax strains, zygotes from 0.5 post-coitus (PC) plugs were obtained by the superovulation of B6 females mated with B6 males. For the B6-Pax6Fey-em4(c.[580TG;591GA])Ems (herein called FeyCor) strain, superovulated Pax6Fey/+ females were setup with B6 males. The day of injection, all females were plug checked, and only those with a PC plug present were harvested. Zygotes were harvested into a EmbryoMax M2 medium (MR-015-D; MilliporeSigma) or Research Vitro Wash (K-RVWA-50; COOK Medical, Bloomington, IN), then treated with a 300 µg/ml solution of Hyaluronidase (H272; Sigma-Aldrich) to remove cumulus cells. Zygotes were then placed into cultured KSOM medium (MR-121-D; MilliporeSigma) or Research Vitro Cleave (K-RVCL-50; COOK Medical) until time of injection. The CRISPR RNP complex was formed immediately prior to microinjection by mixing 2.5 µL of 1 µg/µL Cas9 (CP01-20; PNA Bio) with 3.8 µL of 0.3 µg/µL crRNA:tracrRNA (Genscript) and incubating the mixture on ice for 10 minutes. For 3xFLAG insertion, 4.3 µL of 1.2 µg/µL ssODN (Integrated DNA Technologies) was added to 25 the corresponding complexed RNP and the mixture was topped up to 50 µL using an embryo-grade Tris-EDTA (TE) buffer (CytoSpring, Mountain View, CA). For the Sey germline correction, 5.1 µL of 1 µg/µL ssODN (Integrated DNA Technologies) was used in the final 50 µL reaction. The injection mix was filtered using a 0.45 µm Millex filter (SLHVR04NL; MilliporeSigma) and injections were done into the cytoplasm of one-cell mouse zygotes using the XenoWorks Digital Microinjection System (Sutter Instrument, Novato, CA). Post-microinjection zygotes were placed back into KSOM, and incubated until transferred into 0.5 PC pseudopregnant CD-1 females (Charles River strain code: 022). The sequences of crRNAs and ssODNs used in the microinjection are listed in Table A.2. 2.2.8 Slit lamp imaging Adult mice (2-4 months old) were placed in the induction chamber and induced with isoflurane using a SomnoSuite (Kent Scientific, Torrington, CA). erAft the induction phase, the mice were transferred to the nose cone and eyes were covered with 1% Isopto Tears ophthalmic solution (ALCON, Geneva, Switzerland). Micron IV Retinal Imaging Microscope (Phoenix Research Labs, Pleasanton, CA) with an anterior segment slit lamp attachment was used to image the eyes. 2.2.9 Immunohistological analysis and image processing After slit lamp imaging, mice were sacrificed by cervical dislocation while still under anesthesia. Eyes were immediately enucleated and directly fixed in 4% PFA (P6148; Sigma-Aldrich) in Dulbecco's phosphate-buffered saline (DPBS) (14190-144; Thermo Fisher Scientific) for 2 hours on ice. Subsequently, eyes were immersed in 25% sucrose (S8501; Sigma-Aldrich) in DPBS solution with 0.02% sodium azide (S8032; Sigma-Aldrich) at 4°C overnight. The next day, eyes were orientated in a mold containing Tissue-Tek O.C.T. compound (4583; Sakura 26 Finetek, Torrance, CA) and incubated for 1 hour at room temperature before being frozen using dry ice. The blocks were sectioned at 20 μm with a Microm HM550 cryostat (Thermo Scientific, Waltham, MA). For immunofluorescent staining, sections were processed and mounted as previously described (Korecki et al., 2019), with the difference that antigen retrieval was performed by incubating the slides in freshly made citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) for 1 hour at 55°C, prior to the blocking step that lasted for 1 hour at room temperature. Primary antibodies used were rabbit anti-PAX6 (1:1000; 901301; BioLegend, San Diego, CA), and mouse anti-FLAG (1:500; F3165; Sigma-Aldrich). Secondary antibodies used were anti-rabbit IgG (1:1000; A11037; Thermo Fisher Scientific), and anti-mouse IgG (1:1000; A11029; Thermo Fisher Scientific). Z-stack images were taken at 0.55 steps, and 11 Z-sections (0.5 μm) on a Leica SP8 confocal microscope (Leica Microsystems, Wetzlar, DE) at 40x magnification. Conservative deconvolution of the raw data was carried out using Huygens Professional (Scientific Volume Imaging, Hilversum, Netherlands). The resulting images were stacked at sum slices projection using ImageJ software (http://imagej.nih.gov/ij/, version 1.48). The final images were flattened and exported as tagged image file format (TIFF) files. 2.2.10 Embryonic day 18.5 fetus generation A previously described timed pregnancy protocol was followed (de Leeuw et al., 2016). Heterozygous dams and sires were bred, producing homozygous, heterozygous, and WT offspring for each strain. On embryonic day 18.5 (E18.5), pregnant dams were sacrificed by cervical dislocation and their uteruses were removed. Fetuses were isolated by removing the yolk sac. Fetuses were imaged at 0.8x magnification using Leica MZ125 microscope with a CoolSnap-Pro cf camera (Leica Microsystems). Fetuses were then decapitated and brains were collected. Brains were divided into hemispheres by directly cutting down the longitudinal fissure. 27 Each hemisphere was flash-frozen in liquid nitrogen and stored at -80°C. Lastly, tail tip samples were taken to be used in PCR-based genotyping of the E18.5 fetuses. 2.2.11 Western blot analysis Protein extraction, quantification, and western blotting were performed according to a previously described protocol (Hickmott et al., 2018). Briefly, E18.5 brain hemispheres were homogenized in lysis buffer. The extracted proteins were quantified and 34 μg of each protein sample was run on the gel before transferring to the polyvinylidene difluoride membrane. The blots were blocked and incubated with primary antibodies against PAX6 (1:1000; 901301, BioLegend) and FLAG (1:500; F3165, Sigma-Aldrich). Blots were washed and incubated with anti-rabbit (1:5000; A11037; Thermo Fisher Scientific) and anti-mouse (1:10000; A11029; Thermo Fisher Scientific) secondary antibodies. After the final wash, the fluorescence signals were detected using the LiCOR Odyssey imaging system (Li-COR Biosciences, Lincoln, NE). 2.3 Results 2.3.1 PAX6 and 3xFLAG/PAX6 constructs induced ectopic eyes in Xenopus laevis tadpoles Sey mice produce WT PAX6 protein, so to distinguish the CRISPR-corrected PAX6 protein from WT, a protein tag was necessary. To tag PAX6, FLAG-tag was selected as it is small (Hopp et al., 1988), can be stained with high affinity monoclonal antibodies (Terpe, 2003), and has been previously used for endogenous Pax6 tagging in vivo (Chow et al., 1999). 3xFLAG tag was selected over single FLAG tag, as it is easier to detect with antibodies (Karmakar et al., 2015; Terpe, 2003). PAX6 is highly conserved between diverse species (Wawersik and Maas, 2000), and it is a concern that endogenous gene tagging may cause a functional deficit. However, evidence showing successful use of N-terminally tagged PAX6 in in vitro experiments (Aota et 28 al., 2003; Gan et al., 2014; Jang and Goldman, 2011) supported the possibility of tagging this protein. Over-expression of Pax6 induces the formation of ectopic eyes in X. laevis (Chow et al., 1999). Therefore, to determine if the 3xFLAG-tagged PAX6 remains functional, N- and C-terminally tagged human PAX6 cDNA constructs were tested in a rapid ectopic eye formation assay. X. laevis embryos were injected with mRNA encoding EmGFP, PAX6, N-terminally 3xFLAG-tagged PAX6 (3xFLAG/PAX6), or C-terminally 3xFLAG-tagged PAX6 (PAX6/3xFLAG). The resulting tadpoles were examined 14 days later for the formation of ectopic eye structures (Figure 2.2). PAX6, 3xFLAG/PAX6, or PAX6/3xFLAG constructs, but not EmGFP or water induced ectopic eyes, confirming the functionality of all three constructs. The N-terminally tagged PAX6 construct was selected for therapeutic use, as it produced the greatest number of ectopic eyes. 29 Figure 2.2. FLAG tag does not disrupt the ability of PAX6 to induce ectopic eyes in Xenopus laevis. Xenopus laevis (X. laevis) embryos were injected with mRNA encoding one of: emerald GFP (EmGFP), PAX6, N-terminally or C-terminally 3xFLAG-tagged PAX6 (3xFLAG/PAX6 and PAX6/3XFLAG, respectively), and scored 14 days later for the development of ectopic eyes and retinal pigment epithelium (RPE). A) An example of a non-injected X. laevis tadpole, with one eye on each side of the head. B) An example of a PAX6 mRNA injected X. laevis tadpole, with two additional ectopic eyes (arrows). Insets are magnifications of each ectopic eye. C) Quantification of ectopic eye formation in X. laevis tadpoles. 30 2.3.2 An optimized CRISPR strategy successfully inserted 3xFLAG tag in mouse embryonic stem cells The gRNA and ssODN template designs can tip the balance towards more successful gene editing, and therefore optimizing CRISPR/Cas9 system components prior to in vivo studies is an important step. The N-terminal 3xFLAG-tag location was based on our results with the ectopic eye formation assay in X. laevis tadpoles. To improve the gene editing efficiency, and to readily use the optimized CRISPR strategy in the microinjection, we used the Cas9 RNP complex. The Cas9 RNP complexes with their corresponding ssODNs were electroporated into mouse ESCs and the outcome of genomic manipulation was quantified by site-specific NGS. Figure 2.3A shows the schematic of our best CRISPR strategy for addition of the 3xFLAG tag at Pax6 start codon (ATG) that was able to successfully insert the intact 3xFLAG tag in 1,007/19,573 (5.14%) of NGS reads (Figure 2.3B). In 7,986/19,573 (40.8%) of the reads, Cas9-induced double stranded breaks were repaired by non-homologous end joining (NHEJ) pathway, creating relatively indels in a close vicinity to the cut site. 31 Figure 2.3. Site-specific next generation sequencing revealed the correct insertion of 3xFLAG tag in 5.14% of sequences. Cas9 ribonucleoprotein complexes were assembled in vitro and electroporated into mouse embryonic stem cells. Site-specific next generation sequencing was used to quantify the knock-in. A) Schematic of the best guide RNA (gRNA) and single-stranded oligodeoxynucleotide (ssODN) for CRISPR/Cas9 mediated insertion of a 3xFLAG tag at Pax6 ATG (yellow). In addition to the tag sequence (green), the ssODN contained 60 bp homology arms flanking the Cas9-induced double stranded break. Blue, Pax6 exons; black, Pax6 introns. The diagram is not to scale. B) The top panel shows mutation positions relative to Cas9 cut site. 1,007/19,573 (5.14%) of reads showed the correct 3xFLAG insertion. The bottom panel shows the insertion and deletion (indel) size distribution. 2.3.3 Endogenous Sey tagging created a novel mouse model for aniridia without compromising the protein function in the control mice We used an earlier in vitro optimized CRISPR strategy (Figure 2.3A) to insert a 3xFLAG tag to the Sey allele in mouse zygotes in a rapid manner, creating the 3xFLAG-tagged 32 Sey mouse strain (B6-Pax6Sey-em1(3xFLAG)Ems), called Fey hereafter. Since the Sey mutation causes a pre-mature stop codon and no detectable protein (Hickmott et al., 2018), we were unable to directly characterize the function of the Fey allele. Thus, we created the matched WT control strain (B6-Pax6em2(3xFLAG)Ems), called Fax hereafter. Both strains were backcrossed to WT B6 mice to remove any incidental mutations. As expected, the slit lamp imaging of adult Fey mouse eyes revealed abnormalities comparable to those seen in Sey mice. However, even when required for the entire eye development in a complex and sophisticated mammalian system, the addition of the 3xFLAG tag to the conserved PAX6 in Fax mice resulted in no eye phenotypes (Figure 2.4A). Moreover, we scored eye size and corneal cloudiness by visual inspection in 106 homozygous Fax and 76 WT eyes. We found no significant difference in the ocular phenotype between homozygous Fax and WT mice (one-tailed t-test, p=0.490). Left eyes from a subset of these mice (8 WT and 8 homozygous Fax mice) were examined by slit lamp, predominantly for possible abnormalities in the lens, which could not be assessed by visual inspection (Figure A.1). We found no significant difference in the lens structure between homozygous Fax and WT mouse eyes (one-tailed t-test, p=0.074). Immunohistological analysis was performed to look at the co-localization and expression of PAX6 and 3xFLAG-tagged PAX6 proteins. Sections from Sey, Fey, WT, and Fax retinas showed similar anti-PAX6 staining patterns in the ganglion cell layer (GCL) and inner nuclear layer (INL). Additionally, for Fax retinas, co-labelling of PAX6 and FLAG was detected in both the GCL and INL (Figure 2.4B). 33 Figure 2.4. Fey is a novel mouse model of aniridia and importantly the Fax control mice showed no phenotype. An optimized Cas9 ribonucleoprotein complex and a single-stranded oligodeoxynucleotide containing the 3xFLAG sequence were microinjected into one-cell mouse zygotes to generate transgenic animals in one step. A) Slit lamp analysis of the transgenic mouse eyes. As expected, images of Fey showed small eyes, and corneal opacity, not significantly different from the Sey (Pax6 small eye) mouse model (two-tailed test, p>0.999). Slit lamp images of Fax (FLAG-tagged Pax6) mouse eyes showed normal iris and clear cornea, which were not significantly different from WT eyes (one-tailed t-test, p>0.999). Fax, FLAG-tagged Pax6; Fey, FLAG tagged Sey; Sey, Pax6 small eye; WT, wild type. B) Histological analysis of mouse retinas. Adult mouse eyes were directly fixed and stained with FLAG and PAX6 antibodies. While FLAG staining 34 was absent in the negative controls (Fey and WT retinas), Fax mouse retinas showed positive co-labeling of FLAG and PAX6. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; blue, Hoechst. All images were taken at the same magnification. The scale bar is 34 μm. 2.3.4 Sey mutation was efficiently corrected in mouse ESCs Optimization of CRISPR/Cas9 components in cells enabled us to ensure on-target cleavage activity and specificity prior to in vivo studies. One recent approach that has been quite successful in distinguishing alleles uses Cas proteins with unique PAMs such that correcting the mutation removes the PAM (Christie et al., 2017; Li et al., 2018a; Monteys et al., 2017). However, this approach is restricted by the PAM sequences available, and is currently not applicable to the Sey mutation. We worked with mouse Pax6Sey/Sey and Pax6+/+ ESCs as homozygosity made it easier to interpret modifications from sequencing chromatograms. The on-target cleavage activity was determined by Sanger sequencing of edited whole cell lysates normalized against unedited controls (defined as either Pax6Sey/Sey or Pax6+/+ ESCs electroporated with Cas9 and ssODN template in the absence of gRNA). According to the results, our best CRISPR strategy corrected the Sey mutation with a 42.9% success rate and modified the PAM with a rate of 41.4% to avoid repeated targeting of edited alleles (Figure 2.5A). Upon repeating the in vitro gene editing in triplicate, the average correction rate for Sey mutation was 34.8 ± 4.6 (mean ± standard deviation), while the average PAM change rate was 42.9 ± 3.2. In a head-to-head experiment, our best CRISPR strategy was then tested in both Pax6Sey/Sey and Pax6+/+ ESCs to determine its specificity. Quantification of the PAM change in these cells revealed a 2-fold enrichment of gRNA interaction with the mutant allele. CRISPR/Cas9-mediated gene editing is known to lead to sequence heterogeneity among cells from the same sample (Hough et al., 2016). Hence, to look at the samples at a single 35 molecule level, we sent Pax6Sey/Sey and Pax6+/+ whole cell lysates edited with our best CRISPR strategy and the unedited controls for site-specific NGS, which enabled us to observe the various on-target mutations CRISPR created. As expected, in the unedited Pax6Sey/Sey sample the majority of the NGS reads (11,404/11,458; 99.5%) aligned completely to the reference sequence. The remainder of reads in the unedited sample had single nucleotide substitutions, which could be explained by the error rate associated with this sequencing technique (Pfeiffer et al., 2018). Among the 18,291 aligned reads for the edited Pax6Sey/Sey sample, 3,644 reads (19.9%) had both knock-in variants with no further changes in Pax6 exon 8; 5,162 reads (28.2%) had both knock-in variants and an undesired change within a 20 bp window; 6,608 reads (36.1%) showed the correction of Sey mutation; 6,893 reads (37.7%) had the PAM change; and lastly 7,855 reads (42.9%) remained unmodified (i.e., still had the Sey mutation). The indel profile analysis revealed various sizes of NHEJ-induced indels around the Cas9 cut site (Figure 2.5B). Consistent with the earlier Sanger sequencing results, the indel frequency analysis of edited Pax6Sey/Sey and Pax6+/+ samples showed a 2-fold enrichment of gRNA interaction with the mutant allele. 36 Figure 2.5. In vitro CRISPR-mediated gene editing corrects the Sey mutation in Pax6Sey/Sey embryonic stem cells. Sey is a point mutation where the wild type G nucleotide is mutated to a T, resulting in a premature stop codon. Various CRISPR-mediated editing strategies to correct the Sey mutation were tested in mouse embryonic stem cells and the editing efficiencies were quantified by Sanger sequencing and site-specific next generation sequencing (NGS). A) Sanger sequencing chromatogram of the best CRISPR strategy. Sanger sequencing revealed a correction rate from T to G of 42.9% (red box). To prevent repeat targeting of previously edited alleles, the single-stranded oligodeoxynucleotide (ssODN) also included a silent base change in the protospacer adjacent motif (PAM) sequence, giving an alteration rate from G to A of 41.4% (green box). Relative fluorescent units (RFU). B) Site-specific NGS histograms of the best CRISPR strategy. 11,404/11,458 (99.5%) of reads from the unedited cells (negative control) matched the reference sequence. For the cells edited with the best strategy, 3,644/18,291 reads (19.9%) had both the correction and the PAM change with no other changes in Pax6 exon 8. 7,855/18,291 reads (42.9%) were unmodified. 37 2.3.5 In vivo gene editing corrected the Sey mutation with a 25% success rate Rescuing the mutant phenotype by correcting the Sey mutation in the mouse germline proves causality and serves as a proof-of-principle experiment for a future somatic gene therapy. The corrected mice were generated by microinjecting the in vitro optimized CRISPR gene editing strategy into Pax6Fey/+ and Pax6+/+ zygotes. Fifteen potential founder mice were born. Summary of the phenotyping, genotyping, and sequencing results of these mice is presented in Table 2.1. The proportion of animals with eye abnormalities observed by visual inspection was 46.7% (7/15), which was not significantly different from the expected 50:50 phenotypic ratio of Sey to WT. Moreover, the FLAG allele was present at 26.7% (8/30), which again was not significantly different from the expected 25%. Mice 1-9 and 1-12 were positive for 3xFLAG, which was genetically linked to the Sey allele, but had normal eye phenotype. Sequencing results of ear notches from these two mice confirmed the correction of Sey mutation and presence of the silent base change at the original PAM site. On the allelic level, we achieved a correction rate of 25% (2/8) for the Fey allele. Sequencing results also revealed that the WT allele was hit at a rate of 22.7% (5/22), while only 13.6% (3/22) of this rate represents a deleterious hit. All 8 FLAG alleles were hit by our CRISPR strategy, whereas 77.3% (17/22) of WT alleles remained unmodified. 38 Table 2.1. The Sey phenotype was rescued in two mice by germline correction. Mouse ID Eye Pheno- type 3xFLAG Geno- type Fey Allele Cor. & PAM Change Fey Allele Indel Fey Allele Un-edited WT Allele Changea WT Allele Indel WT Allele Un- edited 1-1 Abnor. F/+ NA ✓ NA NA NA ✓ 1-2 Abnor. F/+ NA ✓ NA NA NA ✓ 1-3 Normal +/+ NA NA NA NA ✓ ✓ 1-4 Normal +/+ NA NA NA NA ✓ ✓ 1-5 Abnor. F/+ NA ✓ NA NA NA ✓ 1-6 Abnor. F/+ NA ✓ NA NA NA ✓ 1-7 Abnor. F/+ NA ✓ NA NA NA ✓ 1-8 Normal +/+ NA NA NA NA NA ✓✓ 1-9 Normal F/+ ✓ NA NA NA NA ✓ 1-10 Normal +/+ NA NA NA ✓ NA ✓ 1-11 Normal +/+ NA NA NA NA NA ✓✓ 1-12 Normal F/+ ✓ NA NA ✓ NA NA 1-13 Abnor. +/+ NA NA NA NA ✓ ✓ 1-14 Abnor. F/+ NA ✓ NA NA NA ✓ 1-15 Normal +/+ NA NA NA NA NA ✓✓ 7/15 (46.7%)b 8/30 (26.7%)c 2/8 (25.0%)d 6/8 (75.0%)d 0/8 (0%)d 2/22 (9.10%)e 3/22 (13.6%)e 17/22 (77.3%)e aThis refers to either a PAM change or point mutations with no effect on the phenotype. bAbnor./Total ratio. cAllele of interest/Total alleles. dAllele of interest/Total Fey alleles. eAllele of interest/Total WT alleles. Abnor., Abnormal; Cor., Corrected; F, 3xFLAG-tagged Pax6 allele; +, ID, identifier; Indel, insertion or deletion; WT Pax6 allele; ✓, present in one allele; ✓✓, present in both alleles; NA, not applicable. 2.3.6 Germline correction of the Sey mutation restored 3xFLAG-tagged PAX6 expression and normal eyes Upon breeding, founders 1-9 and 1-12 were shown to stably transmit the transgene to their progeny. A first pedigree mouse was chosen for each founder; resulting in establishment of two new transgenic strains, B6-Pax6Fey-em4(c.[580TG;591GA])Ems and B6-Pax6Fey-em5(c.[580TG;591GA])Ems, which are called hereafter as Fey Correctionem4 (FeyCorem4) and Fey Correctionem5 (FeyCorem5), respectively. Both strains were backcrossed to WT B6 mice to remove any incidental mutations. 39 Excitingly, unlike Fey mice, FeyCorem4 and FeyCorem5 mice eyes were not significantly different from WT by slit lamp exam (one-tailed t-test, p>0.999) (Figure 2.6A). Additionally, FLAG and PAX6 co-labelling in the immunohistological analysis of mouse retinas from both FeyCor strains confirmed the expression of 3xFLAG-tagged PAX6 in GCL and INL (Figure 2.6B). Figure 2.6. Germline correction of the Sey mutation rescued the mutant eye phenotype. To correct the Sey mutation in vivo, an optimized CRISPR-mediated editing strategy was microinjected into one-cell mouse zygotes. Progeny of founders with inheritable transmission of the transgene were subjected to phenotypical characterization and Pax6 expression analysis. A) Slit lamp images of Fey corrected (FeyCor) mouse eyes demonstrated clear cornea and normal iris comparable to WT mouse eyes, and distinctly none of the Fey eye abnormalities. Fey, FLAG-tagged Sey; Sey, Pax6 small eye; WT, wild type. B) Positive co-labeling of FLAG and PAX6 was observed in the histological analysis of mouse retinas from both FeyCor strains. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; blue, Hoechst. All images were taken at the same magnification. The scale bar is 34 μm. 40 2.3.7 PAX6 level was increased in the brain of germline-corrected mice Being homozygous for a Pax6 or PAX6 null mutation causes anophthalmia and is neonatal lethal in both mouse and human, respectively (Glaser et al., 1994; Hogan et al., 1986). Therefore, to examine PAX6 levels in an expressing tissue, we decided to perform western blot analysis on brain samples isolated from fetuses at E18.5. PAX6 level influenced the phenotype of fetuses as expected, with Pax6Fey/+ and Pax6Fey/Fey fetuses showing microphthalmia and anophthalmia, respectively, while Pax6Fax/+ and Pax6Fax/Fax control fetuses had normal eyes. The germline correction of Sey mutation led to Pax6FeyCor/+ and Pax6FeyCor/FeyCor fetuses having normal eyes that were not significantly different from WT (one-tailed t-test, p>0.999) (Figure A.2). Next, we looked at PAX6 levels using western blotting. Immunoblots of E18.5 brains showed co-staining of 3xFLAG-tagged PAX6 and PAX6 proteins for the FeyCor genotype; suggesting the rescue of 3xFLAG-tagged PAX6 expression in the brain of these fetuses (Figure 2.7A). PAX6 protein levels were determined from two technical replicates with understanding of semi-quantitative nature of these results. For each immunoblot, PAX6 protein levels were normalized against a non-specific protein revealed by the secondary antibody (Figure 2.7B). See Table A.1 for relative PAX6 levels in each sample. Statistical analysis revealed a significant decrease in PAX6 levels in the brains of heterozygous Fey fetuses compared to homozygous FeyCor fetuses (one-tailed t-test, p=0.017), while no significant difference was observed in the mean PAX6 levels between homozygous FeyCor and WT genotypes (two-tailed t-test, p=0.999). As expected, statistical analysis also revealed similar PAX6 levels in the brains of homozygous Fax and WT fetuses (two-tailed t-test, p=0.937), which was significantly more than brains from heterozygous Fey fetuses (one-tailed t-test, p=016), confirming the observation that the Pax6 genotype, but not the endogenous tagging, influences PAX6 protein levels. 41 Figure 2.7. Expression of 3xFLAG-PAX6 was confirmed in Fax and FeyCor mice. Total protein was collected from mouse brains for western blot analysis with PAX6 (red) and FLAG (green) antibodies at embryonic day 18.5 (E18.5). A) Anti-PAX6 detected a doublet of both 3xFLAG-PAX6 (53 kDa), and PAX6 (50 kDa) proteins in lanes 3, and 6. Anti-FLAG identified the 3xFLAG-PAX6 protein. Merge showed positive co-staining with both antibodies. B) Secondary antibody control. In the absence of the primary antibody, the secondary antibody detected non-specific mouse proteins at ~75 kDa and ~55 kDa, the former stronger band was used as the loading control. FeyCor, Fey corrected; Fax, FLAG-tagged Pax6; Fey, FLAG-tagged Sey. 2.4 Discussion Aniridia was first described in 1818 (Samant et al., 2016). Despite considerable research effort over the past 200 years, there remains a therapeutic gap for aniridia patients around the world. Therapy development for rare diseases such as aniridia relies heavily on efficacy and safety studies performed on animal models. In this study, we developed a new mouse model of aniridia, Fey, with the addition of 3xFLAG to the Sey allele, that can be used to explore gene-editing therapies for this debilitating disease. Prior to making Fey, we tested the impact of the 3xFLAG tag on WT Pax6, naming this allele Fax. We were surprised that the heterozygous, and even homozygous addition of 3xFLAG 42 to Pax6 in vivo, did not result in any observed undesired phenotypes in the “control” Fax mice; suggesting that our endogenous gene tagging did not influence the function of PAX6. This is critical since it shows that when the Fey mouse is “corrected” the new allele will be fully functional. However, it should be noted that we focused our characterization on the ocular phenotype, since aniridia is primarily an eye-related disorder, and thus cannot rule out the possibility of other subtle phenotypes in non-ocular Pax6-expressing tissues. Sey is an old mutation (Roberts, 1967), thus this non-functional allele could have undergone random genetic drift over the at least 52 years of continuous breeding since discovery. Such additional mutation(s) could be difficult to detect even by sequencing if located in non-coding Pax6 regulatory elements (Fahey et al., 2013). The success of our mutation-dependent CRISPR strategy relied on the causality of the Sey mutation. Therefore, as an important step, we validated the causality of the Sey mutation in the Fey mouse through a CRISPR-based germline correction experiment and demonstrated that correction of just this one base pair fully restored the ocular phenotype to WT. These results further validate Fey as a novel mouse model for the development of CRISPR-based mutation correcting therapies. The Fey mouse will also be valuable for the development and quantification of efficacy for numerous other therapeutic approaches. For example, nonsense suppression therapies using read-through compounds such as G418, amlexanox, and Ataluren to overcome the presence of nonsense mutations in aniridia. G418 has shown read-through activity both in vitro and in vivo (Barton-Davis et al., 1999; Brasell et al., 2019; Heier and DiDonato, 2009), and amlexanox has shown activity in vitro (Atanasova et al., 2017; Gonzalez-Hilarion et al., 2012). Ataluren has shown activity in mice (Sahel and Marazova, 2013; Wang et al., 2017), and has been in clinical trial for aniridia (ClinicalTrials.gov: NCT02647359). Fey mice can be also used to design and 43 explore CRISPR/Cas13 strategies that target Pax6 mutations at the RNA level without permanently altering the genome (Cox et al., 2017). Additionally, mutation independent strategies such as the intronic insertion of Pax6 cDNA sequence immediately downstream of the AUG, and the use of spliceosome-mediated RNA trans-splicing (SMaRT) technique will benefit from having the new protein FLAG tagged. The former and the latter therapeutic approaches have been shown effective in treating mouse models of hemophilia (Ohmori et al., 2017), and retinitis pigmentosa (Berger et al., 2015), respectively. We successfully achieved both in vitro and in vivo germline correction of the Sey and Fey mutations, respectively, restoring the mutant protein expression. In vitro delivery by electroporation into ESCs resulted in an average 34.8 ± 4.6% SD correction, while in vivo delivery by microinjection into embryos resulted in 25% correction. Furthermore, the bias towards the mutant allele was 2-fold and 4-fold higher in our in vitro and in vivo experiments, respectively. Thus, although both successful, our allele-distinguishing CRISPR strategy performed quite differently dependent upon the delivery method and recipient cell. Therefore, we now hypothesize that the effectiveness of this strategy when delivered by viral or non-viral delivery vectors to somatic cells will need empirical testing. PAX6 plays a critical role in maintaining corneal limbal stem cell (LSC) niche in both human and mouse eye. LSC deficiency is the underlying mechanism responsible for cornea opacification and ultimately blindness in aniridia (Li et al., 2015a; Mort et al., 2011; Ramaesh et al., 2005a; Roux et al., 2018). Hence, in future studies, we aim to deliver our CRISPR strategy to LSCs of post-wean Fey mice to model gene therapy in recently diagnosed children with aniridia. Taken together, we have created a novel mouse model for testing CRISPR-based genome editing, and other, therapeutic approaches for preclinical assay development for aniridia. We 44 demonstrated that germline correction of the Sey mutation alone rescues the mutant phenotype. Finally, we have developed an allele-distinguishing CRISPR strategy that corrected the Sey mutation in both cells and the mouse germline. 2.5 Acknowledgments We thank Tom W. Johnson, of the Mouse Animal Production Service (MAPS), for making the Fey mouse. We also thank Dr. Jingsong Wang at the British Columbia Children’s Hospital Research Institute (BCCHRI) Imaging Core Facility for access to the confocal microscope and Dr. Elizabeth Hui at the BCCHRI Sequencing and Bioanalyzer Core for Sanger sequencing support. This work was funded by a Canadian Institutes of Health Research (CIHR) grant (2OR77895) awarded to E.M.S. The Micron IV Retinal Imaging Microscope and SomnoSuite were funded by the Canada Foundation for Innovation (CFI) (grant 2OR76139) and BCCHRI (grant CRG74760), respectively. Additionally, salary support was provided to S.Z.M.M. by Brain Canada Foundation through the Canada Brain Research Fund, with the financial support of Health Canada and BCCHR institute. The views expressed herein do not necessarily represent the views of the Minister of Health or the Government of Canada. 45 Chapter 3: LNP-Mediated Delivery of CRISPR RNP for Wide-Spread In Vivo Genome Editing in Mouse Cornea2 3.1 Introduction CRISPR-mediated genome editing based therapies hold great promise for treating many genetic diseases (Li et al., 2020). However, delivery of CRISPR components generally demands methodology with little size restriction (Peng et al., 2016). To date, three in vivo CRISPR-based gene therapies have been approved to proceed to clinical trial. Two of these therapies, EDIT 101 (NCT03872479) and EBT-101 (NCT05144386) use a relatively small Cas9 ortholog from Staphylococcus aureus and are delivered with recombinant adeno-associated viruses (rAAVs). The third, NTLA-2001 (NCT04601051) uses the classical Streptococcus pyogenes Cas9 (SpCas9), which is the best-characterized and most widely-used CRISPR endonuclease. Notably, NTLA-2001 uses a lipid nanoparticle (LNP) delivery system (Gillmore et al., 2021). LNPs are non-immunogenic, scalable, and more importantly unlike rAAVs (Wu et al., 2010), they are not restricted in their packaging size (Finn et al., 2018). Two other aspects of LNP are desirable for CRISPR-mediated genome editing therapy delivery. Using the ribonucleoprotein (RNP) format of Cas9 considerably reduces the off-target activity due to its transient expression compared to plasmid or viral-based expression of CRISPR components (Jang et al., 2021; Kim et al., 2014; Liang et al., 2015; Vakulskas and Behlke, 2019). In addition, the engineered nature of LNPs allows for repeated treatments that are therapeutically effective through cumulative genome editing (Finn et al., 2018; Kenjo et al., 2021; Rothgangl et al., 2021). However, repeated 2 This chapter has been published in Journal of Controlled Release as: Mirjalili Mohanna et al. (2022). LNP-Mediated Delivery of CRISPR RNP for Wide-Spread In Vivo Genome Editing in Mouse Cornea. J Control Release. 350:401-413. PMID: 36029893. See Preface for details of my contributions. 46 treatments could be potentially costly and inconvenient to the patients. In comparison, most viral-based strategies are limited to a single treatment due to neutralizing antibodies (Jiang et al., 2006; Riviere et al., 2006). Majority of current studies focus on LNP delivery of Cas9 messenger RNA (mRNA) and guide RNA (gRNA) to the liver (Finn et al., 2018; Gillmore et al., 2021; Han et al., 2022; Qiu et al., 2021). Thus, even though LNP delivery of pre-complexed RNP complexes is an attractive approach for CRISPR-mediated therapies, further optimization of LNP composition and their RNP cargo is required to expand their extrahepatic target tissues and disease applicability. Vision impairment and blindness are diseases with important economic impact, which is felt in reduced education achieved, work years lost, and life-long health-care needs (Koberlein et al., 2013; Marques et al., 2021). The eye is an excellent target tissue for in vivo gene therapy, given its relative isolation from the rest of the body, accessibility, and comparative immune privilege (Zhou and Caspi, 2010). Breakthroughs in gene therapy are saving vision in people suffering from rare genetic eye diseases (Sahel et al., 2021; Testa et al., 2013), and may provide new treatment options for other ocular diseases with unmet therapeutic needs such as aniridia. Aniridia is a rare eye disorder that is caused by haploinsufficiency of paired box 6 (PAX6) gene (Davis-Silberman et al., 2005; Kokotas and Petersen, 2010; Vincent et al., 2003). Although named for the lack of iris, aniridia is actually a panocular disorder in which children are born with low vision due to retinal fovea hypoplasia. Patients typically become legally blind in their twenties to thirties regardless of symptomatic treatments (Hingorani et al., 2012). Progressive corneal clouding is a major cause of blindness in aniridia and occurs in up to 90% of patients (Landsend et al., 2017). This makes the cornea a key therapeutic target for developing new vision-saving therapies for aniridia. A well-characterized and widely-used mouse model of 47 aniridia is called Small eye (Sey) (Gregory-Evans et al., 2013; Hickmott et al., 2018; Hill et al., 1991; Hogan et al., 1988; Li et al., 2007; Mirjalili Mohanna et al., 2020; Wang et al., 2017), which carries the nonsense c.580G>T (p.G194X) variant in the Pax6 gene that has been also reported in a patient (Hever et al., 2006; Kit et al., 2021). The Sey mouse is an excellent model to test new therapeutics for aniridia, since it mimics the corneal abnormalities seen in patients (Ramaesh et al., 2003). Gene disruption via non-homologous end joining (NHEJ) repair pathway was previously demonstrated in the corneal epithelial cells following intrastromal injection of CRISPR/Cas9 plasmids to the mouse eye (Christie et al., 2017; Courtney et al., 2016). Building on this work, a possible advantage of RNP is that all required individual components can be pre-assembled, ensuring that upon delivery the active CRISPR complex can immediately target the DNA. Unbound Cas9 is not encapsulated into the LNP, nor is Cas9 produced in the cell, and the pre-complexed RNPs are rapidly degraded after cellular delivery, minimizing the potential for off-target effects (Kim et al., 2014; Liang et al., 2015). Furthermore, unlike CRISPR plasmids, RNPs do not pose the risk of unwanted integration at either on-target or off-target sites in the genome (Kim et al., 2014). Lastly, the gRNA in the RNP format can be chemically modified, which in turn stabilizes the gRNA by protecting it from degradation by exonucleases (Hendel et al., 2015). Given these benefits, the RNP format of the CRISPR system can be a more efficient and safer alternative to plasmids for targeting the cornea. The ability to package and deliver the large SpCas9 protein, gRNA, and DNA template within one LNP and the flexibility to repeatedly treat with LNP-RNPs to achieve titratable genome editing while minimizing potential toxicity, are attractive options for gene therapy of ocular diseases. Moreover, no previous studies have used LNPs to target the cornea but several 48 groups have achieved limited targeting to a subset of cells in the retina using lipid-based transfection approaches (Huang and Chau, 2021; Jang et al., 2021; Patel et al., 2019; Ryals et al., 2020). Only one of these studies delivered CRISPR-based genome editing by co-delivering Lipofectamine 2000, adenine base editor, and gRNA to the mouse retina via subretinal injection (Jang et al., 2021). In that study, they focused on targeting retinal pigment epithelial (RPE) cells and reported 5.7% correction as their most successful genome editing event. However, Lipofectamine 2000 is not an LNP, nor amenable to therapeutic translation due to its high toxicity (Wang et al., 2018). Thus, the field of genetic eye diseases lacks a translatable LNP delivery method for CRISPR/Cas9 systems. Here we describe the use of the novel Incisive Delivery System (DS), which is an LNP-based platform that allows for the delivery of pre-complexed active Cas9/gRNA RNPs into target cells. As an initial proof-of-principle, the efficiency of our LNP-RNP delivery platform was evaluated in the loxP-3xStop-loxP-tdTomato reporter system. In this reporter system, CRISPR-mediated generation of double stranded breaks near both loxP sites disrupts the transcriptional stop cassette located in the genome between the ubiquitous CAG promoter and tdTomato open reading frame at the Rosa26 locus, turning on tdTomato expression (Tabebordbar et al., 2016). This is a high-throughput and sensitive reporter system, allowing detection of a single positive genome edited cell by epifluorescence. This system was assessed in both ex vivo cortical neurons isolated from embryonic mice, and the mouse eye following direct in vivo delivery to the cornea. Secondly, our LNP-RNP delivery platform was tested in a system with therapeutic relevance. We previously created a novel mouse model for CRISPR-based therapy development for aniridia by FLAG-tagging the Sey allele to enable efficient detection of corrected protein after successful genome editing (Mirjalili Mohanna et al., 2020). This system 49 was also used for targeted genome editing both in ex vivo embryonic cortical neurons and for in vivo delivery to the cornea of mice. Thus, the study moved from ex vivo to in vivo and from a reporter system to a mouse model of a pathogenic human variant and disease. Our novel results demonstrate the first evidence of successful genome editing in the mouse cornea using an LNP-RNP CRISPR strategy, and highlights the potential of this approach for development of CRISPR-based therapeutics for a variety of corneal diseases. 3.2 Materials and Methods 3.2.1 Mouse husbandry and breeding All mice were bred and maintained in the pathogen-free mouse core facility of the Centre for Molecular Medicine and Therapeutics (CMMT) at The University of British Columbia (UBC). Animal work was performed in accordance with the guidelines set by the Canadian Council on Animal Care. All work was done following protocols approved by the UBC Animal Care Committee (protocol A17-0204, A17-0205, A21-0140, and A21-0184). For the ex vivo experiments, one mouse strain used was B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J (JAX Strain #:007914, aka Ai14), for clarity we will call this allele loxP-3xStop-loxP-tdTomato. A cohort of age-matched mice was generated per month, for three consecutive months. For each of the three cohorts, six C57BL/6J-loxP-3xStop-loxP-tdTomato females were crowded for 8 days, with two additional female mice added to the cage to achieve necessary density for suppressing the estrus cycle (Van Der Lee and Boot, 1955, 1956). The crowded females were then mated in pairs with six 129S1/SvImJ-Pax6Sey-em1Ems studs, for clarity we will call this allele FLAG-tagged Sey (Fey). Note: this allele is available from JAX/MMRRC in Strain #:66963. 129S1/SvImJ-Fey studs were experienced with other females prior to mating. Females were checked for the presence of copulatory plugs to determine the date of fertilization, 50 which was then used to estimate the stage of the embryos. Once the copulatory plug was found, the female was removed from the cage and singly housed. Pregnancy was visually confirmed at embryonic day 16.5 (E16.5). For the in vivo experiments, one cohort of approximately 60 mice was generated by timed-pregnancies. Eight C57BL/6J-loxP-3xStop-loxP-tdTomato females were crowded for 8 days and then mated in pairs with eight 129S1/SvImJ-Fey studs. After one week, females were removed from cages and singly housed. Cages were checked for birth daily 18-25 days after mating. Age-matched (±3 days) pups were weaned and ear notched at postnatal day 21 (P21). Collected ear notches were digested in tissue homogenization buffer with Proteinase K according to a previously described protocol (Yang et al., 2009). PCR-based genotyping was performed to detect the presence of 3xFLAG tag using Taq DNA Polymerase (Cat# 18038042, Invitrogen, Waltham, MA), with primer pair oligonucleotide Elizabeth M. Simpson (oEMS) 6145 (5′-GATTCTCCAGTTCAGGCACC-3′) and oEMS6146 (5′-CTCAGTGTTGCCCGGATAGA-3′). 3.2.2 Isolation and culture of ex vivo mouse cortical neurons On E17.5, pregnant females were sacrificed by cervical dislocation and their uteri were removed. Tail tip samples were taken from fetuses for the purpose of genotyping, followed by the dissection of cortices in ice cold Hank's balanced salt solution (HBSS) (Cat# 14175095, Gibco, Thermo Fisher, Waltham, MA). DNA was extracted from tail tips using Extract-N-Amp™ Tissue PCR Kit (Cat# XNAT2-1KT, Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s protocol. PCR-based genotyping was performed to detect the presence of 3xFLAG tag using LongAmp Taq 2X Master Mix (Cat# M0287S, New England Biolabs, Ipswich, MA), with primer pair oEMS6145 and oEMS6146. Cortices were pooled by genotype and incubated in HBSS with 0.05% trypsin (Cat# 25300062, Gibco) for 12 minutes at 37˚C with 51 gentle agitation. The trypsin reaction was halted using an equal volume of 30% Fetal Bovine Serum (Cat# SH3039603, Cytiva, Marlborough, MA) in HBSS. Cortices were subsequently triturated 3 times with a 5 ml serological pipette, followed by an additional 4-6 times with a 200 µl tip added to the end of the pipette. Cell suspensions were passed through a 40 µm nylon strainer (Cat# C352340, Falcon) and pelleted via centrifugation. The supernatant was removed, and the cell pellet was washed once in HBSS and pelleted again. The pellet was re-suspended in neurobasal media (Cat# 21103049, Gibco) supplemented with B27 (Cat# 17504044, Gibco), 2 mM L-glutamine (Cat# SH3003401, Hyclone, Logan, UT) and 1% penicillin/streptomycin (Cat# SV30010, Hyclone). Cells were plated onto poly-D lysine (Cat# P6407, Sigma-Aldrich) coated 24-well plates with or without 12 mm glass coverslips at a density of 1.5 x 105 cells/well in 1 ml of media. Cells were maintained at 37˚C in an incubator with 5% CO2. 3.2.3 Preparation of Incisive DS LNP and encapsulation of RNP and DNA template Ethanolic solutions of the following lipids were prepared at 10 mM: 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (lipids from Avanti Polar Lipids, Alabaster, AL), and cholesterol (Cat# C3045, Sigma-Aldrich). DODMA/DOPE/DSPC/Cholesterol lipids were mixed at the ratio 20/30/10/40 mole %. The solution of lipids was then mixed with an aqueous solution of 25 mM sodium acetate, pH 4, through an Ignite microfluidic mixer (Cat# NIN0001, Precision Nanosystems, Vancouver, Canada) at a total flow rate of 20 ml/min and a ratio of 3:1 v/v (aqueous/lipid mixture). The resultant solution was dialyzed 1:1000 in aqueous solution of 25 mM sodium acetate, pH 4, overnight at 4˚C. Following dialysis, empty LNPs were concentrated using the Amicon (Cat# UFC801008, Sigma-Aldrich) centrifugation filter and the 52 final lipid concentration was adjusted to 10 mM, as assessed by cholesterol assay (Cat# CA97000-018, WAKO, Richmond, VA). Encapsulation of RNP complex and DNA template was as follows: 10 µM Alt-R S.p. Cas9 Nuclease V3 (Cat# 1081059, Integrated DNA Technologies, Coralville, IA) was mixed briefly by pipette and incubated with an equimolar mixture of tdTomato-targeting gRNAs, single Pax6-targeting gRNA, or single luciferase non-targeting gRNA for 5 minutes at room temperature. All gRNAs in this study were ordered from Synthego (Menlo Park, CA) as chemically modified single gRNAs (2'-O-Methyl at 3 first and last bases, 3' phosphorothioate bonds between first 3 and last 2 bases). Alt-R HDR Donor Oligos (Integrated DNA Technologies) were added in equimolar (tdTomato targeting and Sey correction strategies) or 0.5x molar (Sey correction strategy) ratio and incubated for a further 5 minutes at room temperature. Empty LNPs were added at a 0.55 v/v (tdTomato cargo/LNP) or 0.33 v/v (Pax6 cargo/LNP) ratios, mixed briefly by pipette, and incubated for a final 5 minutes. The sequences of gRNAs and templates used in this study are listed in Table B.1. The size distribution and the polydispersity index (PDI) of LNPs encapsulating luciferase non-targeting RNP strategy were measured using a Malvern Zetasizer Nano S instrument (Worcestershire, UK) (He-Ne laser, λ = 632 nm, detection angle = 173°). The average diameter distribution of RNP loaded LNPs was 151.6 ± 0.8 nm (Figure B.1) and the average PDI was 0.39 ± 0.07. 3.2.4 Ex vivo genome editing Embryonic cortical cells were treated on day ex vivo (DEV) 3 (first of two treatments) and/or DEV 6 (single or repeated treatment). LNP-encapsulated RNP and DNA mixtures were prepared immediately prior to treatment. Complete neurobasal media containing 2 µg/ml recombinant human ApoE4 (Cat# 350-04, Peprotech, Cranbury, NJ) was added to bring the final 53 concentration to 100 nM RNP for tdTomato targeting and luciferase non-targeting strategies, or 200 nM RNP for Sey correction strategy. On DEV 3 and/or DEV 6, half (500 µl) of the conditioned media was removed from each well and saved at 37˚C. Media containing LNP-encapsulated RNP and DNA template was added to bring the volume back to 1 ml and a final concentration of 50 nM RNP (tdTomato targeting and Sey correction strategies) or 100 nM RNP (Sey correction strategy) in each well. Cells were returned to the incubator for 2 hours. After incubation, the total media was removed from each well and replaced immediately with 500 µl of the saved conditioned media. Each well was then topped up with 500 µl of fresh complete neurobasal media and returned to the incubator for 72 hours, until the next treatment or time of collection on DEV 9. 3.2.5 Immunocytochemistry (ICC) Cells cultured on 12 mm glass coverslips were washed once in phosphate-buffered saline (PBS) and fixed using 4% paraformaldehyde (PFA) (Cat# P6148, Sigma-Aldrich) for 10 minutes. For cells treated with tdTomato targeting strategies, coverslips were exposed to Hoechst nuclear stain (1:1000) in PBS for 10 minutes, before washing twice with PBS. Coverslips were then mounted in ProLong Gold Antifade reagent (Cat# P10144, Invitrogen) and stored in the dark until visualization. For cells treated with Sey correction strategies, cover slips were blocked in PBS + 0.1% Triton X-100 (Cat# T8787, Sigma-Aldrich) + 0.3% bovine serum albumin (Cat# A2058, Sigma-Aldrich) + 10% goat serum (Cat# G9023, Sigma-Aldrich) at room temperature for 1 hour, and subsequently incubated in primary antibodies at 4°C overnight. Primary antibodies used were rabbit anti-PAX6 (1:1000; Cat# 901301, BioLegend, San Diego, CA) and mouse anti-FLAG (1:500; Cat# F1804, Sigma-Aldrich). Cover slips were gently washed three times with PBS + 0.1% Triton X-100 and incubated with secondary antibodies and Hoechst 54 (1:1000) at room temperature for 2 hours. Secondary antibodies used were anti-rabbit immunoglobulin G (IgG) (1:1000; Cat# A11037, Thermo Fisher Scientific) and anti-mouse IgG (1:1000; Cat# A11029, Thermo Fisher Scientific). Cover slips were gently washed three times with 0.1 M phosphate buffer (PB) buffer, and once in 0.01 M PB buffer and then mounted in ProLong Gold Antifade reagent. 3.2.6 Stereological analysis Cell counting was performed using the Zeiss Axioplan 2 fluorescence microscope (Carl Zeiss Microscopy, Thornwood, NY) and StereoInvestigator software (MBF Bioscience, Williston, VT). 3.2.7 Sanger sequencing and peak quantification The region around Sey variant was PCR amplified using Taq DNA Polymerase (Cat# 18038042, Invitrogen), with primer pair oEMS6071 (5′-TCACTCTATTTTCCCAACACAGCC-3′) and oEMS6075 (5′-GCAAAATATAAGCATCCCAGTGCAT-3′). DNA was extracted from agarose gel and subsequently purified using a QIAquick gel purification kit (Cat# 29706, QIAGEN, Germantown, MD). Unidirectional Sanger sequencing was carried out in the forward direction with primer oEMS6299 (5′-ATGCCGTTCCGATTTCTC-3′) at CMMT DNA Sequencing Core Facility. The sequencing operator was blinded to the samples submitted to avoid bias. Chromatograms were viewed using SnapGene® software (Insightful Science; available at snapgene.com). A free cloud-based version of Analysis Module Variant Analysis Software (Thermo Fisher Scientific) was used to extract the peak height data from Sanger sequencing chromatograms. The peak height data was then normalized against the non-targeting luciferase negative control to determine the percentage of Sey correction and PAM modification in the experimental samples. No samples were excluded from this analysis. 55 3.2.8 Statistical analysis GraphPad Prism version 9.1.1 for Windows (GraphPad Software, San Diego, CA, www.graphpad.com) was used for all statistical analysis and plotting of graphs. Statistical analysis was performed using one-tailed t-test, two-tailed t-test, or one-way ANOVA and p< 0.05 was accepted as statistically significant. Each dot on the graph represents data from a biological replicate. Error bars represent mean ± standard deviation, expressed in percent from 100% baseline. 3.2.9 In vivo genome editing Using data from a small pilot study, it was estimated that if a difference existed, 8 eyes per group would provide a p-value below 0.05 with a power level above 90% (Charan and Kantharia, 2013). Mice were randomly assigned to each group a day prior to the injections by a technician who did not participate in the injections or any of the subsequent analyses. Three Fey mice were excluded from the injections due to either having glaucoma or an unopened eye. LNP-encapsulated RNP and DNA mixtures were prepared immediately prior to injections. 3-month old hybrid B6129F1-loxP-3xStop-loxP-tdTomato WT and Fey mice of both sexes were weighed and anesthetized by isoflurane inhalation in an induction chamber before being moved to the nose cone. A 5 mg/ml working solution of Metacam (DIN: 02240463) was prepared and mice were injected subcutaneously based on their weight (5 mg/kg). Once the surgical plane of anesthesia was reached, Alcaine eye drop (DIN: 00035076) was instilled in the eye to be injected, while Optixcare (Aventix, Burlington, Canada) lubricating eye drop was applied to the contralateral eye. A 2.5 μl syringe (Cat# 7632-01, Hamilton Company, Reno, NV) and Hamilton custom-made 34-gauge removable Small Hub RN needles (length: 9.53 mm; point style: 4; bevel angle: 30°) were used for intrastromal injections. The injections were done according to a 56 previously described method (Basche et al., 2018). In brief, the needle entered the stroma at the 3 o'clock position near corneascleral junction and was slowly moved through the stroma towards the center of cornea where the LNPs were dispensed. Once the needle was carefully removed, Isathal ophthalmic gel (DIN: 02427869) was applied to the injected eye. Mice were examined and weighed daily for 5 days after the injection. During the monitoring period, only one Fey mouse injected with the Sey correction strategy showed an irregular corneal surface and was sacrificed. The final number of mice in each group is shown in Table B.2. 3.2.10 Immunohistochemistry (IHC) Mice were sacrificed 21 days post-injection. Eyes were immediately enucleated and directly fixed in 4% PFA for 2 hours on ice. Cryoprotection, embedding and sectioning of the eyes were carried out according to a previously described method (Mirjalili Mohanna et al., 2020). Long term storage of eye sections in -20°C was avoided, as both epifluorescence and immunofluorescence signals are strongest on fresh sections. For tdTomato epifluorescence, eye sections were washed twice in PBS and counterstained with Hoechst (1:1000) in PBS for 10 minutes. The sections were washed two more times with PBS, before being mounted in ProLong Gold Antifade reagent. Immunofluorescent staining was performed according to a previously described method (Mirjalili Mohanna et al., 2020). For antigen retrieval, sections were submerged in freshly-diluted 1x Target Retrieval Solution (Cat# S1699, Agilent Technologies, Santa Clara, CA) for 10 minutes at 95°C. Primary and secondary antibodies used in IHC were the same as ICC analysis, except all antibody concentrations were doubled. No eyes were excluded from this analysis. Results were scored by a person blinded to the treatment. 57 3.2.11 Microscopy and image processing Cover slips with tdTomato epifluorescence were viewed and imaged at 20x magnification using Zeiss Axioplan 2 fluorescent microscope. Eye sections with tdTomato epifluorescence were viewed and imaged as tile scans at 10x magnification using an Olympus BX61 fluorescent microscope. Immunofluorescence imaging of cover slips and eye sections were carried out at 20x or 40x magnifications using a Leica TCS SP8 Super-Resolution Confocal Microscope (Leica Microsystems, Wetzlar, Germany). For the confocal microscope, Z-stack images were taken at 0.5 μm intervals and subsequently deconvoluted using Huygens Professional Software (Scientific Volume Imaging, Hilversum, Netherlands). The resulting images were processed using ImageJ (http://rsbweb.nih.gov/ij/), and Illustrator (Adobe, Mountain View, CA). 3.2.12 Quantification of tdTomato expression in the cornea Three sections from the two highest tdTomato-expressing Fey and WT eyes were chosen for this analysis. The sections were at least 480 μm apart. For each section, a 400 μm-wide box spanning all three layers of the cornea was drawn in the center of the cornea in ImageJ. Stromal and endothelial cells within the box were manually counted using the ImageJ cell counter plugin. 3.3 Results 3.3.1 LNP delivery of an effective CRISPR RNP strategy turned on tdTomato expression in ex vivo cortical neurons Deleting a sequence using CRISPR is easier than correcting a sequence, as NHEJ repair occurs more frequently than homology-directed repair (HDR) (Frit et al., 2014). Similarly, ex vivo nanoparticle delivery is easier than in vivo. Thus, we decided to start with ex vivo embryonic cortical neurons carrying the loxP-3xStop-loxP-tdTomato reporter allele to evaluate the LNP-RNP delivery platform and optimize the CRISPR cargo. In the first step, we encapsulated 58 recombinant SpCas9 pre-complexed with two previously described (Tabebordbar et al., 2016; Yang et al., 2017) and two newly designed gRNAs, either individually or in combination, using the Incisive DS LNP platform (Figure 3.1a). All gRNA target sites are followed by a canonical SpCas9 NGG protospacer adjacent motif (PAM), except the 5′ extended loxP (eloxP). Next, we tested three DNA templates with various homology arm lengths to facilitate the ligation of the broken DNA ends after the deletion. Our ex vivo studies showed that one of the most successful genome editing results was achieved when cortical neurons were treated with LNPs carrying Cas9, a mixture of all gRNAs, and the shortest DNA template (oEMS6469) (Figure 3.1b, c). The results of stereological analysis demonstrated that tdTomato was expressed at an average 4.3% ± 1.5% SD of total cells (Figure 3.1d). This CRISPR strategy was therefore chosen for all future ex vivo and in vivo experiments targeting the transcriptional stop cassette upstream of tdTomato. 59 Figure 3.1. An optimized CRISPR RNP genome editing strategy resulted in the successful disruption of stop signals and turn on of tdTomato expression in ex vivo cortical neurons. a Schematic of loxP-3xStop-loxP-tdTomato reporter allele with three upstream transcriptional stop signals that are flanked by loxP sites. Several Cas9/gRNA ribonucleoprotein complexes were tested for the disruption of the stop signals and turn on of tdTomato expression. The gRNA target sites are shown by arrows. Except the 5′ eloxP (orange arrow), all other target sites have the canonical NGG protospacer adjacent motif (PAM) next to them. b Schematic of a single-stranded DNA template with 40-bp homology arms used to facilitate the end joining process after the Cas9 cut. c Representative images of embryonic mouse cortical neurons. Cortical neurons treated with all gRNAs and oEMS6469 DNA template showed one of the most successful tdTomato expression. Cortical neurons treated with a luciferase non-targeting CRISPR strategy served as the negative control. tdTomato expression was absent in the negative control, as the two solitary red dots were not associated with Hoechst nuclear stain. d Quantification of tdTomato expression in cortical neurons treated with all gRNAs and oEMS6469 DNA template. tdTomato was expressed at an average 4.3% ± 1.5% SD of total cells. Error bars represent the standard deviation of three biological replicates. cgEMS, CRISPR guide EMS; eloxP, extended loxP; oEMS, oligonucleotide EMS; orange arrow, gRNA target site next to a non-canonical PAM; Pos, positive; Stop, a transcriptional stop signal in the DNA; Sp_Ai9_L, SpCas9_Ai9_Left. Images were taken at 20X magnification. Scale bar = 100 μm. 3.3.2 LNP delivery of the CRISPR RNP strategy corrected the Sey variant and restored FLAG-tagged PAX6 expression in 38% of ex vivo cortical neurons Next, we evaluated LNP-mediated delivery of our pre-validated Sey correction CRISPR strategy (Mirjalili Mohanna et al., 2020) in embryonic cortical neuron cultures established from the 3xFLAG-tagged Sey mouse strain (called Fey hereafter). The Sey correction strategy is more challenging than the tdTomato targeting strategy as it relies on HDR for the precise correction of a single nucleotide variant and subsequent restoration of FLAG-tagged PAX6 expression (Figure 3.2a). The SpCas9 RNP complex was used at equimolar (50:50) or 2:1 (100:50) molar ratio to DNA template in the tested conditions and the genome editing outcome was assessed at the protein level by immunocytochemistry (ICC) analysis. Co-labeling of PAX6 and FLAG proteins was observed in the cortical neurons treated with either condition, confirming successful genome editing and the expression of FLAG-tagged PAX6 (Figure 3.2b). The weak background staining observed in the negative control was addressed in future ICC analysis. For the 50:50 nM condition, the FLAG tag was expressed at an average 15% ± 1.7% SD of total cells, and 28% ± 60 2% SD of PAX6 positive cells. For the 100:50 nM condition, the FLAG tag was expressed at an average 22% ± 2.6% SD of total cells, and 38% ± 5.1% SD of PAX6 positive cells (Figure 3.2c, d). Hence, according to stereological analysis, percentage of FLAG positive cells was significantly increased (two-tailed t-test, p=0.019 and 0.031, respectively) when a higher concentration of the RNP complex was used. In all conditions tested, some cells had condensed nuclei and some had uncondensed or normal nuclei. Only healthy neurons (uncondensed nuclei) expressed PAX6 protein. 61 62 Figure 3.2. A pre-established CRISPR RNP genome editing strategy resulted in FLAG-tagged PAX6 expression in 38% of ex vivo cortical neurons. a Fey cells have a 3xFLAG tag after the Pax6 start codon and carry the Sey nonsense variant in exon 8. Using these cells enables us to visualize the edited neurons via FLAG tag detection in immunocytochemistry analysis, thereby providing a quantitative assessment for our Pax6 genome editing experiments. b Representative images of embryonic mouse cortical neurons. FLAG (red) was expressed in cortical neurons treated with different concentrations of LNP-encapsulated RNP to template (50:50 nM and 100:50 nM). As expected, co-labelling of PAX6 (green) and FLAG (red) (which indicates correction of the Sey variant) was observed in both treatment groups (merge, yellow). FLAG expression was absent in negative control, which was cortical neurons treated with a luciferase non-targeting CRISPR strategy. c & d Quantification of FLAG expression in cortical neurons treated with different concentration ratios of RNP to template. Condition 100:50 nM resulted in a significantly higher FLAG expression. Two-tailed t-test was used to determine the significance of data. Error bars represent the standard deviation of three biological replicates. Fey, 3xFLAG-tagged Sey; Sey, Pax6 small eye; Pos, positive; WT, wild type. Images were taken at 20X magnification. Scale bar = 100 μm. 3.3.3 LNP delivery of the CRISPR RNP strategy corrected the Sey variant in 17% of ex vivo cortical neurons The on-target cleavage activity of the LNP-encapsulated Sey correction CRISPR RNP strategy was quantified by Sanger sequencing to confirm the genomic manipulations at the DNA level. In the Sanger sequencing chromatogram of non-targeting luciferase negative control, the dominant peak at the site of Sey corresponded to the pathogenic nucleotide variant (T). Successful genome editing changed the dominant peak to the WT nucleotide (G) in the Fey cortical neurons treated with 100:50 nM of Sey correction strategy. Additionally, the blocking change that was introduced in the original PAM to prevent re-cutting of the edited sequences was observed in the chromatogram. Our most successful experimental result showed the variant correction and PAM modification were 17% and 28%, respectively, after a single treatment of Fey cortical neurons with the 100:50 nM condition (Figure 3.3a). A lower percentage of Sey correction of the variant nucleotide than PAM modification was expected. It is explained by the fact that Fey embryonic cortical neurons are heterozygous and thus have both WT and Sey alleles at the variant nucleotide position, but are homozygous at the PAM modification. We have 63 previously shown that while our Sey correction strategy favors targeting of the Sey allele changing both the variant and PAM, the WT allele can also be hit in some cases resulting in only the modification of the PAM (Mirjalili Mohanna et al., 2020). Finally, consistent with our earlier observations, Sanger sequencing results from three biological replicates demonstrated higher average variant correction (14% ± 2.5% SD) and PAM modification (22% ± 6% SD) for the 100:50 nM condition compared to the 50:50 nM condition (11% ± 2.9% SD for the variant correction and 20% ± 3.8% SD for PAM modification) (Figure 3.3b, c). However, the difference between groups did not reach statistical significance. Nevertheless, condition 100:50 nM was selected for future in vivo studies, as it outperformed the other condition in both ICC and DNA analyses. 64 Figure 3.3. An optimized CRISPR RNP genome editing strategy corrected the Sey variant in 17% of ex vivo cortical neurons. a The Sey variant (red box) changes a G nucleotide to a T, creating a premature stop codon. Top panel shows a representative Sanger sequencing chromatogram around the heterozygous Sey variant in the negative control. The negative control was Fey embryonic cortical neurons treated with a luciferase non-targeting CRISPR strategy. Bottom panel shows a representative chromatogram from Fey cortical neurons treated with LNP-encapsulated 100:50 nM of Sey correction strategy. The dominant peak in the negative control is a T nucleotide, while the dominant peak in the edited cells has shifted to a G nucleotide, reflecting the correction of the Sey variant from T back to G. To prevent repeat targeting of edited sequences, a blocking change was introduced by the homology-directed repair template in the protospacer adjacent motif (PAM) sequence. The blocking change (green box) was observed as the secondary A peak in the chromatogram of the edited cells. b & c Quantification of Sey variant correction and PAM modification in Fey cortical neurons revealed no significant difference between the 50:50 nM and 100:50 nM treatments. Two-tailed t-test was used to determine the significance of data. Error bars represent the standard deviation of three biological replicates. Fey, 3xFLAG-tagged Sey; ns, non-significant; Sey, Pax6 small eye. 65 3.3.4 Repeated treatment with LNP-RNP significantly increased the expression of both tdTomato (30%) and FLAG-tagged PAX6 (35%) proteins in ex vivo cortical neurons One advantage of non-viral delivery systems is the ability to use repeated dosing to increase the transfection level without inducing immunogenic effects. On this front, we compared the efficiency of single treatment and repeated treatment with our best LNP-encapsulated tdTomato targeting CRISPR RNP strategy in embryonic cortical neurons. Interestingly, elevated expression of tdTomato was observed in cortical neurons after repeated treatment (Figure 3.4a). Stereological analysis revealed a significant increase in tdTomato levels from 7% ± 4.7% SD to 30% ± 11% SD following the second treatment (two-tailed t-test, p=0.026) (Figure 3.4b). To strengthen our observations, we also assessed repeated treatment with our Sey correction LNP-RNP strategy. This time however, we decided to improve the suboptimal 50:50 nM condition to avoid a potential ceiling effect with our 100:50 nM condition. Co-labeling of PAX6 and FLAG proteins in cortical neurons was observed after both treatments (Figure 3.4c). In the absence of primary antibodies, the anti-mouse secondary antibody detected the endogenous mouse IgG antibodies and caused a weak background staining, which was distinctively different from the FLAG signal in the experimental samples (Figure 3.4c). On average, 8% ± 1.8% SD of total cells, and 16.5% ± 2.1% SD of PAX6-positive cells expressed FLAG after a single treatment, whereas these numbers increased to 14.5% ± 3% SD and 35% ± 5.2% SD, respectively, with repeated treatment (Figure 3.4d, e). Therefore, consistent with our earlier observations, repeated treatment resulted in a significant increase in the number of FLAG positive cells compared to a single treatment (two-tailed t-test, p=0.01 and 0.0005). To explore the potential cytotoxicity effects of our LNPs, we next quantified the percentage of PAX6 positive cells in untreated Fey cortical neurons, as well as Fey cortical neurons that received 66 either a single treatment or repeated treatment of luciferase non-targeting strategy (Figure 3.4f). Our statistical analysis showed no difference in the mean PAX6 expression among the three groups (one-way ANOVA, p=0.078); suggesting a lack of LNP-induced cytotoxicity within the concentration and parameters tested. 67 68 Figure 3.4. Repeated treatment significantly increased both tdTomato (30%) and FLAG-tagged PAX6 (35%) proteins in ex vivo cortical neurons. a Representative images of loxP-3xStop-loxP-tdTomato embryonic mouse cortical neurons. tdTomato expression was observed in a greater number of cells after repeated treatment with our best LNP-encapsulated tdTomato targeting CRISPR strategy. tdTomato expression was absent in the negative control, which was cortical neurons treated with a luciferase non-targeting strategy. b Quantification of tdTomato expression revealed a significant increase in tdTomato levels after repeated treatment. Two-tailed t-test was used to determine the significance of data. Error bars represent the standard deviation of three biological replicates. c Representative images of Fey embryonic mouse cortical neurons. FLAG (red) was expressed in cortical neurons treated once or twice with 50:50 nM of RNP to template. As expected, co-labelling of PAX6 (green) and FLAG (red) (which indicates correction of the Sey allele) was observed in both treatment groups (merge, yellow). FLAG was not expressed in negative control, which was cortical neurons treated with a luciferase non-targeting strategy. In contrast to experimental samples that expressed FLAG, only a weak background staining was observed in the no primary Ab control. d & e Quantification of FLAG expression in cortical neurons treated either once or twice with 50:50 nM of RNP to template. Repeated treatment resulted in higher FLAG expression. Two-tailed t-test was used to determine the significance of data. Error bars represent the standard deviation of four biological replicates. f Quantification of PAX6 expression in Fey cortical neurons assessing cytotoxicity after single treatment or repeated treatment with a luciferase non-targeting strategy. There was no significant difference in the mean PAX6 expression among the three groups. One-way ANOVA test was used to determine the significance of data. Error bars represent the standard deviation of three biological replicates. Ab, antibody; Fey, 3xFLAG-tagged Sey; ns, non-significant; Pos, positive. Images were taken at 20X magnification. Scale bar = 100 μm. 3.3.5 Repeated treatment with LNP-RNP increased, but not significantly, the correction of the Sey variant in ex vivo cortical neurons Similar to earlier analysis, the effect of repeated treatment on the on-target cleavage activity of the Sey correction LNP-RNP CRISPR strategy was investigated by Sanger sequencing to confirm the genomic manipulations at the DNA level. When compared to the negative control, the variant correction and PAM modification were both evident in the chromatograms of Fey embryonic cortical neurons with either single treatment or repeated treatment of the 50:50 nM Sey correction strategy (Figure 3.5a). Sanger sequencing results from six biological replicates indicated an average 14% ± 4% SD correction and 26.7% ± 11.7% SD PAM modification after a single treatment versus 16.7% ± 3.8% SD correction and 27.3% ± 6.4% SD PAM modification after the repeated treatment (Figure 3.5b, c). Thus, repeated treatment resulted in higher 69 correction of the variant, but the increase was not statistically significant (one-tailed t-test, p=0.133). Figure 3.5. Repeated treatment with LNP-RNP increased, but not significantly, the correction of the Sey variant in ex vivo cortical neurons. 70 a The effect of repeated treatment on genome editing levels in Fey embryonic mouse cortical neurons. Top panel demonstrates the Sanger sequencing chromatogram around the Sey variant in the negative control. The negative control was cortical neurons treated with an LNP-encapsulated luciferase non-targeting CRISPR strategy. Middle and bottom panels, respectively, demonstrate the Sanger sequencing chromatograms of cortical neurons treated either once or twice with 50:50 nM of RNP to template. The dominant peak in the Sey location (red box) has changed to a G nucleotide in both treatment groups compared to the negative control. The introduced blocking change (green box) in the protospacer adjacent motif (PAM) sequence was observed in both treatment groups. b & c Quantification of data revealed no significant difference in average correction and PAM modification between single treatment and repeated treatment groups. One-tailed t-test was used to determine the significance of data. Error bars represent the standard deviation of six biological replicates. Fey, 3xFLAG-tagged Sey; ns, non-significant; Sey, Pax6 small eye. 3.3.6 In vivo intrastromal delivery of the LNP-RNP CRISPR strategy turned on wide-spread tdTomato expression in the cornea We used the loxP-3xStop-loxP-tdTomato reporter mice to evaluate the delivery and editing efficiency of our intrastromally-delivered LNP-RNP CRISPR strategy. Fey and WT mice were injected with either the LNP-encapsulated tdTomato targeting RNP or luciferase non-targeting RNP strategy. Intrastromal injections of our tdTomato targeting LNP-RNP strategy led to wide-spread expression of tdTomato in the cornea of both the Fey and WT experimental mice, whereas no tdTomato expression was observed in the corneas of mice that received luciferase non-targeting strategy (Figure 3.6a). In some instances, the corneas showed minor damage where the needle entered (Figure 3.6b). Importantly, there was no correlation between corneal damage and tdTomato expression, as tdTomato expression was not always observed at the site of damage. Upon confocal examination, it was evident that tdTomato was expressed in two of the three corneal cell layers. It was widely expressed in stromal cells, which is where the injection occurred, as well as some endothelial cells, but not in the corneal epithelial cells (Figure 3.6c). On average, the in vivo genome editing in the Fey corneas resulted in tdTomato expression in 6.2% ± 2.9% SD of stromal and endothelial cells. This number was increased to 13.1% ± 2.2% 71 SD for the WT corneas, although there was no significant difference in the tdTomato expression between the two genotypes (Figure 3.6d). Interestingly, in 4 out of 6 WT mice, we also observed transfection of the iris (Figure 3.6e). We hypothesize that the iris transfection is not due to the perforation of the endothelium, but rather due to leakiness at the entry site of the needle near the corneoscleral junction. Despite this dispersal of LNPs, tdTomato expression was not observed in the retina or elsewhere in the eye. 72 Figure 3.6. Intrastromally-delivered CRISPR genome editing resulted in wide-spread tdTomato expression in vivo in the cornea. a Representative fluorescence images of mouse corneas. Fey and WT mice injected with the LNP-encapsulated tdTomato targeting CRISPR strategy demonstrated wide-spread tdTomato expression in their corneas. tdTomato expression was absent in the cornea of negative control mice that were injected with a non-targeting luciferase strategy. b Eye showing damage at the site of intrastromal injection but expression beyond the damaged location. c Representative confocal images of mouse corneas. Stromal 73 and endothelial cells were both positive for tdTomato expression, whereas no tdTomato expression was observed in the epithelial cells. d Quantification of tdTomato expression in the cornea revealed no significant difference in the average tdTomato positive cells between Fey and WT mice. Two-tailed t-test was used to determine the significance of data. Error bars represent the standard deviation of two biological replicates. e tdTomato expression observed in the iris of WT mice. Blue, Hoechst; End, endothelium; Epi, epithelium; Fey, 3xFLAG-tagged Sey; ns, non-significant; Pos, positive; red, tdTomato epifluorescence; Str, stroma; WT, wild type. Confocal images were taken at 40X magnification. Scale bar = 50 μm. 3.3.7 In vivo intrastromal delivery of the LNP-RNP CRISPR strategy did not result in detectable correction of the Sey variant in the cornea To further explore the targeting ability of our delivery platform, Fey and WT mice were injected intrastromally into the cornea with LNPs encapsulating the Sey correction CRISPR strategy. Since, PAX6 expression is limited to the corneal epithelial layer (Li et al., 2015a) (Figure 3.7), and our earlier in vivo experiment demonstrated no tdTomato expression in the epithelial cells (Figure 3.6c), we did not expect, nor did we see, turn on of FLAG-tagged PAX6 expression (Figure 3.7). We used homozygote 3xFLAG-tagged Pax6 (Fax) mice, which express FLAG tag in all PAX6-expressing cells of the eye, as positive controls (Mirjalili Mohanna et al., 2020). Nevertheless, we did anticipate obtaining evidence of genome editing at the DNA level in the stromal and endothelial cells as our earlier in vivo experiment demonstrated expression in those cells (Figure 3.6c). However, Sanger sequencing did not detect any genomic modifications in the analyzed corneas, implying that the genome editing events, if any, were below the detection threshold of this technique. 74 Figure 3.7. Intrastromally-delivered CRISPR genome editing strategy did not target PAX6-expressing corneal epithelial cells in vivo. Representative confocal images of mouse corneas. In the positive control 3xFLAG-tagged Pax6 (Fax) mice, FLAG (red) expression was detected in the PAX6-expressing (green) corneal epithelial cells (merge, yellow). However, FLAG expression was not observed in the Fey mice corneas that were injected with the LNP-encapsulated Sey-correction strategy at 100:50 nM. As expected, FLAG expression was absent in the cornea of negative control mice that were injected with a non-targeting luciferase strategy. End, endothelium; Epi, epithelium; Str, stroma. Images were taken at 40X magnification. Scale bar = 50 μm. 3.4 Discussion Here we first demonstrated and optimized the effectiveness of our novel LNP platform for delivering RNPs and template DNA to ex vivo mouse cortical neurons. We then showed high genome editing levels in vivo in mouse cornea. Importantly, this is the first time LNP delivery of RNP complex and DNA template has been achieved in the eye. Given the therapeutic importance of genome editing, and the clinical importance of the cornea as a therapeutic target, our results highlight the potential use of this delivery strategy in treating corneal diseases. These conditions may include genetic eye diseases such as macular corneal dystrophy (MCD) (Di Iorio et al., 2010), Fuchs' endothelial corneal dystrophy (FECD) (Uehara et al., 2021), and of course the 75 congenital blindness aniridia (Mirjalili Mohanna et al., 2020), or infectious diseases such as herpetic stromal keratitis (HSK) (Yin et al., 2021). In recent years, we and others have shown that aniridia (Mirjalili Mohanna et al., 2020), FECD (Uehara et al., 2021), and HSK (Yin et al., 2021) are good candidates for CRISPR-mediated genome editing therapies. LNPs come in a staggering complexity of formulations. However, to date, LNP encapsulation of RNPs remains challenging due to their complex composition and overall charge (Wei et al., 2020). In this study, we demonstrated the efficiency of the Incisive LNP delivery platform to encapsulate the active SpCas9 protein, multiple gRNAs, and DNA template within LNPs, allowing their delivery and release into target cells for precise genome editing. This platform gives the advantage to perform a one-step encapsulation enabling testing various CRISPR strategies in the shortest period of time. In contrast to viral vectors, LNPs have the potential to be administered multiple times with the goal to increase their therapeutic efficiency. Onpattro (Patisiran) (NCT01960348) is the first FDA approved LNP-based RNAi therapeutic that demonstrated the safety and efficiency of using multiple dose regiments to treat polyneuropathy of hereditary transthyretin-mediated amyloidosis in adults (Adams et al., 2018). More recently, research and real-world data has shown that a third dose of the Pfizer-BioNTech lipid-based mRNA Covid-19 vaccine helps to prevent severe disease in adults (Andrews et al., 2022; Bar-On et al., 2021). Animal studies have reported cumulative in vivo genome editing effects upon repeated treatment with LNP-based CRISPR strategies (Finn et al., 2018; Kenjo et al., 2021; Rothgangl et al., 2021). In this study, our stereological analysis showed a higher percentage of tdTomato and FLAG-tagged PAX6 proteins in ex vivo cortical neurons after repeated treatment. Although supportive of this result, the difference in genome editing levels in DNA analysis after repeated treatment did not reach 76 statistical significance. This is possibly due to smaller sample size available for Sanger sequencing compared to stereological analysis, since in the latter, cells were counted from many randomly sampled sites to obtain a reliable quantitative data per cover slip. Unfortunately, we were unable to build on our data by repeat dosing in vivo, since our current protocol permits only a single intrastromal injection per animal. This will be an important topic for future studies. In this work, we showed both ex vivo tdTomato targeting and precise correction of Sey pathogenic variant with restoration of FLAG-tagged PAX6 expression. However, while our in vivo studies with the tdTomato targeting were also highly successful, the in vivo Sey correction strategy was not. This was surprising especially in light of a study that showed a single intrastromal injection of CRISPR plasmids to the eyes of Krt12+/luc2 reporter mice resulted in significant knockdown of luciferase expression in the cornea (Christie et al., 2017). On the other hand, this may simply have been a detection issue. The transduced corneal stromal and endothelial cells do not naturally express PAX6, so no FLAG-tagged expression could be detected. Further, in future studies Sanger sequencing could be replaced by the more sensitive pyrosequencing (Courtney et al., 2016) or next generation (Mirjalili Mohanna et al., 2020) sequencing techniques. Additionally, it may be possible to improve the in vivo HDR efficiency by further optimizing the Sey correction CRISPR strategy, and applying a repeat dosing protocol. In humans, intrastromal injection is routinely used to administer antibiotics and antifungals directly into the cornea (Hu et al., 2016; Vieira et al., 2012). In contrast to topical delivery, intrastromal injection offers the advantage of concentrating therapeutic agents in the stromal layer by bypassing the corneal epithelium that acts as a physical barrier, as well as reducing the need for frequent administration. However, it is known that intrastromal injections are technically difficult to perform and can damage both the mouse and human eye (Hippert et 77 al., 2012; Soleimani et al., 2020). The damage observed in some of the corneas in our study is attributable to the small size of the mouse eye and technical difficulties during injection independent of the LNP composition or its cargo. This can be mitigated by training and frequently changing the rapidly dulled needles. Intrastromal injection continues to be a preferred choice in our work as it allows for concentrated delivery to the cornea, with minor impact on the retina and other eye structures. It is known that Apolipoprotein E4 (ApoE4), a protein involved in lipid transport, provides an important mechanism for facilitating the uptake and subsequent endocytosis of LNPs in cultured neurons (Rungta et al., 2013; Sebastiani et al., 2021). Hence, for the ex vivo experiments, we supplemented the cortical neuron cultures with recombinant ApoE4 and had a strong positive result. Thus, we were concerned when we could not find evidence of ApoE4 presence in the corneal stromal layer. Nevertheless, our in vivo genome editing with tdTomato targeting strategy resulted in wide-spread tdTomato expression in the cornea. Interestingly, previous work has shown comparable LNP uptake in the RPE of apoE-/- and C57BL/6 control mice, indicating that ApoE4 might not augment the uptake of LNPs in every tissue (Ryals et al., 2020). Our data provides further support for this observation. The cornea is made up of three cell layers, the middle stroma where injection was directed, underlain by the endothelial layer, and overlain by the epithelial cell layer (Sridhar, 2018). Interestingly, in all WT eyes, intrastromal injection of tdTomato targeting strategy resulted in transfection of not only the stromal cells, but also the endothelial cells. Corneal stroma is separated from the single cell-layer thick endothelium by a thin acellular matrix called Descemet’s membrane. This indicates the probable ability of our LNPs to pass through Descemet’s membrane and transfect endothelial cells. Structural abnormalities such as 78 keratolenticular adhesions (Ramaesh et al., 2003) made it difficult to analyze endothelial layer transfection in the Fey eyes. In the time course of 21 days, we did not detect either tdTomato or FLAG expression in the epithelial layer, but this does not absolutely rule out the ability of our LNPs to reach epithelium. One possible explanation for this could be the 21 day harvest time, as most basal corneal epithelial cells adjacent to the stroma become terminally differentiated cells and shed from the epithelium every 14 days (Douvaras et al., 2013). On the other hand, 21 days certainly was too early to see the impact of transduction of limbal stem cells, which take months for the daughter cells to populate the corneal epithelial layer (Collinson et al., 2004). Thus, further shorter and longer-term studies will be needed to explore broadening the therapeutic impact of this approach to also include corneal epithelial disease (Roux et al., 2018). 3.5 Acknowledgments We thank Dr. Jingsong Wang at the British Columbia Children’s Hospital Research Institute (BCCHRI) Imaging Core Facility for microscope support, Ms. Marketa Hlavon at the BCCHRI Sequencing and Bioanalyzer Core for Sanger sequencing support, and Dr. Ainsley Coquinco of Incisive Genetics Inc. for technical support. This work was funded by Incisive Genetics Inc., Vancouver, BC, Canada. 79 Chapter 4: rAAV-PHP.B Escapes the Mouse Eye and Causes Lethality Whereas rAAV9 can Transduce Aniridic Corneal Limbal Stem Cells Without Lethality3 4.1 Introduction Gene therapy is undergoing a successful revival for disorders with unmet therapeutic needs. To date, recombinant adeno-associated viruses (rAAVs) are one of the most frequently used vectors for in vivo delivery of gene therapies (Wang et al., 2019). rAAV-based gene therapies have shown acceptable long-term safety and efficacy profiles in several clinical trials, leading to FDA-approval of Luxturna® in 2017 and Zolgensma® in 2019 (Kuzmin et al., 2021). However, recent studies have raised safety concerns associated with high-doses of rAAV in both preclinical studies (Hinderer et al., 2018; Hordeaux et al., 2020; Hordeaux et al., 2018) and clinical gene therapy trials (Agarwal, 2020; Kishimoto and Samulski, 2022). Therefore, when developing a new rAAV gene therapy, elements such as viral capsid, route of administration, dose, and promoter need to be carefully considered. Among these elements, we will explore the choice of capsid and route of administration in our study to pave the way for developing a new rAAV-based therapy for the eye disorder aniridia. Aniridia is a rare congenital eye disorder, which is caused by autosomal dominant variants in the paired box 6 (PAX6) gene. It clinically manifests as reduced visual acuity and photophobia in childhood due to hypoplasia of the retinal fovea and iris, and gradually leads to blindness by the second to third decade of life due to glaucoma and/or slowly progressing 3 This chapter has been published in Gene Therapy as: Mirjalili Mohanna et al. (2023). LNP-Mediated Delivery of CRISPR RNP for Wide-Spread In Vivo Genome Editing in Mouse Cornea. Gene Ther. Epub ahead of print. PMID: 37072572. See Preface for details of my contributions. 80 corneal vascularization and clouding (Hingorani et al., 2012; Shortt et al., 2014). This time course means there is a therapeutic window available to undertake treatment. To date, various symptomatic interventions are available for aniridia patients to slow the disease progress, but none prevent the eventual blindness. Limbal stem cell (LSC) deficiency has been proposed as the underlying mechanism responsible for corneal clouding in aniridia (Li et al., 2015b; Mort et al., 2011; Ramaesh et al., 2005a) and as an important contributing factor to the corneal epithelial thinning observed in this disease (Davis et al., 2003; Hickmott et al., 2018; Ramaesh et al., 2003; Ramaesh et al., 2005b). In a healthy cornea, the slow-dividing LSCs are present throughout life and continuously replenish the epithelium with new cells to maintain the transparency of the cornea (Mort et al., 2012). This makes LSCs of the cornea important targets for intervention in patients with aniridia. The rAAV capsid controls tropism and together with the route of administration has the ability to greatly influence the safety and efficacy of gene therapy (Colon-Thillet et al., 2021). To date, 13 naturally occurring AAV serotypes have been identified, and numerous variants within these serotypes have been isolated from various species including humans and non-human primates (NHPs) (Sabatino et al., 2022). rAAVs were generated on the foundation of these naturally occurring capsids and have been studied by many groups to determine their tissue tropism (Becker et al., 2022). rAAV9 is among the most-commonly used capsids for systemic delivery to the central nervous system (CNS), owning to its natural ability to cross the blood-brain barrier (BBB) after intravenous injection leading to neuronal tropism in neonate pups, and astrocytic tropism in adult mice (Foust et al., 2009) as well as in NHPs (Samaranch et al., 2012). In the last decade, it has become increasingly popular to apply capsid engineering approaches such as directed evolution to further improve the transduction efficiency of rAAVs. In 2016, 81 capsid engineering studies led to the development of rAAV-PHP.B (hereafter referred to as PHP.B) with superior BBB-crossing ability after intravenous injection in adult mice compared to its parental rAAV9 capsid (Deverman et al., 2016). The following year, a directed evolution strategy led to the discovery of a second-generation capsid, rAAV-PHP.eB, with further enhanced CNS transduction efficiency after systemic delivery in adult mice (Chan et al., 2017), creating new opportunities for capsid selection when developing gene therapy. However, shortly after, researchers discovered that the excellent properties of PHP.B capsids for systemic delivery to the CNS depends on interaction with the LY6A receptor and therefore are mouse strain (Hordeaux et al., 2018; Hordeaux et al., 2019; Huang et al., 2019; Matsuzaki et al., 2019) and species-specific (Hordeaux et al., 2018; Liguore et al., 2019; Matsuzaki et al., 2018). In spite of this, the PHP.B family of capsids is still widely used in preclinical gene therapy studies (Chen et al., 2022b; Gyorgy et al., 2019; Hana et al., 2021; Shubina-Oleinik et al., 2021; Taiber et al., 2021; Wu et al., 2021) and are valuable in demonstrating efficacy in a mouse model prior to NHP studies, recognizing that the final capsid selection must be optimal for humans. Intravitreal injections of rAAV9 have previously proven effective for the transduction of various PAX6-expressing retinal cells in wild-type (WT) mouse eyes (Hickmott et al., 2016; Korecki et al., 2021; Simpson et al., 2019). These studies used a “current” marker (e.g. green fluorescent protein) showing where the virus is currently, which may be suitable for modeling traditional gene-augmentation therapies. In contrast, much less work has been done with rAAV carrying a “historical” marker (e.g. Cre, rearranging the genome), which aims to show all cells transduced over time and thus is better suited for modeling newer gene editing therapies such as CRISPR. Cre has been previously used in the cornea to show the ability of intrastromal injection of rAAV8[Y733F] to reach PAX6-expressing corneal epithelial cells, as well as the 82 therapeutically significant corneal LSCs in WT mouse eyes (Basche et al., 2018). Now it is important to build on these works and explore the transduction patterns of rAAV9 and the novel PHP.B capsids in the aniridic retina and cornea using a historical marker to make the findings translatable, especially to gene editing strategies for this disease. There is a good mouse model of aniridia known as Small eye (Sey), which is also caused by a dominant variant in the Pax6 gene (Hill et al., 1991; Ramaesh et al., 2003). The corneal abnormalities seen in these mice mimic those of aniridia patients (Ramaesh et al., 2003) and have been similarly attributed to defects in LSCs (Collinson et al., 2004). The Sey mice used in this study have been extensively characterized (Hickmott et al., 2018) and bred to additionally carry a loxP-3xStop-loxP-tdTomato Cre-reporter allele (Madisen et al., 2010). The resulting mice have previously been used for testing and optimizing gene editing delivery methods for aniridia (Mirjalili Mohanna et al., 2022). Injecting these reporter mice with rAAV expressing improved Cre (iCre) removes the transcriptional stop signal upstream of tdTomato gene, resulting in tdTomato epifluorescence expression that can be visualized at the single cell level in histological analysis, allowing characterization of vector target-cell specificity. This has been demonstrated for corneal LSCs in WT mice when transduction of LSCs led to tdTomato expression, the cells divided and gave rise to tdTomato positive transient amplifying cells, that became terminally differentiated epithelial cells of the central cornea (Basche et al., 2018). This cell movement in the corneal epithelial layer generated a red stripe-like banding pattern and indicated the successful targeting of functional LSCs (Basche et al., 2018). Here we present our findings from testing two capsids (rAAV9 and PHP.B), in two mouse strains (WT and Sey), by three different administration routes (intrastromal, intravitreal, and intravenous). We recommend the best capsid and administration routes for future gene 83 therapy for aniridia. In addition, these findings deliver an important message of rAAV lethality for all rAAV ocular gene therapies in development. 4.2 Materials and Methods 4.2.1 Mouse husbandry and breeding Experimental mice were primarily B6129F1-loxP-3xStop-loxP-tdTomato, Pax6Sey/+ or B6129F1-loxP-3xStop-loxP-tdTomato, Pax6+/+, of both sexes, produced as the first generation by crossing homozygous B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J (JAX strain: 007914, aka Ai14) dams to 129S1/SvImJ-Pax6Sey/+ (MMRRC Stock No: 050624-MU) studs. A subset of experimental mice was B6129F1-loxP-3xStop-loxP-tdTomato, Hmgcr-lacZ, Pax6Sey/+ or B6129F1-loxP-3xStop-loxP-tdTomato, Hmgcr-lacZ, Pax6+/+, of both sexes, produced by a two-generation breeding strategy. Initially, homozygous B6-loxP-3xStop-loxP-tdTomato dams were crossed to B6-Hmgcr-lacZ (JAX strain: 024050) studs. The resulting homozygous B6-loxP-3xStop-loxP-tdTomato, Hmgcr-lacZ dams were then crossed to 129S1/SvImJ-Pax6Sey/+ studs. (Note: Hmgcr-lacZ was not used in these experiments.) The non-reporter control mice (i.e. without the loxP-3xStop-loxP-tdTomato, or Hmgcr-lacZ alleles) were either B6129F1-Pax6Sey/+ or B6129F1-Pax6+/+, of both sexes, produced as the first generation by crossing C57BL/6J (JAX strain: 000664) dams to 129S1/SvImJ-Pax6Sey/+ or 129S1/SvImJ (JAX strain: 002448) studs. Mice were maintained in the pathogen-free Centre for Molecular Medicine and Therapeutics (CMMT) mouse facility on a 7 am–7 pm light cycle, 20 ± 2°C with 50 ± 5% relative humidity, and had food and water ad libitum. All procedures involving mice were in accordance with the Canadian Council on Animal Care and UBC Animal Care Committee (Protocols A17-0204, A17-0205, A21-0140, and A21-0184). 84 Pups were weaned and ear notched at postnatal day (P) 21. Collected ear notches were digested in 200 µl of tissue homogenization buffer with Proteinase K (Cat #P2308, Sigma-Aldrich, Burlington, MA) according to a previously described protocol (Yang et al., 2009). PCR-based genotyping was carried out to determine the presence of the Sey variant or Hmgcr-lacZ using Taq DNA Polymerase (Cat #18038042, Invitrogen, Waltham, MA). Primer pair oEMS6071 (5′-TCACTCTATTTTCCCAACACAGCC-3′) and oEMS6073 (5′-CTGAGCTTCATCCGAGTCTTCTTA-3′) was used for identifying Sey mice. Primer pair oEMS6087 (5′-AAAGCAGGATTTATGGCAAGGTG-3′) and oEMS6088 (5′-AGTTTGAGGGGACGACGACAGTA-3′) was used for identifying Hmgcr-lacZ-positive mice. 4.2.2 rAAV Production Small chicken beta actin promoter/CMV enhancer (smCBA) was ordered for direct DNA synthesis (GenScript, Piscataway, NJ), and cloned into the multiple cloning site (AvrII, FseI, MluI, and AscI) of our “plug and play” rAAV2 backbone plasmid (pEMS1987) (de Leeuw et al., 2016). This genome plasmid includes: an intron (optimized chimeric; 173 bp) (Promega, Madison, MI) (Sakurai et al., 2010), NotI flanked iCre recombinase (1032 bp) (Shimshek et al., 2002), AsiSI flanked woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) mut6 (587 bp) (Zanta-Boussif et al., 2009), and SV40 polyA (222 bp) (Promega) sequences. The resulting plasmid, pEMS2118 (Addgene plasmid #49143), was propagated in E.coli SURE cells (Cat #200227, Agilent Technologies, Santa Clara, CA). DNA was prepared by QIAGEN Spin MiniPrep Kit (Cat #27104, QIAGEN, Germantown, MD) and plasmid was confirmed free of rearrangements by AhdI digest, inverted terminal repeats verified by SmaI digest, and cloning sites verified by Sanger sequencing. Confirmed plasmid was then sent to the University of Pennsylvania Vector Core (Philadelphia, PA) for large-scale DNA amplification using the 85 EndoFree Plasmid Mega Kit (Cat #12381, QIAGEN, Hilden, Germany). Quality control on the plasmid preparation was done by SmaI, PvuII, and SnaBI digests, and the confirmed plasmid was then packaged into either rAAV9 or PHP.B capsid by the University of Pennsylvania Vector Core. The rAAV genome titer was determined by droplet digital PCR using primers that recognize inverted terminal repeats. 4.2.3 Intravenous injections Sample sizes (number of mice per group) were based on a previously published similar experiment (de Leeuw et al., 2016). Pups were randomly grouped based on the litter and all were injected regardless of eye phenotype. P21 injections were into the tail vein. A 31-gauge needle on a 0.33 cc syringe (Cat #320440, BD, Franklin Lakes, NJ) was inserted into the tail vein and 50 µl of virus at a titer of either 1 x 1013, 3 x 1012, 1.5 x 1012, 6 x 1011, or 3 x 1011 viral genome/ml (vg/ml) of PHP.B ssAAV-smCBA-iCre-WPRE (hereafter referred to as PHP.B iCre) in phosphate buffer saline (PBS) and 0.001% Pluronic acid (Cat #24040032, Gibco, Thermo Fisher, Waltham, MA) (together, PBS+P) was slowly injected. Final injection doses were: 5 x 1011 (16 mice), 1.5 x 1011 (16 mice), 7.5 x 1010 (7 mice), 3 x 1010 (7 mice), and 1.5 x1010 (7 mice) vg/mouse. Mice were observed daily for 5 days following injection, followed by weekly observations. 4.2.4 Intravitreal and intrastromal injections Sample sizes (number of mice per group) were based on a previously published similar experiment (de Leeuw et al., 2016). Mice were genotyped and grouped by one person and injections were done on a separate day by a different person who did not re-examine the eyes. Adult mice (2 months old for intravitreal injections and 3 months old for intrastromal injections) were weighed, anesthetized with isoflurane inhalation, and injected with 5 mg/kg Metacam 86 (DIN: 02240463) for pain management. All procedures were performed while mice were at surgical plane of anesthesia. Alcaine eye drops (DIN: 00035076) were applied to numb the eye receiving the injection, and Optixcare (Aventix, Burlington, Canada) lubricating eye gel was applied to the contralateral eye to prevent dryness. Custom-made 33- and 34-gauge removable Small Hub RN needles (length: 9.53 mm; point style: 4; bevel angle: 30°) (Hamilton Company, Reno, NV) were used for intravitreal and intrastromal injections, respectively. For rAAV injections, 2 μl of undiluted virus at a titer of either 4.2 x 1013 vg/ml of PHP.B iCre or 3.8 x 1013 vg/ml of rAAV9 ssAAV-smCBA-iCre-WPRE (hereafter referred to as rAAV9 iCre) in PBS+P was loaded into a 2.5 μl syringe (Cat #7632-01, Hamilton Company). For vehicle-only injections, 2 μl of PBS+P was loaded into the syringe. Final injection doses were: 8.4 x 1010 vg/mouse for PHP.B iCre and 7.6 x 1010 vg/mouse for rAAV9 iCre. Intravitreal injection was performed by inserting the needle perpendicular to the sclera close to the limbus region and slowly advancing within sclera to vitreous chamber before dispensing the virus. Intrastromal injection was performed following a previously described method (Basche et al., 2018; Mirjalili Mohanna et al., 2022). Briefly, the needle was inserted into the stroma near the corneoscleral junction and was carefully advanced to the center of the stroma where the payload was dispensed. The central injection method in combination with the injection volume were chosen with the goal of reaching as much of the cornea as possible after a single injection. Injection success was a predetermined inclusion criterion for intrastromal injections, as this is a challenging technique to perform on the small mouse eye. Mice that did not receive a successful intrastromal injection were immediately euthanized. Mice were weighed and eyes closely examined daily for five days following injection. After that, mice were monitored weekly until 87 the first lethality was observed, leading to careful examination of all the remaining mice twice a week until they were euthanized and tissues were harvested from a subset. 4.2.5 Slit lamp imaging Slit lamp imaging was performed according to a previously described method (Mirjalili Mohanna et al., 2020). Briefly, 3-month-old mice were placed under general anesthesia. The Phoenix Micron Anterior Segment Imaging system (Phoenix Research Labs, Pleasanton, CA) was used to visualize the corneal phenotype of the mouse left eye. 4.2.6 Histology Intraocularly-injected mice were initially planned to be harvested at 1 month and 7 months post-injection, but due to the unforeseen lethality in PHP.B iCre mice, the experimental mice were euthanized and tissues were harvested once they reached a humane endpoint and the non-reporter control mice were euthanized without harvesting. Despite the absence of phenotype in rAAV9 iCre mice, harvest time for these mice was also adjusted to match the PHP.B iCre mice for comparison purposes. However, in the case of intrastromal rAAV9 iCre injections, a subset of injected mice was aged and harvested at 7 months post-injection to allow for the formation of LSC stripes. At the time of harvest, mice were euthanized by anesthetic avertin (Cat #T48402, Sigma-Aldrich) overdose, and transcardially perfused according to a previously described method (Korecki et al., 2019). Tissues were then harvested and post-fixed overnight in 4% paraformaldehyde (PFA) in PBS at 4°C. On the following day, for cryosections, tissues were transferred to 25% sucrose in PBS for a minimum of 24 hours at 4°C or until they were processed. Tissues were embedded in Tissue-Tek optimal cutting temperature compound (Cat #4583; Sakura FineTek, Torrance, CA) in cryomolds on dry ice. Next, 20 μm thick cryosections were cut and directly mounted onto Superfrost Plus slides (Cat #1255015, Thermo Fisher). 88 Slides were stored at -80°C until staining was performed. For staining, slides were warmed to room temperature for 5 minutes and pressed underweight for 30-60 minutes. Cryosections were then washed twice in PBS and incubated with Hoechst nuclear stain (1:1000) in PBS for 10 minutes. Following incubation, cryosections were washed twice more with PBS, before being mounted with ProLong Gold Antifade reagent (Cat #P10144, Invitrogen). Slides were kept in dark at 4°C until visualization. For corneal flat mounts, fixed eyes were stored in PBS until they were processed. Eyes were placed in a petri dish filled with PBS. Corneas were dissected using surgical scissors (Cat #15000-03, Fine Science Tools, Foster City, CA) and forceps (Cat #11251-33, Fine Science Tools) under a Leica MZ APO dissecting microscope (Leica Microsystems, Wetzlar, DE). Four radial cuts were made into the corneas before transferring them onto slides (one cornea per slide). Corneas were then mounted with ProLong Gold Antifade reagent with DAPI (Cat #P36935, Invitrogen) and flattened with a cover slip. Slides were kept in dark at 4°C overnight before imaging. All injected eyes were processed and analyzed, whereas non-ocular tissues from a minimum of 2 mice per genotype were processed and analyzed for each group at each harvest time point. Results were confirmed by an individual blinded to treatment groups. 4.2.7 Microscopy and image processing tdTomato epifluorescence was visualized in cryosections and corneal flat mounts using an Olympus BX61 fluorescent microscope (Olympus, Tokyo, Japan) at either 4× magnification (cerebellum, spinal cord, liver, heart, and pancreas) or 10× magnification (eye cryosections and corneal flat mounts). The cerebellum and eye images were taken as Tile scans. Corneal flat mounts were re-imaged as Tile scans using a Leica SP8 confocal microscope (Leica Microsystems) at 20× magnification by z-projection through the epithelium that was defined at the center of each cornea. The resulting images were processed using Image J 89 (http://rsbweb.nih.gov/ij/), and Illustrator (Adobe, Mountain View, CA). In some images, Hoechst/DAPI was enhanced consistently throughout the entire image for better visualization. 4.2.8 Statistical analysis GraphPad Prism version 9.1.1 for Windows (GraphPad Software, San Diego, CA, www.graphpad.com) was used for all statistical analysis and plotting Kaplan–Meier survival curves with 95% confidence intervals (based on the asymmetrical method) (Machin et al., 2006). Log-rank test was performed for statistical comparison of the survival curves and p< 0.05 was accepted as statistically significant. 4.3 Results 4.3.1 PHP.B iCre injections caused lethality in mice, regardless of administration route Our previous work with rAAV9 shows its favorable tropism for the retina and cornea after intraocular injections in WT mice (Hickmott et al., 2016; Korecki et al., 2021; Simpson et al., 2019). However, rAAV9 is known to be inefficient in transducing the retina after intravenous injection in adult mice (Bostick et al., 2007). At the time this study began, PHP.B was chosen as the most advanced capsid for non-invasive systemic delivery to the retina in adult mice (Deverman et al., 2016). Therefore, the PHP.B capsid was used for all three administration routes (intravenous, intravitreal, and intrastromal), whereas the use of rAAV9 was limited to the intraocular injections. Intravenous injections were carried out at five doses of PHP.B iCre virus, with mice injected at the three highest doses showing a progressive phenotype leading to lethality (Figure 4.1A). Uninjected mice served as controls. Mice injected with the highest dose of 5 x 1011 vg/mouse were first observed to have a mild “clasping” phenotype for their hind legs 33 days post-injection. The clasping phenotype progressed to a hind leg paralysis and these animals were 90 euthanized 43 days post-injection. Mice injected with the second highest dose of 1.5 x 1011 vg/mouse were first observed to have a mild clasping phenotype for their hind legs 58 days post-injection, with the phenotype progressing quickly, the remaining mice were euthanized 65 days post-injection. Mice injected with the third highest dose of 7.5 x 1010 vg/mouse were first observed to have a very mild clasping phenotype in their hind legs at 69 days post-injection, with the phenotype progressing to severe and mice beginning to have difficulty moving around the cage due to hind leg paralysis at 153 days post-injection, at which point the mice were euthanized. Mice injected with the fourth highest dose (second lowest) of 3 x 1010 vg/mouse showed a mild clasping phenotype seven months (215 days) post-injection. Mice injected with the lowest dose of 1.5 x 1010 vg/mouse showed no phenotype at seven months (215 days) post-injection, which was the experimental end point. The Kaplan-Meier survival curves were significantly different (p<0.0001) among the five groups according to the log-rank test. Although the lowest intravenous dose (1.5 x 1010 vg/mouse) did not result in any observable phenotype, we concluded that this dose was probably too low to have a therapeutic impact. Therefore, to continue to compare both capsids we chose to focus on intraocular (intravitreal or intrastromal) injections. To maximize transduction, and because we did not anticipate any deleterious impact of intraocular injections, undiluted PHP.B and rAAV9 iCre viruses were used for intravitreal injections. Throughout these experiments, non-reporter virus injected mice and vehicle-only injected mice served as controls to account for the potential impact of transgene expression or delivery method on the mice wellbeing. Experimental mice and non-reporter control mice were intravitreally injected with 8.4 x 1010 vg/mouse of PHP.B iCre. Surprisingly, we found a dead mouse among the experimental mice 13 days post-injection. Upon careful examination of the 91 remaining injected animals, mice in both the experimental and non-reporter control groups, displayed ataxia and weight loss and were subsequently harvested between 17-62 days post-injection when they reached a humane endpoint. The ataxia phenotype observed was characterized by motor coordination and balance deficits, as well as head and body tremors. Overall, this phenotype had a more rapid onset, was faster progressing, and more severe than what we observed after the intravenous injections. It is worth noting that ataxia is not part of the aniridia phenotype in either patients with heterozygous PAX6 loss-of-function variants (Hall et al., 2018) or Sey mice (Hickmott et al., 2018). Importantly, in a parallel study, experimental mice and non-reporter control mice were intravitreally injected with 7.6 x 1010 vg/mouse of, not PHP.B, but rAAV9 iCre. At the time of harvest (62 days post-injection), these mice did not display any adverse events. As expected, no phenotype was observed in mice that received vehicle-only injections. Similar to intravitreal injections, to maximize transduction, PHP.B and rAAV9 viruses were used at their highest possible dose for intrastromal injections. These injections were initiated at the time when we did not anticipate any deleterious impact of intrastromal injection. Non-reporter virus injected mice and vehicle-only injected mice served as controls. Experimental mice and non-reporter control mice were intrastromally injected with 8.4 x 1010 vg/mouse of PHP.B iCre. Surprisingly, a dead mouse was observed at 16 days post-injection. Except for one mouse in the experimental group, all remaining mice in both experimental and non-reporter control injected groups displayed varying degrees of ataxia and weight loss after the injections and were subsequently harvested between 16-68 days post-injection when they reached a humane endpoint. The fast-progressing ataxia in these mice was similar to our observations after intravitreal injections. Importantly, in a parallel study, experimental mice and non-reporter 92 control mice were intrastromally injected with 7.6 x 1010 vg/mouse of, not PHP.B, but rAAV9 iCre. At the time of harvest (68 days post-injection), these mice did not display any adverse events. As expected, no phenotype was observed in mice that received vehicle-only injections. Kaplan-Meier survival analysis showed significantly reduced survival in mice intraocularly injected (intravitreal or intrastromal) with PHP.B iCre compared to those injected with rAAV9 iCre (log-rank test, p=0.0005 for intravitreal injections and p<0.0001 for intrastromal injections) (Figures 4.1Bi and Ci). Survival curves were not significantly different between WT and Sey mice that received intraocular injections of PHP.B iCre (log-rank test, p=0.97 for intravitreal injections and p=0.42 for intrastromal injections) (Figures 4.1Bii and Cii). This demonstrates that survival of these mice was not influenced by Pax6 genotype. Lastly, survival curves were not significantly different between experimental and non-reporter control groups that received intraocular PHP.B iCre injections (log-rank test, p=0.15 for intravitreal injections and p=0.10 for intrastromal injections) (Figures 4.1Biii and Ciii). Hence, the observed lethality was attributed to the PHP.B iCre virus, rather than tdTomato or lacZ expression. 93 Figure 4.1. Mice injected with PHP.B or rAAV9 iCre viruses had different survival curves. Cages were closely examined for phenotype once or twice a week. Animals were euthanized once they reached a humane endpoint and tissues were harvested from a subset. Kaplan-Meier survival curves (solid or dashed lines) were generated with 95% confidence intervals (dotted lines). A Intravenous injections of high doses of PHP.B iCre (5 x 1011, 1.5 x 1011, and 7.5 x 1010 vg) caused severe adverse events leading to euthanasia of the entire dose group on the same day. Intravenous injections of high doses of PHP.B iCre significantly reduced the probability of survival compared to the groups that received lower doses (3 x 1010 and 1.5 x 1010 vg). Bi & Ci Intravitreal and intrastromal injections of PHP.B iCre significantly reduced the probability of survival compared to rAAV9 iCre injected mice. Bii & Cii The probability of survival was not significantly different between the PHP.B iCre injected WT and Sey mice. Biii & Ciii The probability of survival was not significantly different between the PHP.B iCre injected tdTomato reporter and non-reporter mice. Log-rank test was used to determine the significance of data. iCre, improved Cre; N. Sig, non-significant; Sey, Pax6 small eye; Sig, significant; vg, viral genome; WT, wild type. 94 4.3.2 Intraocular injected rAAVs escaped from the eye To determine the rAAV transduction in non-target cells after intraocular injections (intravitreal or intrastromal), we evaluated cerebellum, spinal cord, liver, heart, and pancreas for the presence of tdTomato expression (Figures 4.2 and 4.3). Histological analysis of early-harvested (16-30 days post-injection) tissues revealed tdTomato expression in the liver and heart, but was almost absent in pancreas, in both PHP.B and rAAV9 iCre injected WT and Sey mice. This demonstrates the ability of both these capsids to escape from the eye, regardless of the route of administration. Nevertheless, no tdTomato expression was observed in the cerebellum and spinal cord of mice intraocularly injected with rAAV9 iCre, which were the mice that did not display any adverse events. Whereas the PHP.B iCre injected mice demonstrated strong tdTomato expression in these tissues, regardless of the route of administration, and demonstrated severe ataxia and lethality. Histological analysis of tissues from vehicle-only control mice did not show any tdTomato expression. 95 Figure 4.2. Intravitreally-delivered PHP.B and rAAV9 iCre viruses escaped the eye. Fluorescence images of mouse tissues (cerebellum, spinal cord, liver, heart, and pancreas). Injection doses were: 8.4 x 1010 vg/mouse for PHP.B iCre and 7.6 x 1010 vg/mouse for rAAV9 iCre. Mice were perfused 17-30 days after intravitreal injection of iCre viruses (see Methods for more detail). Tissues from a 96 minimum of 2 mice per genotype were analyzed. The sectioned tissues were evaluated for tdTomato epifluorescence. The tdTomato signal was observed in the central nervous system cerebellum, and spinal cord of PHP.B iCre injected WT and Sey mice, but not in rAAV9 iCre injected mice. In contrast, tdTomato signal was detected in the peripheral tissues liver and heart, but was almost absent in pancreas, in both PHP.B iCre and rAAV9 iCre injected WT and Sey mice. tdTomato expression was not observed in age-matched vehicle-only injected mice (negative controls). The number in brackets refers to the mouse ID. Scale bar = 500 μm. Blue, Hoechst nuclear stain; iCre, improved Cre; red, tdTomato epifluorescence; Sey, Pax6 small eye; WT, wild type. 97 Figure 4.3. Intrastromally-delivered PHP.B and rAAV9 iCre viruses escaped the eye. Fluorescence images of mouse tissues (cerebellum, spinal cord, liver, heart, and pancreas). Injection doses were: 8.4 x 1010 vg/mouse for PHP.B iCre and 7.6 x 1010 vg/mouse for rAAV9 iCre. Mice were perfused 98 16-30 days after the intrastromal injection of iCre viruses (see Methods for more detail). Tissues from a minimum of 2 mice per genotype were analyzed. The sectioned tissues were evaluated for tdTomato epifluorescence. The tdTomato signal was observed in the central nervous system cerebellum, and spinal cord of PHP.B iCre injected WT and Sey mice, but not in rAAV9 iCre injected mice. In contrast, tdTomato signal was detected in the peripheral tissues liver and heart, but was almost absent in pancreas, in both PHP.B iCre and rAAV9 iCre injected WT and Sey mice. tdTomato expression was not observed in age-matched vehicle-only injected mice (negative controls). The number in brackets refers to the mouse ID. Scale bar = 500 μm. Blue, Hoechst nuclear stain; iCre, improved Cre; red, tdTomato epifluorescence; Sey, Pax6 small eye; WT, wild type. 4.3.3 In PHP.B ocular-injected mice tdTomato signal directly correlated with severity of ataxia We compared tissues from early-harvested (16-17 days post-injection) mice that had severe ataxia with late-harvested (62-68 days post-injection) mice to better understand the correlation between tdTomato expression and the severity of phenotype. For the intravitreal PHP.B iCre injections of experimental mice, we had two healthy WT and three healthy Sey mice at the end of the study. For the intrastromal injections we only had one healthy Sey mouse at the end of the study, as the only WT mouse that made it to 68 days had moderate ataxia, it was included in this analysis. In order to do a head-to-head comparison, tissues from early-harvested PHP.B iCre experimental mice (Figures 4.2 and 4.3) were re-analyzed together with late-harvested tissues under the same histology and imaging conditions. We found that for both WT and Sey mice, tdTomato signal intensity and spread was higher in the cerebellum, spinal cord, liver, and heart of mice with severe ataxia compared to healthy mice after intravitreal PHP.B iCre injections (Figure 4.4A). Similarly, for the intrastromal PHP.B iCre injections, tdTomato signal intensity and spread was higher in the cerebellum, spinal cord, liver, and heart of mice with severe ataxia compared to healthier mice (Figure 4.4B). 99 100 Figure 4.4. In PHP.B ocular-injected mice tdTomato signal was higher in ataxic versus healthy mice. Fluorescence images of tissues from early-harvested mice (17 days post-intravitreal injection and 16 days post-intrastromal injection) with severe ataxia compared to late-harvested mice (62 days post-intravitreal injection and 68 days post-intrastromal injection). A Intravitreal PHP.B iCre injections: Tissues from WT and Sey mice with severe ataxia exhibited a higher tdTomato intensity and spread in comparison to healthy mice. B Intrastromal PHP.B iCre injections: Tissues from WT and Sey mice with severe ataxia exhibited a higher tdTomato intensity and spread in comparison to healthier mice. The number in brackets refers to the mouse ID. A subset of mice presented in Figures 4.2 and 4.3 were re-analyzed for this comparison. Scale bar = 500 μm. Blue, Hoechst nuclear stain; iCre, improved Cre; red, tdTomato epifluorescence; Sey, Pax6 small eye; WT, wild type. 4.3.4 Intravitreal PHP.B and rAAV9 iCre injections resulted in transduction of a variety of retinal cells Gene therapy for aniridia will be focused on delivering virus to the PAX6-expressing cells of the retina (ganglion, amacrine, horizontal, and Müller glia) and cornea (LSCs and epithelial). Intravitreal PHP.B iCre injections resulted in tdTomato expression in all four PAX6-expressing cells of the retina (identified by location and morphology), in both WT and Sey mice (Figures 4.5 and C.1). Whereas, intravitreal injection of rAAV9 iCre into WT mice led to tdTomato expression in all but the Müller glia. In contrast, in Sey mice tdTomato expression was observed in all four PAX6-expressing cells. Interestingly, tdTomato expression was not restricted to the retina despite the intravitreal injection site being close to the retina (Figure C.1). Expression was also observed in the corneal stromal and endothelial cells, but was almost absent from the epithelial layer in both WT and Sey mice, regardless of rAAV capsid. tdTomato expression was absent in the vehicle-only injected eyes (negative controls). 101 102 Figure 4.5. Intravitreally-delivered iCre viruses successfully transduced a variety of retinal cells and some corneal cells. Fluorescence images of injected mouse retinas and corneas. Injection doses were: 8.4 x 1010 vg/mouse for PHP.B iCre and 7.6 x 1010 vg/mouse for rAAV9 iCre. Intravitreal injection of PHP.B iCre into WT mice resulted in tdTomato expression in all four PAX6-expressing cells of the retina, ganglion, amacrine, horizontal, and Müller glia. rAAV9 iCre injection resulted in tdTomato expression in three PAX6-expressing cells, excluding Müller glia. But, intravitreal injections of PHP.B and rAAV9 iCre into Sey mice resulted in the expression of tdTomato in all PAX6-expressing cells of the retina. Intravitreal injections of PHP.B and rAAV9 iCre viruses also resulted in tdTomato expression in the stromal and endothelial layers of the cornea, but tdTomato expression was almost absent from the epithelial layer in both WT and Sey mice. tdTomato expression was not observed in age-matched vehicle-only injected eyes (negative controls). Retinal cells were identified by their location and morphology. Images with the same Roman numeral belong to one mouse, and correspond to overview images in Figure C.1. For the PHP.B iCre WT and Sey groups, each fluorescence image is a representative from 11 eyes. For the rAAV9 iCre WT and Sey groups, each fluorescence image is a representative from 4 and 8 eyes, respectively. Scale bar = 100 μm. A, amacrine; blue, Hoechst nuclear stain; End, endothelium; Epi, epithelium; G, ganglion; GCL, ganglion cell layer; H, horizontal; iCre, improved Cre; INL, inner nuclear layer; IPL, inner plexiform layer; M, Müller glia; ONL, outer nuclear layer; OPL, outer plexiform layer; red, tdTomato epifluorescence; Sey, Pax6 small eye; Str, stroma; WT, wild type. 4.3.5 Intrastromal PHP.B and rAAV9 iCre injections resulted in transduction of all three corneal cell layers Intrastromal PHP.B and rAAV9 iCre injections resulted in tdTomato expression in all three corneal cell layers, encompassing corneal epithelial cells (basal cells, wing cells, and squamous cells), stromal cells (keratocytes), and endothelial cells, identified by location and morphology in both WT and Sey mice (Figures 4.6 and C.2). Additionally, sectioning demonstrated that the central intrastromal injection method used resulted in even transduction throughout the cornea (Figure C.2). tdTomato was only expressed in the retinal blood vessels of intrastromally-injected mice (Figure C.2). tdTomato expression was absent in the vehicle-only injected eyes (negative controls). 103 Figure 4.6. Intrastromally-delivered iCre viruses successfully transduced all three corneal cell layers. Fluorescence images of injected mouse corneas. Injection doses were: 8.4 x 1010 vg/mouse for PHP.B iCre and 7.6 x 1010 vg/mouse for rAAV9 iCre. Intrastromal injections of PHP.B and rAAV9 iCre showed successful transduction of cells in all three layers of the cornea (Epi, Str, and End) in WT and Sey mice. In the corneal epithelial layer, tdTomato expression was seen in basal cells, wing cells, and squamous cells. Sections were chosen to show all the transduced cell types in one image. Overall, the results did not show a bias in transduction of corneal cell types between PHP.B and rAAV9. tdTomato expression was not observed in age-matched vehicle-only injected eyes (negative controls). Corneal cells were identified by their location and morphology. Images with the same Roman numerals belong to one mouse, and correspond to overview images in Figure C.2. For the PHP.B iCre WT and Sey groups, each fluorescence image is a representative from 6 and 10 eyes, respectively. For the rAAV9 iCre WT and Sey groups, each fluorescence image is a representative from three eyes. Scale bar = 100 μm. B, basal cell; blue, Hoechst nuclear stain; E, endothelial cell; End, endothelium; Epi, epithelium; iCre, improved Cre; K, keratocyte; red, tdTomato epifluorescence; S, squamous cell; Sey, Pax6 small eye; Str, stroma; W, wing cell; WT, wild type. 104 4.3.6 Intrastromal rAAV9 iCre injections successfully transduced corneal LSCs in adult aniridic mouse eyes In the Sey eyes, the exact age at which the LSC deficiency manifests is unknown. In this study, all intrastromal injections were performed when mice were 3 months old. By 3 months, the cloudy cornea phenotype of aniridia is already apparent in the Sey eyes (Figure 4.7). The success of an rAAV gene therapy targeted towards LSC deficiency may depend on whether any functional LSCs are still present in the damaged Sey corneas at the time of injection. To investigate this, we looked at WT and Sey corneas 7 months after intrastromal rAAV9 iCre injections. It must be noted that tdTomato expression at 7 months post-injection is not a reflection of continuing virus presence, but is due to the permanent genomic rearrangement by the iCre/loxP recombination system that makes the transduced cells, and any cells descending from them, to persistently express tdTomato. Fluorescence microscopy demonstrated widespread tdTomato expression in the cornea of both WT and Sey mice (Figure 4.8). Confocal microscopy confirmed that the tdTomato stripes are located in the corneal epithelium. In general, in the Sey eyes, the tdTomato stripes that originated from the limbus, as expected did not reach the center of the cornea (Collinson et al., 2004). Overall, two out of three WT corneas and five out of eight Sey corneas showed tdTomato stripes in the epithelium, indicating that functional LSCs are present in 3-month-old Sey corneas, which can be successfully transduced with rAAV9. tdTomato expression was absent in the cornea of vehicle-only injected mice (negative controls). 105 Figure 4.7. Corneal clouding was present in 3-month-old Sey corneas. Representative slit lamp images of uninjected Sey and WT eyes at 3 months old. Sey eyes were imaged at two light settings to better capture the cloudiness of the corneas. All Sey eyes displayed severe corneal clouding, whereas age-matched WT eyes had transparent corneas. Sey, Pax6 small eye; WT, wild type. 106 107 Figure 4.8. Intrastromal injections of rAAV9 iCre successfully transduced corneal LSCs in Sey mice. Representative fluorescence and confocal images of WT and Sey corneal flat mounts 7 months after injection. Corneal flat mounts were first examined under the fluorescence microscope to assess tdTomato expression in all three layers of the cornea, and then subjected to confocal microscopy to focus specifically on the entire corneal epithelial layer. Intrastromal injections of rAAV9 iCre resulted in tdTomato stripes indicative of LSC transduction in both WT and Sey corneas. tdTomato expression was not observed in age-matched vehicle-only injected corneal flat mounts (negative controls). Blue, DAPI nuclear stain; red, tdTomato epifluorescence; Sey, Pax6 small eye; WT, wild type. 4.4 Discussion For ocular gene therapy, rAAV has been a popular choice (Wang et al., 2019). Given the deteriorating state of the aniridic eye (Lee et al., 2008a), a non-ocular delivery system such as intravenous was quite attractive. However, recent news of several deaths associated with intravenous delivery of high-dose rAAV therapies (Agarwal, 2020; Kishimoto and Samulski, 2022) represents a major setback for the field and emphasizes the importance of establishing maximally-tolerated viral dose during preclinical gene therapy studies. Thus, the primary goal of our intravenous rAAV dose-response studies was to find the maximally-tolerated viral dose. Unfortunately, we concluded that the only dose of PHP.B iCre that did not cause toxicity in our mice was probably too low to be a viable option for our gene therapy. Thus, to continue to compare both capsids we shifted to the option of intraocular delivery. Luxturna®, an rAAV2-based augmentation gene therapy delivered intraocularly was approved in 2017 by the FDA for treating patients with a rare form of inherited vision loss (Russell et al., 2017). Given this, and the intravenous toxicity, we shifted our focus to intraocular delivery with an aim to concentrate the rAAV in the eye and improve the safety and efficacy profiles. We were surprised to see severe ataxia and early lethality in our intravitreally-injected mice when the PHP.B capsid was used. It was even more shocking to see severe ataxia and early lethality after intrastromal PHP.B iCre injections, considering that the injection was done into the 108 cornea, an avascular tissue in WT eyes. These observations were especially unexpected since in the literature ataxia has not been reported with the use of PHP.B in mice, and currently the only report of PHP.B-associated ataxia comes from the study in piglets that received high dose of intravenous injection of this virus (Hinderer et al., 2018). Thus, this study is the first report of ataxia and lethality after intraocular injections of PHP.B. Furthermore, the adverse events that we observed in our study after intravitreal and intrastromal injections of PHP.B were substantially more severe than the local ocular toxicity (defined as morphological changes to the retina and retinal pigment epithelium) that another group observed in mice after subretinal injections of multiple rAAVs (although not PHP.B) (Xiong et al., 2019). Interestingly, the PHP.B capsid was used by an independent group for successful intravitreal delivery of CRISPR/Cas9 components to the retina of mouse pups (1 x 109 vg/eye) (Giannelli et al., 2018). The absence of toxicity could be due to lower virus dose, different promoter and regulatory elements, but is most likely due to the very short harvest time of 2 weeks. In our work, we are confident that the observed lethality is not due to promoter, viral genome dose, or vector preparation, as both PHP.B (lethal) and rAAV9 (non-lethal) iCre viruses had the exact same viral genome; were used at comparable doses; and were prepared in the same facility. Our findings are consistent with a group that showed intravenous injection of PHP.B virus at a high dose in an adult NHP resulted in severe acute toxicity and ultimately euthanasia, while the animal that received the same dose of rAAV9 showed no clinical symptoms (Hordeaux et al., 2018). Together, these results highlight the inherent differences in tropism in even closely related capsids (PHP.B and rAAV9) that can dramatically impact safety. The eye is an easily accessible and contained structure, and therefore is widely considered to be a safe target for gene therapy (Liu et al., 2011). Typically, tissue transduction 109 after escape from the eye has not been reported, even for the PHP.B capsid (Giannelli et al., 2018). However, several studies have performed biodistribution analysis after intraocular rAAV2 (Tchedre et al., 2021), rAAV8 (Chung et al., 2021; Marangoni et al., 2016; Seitz et al., 2017; Song et al., 2020), or rAAV8G9 (Miyadera et al., 2020) injections and detected vector genome in extraocular tissues. A limitation of these studies is that they do not make it clear whether or not extraocular cells had been transduced. In the current study, histological analysis demonstrated that both PHP.B and rAAV9 viruses escaped from the eye after intravitreal and intrastromal injections, and then transduced peripheral tissues. The “historical” reporter used in this study (a genomic rearrangement turning on tdTomato), may have more effectively captured the true extent of the viral escape since transduced cells are permanently and clearly marked. This is compared to the more commonly used “current” reporters (e.g. green fluorescent protein), for which signal is determined by viral copy number, and there is no signal if the virus is lost from the cell. Applying Occam’s razor to explain both intravitreal and intrastromal escapes, we suggest that PHP.B and rAAV9 capsids have crossed the adult blood-ocular barrier from the eye side into the bloodstream, resulting in extensive transduction of non-ocular tissues. In the future, we would recommend replacing a ubiquitous promoter as used here, with a target-cell-specific promoter to minimize transgene expression in off-target tissues (de Leeuw et al., 2016; Hickmott et al., 2016; Korecki et al., 2021; Simpson et al., 2019). In this study, we must consider the mechanism whereby PHP.B and rAAV9, which both reached the bloodstream regardless of route of administration, differentially resulted in ataxia and death (PHP.B lethal and rAAV9 non-lethal). It is important to note that in adult mice, PHP.B capsids outperform rAAV9 in crossing the BBB and transducing the CNS (Deverman et al., 2016; Huang et al., 2019). Our results support this in that we saw transduction of cerebellum and 110 spinal cord only with the lethal PHP.B capsid. Another candidate region from NHP studies would be the dorsal root ganglia (DRG) (Buss et al., 2022; Hinderer et al., 2018; Hordeaux et al., 2020), however DRG are not protected by the BBB and both PHP.B and rAAV9 capsids have been shown in NHP to have similar tropism for DRG (4). Thus, in our study, we conclude that the ataxia phenotype observed with PHP.B is most likely due to differential transduction of the cerebellum and/or spinal cord. LSC deficiency has been proposed as the underlying mechanism responsible for corneal clouding, a major contributor to blindness in aniridia (Li et al., 2015b; Mort et al., 2011; Ramaesh et al., 2005a). Thus, LSC are a critical therapeutic target. Intrastromal injection of rAAV8[Y733F] has been shown to transduce LSCs in adult WT mice (Basche et al., 2018), but similar data was not available for Sey mice. We know from other work that PAX6 haploinsufficiency results in fewer functional LSCs compared to WT corneas (Collinson et al., 2004). Nevertheless, the exact age of onset for LSC deficiency in the Sey cornea is unclear and therefore there was uncertainty whether rAAVs would be able to transduce these therapeutically important cells in an already cloudy Sey cornea. Here we have shown for the first time that 3-month-old cloudy Sey corneas still have functional LSCs and that they can be transduced with rAAV9. As expected, the tdTomato stripes from these LSCs fail to reach the center of the Sey corneas, consistent with the previously observed defective migration (Collinson et al., 2004). Regarding route of administration, we were surprised to see that intravitreal injection of rAAV9 was not only excellently suited for transducing the therapeutically important retinal cells, but it also reached the cornea. Since it would be exciting if one injection could transduce both the therapeutic retinal and corneal LSC targets, this observation deserves further study. Overall, our findings are most promising in the context of a future gene therapy for aniridia, which can 111 transduce LSCs and may restore the normal migration pattern from these cells, thereby eliminating corneal clouding and the resulting blindness. In this study, we conclude that PHP.B iCre can cause lethality in mice regardless of the administration route (intravenous, intravitreal, or intrastromal). Furthermore, we hypothesize the mechanism of escape from the eye by both capsids lies in their ability to cross adult blood-ocular barrier from the eye to the bloodstream, leading to the transduction of non-ocular tissues. Their differential tropism for CNS is the likely explanation for the observed lethality phenotype with PHP.B only. Most importantly, we have demonstrated that functional LSCs are present in the cloudy cornea of 3-month-old Sey mice. Lack of adverse events and successful transduction of the therapeutically important corneal LSCs and retinal cells with rAAV9 in the Sey eyes makes this capsid a good candidate for use in future aniridia gene therapy development. Finally, our findings regarding rAAV lethality will be impactful for other researchers working on developing rAAV-based gene therapies for ocular disease. 4.5 Acknowledgments The authors would like to thank Ms. Sabrina Li who during her Co-op Program sectioned some of the tissues used in this study. Additionally, the authors would like to thank Ms. Siu Ling Lam and Ms. Diana Djaksigulova for preparing the pEMS2118 plasmid and corneal flat mounts, respectively. The authors also thank Dr. Jingsong Wang at the BCCHRI Imaging Core Facility for microscope support. 112 Chapter 5: Discussion In this thesis, I have described my work towards a CRISPR-based gene therapy to prevent blindness in a novel mouse model of aniridia. In my discussion, I will provide a summary of major findings in each data chapter, point out the study significance and shortcomings, as well as propose future directions for the field. 5.1 Overview of major findings In Chapter 2, we created a novel mouse model for aniridia by adding a 3xFLAG tag after the start codon of Pax6 on the Sey allele. Since the Sey mouse is a heterozygote and still expresses WT PAX6, addition of a protein tag allows for differential detection of CRISPR-corrected and WT proteins by antibody labeling and therefore provides a quantitative means for testing CRISPR-based gene editing strategies for preclinical assay development for aniridia. Next, we performed gene editing of the novel Fey mouse embryos and completely rescued the aniridia phenotype. Our results confirmed the sole causality of the Sey variant for the classical aniridia phenotype observed in mice and validated our CRISPR-based gene editing approach for correcting only one base to treat aniridia. Finally, we developed an allele-distinguishing CRISPR strategy and demonstrated its success in correcting the Sey variant in vitro in mouse embryonic stem cells, and in vivo in the mouse germline. In Chapter 3, we built on our previous work by collaborating with industry to develop a novel LNP platform for CRISPR RNP delivery and tested it for ex vivo and somatic gene editing using both a newly designed CRISPR strategy, as well as our pre-established Sey correction CRISPR strategy from Chapter 2. For the experiments in this chapter we bred Ai14 reporter mice with our Fey mice (created in Chapter 2) to develop a robust and sensitive reporter model that enabled us to evaluate the efficacy of our LNP-RNP delivery platform. We have also included 113 WT reporter mice in our study to be able to extend our findings to other corneal diseases in need of a delivery platform for their CRISPR therapeutics. Our characterization work showed successful gene editing of the reporter gene and the correction of the Sey variant in ex vivo cortical neurons after a single treatment. Moreover, we showed the value of repeated treatment in improving the gene editing outcomes. For the first time, we demonstrated wide-spread gene editing in the corneal stroma and endothelium, but not the epithelium, following in vivo intrastromal injection of our LNP-RNPs to the mouse eye. In Chapter 4, we took our work one step further by evaluating rAAV, a frequently-used viral vector, for CRISPR delivery for aniridia. Corneal clouding leading to blindness has been reported in the majority of aniridia patients (Dyer et al., 2021) and it is speculated to be due to LSC deficiency (Li et al., 2015a; Mort et al., 2011; Ramaesh et al., 2005a). Therefore, we focused our work on clarifying some uncertainties in the field by answering whether functional LSCs are present in the damaged aniridic corneas and if rAAV can transduce these therapeutically important cells. We tested two capsids (rAAV9 and rAAV-PHP.B) in the reporter mouse model of aniridia (first bred in Chapter 3) and WT mice by three different administration routes (intrastromal, intravitreal, and intravenous). We have shown that rAAV-PHP.B iCre injections led to lethality in mice, regardless of the route of administration, whereas rAAV9 did not cause any lethality. Furthermore, rAAV9 successfully transduced functional LSCs, as well as all four PAX6-expressing retinal cells in aniridic eye, after intrastromal and intravitreal delivery, respectively. Our work identified rAAV9 as a promising gene delivery vector for future gene editing or gene augmentation therapies for aniridia. 114 5.2 Significance beyond the scope of the current thesis In preclinical studies, researchers rely on animal models to assess the safety and efficacy of their drug candidates or gene therapy approaches for their disease of interest. The Fey mouse model of aniridia that we developed in Chapter 2 is very useful for testing various nonviral and viral gene delivery vectors and administration routes and ultimately identifying the best approach for aniridia. A good example of such use is studies done with these novel mice in Chapter 3. However, the utility of this mouse model is not limited to CRISPR therapeutics and can be extended to other therapeutic strategies such as nonsense suppression therapies. These variant-independent drug-based therapies can act on premature stop codons that are introduced into the PAX6 open reading frame as a result of nonsense variants, splice site variants, or frameshift variants. These three variant classes account for 77% of all pathogenic variants associated with aniridia (Tzoulaki et al., 2005) and therefore highlight the importance of variant-independent treatment strategies for this disease. A nonsense suppression drug, Ataluren, showed its initial success in treating aniridia in the Sey mice (Gregory-Evans et al., 2013; Wang et al., 2017) before entering clinical trial. Our Fey mouse model is more advantageous than other existing mouse models of aniridia for testing different nonsense suppression drugs, as it allows for precise quantification of the treatment outcomes using antibodies against FLAG. CRISPR-mediated gene editing is a promising approach for treating corneal diseases and dystrophies, but lack of safe delivery methods that can effectively target corneal cells has greatly hindered clinical applications. LNPs are the most clinically advanced nonviral vector (Zu and Gao, 2021), but no attempts in the literature were made to optimize this vector for the delivery of active Cas9 RNPs to the eye. Therefore, in Chapter 3 we focused on bringing LNP-mediated delivery of Cas9 RNPs one step closer to clinical trials. We have shown the value of repeated 115 treatment in increasing the gene editing efficiency in cortical neurons and added to the growing body of evidence that repeated treatment with LNPs can be beneficial. Furthermore, our successful gene editing results in the stromal and endothelial corneal layers support the prospect of using our novel LNP-RNP platform for CRISPR-based therapy development for a variety of well-characterized stromal and endothelial corneal dystrophies such as macular corneal dystrophy and Fuchs endothelial corneal dystrophy, or even infectious diseases such as herpetic stromal keratitis. Lastly, even though in Chapter 3 we argued in favour of repeated treatment, a single treatment might be sufficient to achieve the desired result depending on the turnover time of the target cells. In our case, the wide-spread gene editing we observed in the cornea was the outcome of a single LNP-RNP injection to the eye. This is especially important since unlike terminally-differentiated corneal epithelial cells, the turnover time of stromal cells is very slow (Lagali, 2020; West-Mays and Dwivedi, 2006) and therefore the gene edited cells can last for at least several years. Similarly, corneal endothelial cells are not known to divide post-developmental stage of the cornea and hence the gene edited cells can be retained throughout life (Joyce, 2012). A one-and-done treatment is more cost-effective, puts less burden on the healthcare system, and is more appealing to the patients. In the clinic, ophthalmic applications have been leading the gene therapy field, with 42 ongoing clinical trials of rAAV-based ocular therapies (www.clinicaltrials.gov). There is a misconception in the field that the eye is quite contained and gene therapy for ocular diseases are generally safe. However, typically, preclinical studies with rAAV in the mouse eye does not include evaluating virus transduction in non-ocular tissues. In Chapter 4, we reported lethality and virus escape after intraocular rAAV injections. Our findings highlight the need for careful consideration of several elements (such as viral capsid, dose, and promoter) during preclinical 116 studies and emphasize the importance of harvesting and analyzing non-ocular tissues even in the absence of any adverse events. Our conclusions are impactful for researchers developing rAAV-based therapeutics for any ocular diseases. Additionally, based on our intraocular injections data in Chapter 4, we proposed rAAV9 as the capsid of choice for future gene therapy development for aniridia. rAAV9 offers certain advantages compared to some other well-known capsids such as rAAV2, rAAV8, and rAAV5 that are usually preferred for intraocular injections and have shown efficacy in many proof-of-concept experiments (Ali et al., 1996; Allocca et al., 2007; Boye et al., 2012; Boye et al., 2011; Hippert et al., 2012; Lotery et al., 2003; Mussolino et al., 2011; Pang et al., 2011; Pang et al., 2012; Petersen-Jones et al., 2009; Stieger et al., 2008; Vandenberghe et al., 2011; Yang et al., 2002; Yiu et al., 2020), as well as in clinical trials (ClinicalTrials.gov identifier: NCT01024998, NCT00749957, NCT03872479, NCT02781480, NCT04671433, NCT03758404, NCT05616793). Using rAAV9 opens up the opportunity to treat non-ocular symptoms associated with aniridia either by systemic injection of the rAAV9-packaged gene therapy to target pancreas (Korecki et al., 2021) or by direct injection to the brain (Huda et al., 2014) to address some of the underlying issues. The latter would be especially interesting, since a recent study in Pax6-deficient mice suggested a role for PAX6 in the adult brain neurogenesis and neural plasticity (Grant et al., 2020). Hence, a multi-functional rAAV9 gene therapy can be a valuable option for other researchers in the field who work on the systemic symptoms associated with aniridia. 5.3 Limitations and caveats Developing CRISPR strategies for autosomal dominant diseases such as aniridia is more challenging due to the present of both WT and mutant alleles. Therefore, an ideal CRISPR strategy must be able to distinguish between WT and mutant alleles without sacrificing 117 efficiency and safety. This specificity is particularly hard to achieve when there is only a single base-pair mismatch between the two alleles. Although the CRISPR strategy we developed in Chapter 2 was shown to have a bias towards interacting with the mutant allele, the less-frequently observed deleterious hits on the WT allele cannot be simply overlooked. Consistent with our findings, others have reported that a single base-pair mismatch is not enough to always protect the DNA from non-specific targeting by the gRNA and subsequent cleavage by Cas9 nuclease, which can create undesired indels (Anderson et al., 2015; Cho et al., 2014; Fu et al., 2013; Hsu et al., 2013; Josephs et al., 2015). Hence, to further enhance the safety profile of CRISPR-mediated gene editing for aniridia, we need to develop allele-specific strategies that limit the interaction of CRISPR components to the mutant allele. In recent years, allele-specific CRISPR strategies have gained popularity and been successfully implemented in animal models to treat several autosomal dominant conditions including Meesmann epithelial corneal dystrophy (Courtney et al., 2016), retinitis pigmentosa (Li et al., 2018b; Patrizi et al., 2021), Huntington disease (Monteys et al., 2017), and Hutchinson-Gilford progeria syndrome (Koblan et al., 2021). The in-the-PAM approach (i.e. targeting PAM-generating variants) in particular has been quite successful in making a CRISPR strategy allele-specific (Courtney et al., 2016; Li et al., 2018b; Monteys et al., 2017). In this approach, only the variant allele harbours the PAM sequence and therefore correcting the variant removes the PAM, leading to allele-restricted targeting. However, this approach is restricted to PAM-generating variants, and it is currently not applicable to the Sey variant and many other PAX6 variants that cause aniridia. CRISPR base editors are another novel approach that can be used for targeting alleles that differ by a single base-pair (Koblan et al., 2021). Base editing does not create a double-stranded break (Komor et al., 2016) and therefore is deemed to have a better safety profile than the classic homology-118 directed repair when it comes to correcting single base variants (Tao et al., 2022). Current base editors fall in two categories: 1) cytosine base editors (Komor et al., 2016) that enable conversion of a cytosine-guanine base-pair into a thymine-adenine, or 2) adenine base editors that enable conversion of an adenine-thymine base-pair to a guanine-cytosine (Gaudelli et al., 2017). Although the CRISPR base editing approach is very attractive for precise gene editing, neither of the well-established base editors are applicable to the Sey variant, as correcting this variant requires a base editor that can convert a thymine-adenine base-pair to a guanine-cytosine. Expansion of the repertoire for base editing in the future will allow the use of this approach in targeting a wider spectrum of single base variants including the Sey variant. Effective delivery of therapeutics to the target cells remains a major obstacle for many gene therapies. Despite the overall success with our LNP-RNP delivery platform in Chapter 3 in reaching cells in 2 of 3 corneal cell layers, there was no evidence of transducing PAX6-expressing corneal epithelial cells at harvest time (21 days after injection). Our study was not designed to fully assess the ability or inability of our LNPs to reach epithelium, since the entire epithelium is turned over every 14 days (Douvaras et al., 2013) and therefore the transduced cells might have shed into the tear film and been lost before the histological analysis. The renewal time, which is defined as the time required for corneal LSCs to re-populate the epithelium is approximately 9 weeks in WT mice and 28 weeks in aniridic mice (Collinson et al., 2004). Considering the early harvest time in our study, we could also not investigate whether our LNPs can transduce PAX6-expressing corneal LSCs that reside adjacent to cornea in the limbus. Therefore, the biggest caveat of our study was having only one harvest time point, which limited us from identifying all possible targets of our LNP delivery platform in the cornea. Having found initial positive results, this study could be repeated with a bigger cohort that allows multiple 119 harvest time points at 7 days, 14 days, 21 days, 3 months, and 7 months after intrastromal injection of our LNP-RNPs. It would be even more ideal to conduct a longitudinal study using a slit lamp with a specialized filter to determine the dynamics of tdTomato expression over a 7-month period following the intrastromal injections. Purchasing such a slit lamp should be planned for the future. Intrastromal injections are used for targeting cells of the cornea in gene therapy (Miyadera et al., 2020). A good injection will not only affect efficacy of the treatment, it can also lessen the potential complications associated with the procedure. We used this administration route in Chapter 4 for the local delivery of rAAVs to the cornea and limbus of WT and Sey mice. The quality of an intrastromal injection is usually assessed by the extend of payload dispersion in the corneal stromal layer after a single injection. In a clear cornea, the payload dispersion can be visualized as a temporary opacity in the cornea that appears immediately after the intrastromal injection and clears up on its own within 24 hours. This phenomenon has been also reported in other studies that involve intrastromal rAAV injections to the dog (Miyadera et al., 2020) and rabbit eyes (Song et al., 2020). One of the caveats of doing intrastromal injections on the Sey eyes is the pre-existing corneal clouding in these eyes. The aniridia-related corneal clouding in the Sey eyes makes it harder to assess the quality of intrastromal injections, especially in the extremely cloudy eyes. Mixing a very small amount of fluorescein with the viral payload is one possible approach to address this caveat in future studies. Similar approach has been used in the literature for better monitoring of rAAV diffusion inside the vitreous body after intravitreal injection (Looser et al., 2018). Typically, intrastromal injection is hard to perform in a mouse due to the small eye size (Matthaei et al., 2012; Mirjalili Mohanna et al., 2022). It is even more challenging to do this 120 injection in the Sey mice, considering the reduced eye size compared to WT mice (Hickmott et al., 2018). The mouse eye still grows in size during the first three months of postnatal development (Tkatchenko et al., 2010), making it very hard to perform intrastromal injections on younger mice. Therefore, the ability to successfully intrastromally inject Sey eyes greatly affected our decision when choosing the age of mice. Although, we have shown in Chapter 4 that the cloudy cornea of 3-month-old Sey mice still have functional LSCs, it would be ideal to intervene earlier to maximize the likely success rate of gene therapy. Supporting this are results from two studies that showed treating younger Pax6-deficient mice results in a better rescue of ocular abnormalities (Rabiee et al., 2020; Wang et al., 2017). A 3-months old mouse is analogous to a 23 years old human (Dutta and Sengupta, 2016). In the literature, intrastromal injections have been successfully performed on children’s and young adults’ eyes (Albab et al., 2020; You et al., 2015). Because a human eye is much bigger than a mouse eye, even the presence of microphthalmia should not interfere with intrastromal injections. Although, microphthalmia happens very infrequently in patients that only carry aniridia-causing PAX6 variants with no additional pathogenic variants in other important ocular genes (Deml et al., 2016; Williamson and FitzPatrick, 2014; Xiao et al., 2012). Hence, overall, we do not anticipate facing any technical challenges during early intervention when gene therapy is administered intrastromally to children’s or even newborn babies’ eyes. 5.4 Future directions 5.4.1 CRISPR/Cas9-mediated somatic gene editing in Fey mouse model of aniridia Based on the results and conclusions presented in this thesis, the immediate next step would be to develop a somatic gene therapy using a dual rAAV9-CRISPR/Cas9 system. A dual rAAV strategy must be adopted as packaging all the necessary components in one vector is not 121 possible due to the limited cargo capacity of rAAV (~4.9 kb). Dual vector strategies have been successfully used for in vivo rAAV-CRISPR gene editing in a variety of tissues (Long et al., 2015; Monteys et al., 2017; Tabebordbar et al., 2016; Yang et al., 2016), including the retina (Hung et al., 2018). A dual rAAV9 delivery system will be implemented by constructing two rAAV plasmids, one containing the sequence for the SpCas9 cDNA, and the other one the sequence for the gRNA and template DNA from Chapter 2, as well as a visual marker. Upon successful in vitro validation of CRISPR plasmids, viral vectors will be commercially produced using rAAV9, which displayed satisfactory tropism for therapeutically important aniridic corneal and retinal cells in Chapter 4. As sex does not have any known effect on development of aniridia (Netland et al., 2011), it will be important to develop therapeutics that will be effective for both sexes. The rAAV9-CRISPR gene therapy will be delivered to adult B6129F1 Fey mice of both sexes by intravitreal and intrastromal injections. The structure and function of the eye in the CRISPR treated mice will be compared to vehicle-only Fey and WT control mice using slit lamp imaging to longitudinally examine corneal structure, before administration of the therapy, and again once a week over a period of 4 weeks after treatment with the frequency of imaging being dropped to once a month for the remaining period until the harvest time (7 months post-treatment). Improvement in corneal transparency will be the main endpoint for this examination. Slit lamp imaging will be accompanied by optical coherence tomography to examine the fine structures of the eyes in vivo. Additionally, transepithelial electrical resistance measurement, as well as optokinetic tracking will be carried out to assess how gene therapy influences corneal epithelial barrier function and vision, respectively. At the end of the study, mice will be euthanized and eyes will be processed for histological analysis and ELISA. This will allow us to 122 assess the structure of the cornea following treatment, and to look at the expression and levels of the CRISPR-corrected FLAG-tagged PAX6 in the cornea and the retina. 5.4.2 Prospect of an in vivo CRISPR-based gene therapy for aniridia patients As shown in Chapter 2 and 3, Fey mouse is an excellent model to test new CRISPR therapeutics for aniridia. However, rescuing the mutant phenotype in these mice by somatic gene editing only serves as a proof-of-principle experiment, since the developed CRISPR therapy is not directly translatable to patients. This is because the PAM sequence used for cgEMS4 is specific to mice and does not exist in human genome. Therefore, even though the nonsense c.580G>T (p.G194X) variant has been reported in the PAX6 gene of an aniridia patient (Hever et al., 2006; Kit et al., 2021), our CRISPR strategy cannot be used to treat this patient due to the genomic sequence variation between species. To accelerate the discovery phase and bring the CRISPR gene editing for aniridia closer to the clinical use, the next step after curing the Fey mouse model should be to cure a humanized mouse model. Experiments on humanized animal models provide the most accurate data for any pre-clinical CRISPR-based gene therapy studies, as the success of such therapies rely on the target sequence identity. Currently, aniridia does not have such animal model and therefore the first step would be to create a humanized mouse model for this disease by leveraging the power of CRISPR/Cas9 system to swap the mouse exon with the human counterpart that has a PAX6 loss-of-function variant. This approach has been successfully implemented by others to create a humanized mouse model for Duchenne muscular dystrophy (Zhang et al., 2022). Once a humanized mouse model of aniridia is available, CRISPR therapy elements need to be designed to correct the pathogenic variant in the humanized mice and prove efficacy before being able to potentially help patients. 123 CRISPR gene therapy is a form of precision medicine because it is tailored to correct a specific genetic variant. In the case of PAX6, over 500 variants have been reported in the literature (Lima Cunha et al., 2019) and hence a CRISPR therapy developed for a specific variant is not applicable to other PAX6 variants that cause aniridia. Moreover, while feasible, it would be very costly to create humanized mouse models for every PAX6 variant and subsequently develop therapeutic assays for each of them. To circumvent this potential obstacle and broaden the utility of such therapies, researchers can identify the four highest-recurring causal-coding PAX6 variants in aniridia patients (reported in 24% of aniridia cases according to Leiden Open Variation Database) and create humanized mice for each of those variants. The resulting mice can be then used to develop and optimize the molecular tools needed to create new CRISPR-based therapies for aniridia. 5.4.3 Prospect of an ex vivo CRISPR-based gene therapy for aniridia patients Ex vivo CRISPR-mediated gene editing of cultured autologous corneal LSCs might be a viable option for treating genetic eye disorders that involve unilateral LSC deficiency (Guerin et al., 2022; Pellegrini et al., 1997). However, in the case of the bilateral LSC deficiency in aniridia, performing peripheral corneal biopsies is likely to exacerbate the poor ocular surface condition in the patients (Roux et al., 2018), and therefore it is deemed a risky procedure that has shown limited success in the literature (Latta et al., 2017). Differentiated human induced pluripotent stem cells (iPSCs) can be an alternative source of cells for the ex vivo gene editing. In this approach, skin biopsies are taken from molecularly-confirmed patients and reprogrammed to become iPSCs. iPSCs harboring the pathogenic PAX6 variant can be then corrected with a CRISPR strategy, such as the one developed in Chapter 2, and subsequently be differentiated into corneal LSCs that can be transplanted back onto the patient’s eyes to restore the ocular 124 surface health. Supporting this approach is the evidence of straightforward and efficient CRISPR gene editing of iPSCs (Geng et al., 2020), as well as the recent success in generating corneal LSCs using iPSCs of patients with aniridia (Lima Cunha et al., 2023). The main advantage of iPSC-derived transplants is the use of autologous cells in this therapy, which eliminates the concerns around lack of a suitable donor or immune-mediated allograft rejection. As exciting as this approach seems, the safety and efficacy of it must be fully assessed in future studies. 5.4.4 Finding a phenotype in the aniridic mouse retina The Pax6 genotype has shown to affect the thickness of the cornea, with Sey mice having thinner corneas compared to WT mice (Hickmott et al., 2018). Therefore, measuring the corneal thickness can be used as an endpoint for gene therapy studies in mice with aniridia. Furthermore, as mentioned earlier, corneal transparency is another key endpoint for such studies. However, unlike cornea, currently there is no consensus among researchers regarding a retinal phenotype in Sey mice (Gregory-Evans et al., 2013; Hickmott et al., 2018; Wang et al., 2017). Finding a therapeutically significant phenotype in the mouse retina is especially challenging since the mouse retina does not have a fovea (Huberman and Niell, 2011), making it hard to draw any appropriate conclusions parallel to patients’ foveal hypoplasia using this species. Interestingly, a recent study examined the structure of the retina in Pax6Sey-Neu/+ mice on BALB/c genetic background and identified areas of cell disorganization and cell clumping, which they termed “hotspots” (Cole et al., 2022). Within these hotspots they observed a difference in retinal thickness between mutant and WT mice, and used this as one of the outcome measures to assess the efficacy of their drug. Hence, it is important for the researchers in the field to examine the retina of other mouse models of aniridia including Sey mice and potentially take advantage of this retinal phenotype in their preclinical studies. The best approach to do this 125 would be to harvest eyes from a large number of aniridic and WT mice and carefully examine the eye sections for the presence of hotspots while the investigator is blinded to the genotypes. 5.4.5 Evaluating the potential of LNP delivery platform in the retina Many gene editing experiments are limited by the ability to deliver the functional gene editing machinery to the target site. Given our success in Chapter 3 with the LNP-mediated delivery of Cas9 RNPs to the cornea and the importance of the retina as a therapeutic target for a wide range of ocular diseases such as aniridia (Daruich et al., 2022), glaucoma (Munemasa and Kitaoka, 2012), diabetic retinopathy (Kern and Barber, 2008), age-related macular degeneration (Grisanti and Tatar, 2008) and retinitis pigmentosa (Ferrari et al., 2011), further studies must be undertaken to determine whether this platform will be suitable for therapy delivery to the retina. Following the same path as Chapter 3, the initial optimization experiments can be carried out in cortical neurons. In the absence of primary retinal cells, cortical neurons are a relevant choice, since the retina consists of several neuronal layers (Masland, 2012). For the in vivo experiments, the delivery can be done through both intravitreal and subretinal injections. However, for aniridia, intravitreal delivery is the preferred route of administration to reach PAX6-expressing cells of the inner retina (Hickmott et al., 2016). Nevertheless, the presence of vitreous body and inner limiting membrane (ILM) (an acellular membrane that separates vitreous body from the retina) (Halfter et al., 2008) are the biggest hurdles for effective intravitreal delivery of LNPs to the retina. LNP size and surface charge play important roles for bypassing these hurdles (Huang and Chau, 2019a). Since both vitreous and ILM are negatively charged, high net positive or negative surface charge of LNPs considerably limits the passive diffusion of these particles from the vitreous to the retina (Huang and Chau, 2019b). Unfortunately, we did not have access to equipment to measure the zeta potential of our LNPs in Chapter 3. Therefore, it would be 126 important to determine this value in future studies. With regard to the LNP size, currently there is no definite size range that can guarantee successful permeability across ILM (Jackson et al., 2003; Kamei et al., 1999). This is in part due to interspecies variation and therefore must be determined empirically in future work. 5.4.6 Investigating the potential role of exogenous Apolipoprotein E in cellular uptake of LNPs in the cornea In Chapter 3, we briefly discussed the importance of Apolipoprotein E (ApoE) for facilitating LNP uptake in cultured neurons (Mirjalili Mohanna et al., 2022; Rungta et al., 2013; Sebastiani et al., 2021) and pointed out that we could not find report of ApoE expression in the mouse cornea in the literature. Results from in vivo studies in rodents suggest that cellular uptake of ApoE-bound LNPs is cell-specific and strongly depends on the presence of low-density lipoprotein (LDL) receptors (Akinc et al., 2010; Rungta et al., 2013). We know from the literature that rat corneal epithelial cells express LDL receptor-related protein 1 (LRP1) (Higuchi et al., 2016), which recognizes ApoE (Kanekiyo et al., 2013). Hence, additional experiments must be designed to first determine whether WT and Sey mouse corneal cells express LRP1 and next investigate the significance of ApoE presence for facilitating LNP uptake into the corneal cells that express this receptor. The first objective can be readily achieved by immunostaining mouse corneal sections with an anti-LRP1 antibody and looking for LRP1 expression in the cells. The second objective can be achieved by spiking the LNP payload with exogenous ApoE during intrastromal injections and comparing the transduction efficiency between the experimental group (LNP + ApoE) and positive controls (LNP only). Building on the results of animal studies, supplementing LNP-based therapies with exogenous ApoE might also enhance 127 uptake in human corneas, even though LRP1 and its ligand ApoE are naturally expressed in all three corneal layers (Dyrlund et al., 2012; Mogensen et al., 2022). 5.4.7 Improved administration routes for targeting the cornea 5.4.7.1 Central vs. edge intrastromal injections In the corneal limbus of human eyes, there are structures known as limbal crypts that act as a niche for LSCs (Shortt et al., 2007). Interestingly, these limbal crypts were shown to be not uniformly distributed in the corneal limbal regions and were mainly located at 12 and 6 o’clock positions (the superior and inferior limbal regions) (Grieve et al., 2015; Shortt et al., 2007). Administering the therapeutics as close to these crypt-rich LSC niches as possible may maximize the number of LSCs the therapy can target. In both Chapter 3 and 4, the intrastromal injections were done by inserting the needle directly into the stroma at 3 o'clock position near the corneal limbus border. The needle was then slowly advanced towards the central stroma where the injection occurred. Considering the location of the crypt-rich LSC niches, an alternative injection method would be to carefully insert the tip of the needle into the stroma at either 12 or 6 o'clock position and dispense the payload very close to the edge of the limbus. Additionally, in theory, the edge intrastromal injection should be less invasive than the central intrastromal injection where the needle must move quite a bit until it reaches the center of the stroma. This advantage may enable the injector to perform two edge intrastromal injections in one session to reach limbal crypts residing at both 12 and 6 o’clock positions. The main challenge in testing the efficacy of these injection methods in targeting LSCs is that the morphology of corneal LSC niche is different between species and unlike the human cornea, limbal crypts have not been observed in the mouse cornea (Grieve et al., 2015; Li et al., 2017). Therefore, a better animal model that has a closer corneal crypt architecture to humans, 128 such as pig (Grieve et al., 2015), is needed for the head-to-head comparison of these injection methods and to ultimately translate new findings into clinical settings. 5.4.7.2 Dissolving microneedle patches Dissolving microneedle patches can be used as a less invasive alternative to intraocular injections for delivering therapeutics to the cornea. These microneedle patches are applied on the cornea by patients in a similar manner to a contact lens. Upon application, the microneedles penetrate the outer layer of the cornea where they gradually dissolve and release their payload over the course of few days. This novel method was initially developed for delivery of drugs in to the skin (Lee et al., 2008b), but later on it gained popularity and was adopted by others for intracorneal delivery of an anti-angiogenic monoclonal antibody (DC101) to a mouse model of corneal neovascularization (Than et al., 2018). The latter resulted in a significant reduction in the neovascular region after a single treatment that was comparable with the outcome achieved by intraocular injection of the drug. To date, these microneedle patches have not been tested in clinical trials for any ocular disease. Scalability issues arising from differences in the surface size of the cornea and its thickness impede direct translation of these eye patches between mouse and human. In the case of aniridia, for this method to be successfully implemented for treating patients, the intervention must be done during the first decade of life when the LSC deficiency has not yet impacted the ocular surface and the cornea is relatively normal (Fries et al., 2022). This is because the small insertion marks left on the corneal epithelium by microneedles may exacerbate the unhealthy ocular surface in older aniridia patients that suffer from a gradual loss of LSCs (Lagali et al., 2013). Moreover, in vivo research must be carried out to investigate whether therapeutics delivered to the cornea by these microneedle patches can reach corneal LSCs. It will be 129 especially important to determine the best location for inserting the eye patch in order to reach target cells in therapeutically beneficial quantities. For instance, inserting the microneedle patch either directly on or as close as possible to corneoscleral junction may increase the likelihood of the therapeutics reaching LSCs, compared to inserting the patch on the central cornea. Thus, as exciting as this minimally invasive delivery route sounds, it requires considerable optimization before being able to help aniridia patients. 5.4.7.3 Topical application Topical application is a non-invasive method for delivering therapeutics to the cornea (Loscher et al., 2022) and it is probably the safest and most straightforward administration route for patients with aniridia, considering the fragility of their corneas (Lee et al., 2008a) and the increased risk of developing secondary ocular complications following any invasive procedures (Tibrewal et al., 2022). Although this delivery route is very attractive, too often the therapeutic is not effectively absorbed by the cornea due to a number of anatomical and physiological barriers. Tear film is a major precorneal barrier and is known to prevent the penetration of 95-99% of topically-applied drugs (Loscher et al., 2022). For the small subset of drugs that successfully penetrate the tear film, the next challenge would be to effectively penetrate the corneal epithelial barrier. Drug size, composition, and charge are some of the parameters that can influence the permeability to the cornea (Loscher et al., 2022). There are successful reports of topically-applied small drugs to the eye of Pax6-deficient mice (Cole et al., 2022; Gregory-Evans et al., 2013; Rabiee et al., 2020; Wang et al., 2017). However, with an approximately 25 nm diameter capsid, rAAVs are large (McIntosh et al., 2022) detergent-sensitive molecules (Kondratova et al., 2019), and it is unknown whether they can penetrate through the tear film and into the aniridic cornea to transduce PAX6-expressing 130 cells when delivered in an eye drop. Similarly, our RNP encapsulated LNPs are quite large with an average diameter size of about 151 nm (Mirjalili Mohanna et al., 2022) and unknown zeta potential. The latter is especially important since under normal physiological conditions, the positive charge of a compound can make the penetration into the negatively-charged cornea easier (Loscher et al., 2022). Therefore, initially ex vivo studies must be conducted with the goal to develop eye drop formulations that do not compromise the ability of rAAVs or LNPs to transduce cells. Once those eye-drop formulations have been established, in vivo experiments must be performed to assess the safety and efficacy of topical delivery of rAAVs and LNPs to the aniridic mouse cornea. Currently, there is no literature that shows LSCs can be targeted by topical application. Hence, if rAAVs and LNPs fail to deliver the therapeutics to the aniridic mouse LSCs with topical application, the study can be focused on targeting the corneal epithelial cells. Because these cells turnover rapidly, substantial studies must be carried out to determine the optimal dose for repeated treatments and the intervals at which the eye drop needs to be administered to improve the disease phenotype and maintain the cornea. 5.5 Concluding remarks Our work towards prevention of blindness in aniridia initiated by creating a novel mouse model that is optimal for CRISPR therapy development (Chapter 2) and was followed by testing both nonviral (Chapter 3) and viral (Chapter 4) vector delivery systems to find a safe and effective approach that is best for in vivo somatic CRISPR gene editing therapy for aniridia. 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Shown here are representative slit lamp images of one eye each of four adult mice (3-4 months old) for each genotype. In addition, examining slit lamp images, the lens structure was not found to be significantly different between homozygous Fax and WT mouse eyes (one-tailed t-test, p=0.074). Fax, FLAG tagged Pax6; WT, wild type. 174 Figure A.2. Eyes of heterozygous and homozygous Fax and FeyCor E18.5 fetuses were not significantly different from WT. Fetuses’ heads were imaged under identical light microscope conditions and the phenotypes were scored based on the absence or presence of the eye, as well as, the size of non-pigmented area. Fey heterozygous (Het) and homozygous (Hom) fetuses showed microphthalmia and anophthalmia, respectively. The observed phenotype in the former was significantly different from FeyCor and Fax Hom fetuses (one-tailed t-test, p<0.001). Fax and FeyCor Het and Hom fetuses’ eyes were not significantly different from WT eyes (one-tailed t-test, p>0.999). Three individual fetuses are presented here for each genotype. Fax, FLAG tagged Pax6; Fey, FLAG tagged Sey; FeyCor, Fey corrected; WT, wild type. 175 Table A.1. Western blot densitometry data generated for two technical replicates. Sample PAX6 Relative O.D.a (Blot A) PAX6 Relative O.D. (Blot B) Mean Standard Deviation FeyCor Hom 9.20 7.59 8.40 1.14 FeyCor Het 11.7 6.26 9.01 3.88 WT 12.1 4.67 8.39 5.26 Fax Hom 8.72 7.31 8.02 0.99 Fax Het 6.67 5.32 6.00 0.95 Fey Hom 0.00 0.00 0.00 0.00 Fey Het 2.51 3.68 3.10 0.83 aRelative O.D. reflects the amount of PAX6 protein normalized against the loading control. Fax, FLAG tagged Pax6; Fey, FLAG tagged Sey; FeyCor, Fey corrected; Het, heterozygous; Hom, homozygous; O.D., optical density; WT, wild type. 176 Table A.2. CRISPR RNAs (crRNAs) and single-stranded oligodeoxynucleotide (ssODN) templates used for in vitro and in vivo gene editing. ID Description Sequence (5′-3′) Stranda cgEMS3 crRNA for in vitro and in vivo 3xFLAG tag insertion after Pax6 start codon in exon 4. UUCUGCAUGCUGGAGCUGGU Antisense cgEMS4 crRNA for in vitro and in vivo correction of the Sey mutation in exon 8. CAGUUCUAACUGAGAAGACU Sense oEMS6349b 189 base ssODN template for cgEMS3. Besides the 66 bp 3xFLAG tag sequence (in bold), there is one unnecessary base change (underlined) in this donor. This ssODN template was used for generating the Fey strain. ACGGCCCGCACGCCTCCAGC CCAACAGTCCAGAGAAAGAT CTGAGACACTTACTGTTCTG CTTGTCATCGTCATCCTTGT AATCGATGTCATGATCTTTA TAATCACCGTCATGGTCTTT GTAGTCCATGCTGGAGCTGG TTGGCAGCCTCCGGATCCCA CGGGGCTCGAATACGGGGCT CTGTTAGGA Antisense oEMS6297 189 base ssODN template for cgEMS3. It contains 3xFLAG sequence (in bold) for the insertion after Pax6 start codon. This ssODN template was used for generating the Fax strain. TCCTAACAGAGCCCCGTATT CGAGCCCCGTGGGATCCGGA GGCTGCCAACCAGCTCCAGC ATGGACTACAAAGACCATGA CGGTGATTATAAAGATCATG ACATCGATTACAAGGATGAC GATGACAAGCAGAACAGTAA GTGTCTCAGGTCTTTCTCTG GACTGTTGGGCTGGAGGCGT GCGGGCCGT Sense oEMS6300 160 base ssODN template for cgEMS4. Besides correcting the Sey mutation (in bold), this ssODN contains one silent base change in the Cas9 protospacer adjacent motif (PAM) (underlined). AATCTGCTCTTGGGTAAAAG ATGTTCTATTTCTTTGCAGC TTCCGCTTCAGCTGAAGTCG CATCTGAGCTTCATCTGAGT CTTCTCCGTTAGAACTGATG GAGTTGGTGTTCTCTCCCCC TCCTTCCTGTTGCTGGCAGC CATCTGCAACAAAAAGAATA Antisense aStrand relative to mouse Pax6. bThis donor was designed to test three ribonucleoprotein (RNP) complexes in vitro. The unnecessary base change was intended to prevent one of the RNP complexes from re-targeting the edited sequence. However, this base change was not needed for cgEMS3 as the PAM for this crRNA was destroyed after the correct insertion of the tag. cgEMS, CRISPR guide Elizabeth M. Simpson; Fax, FLAG tagged Pax6; Fey, FLAG tagged Sey; FeyCor, Fey corrected; ID, identifier; oEMS, oligo Elizabeth M. Simpson; Sey, small eye. 177 Appendix B Supplementary Materials for Chapter 3 Figure B.1. The average diameter distribution of RNP loaded LNPs was 151.6 ± 0.8 nm. Size distribution of LNPs encapsulating luciferase non-targeting RNP strategy was measured using a Malvern Zetasizer Nano S instrument. 178 Table B.1. CRISPR guide RNAs (gRNAs) and DNA templates used for ex vivo and in vivo genome editing. ID Description Sequence (5'-3') Sp_Ai9_La gRNA for removal of the transcriptional stop cassette and turn on of tdTomato AAAGAAUUGAUUUGAUACCG eloxPb gRNA for removal of the transcriptional stop cassette and turn on of tdTomato GUAUGCUAUACGAAGUUAUU cgEMS48 gRNA for removal of the transcriptional stop cassette and turn on of tdTomato GUCUGGAUCUGCGACUCUAG cgEMS49 gRNA for removal of the transcriptional stop cassette and turn on of tdTomato UCAAUGUAUCUUAUCAUGUC cgEMS4 gRNA for correction of the Sey variant in exon 8 of Pax6 gene. CAGUUCUAACUGAGAAGACU Luciferase gRNA against non-endogenous target (firefly luciferase) with no match in mouse CUUCGAAAUGUCCGUUGGGU oEMS6469 80 base DNA template for tdTomato targeting strategy TTGTGCTGTCTCATCATTTTGGCAAAGAATTGATTTGATAATTAGGTCCCTCGACCTGCAGCCCAAGCTAGATCGAATTC oEMS6460 100 base DNA template for tdTomato targeting strategy GTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTGATTTGATAATTAGGTCCCTCGACCTGCAGCCCAAGCTAGATCGAATTCGGCCGGCCTT oEMS6461 160 base DNA template for tdTomato targeting strategy TTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTGATTTGATAATTAGGTCCCTCGACCTGCAGCCCAAGCTAGATCGAATTCGGCCGGCCTTCACGATCCCGCCACCATGGTGAGCAAGGGC oEMS6300 160 base DNA template for Sey correction strategy. Besides correcting the Sey variant (in bold), this template contains one silent blocking base change in the PAM (underlined) AATCTGCTCTTGGGTAAAAGATGTTCTATTTCTTTGCAGCTTCCGCTTCAGCTGAAGTCGCATCTGAGCTTCATCTGAGTCTTCTCCGTTAGAACTGATGGAGTTGGTGTTCTCTCCCCCTCCTTCCTGTTGCTGGCAGC CATCTGCAACAAAAAGAATA a. This gRNA sequence is taken from literature (Tabebordbar et al., 2016). b. This gRNA sequence is taken from literature (Yang et al., 2017). cgEMS, CRISPR guide EMS; eloxP, extended loxP; ID, identifier; oEMS, oligonucleotide EMS; PAM, protospacer adjacent motif; Sey, Pax6 small eye; Sp_Ai9_L, SpCas9_Ai9_Left. 179 Table B.2. Final mouse numbers and experimental groups for in vivo genome editing. ID Genotype Experimental Group 20564(2)-2-4 WT tdTomato targeting CRISPR RNP strategy 20564(2)-2-7 WT 20571(1)-1-5 WT 20565(2)-1-4 WT 20565(2)-1-7 WT 20564(2)-2-2 WT 20571(1)-1-7 Fey 20571(1)-1-8 Fey 20565(2)-1-5 Fey 20564(1)-1-7 WT Sey correction CRISPR RNP strategy 20564(1)-1-9 WT 20567(1)-1-8 WT 20568(2)-2-1 WT 20564(1)-1-6 Fey 20564(1)-1-8 Fey 20564(1)-1-10 Fey 20567(1)-1-7 Fey 20568(1)-1-5 Fey 20568(2)-2-2 Fey 20570(1)-1-2 Fey 20569(2)-2-5 Fey 20566(2)-1-3 WT Luciferase non-targeting CRISPR RNP strategy 20570(1)-1-6 WT 20568(2)-2-9 WT 20571(1)-1-1 WT 20566(2)-1-5 Fey 20567(2)-2-4 Fey 20570(1)-1-5 Fey 20571(1)-1-4 Fey Fey, 3xFLAG-tagged Sey; ID, identifier; RNP, ribonucleoprotein; Sey, Pax6 small eye; WT, wild type. 180 Appendix C Supplementary Materials for Chapter 4 Figure C.1. A variety of retinal and corneal cells were successfully transduced in WT and Sey mice after intravitreal delivery of PHP.B and rAAV9 iCre viruses. Representative overview images of mouse eyes. The Roman numerals correspond to images in Figure 4.5. Blue, Hoechst nuclear stain; iCre, improved Cre; red, tdTomato epifluorescence; Sey, Pax6 small eye; WT, wild type. 181 Figure C.2. All three corneal cell layers were successfully transduced in WT and Sey mice after intrastromal delivery of PHP.B and rAAV9 iCre viruses. Representative overview images of mouse eyes. The Roman numerals correspond to images in Figure 4.6. Blue, Hoechst nuclear stain; iCre, improved Cre; red, tdTomato epifluorescence; Sey, Pax6 small eye; WT, wild type.
Thesis/Dissertation
eng
Medical Genetics
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Simpson, Elizabeth M.
Successful gene editing in a novel mouse model of aniridia towards prevention of blindness
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