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Subretinal gene delivery using helper-dependent adenoviral vectors Wu, Linda; Lam, Simon; Cao, Huibi; Guan, Rui; Duan, Rongqi; van der Kooy, Derek; Bremner, Rod; Molday, Robert S; Hu, Jim Apr 4, 2011

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METHODOLOGY Open AccessSubretinal gene delivery using helper-dependentadenoviral vectorsLinda Wu1,2, Simon Lam1,2, Huibi Cao1, Rui Guan1, Rongqi Duan1, Derek van der Kooy4, Rod Bremner2,5,Robert S Molday6 and Jim Hu1,2,3*AbstractThis study describes the successful delivery of helper-dependent adenoviral vectors to the mouse retina with longterm and robust levels of reporter expression in the retina without apparent adverse effects. Since these vectorshave a large cloning capacity, they have great potential to extend the success of gene therapy achieved using theadeno-associated viral vector.BackgroundThe eye has several unique features that make it a wellsuited target organ for gene therapy. It has a highlycompartmentalised structure which allows for the effi-cient delivery of a small volume of vector suspension toa specific subset of cells. The precise targeting of a par-ticular cell type minimises viral dissemination andunwanted systemic effects. Additionally, immuneresponses resulting from intraocular administration areattenuated compared to those following systemic admin-istration because the eye has both physical barriers aswell as an internal environment that promotes tissuepreservation and protects against harmful inflammatoryresponses that can limit transgene expression. Lastly, awide range of well characterized animal models areavailable for studies of eye disease progression [1,2].Several recent gene therapy trials have brought clinicalbenefits to patients with Leber’s Congenital Amaurosis,a severe childhood retinal dystrophy [3-6]. Currently,the majority of eye gene therapy trials are carried out byusing the adeno-associated virus (AAV). Although ear-lier work with AAV was shown a lag between viral vec-tor injection and transgene expression, the self-complementary AAV (scAAV) has been shown toexpress the transgene in as little as one-day after injec-tion, and can transfect the photoreceptor cells in addi-tion to the RPE [7]. However, the limited cloningcapacity of the AAV vector (4.7 kb) [8] is a majorobstacle to delivery of large therapeutic genes or geneswith long DNA regulatory elements that has yet to beovercome. Although some have attempted to deliverlarge genes using AAV[9,10], the latest studies haverevealed that the expression of the transgenes was aresult of co-infection and recombination within targetcells [11]. The existence of retinal diseases involvinggenes beyond the AAV’s cloning capacity encouragesstudies of potential viral gene therapy vectors beyondAAV. For example, ABCA4 is a member of the ATP-binding cassette transporter sub-family, mutations ofwhich are linked to Stargardt macular dystrophy. Asanother example, CEP290, a gene encoding a 290 kDacentrosomal protein, is associated with a frequent formof Leber’s Congenital Amaurosis (LCA10). The cDNAsof these genes are 6.8 kb and 7.4 kb respectively, pre-cluding the use of AAV vectors even before considera-tion of regulatory elements. Thus, it is important todevelop alternative vectors that have a large cloningcapacity, the ability to transduce non-dividing cells inthe post-mitotic retina, and a low immunogenicity toallow sustained long-term transgene expression. Thehelper-dependent adenoviral (HD-Ad) vector presentsall these characteristics which make it an ideal candidatefor retinal gene therapy.HD-Ad vector, also known as the gutless, gutted, orhigh-capacity Ad vector, has been developed with signif-icant improvements in the safety and delivery efficiencyafter many changes made to the first generation adeno-viral (FG-Ad) vectors [12-16]. The main difference ingenome composition between the HD-Ad vector and itsparental FG-Ad vector is that the HD-Ad vector is fully* Correspondence: jim.hu@utoronto.ca1Physiology and Experimental Medicine Program, Hospital for Sick Children,555 University Avenue, Toronto, Ontario, M5G1X8, CanadaFull list of author information is available at the end of the articleWu et al. Cell & Bioscience 2011, 1:15http://www.cellandbioscience.com/content/1/1/15 Cell & Bioscience© 2011 Wu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.devoid of all viral coding genes, leaving only the ITRand ψ sequences necessary for vector replication andpackaging, respectively [17,18]. This strategy preventsthe production of any viral proteins which in turn sig-nificantly reduces the cytotoxic T lymphocyte (CTL)response brought upon by viral gene expression [19-21].A minimized immune response reduces toxicity to hostcells, delays vector clearance, and promotes long-termtransgene expression. In fact, upon HD-Ad injectionthrough the tail vein of mice, transgene expression inlivers has been shown to have life-long persistence [22].However, very little is known about the utility of HD-Ad vectors in retinal gene therapy [23,24], especiallyregarding the delivery of HD-Ad into the subretinalspace[19,25]. We hypothesized that the HD-Ad systemcan be used to deliver transgenes into retinal pigmentepithelial (RPE) and photoreceptor (PR) cells.We performed extensive analyses of HD-Ad vectordelivery to mouse subretinal space using LacZ as areporter gene and found that the HD-Ad elicits trans-gene expression for a minimum of 2 months with nosign of decrease in expression. We also observed a doseresponse in reporter gene expression. Our results showthat HD-Ad vectors have great potential to extend thesuccess of eye gene therapy to applications whichrequire vectors for delivering large genes or regulatoryelements.Results and discussionCurrently, retinal gene therapy trials are carried out withAAV vectors [26] and little is known about the feasibil-ity of HD-Ad vector as a vehicle for gene delivery tohuman retinal cells. To examine the potential of HD-Advectors to be used for retinal gene therapy, mice weregiven subretinal injections with the CMV-LacZ HD-Advector. At 4 different time points, the retinal expressionof the reporter gene was determined via X-gal staining.One week following injection with HD-Ad-CMV-LacZat 1 × 108 vector particles (vp)/eye, mice showed robusttransgene expression along the retinal layer (Figure 1A).The X-gal positive areas were seen in the RPE predomi-nantly, with sporadic expression found in the proximalregions of the outer segments (OS). Two months post-injection (Figure 1D), the furthest time point tested forthe HD-Ad-CMV-LacZ injected mice, trends in expres-sion were comparable to the 1 week time point. X-galstaining was present along the retinal layer with themajority of expression in the RPE layer and little expres-sion in the OS of the PR cells.HD-Ad vectors have attributes that make them desir-able in gene therapy trials. Due to their genome beingdevoid of all viral coding genes [17,18], little or no CTLresponse arises [19-21], and the vector can persist inhost cells for a very long time where they stay in episo-mal form [27]. Since retinal cells are terminallyFigure 1 Retinal expression of LacZ reporter. Eyes were processed for X-gal staining and cut at serial sections of 6 μm and counterstainedwith neutral red. Expression was detected at 1 week (A), 2 weeks (B), 1 month (C), and 2 months (D) in the RPE layer of the retina. Someexpression was also detected in the proximal OS. Control (E) with no vector injection reveals no b-galactosidase activity. RPE, retinal pigmentepithelium; OS, outer segments; ONL, outer nuclear layer; INL, inner nuclear layer.Wu et al. Cell & Bioscience 2011, 1:15http://www.cellandbioscience.com/content/1/1/15Page 2 of 7differentiated and non-replicative, dilutional loss of epi-somes is unlikely for HD-Ad vectors. Furthermore, sincethe genome is non-integrating, there is minimal risk ofinsertional mutagenesis [27]. Although we only exam-ined reporter expression up to two months, we predictthat it would continually persist had further time pointsbeen examined.Transgene expression was detected in mouse retinathree days following intraocular injections with HD-Advectors [19,25]. Our results show that onset of maxi-mum gene expression occurred no later than 1 weekpost-injection. The lack of delay for transgene expres-sion makes HD-Ad vectors superior if immediate trans-gene expression is desired. For example, acute damageby physical trauma to the eye resulting in fast retinalcell deterioration will require a vector that quickly deli-vers survival factors to rescue rapidly dying cells. Thedelivery of various neurotrophic factors, growth factors,and cytokines protect neurons from cell death in theseinstances including BDNF, CNTF, neurotrophin-3 and-4, and bFGF [28-31].To examine the effect of viral vector dosage on trans-gene expression, we injected mice with 3 different viralvector doses and evaluated reporter gene expressionlevels. At the lowest vector dose of HD-Ad-CMV-LacZ,1 × 107 vp/eye, transgene expression was detected (Fig-ure 2B), but consistency of expression throughout theentire retina was not observed as only several areasaround the retina showed b-galactosidase activity. Serialsectioning of tissues at this dose revealed that expres-sion was absent from a large proportion of the retina(Figure 2F). Also, expression of the transgene was con-fined to the RPE layer of the retina (Figure 2M) andabsent from PR cells.At 1 × 108 vp/eye, significant LacZ expression wasobserved throughout the retina (Figure 2C and 2G).Serial sectioning of tissues in this group reveals consis-tent and widespread X-gal staining all along the RPElayer (Figure 2G and 2N). The highest vector concentra-tion, 1 × 109 vp/eye resulted in more wide-spread X-galstaining (Figure 2H) without affecting the histology (Fig-ure 2D). Expression at this dose revealed robust stainingin the RPE as well as in photoreceptor inner and outersegments (Figure 2O).As a means to quantitatively evaluate the amount ofreporter gene activity with viral dose, we performed a b-galactosidase activity assay. We found that with anincrease in vector delivery, there was a statistically sig-nificant increase in b-galactosidase activity (Figure 3).Correspondingly, each vector dose group had a statisti-cally significant difference in transgene activity from allother vector dose groups (1-way ANOVA and Bonfer-roni corrected pair-wise t-tests, p < 0.05). Additionally,at each of the vector dose groups, b-galactosidase activ-ity levels were also measured over different time pointsand we found that overall there was no significant asso-ciation between time points and transgene activity levels(2-way ANOVA; dose p < 0.05; time p > 0.05; interac-tion p > 0.05). These results demonstrate that there is asignificant correlation between viral vector dosage andtransgene expression.Our results demonstrate that the viral vector deliveryresulted in a dose response trend in levels of the expres-sion, as determined by histochemical (Figure 2) andquantitative analyses (Figure 3). These results suggestthat the vector dose can be used to control the level oftransgene expression. Another way to regulate theamount of transgene expression is to use cis-actingDNA elements and promoters. AAV vectors are gener-ally unable to carry these large regulatory sequences,but HD-Ad vectors with their large 37 kb cloning capa-city can house multiple transgenes and native regulatoryelements that promote desirable gene expression. Speci-fically, future studies will be directed to examine theefficiency of HD-Ad vectors for targeting transgeneexpression to photoreceptor cells using cell-specificpromoters.Since potential tissue damage with high viral load is aconcern for retinal gene therapy, it is important todetermine the lowest amount of vector required formaximum gene expression. To examine whether HD-Advector delivery could cause tissue damage, we performedH&E staining of the retinas from mice that received dif-ferent doses of HD-Ad-CMV-LacZ. The eyes were firstprocessed for X-gal staining at 1 week post-injection toverify that viral delivery was successful. The X-galstained retinal tissues were then used for H&E staining.The results showed that at three increasing viral vectorconcentrations, there was no visible sign of inflamma-tion (retinal folding, granulations, cell death, and tissuenecrosis) (Figure 4A and 4B and 4C). Likewise, tissuemorphology of all injected mice were similar to theirnegative, no-injection controls (Figure 4D), suggestingthat at the doses we selected, the viral vector concentra-tion does not cause tissue damage. Our results showthat at 1 week following gene delivery, morphology ofthe retina remains normal with little or no sign of tissuedamage or inflammation. Even at our highest viral vec-tor dose, no visible inflammation was detected in theocular space, implying the absence of a strong immuneresponse. This lack of immunogenicity will lead to pro-longed transgene expression in the host.ConclusionsAn ideal vector for retinal gene therapy needs to fulfillthe following criteria. First, it must be able to targetWu et al. Cell & Bioscience 2011, 1:15http://www.cellandbioscience.com/content/1/1/15Page 3 of 7cells in the retina. Second, it must be able to circumventthe immune system from clearance of the vector as wellas prevent an immune reaction that may damage oculartissue. Third, it must be safe by avoiding insertionaltumorigenesis. Finally, it must retain a relatively largecloning capacity for carrying large therapeutic genes aswell as long expression control DNA elements. Whilegreat progress has been made with AAV based vectors,it remains incapable of carrying large therapeutic genes.The results of this study demonstrate that HD-Ad ful-fills these requirements and has great potential forfurther research as a vector for retinal gene therapy.MethodsHD-Ad Vectors and their productionHD-Ad-CMV-LacZ[32] used in this study expresses theLacZ reporter gene under the control of the cytomega-lovirus immediate-early promoter (CMV). The reporterFigure 2 Correlation of transgene expression with viral vector concentration. Mouse eyes were injected with 1 μL of a CMV LacZ vectorand enucleated at 2 weeks and processed for X-gal staining (B-D). Serial sections were cut at 6 μm and tissues were counterstained with neutralred to reveal retinal layers (F-H). Controls with no vector injection (A, E, I) reveal no sign of b-galactosidase activity. Eyes injected at 1 × 107 vp/μL (B, F, J) showed transgene expression to be present, but sporadic in the retina and confined to the RPE (M). At a higher dose of 1 × 108 vp/μL (C, G, K) expression was seen around the entire retinal layer and detected in the RPE predominantly (N). At 1 × 109 vp/μL (D, H, L) significantexpression was detected around the entire retinal layer with staining in both the RPE layer as well as PR segments (O).Wu et al. Cell & Bioscience 2011, 1:15http://www.cellandbioscience.com/content/1/1/15Page 4 of 7gene cassette was cloned into the viral vector pC4HSU[14] and the viral particles were prepared as described[14,15]Animal care and subretinal injectionOne month old female CD-1 mice (Charles RiverLaboratories International) used this study were treatedin strict compliance with the Association for Researchin Vision and Ophthalmology (ARVO) statement on theUse of Animals in Ophthalmic and Vision Research.The animal protocol was approved the Animal CareCommittee of the Hospital for Sick Children. For sub-retinal vector delivery, animals were anaesthetized viaintraperitoneal injections of a mixture (100 μL/10 gbody mass) of ketamine (20 mg/mL; Wyeth AnimalHealth), xylazine (2 mg/mL; Bayer HealthCare) in saline,and pupils were dilated with a topical application of amixture of 0.2% Cyclogyl, 0.5% Mydfrin, and 0.1% Tro-picamide (all from Alcon) in water for 30 seconds.Under an SZX12 dissection microscope (Olympus), asmall incision was made through the cornea, adjacent tothe limbus with a 301/2-gauge needle. A 33 gaugeblunt-end needle (Hamilton) was then inserted throughthe incision with special care to avoid the lens, and waspushed through the retina to the subretinal space wherethe virus was injected very slowly. Each animal received1 μl of virus in the right eye, leaving the left eye as anegative control. Partial retinal detachment wasobserved and recovered in a week post-injection.X-gal staining of whole eyeballEyes were enucleated and fixed with 1% formaldehyde,0.1% glutaraldehyde, 2 mM MgCl2, 5 mM EGTA in 0.1M sodium phosphate buffer, pH 7.8 for 30 minutes at 4°C with rocking. Fixed tissues were washed with 2 mMMgCl2, 0.01% Deoxycholate, 0.02% NP-40 in 0.1 Msodium phosphate buffer at 4°C with rocking andstained with X-gal in the wash solution containing 5mM K4Fe(CN)6, 5 mM K3Fe(CN)6 and 40 mg/ml ofdimethyl formaide) at 37°C with shaking for 3 hours.After staining, samples were washed 3 times with 70%ethanol and post-fixed with 10% formaldehyde at 4°Cfor 4 hours. Samples were then sent to the PathologyDepartment of The Hospital for Sick Children wherethey were embedded in paraffin blocks. 60 serial sec-tions (6 μm thick) were then cut at the horizontal meri-dian and distributed on 10 slides representative of thewhole eye at different levels. Light microscope imageswere taken on a DM IRB microscope (Leica). Four eyeswere examined at 1 week, and 3 eyes at each of 2weeks, 1 month, and 2 months. The un-injected eye ofeach animal was used as controls.Histology analysisFor H&E staining, tissues were deparaffinised and rehy-drated in a series of alcohol rehydration steps. Slideswere stained with hematoxalin (Poly Scientific) for 3minutes and rinsed with deionized water. Tissues weredipped briefly in acidified ethanol (1 mL of concentratedHCl in 700 mL of 70% ethanol) to de-stain and rinsedwith deionized water. Excess water was blotted from theslide before staining tissue with eosin (Poly Scientific)for 1 minute. Tissues were dehydrated in a series ofalcohol dehydration steps and mounted with xylene-based mounting media, Permount (Fisher Scientific) andcovered with a coverslip. For neutral red staining, tissueswere deparaffinised and rehydrated in a series of alcoholrehydration steps. Slides were stained with neutral redstaining solution (0.1% neutral red in 37 mM acetatesolution, pH4.8) for 2 minutes and rinsed in runningtap water until dye has been removed from slides. Tis-sues were dehydrated in a series of alcohol dehydrationsteps and mounted with xylene-based mounting media,Permount (Fisher Scientific) and covered with acoverslip.b-Galactosidase reporter assaysEyes were enucleated and the lens and vitreous wasremoved under a dissection microscope, leaving only theeyecup. Tissue was homogenized in lysis bufferFigure 3 Quantitative analysis of vector dose-dependenttransgene expression. Mouse eyes were injected with 1 μL of aCMV LacZ vector at increasing concentrations of 1 × 107 vp/μL, 1 ×108 vp/μL and 1 × 109 vp/μL. Eyes were enucleated, processed forX-gal staining and measured for b-galactosidase activity levels.Transgene expression was determined by luminescence andreported as a fold increase relative to the control. Statistical analysisrevealed no significant difference between the time points.However, activity levels were determined to be significantly differentwith the increase in viral vector dose suggesting that a dose-dependent relationship exists (2-way ANOVA; dose p < 0.05; time p> 0.05; interaction p>0.05). Error bars indicate standard error of themean (SEM) and 4 mice were used for each time point of eachvector dose.Wu et al. Cell & Bioscience 2011, 1:15http://www.cellandbioscience.com/content/1/1/15Page 5 of 7containing 100 mM potassium phosphate buffer pH 8.0with 0.5 mM DTT, 10% Triton X-100 and proteinaseinhibitor cocktail (Roche Diagnostics). Samples werecentrifuged for 15 minutes at 12,000 RPM in 4°C.Supernatant was collected and either processed immedi-ately or stored at -80°C. The lysate was heat inactivatedat 48°C for 50 minutes and allowed to cool to roomtemperature. The b-galactosidase activity was measuredusing a chemiluminescent assay as described [33]. Foureyes were used for each time point at each dosage level(total of 64 eyes).List of abbreviations usedAAV: adeno-associated virus; CMV: cytomegalovirus; HD-Ad: helper-dependent adenoviral vector; FG-Ad: first generation Adenoviral; CTL:cytotoxic T lymphocyte; H&E: Haematoxylin and Eosin; RPE: retinal pigmentepithelial; PR: photoreceptor; OS: outer segments; ONL: outer nuclear layer;INL: inner nuclear layer.AcknowledgementsThis work was partially supported by the Canadian Institutes of HealthResearch (CIHR) and Foundation Fighting Blindness-Canada (fundingreference numbers MOP-77750, CIHR RMF-92101). LW received a studentshipfrom the CIHR.Author details1Physiology and Experimental Medicine Program, Hospital for Sick Children,555 University Avenue, Toronto, Ontario, M5G1X8, Canada. 2Department ofLaboratory Medicine and Pathobiology, University of Toronto, 1 King’sCollege Circle, Toronto, Ontario, M5S1A8, Canada. 3Department ofPaediatrics, University of Toronto, 555 University Avenue, Toronto, Ontario,M5G1X8, Canada. 4Department of Molecular Genetics, University of Toronto,1 King’s College Circle, Toronto, Ontario, M5S1A8, Canada. 5Toronto WesternResearch Institute, University Health Network, University of Toronto, 399Bathurst Street, Toronto, Ontario, M5T 2S8, Canada. 6Department ofBiochemistry and Molecular Biology, 2350 Health Sciences Mall, University ofBritish Columbia, Vancouver, B.C. V6T 1Z3, Canada.Authors’ contributionsAll authors read and approved the final manuscript. LW carried out most ofthe experiments and participated in the manuscript preparation. SLparticipated in data analysis including the help of statistics and in revisingthe manuscript. HC and RG participated in the design of experiments as wellas experimentation. RD prepared and purified the viral vectors. DV and RBparticipated in data analysis and assisted in the experimental design. RMassisted in the experimental design and participated in revising themanuscript. JH supervised the whole project including manuscriptpreparation.Figure 4 H&E staining of retinal tissue sections. Eyes were processed for X-gal staining at 3 different concentrations of viral vector dose at 1week post-injection and counterstained with H&E. Controls (A) with no injection revealed no b-galactosidase activity and normal physiology. CMVLacZ injection at 1 × 107 vp/μL (B), 1 × 108 vp/μL (C), and 1 × 109 vp/μL (D) all show signs of transgene expression in the RPE layer, without visiblesigns of inflammation or cell toxicity. RPE retinal pigment epithelium; OS outer segments; ONL outer nuclear layer; INL inner nuclear layer.Wu et al. Cell & Bioscience 2011, 1:15http://www.cellandbioscience.com/content/1/1/15Page 6 of 7Competing interestsThe authors declare that they have no competing interests.Received: 14 December 2010 Accepted: 4 April 2011Published: 4 April 2011References1. Chader GJ: Animal models in research on retinal degenerations: pastprogress and future hope. Vision Res 2002, 42:393-399.2. Rivas MA, Vecino E: Animal models and different therapies for treatmentof retinitis pigmentosa. Histol Histopathol 2009, 10:1295-1322.3. 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Yang T, Duan R, Cao H, Lee BH, Xia C, Chang Z, Keith Tanswell A, Hu J:Development of an inflammation-inducible gene expression systemusing helper-dependent adenoviral vectors. J Gene Med 2010, 12:832-839.doi:10.1186/2045-3701-1-15Cite this article as: Wu et al.: Subretinal gene delivery using helper-dependent adenoviral vectors. Cell & Bioscience 2011 1:15.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitWu et al. Cell & Bioscience 2011, 1:15http://www.cellandbioscience.com/content/1/1/15Page 7 of 7


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