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Nonviral gene transfer by chitosan polymer-based nanotechnology Juan, Chih-Yang Michael 2009

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  NONVIRAL GENE TRANSFER BY CHITOSAN POLYMER-BASED NANOTECHNOLOGY by CHIH-YANG MICHAEL JUAN B.Sc., The University of British Columbia, 2007   A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE STUDIES (Physiology)   THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August 2009  © Chih-Yang Michael Juan, 2009  ii ABSTRACT Gene therapy provides a potential alternative to conventional protein drug administration, allowing the body to produce its own drugs based on the exogenously introduced genetic information.  Increasing efforts have been devoted to developing nonviral-based gene transfer systems owing to its attractive safety features.  In this thesis, a nonviral chitosan/ФC31 gene transfer technology was evaluated for the delivery of two transgenes, SEAP and leptin.  While SEAP reporter gene transfer allowed evaluation of delivery methods, leptin gene transfer permitted the assessment of therapeutic efficacy.  Chitosan nanoparticles encapsulating 2 µg SEAP or leptin transgene were able to transfect HEK 293T cells, which secreted SEAP and human leptin proteins that achieved as much as 9414 ± 169 and 74 ± 5 ng/ml, respectively.  In culture, the human leptin protein expressed by the leptin constructs effectively activated the leptin signalling pathway, represented by the phosphorylation of STAT3, demonstrating the protein’s biological activity.  In vivo, SEAP gene transfer, in C57BL6/j mice, was achieved via intrasplenic and i.p. chitosan nanoparticle delivery.  For the i.p. route, it was revealed that, at Day 1 post gene transfer, a 60% decrease in the DNA dose from 625 to 250 µg led to an at least ~7-fold increase in circulating SEAP, detected at 371 ng/ml.  Furthermore, a similar level of SEAP (353 ng/ml) was obtainable with an even lower dose of DNA (50 µg).  Mice receiving a single i.p. injection of chitosan nanoparticles containing 50 µg gWIZTM-SEAP plasmid showed transgene expression kinetics characteristic of those observed with a CMV promoter, with circulating SEAP measurements of 342 ± 60, 108 ± 18, 30 ± 3, and 7 ± 1 ng/ml at Day 1, 3, 5, and 7 post-treatment.  Furthermore, a rare case of successful oral SEAP gene transfer was observed following a single oral feeding of chitosan nanoparticles.  These iii data demonstrate that the use of the chitosan/ФC31 gene transfer system can lead to successful SEAP gene transfer via the i.p. route of administration, although in vivo data did not support effective leptin gene transfer.  Future studies on improving transgene vector design are warranted for further development of this gene transfer system.                    iv TABLE OF CONTENTS ABSTRACT ..................................................................................................................... ii TABLE OF CONTENTS .................................................................................................. iv LIST OF TABLES  .......................................................................................................... vi LIST OF FIGURES  ....................................................................................................... vii LIST OF ABBREVIATIONS  ........................................................................................... ix ACKNOWLEDGEMENTS  .............................................................................................. xi INTRODUCTION  ............................................................................................................ 1 Gene Therapy  ...................................................................................................... 1 Gene therapy clinical trials ............................................................................ 1 Viral-based vectors ....................................................................................... 6 Nonviral-based vectors ................................................................................. 8 Chitosan/ФC31 Gene Transfer Technology  ....................................................... 11 Chitosan  .................................................................................................... 11 ФC31 Integrase ........................................................................................... 20 Gene Transfer Delivery Routes  .......................................................................... 25 Intrasplenic ................................................................................................. 25 Intraperitoneal ............................................................................................. 26 Oral ............................................................................................................. 27 SEAP .................................................................................................................. 28 Leptin .................................................................................................................. 28 Clinical Leptin Therapy ........................................................................................ 31 Leptin Gene Therapy .......................................................................................... 37 THESIS INVESTIGATION ............................................................................................ 39 MATERIALS AND METHODS ....................................................................................... 40 Cloning of Plasmid Constructs ............................................................................ 40 Transfection of HEK 293T Cells Using LipofectamineTM 2000 ............................ 47 Testing of Leptin Plasmid Construct Using Cell-Based Bioassay........................ 48 Western Blot Analysis of Phospho-STAT3 in Stimulated CHO-ObRb Cells ........ 51 Synthesis of Chitosan Nanoparticles ................................................................... 52 v Gel Motility Test .................................................................................................. 55 Measurement of Size, Polydispersity Index, and Zeta Potential of Chitosan Nanoparticles ...................................................................................................... 56 Transfection of HEK 293T Cells Using Chitosan Nanoparticles .......................... 56 Long Term In Vitro Studies with ФC31 Integrase ................................................ 57 In Vivo Delivery of Nanoparticles (Intrasplenic and Intraperitoneal) - Preliminary Studies ................................................................................................................ 59 SEAP Reporter Gene Transfer and Long Term Circulating SEAP Tracking ....... 61 SEAP Chemiluminescent Assay ......................................................................... 62 Human Leptin Gene Transfer and Body Weight and Blood Glucose Monitoring ........................................................................................................... 63 Human Leptin Radioimmunoassay and Enzyme-Linked Immunosorbent Assay .................................................................................................................. 66 DATA ANALYSIS........................................................................................................... 70 RESULTS ...................................................................................................................... 71 Study 1:  Cloning and In Vitro Characterization of Plasmid Constructs ............... 71 Study 2:  Synthesis and Quality Control of Chitosan Nanoparticles .................... 80 Study 3:  In Vivo Gene Transfer of SEAP Reporter Gene ................................... 90 Study 4:  In Vivo Gene Transfer of Human Leptin Transgene ........................... 101 DISCUSSION .............................................................................................................. 115 Promoter Selection for Transgene Expression In Vivo ...................................... 115 Evaluation of Potential Long Term Transgene Expression Conferred by Nonviral Gene Transfer ................................................................................................... 117 Nonviral Gene Transfer: Delivery Routes and Barriers to Overcome ................ 121 Optimization of the Chitosan/ФC31 Gene Transfer Technology ....................... 127 CONCLUSIONS .......................................................................................................... 134 REFERENCES ............................................................................................................ 135 vi LIST OF TABLES Table I.  Comparison of vector elements and their relative positions in gWIZTM-SEAP and CMV-Lep-WPRE-attB ............................................................................................. 46 Table II.  Human leptin assay comparison between Alpco Human Leptin (Ultrasensitive) RIA and Invitrogen Human Leptin ELISA ....................................................................... 68                   vii LIST OF FIGURES Figure 1.  Schematic illustration of the structure and transmission electron micrographs of chitosan ..................................................................................................................... 12 Figure 2.  Mechanism of chitosan-based gene transfer and ФC31-mediated plasmid genomic integration ....................................................................................................... 18 Figure 3.  EF1α-Lep-WPRE-attB (A) cloning procedures and (B) plasmid construct map ............................................................................................. 41 Figure 4.  Plasmid construct map of CMV-Lep-WPRE-attB .......................................... 43 Figure 5.  Plasmid construct map of gWIZTM-SEAP ...................................................... 44 Figure 6.  Schematic representation of the leptin signalling pathway............................ 49 Figure 7.  Flow chart of the (A) production and (B) quality control steps of chitosan nanoparticles synthesis ................................................................................................. 53 Figure 8.  Evaluation of human leptin ELISA with in-house positive controls ................ 69 Figure 9.  Human leptin protein secretion from (A) STC-1 and (B) HEK 293T cells transfected with human leptin-expressing plasmid constructs ....................................... 72 Figure 10.  Leptin bioassay results shown by pSTAT3 western blot and densitometric signal quantification ....................................................................................................... 75 Figure 11.  SEAP protein secretion from HEK 293T cells transfected with different SEAP-expressing plasmids ........................................................................................... 77 Figure 12.  ФC31-mediated long term SEAP gene expression in LipofectamineTM- transfected cells ............................................................................................................. 79 Figure 13.  Gel motility test showing complete encapsulation of plasmid DNA by chitosan ......................................................................................................................... 81 Figure 14.  Size, polydispersity index, zeta potential, and in vitro testing of LMW (5 kDa) chitosan nanoparticles encapsulating EF1α-SEAP/ CMV-Int ......................................... 83 Figure 15.  Size, polydispersity index, and zeta potential of LMW (5 kDa) chitosan nanoparticles synthesized for long term SEAP gene transfer follow-up study ............... 85 Figure 16.  Comparison of size, polydispersity index, zeta potential, and in vitro transfection efficiency between HMW (226 and 429 kDa) and LMW (5 kDa) chitosan nanoparticles ................................................................................................................. 87 Figure 17.  ФC31-mediated long term SEAP gene expression in nanoparticle- transfected cells ............................................................................................................. 89 Figure 18A.  Preliminary method study of SEAP gene delivery through intrasplenic (i.s.) and intraperitoneal (i.p.) administration of SEAP plasmid-containing LMW chitosan nanoparticles ................................................................................................................. 92 viii Figure 18B.  Preliminary dosing study of SEAP gene delivery through intraperitoneal (i.p.) administration of SEAP plasmid-containing LMW chitosan nanoparticles ............. 92 Figure 19.  Intraperitoneal (i.p.) administration of LMW chitosan nanoparticles containing gWIZTM-SEAP – short and long term SEAP tracking .................................... 94 Figure 20.  Oral administration of LMW chitosan nanoparticles containing gWIZTM- SEAP and circulating SEAP tracking ............................................................................. 96 Figure 21.  Intraperitoneal (i.p.) administration of LMW chitosan nanoparticles containing EF1α-SEAP and CMV-Int – short and long term SEAP tracking .................. 98 Figure 22.  Follow-up study of intraperitoneal (i.p.) administration of LMW chitosan nanoparticles containing EF1α-SEAP and CMV-Int ..................................................... 100 Figure 23.  Oral administration of human leptin plasmid-containing LMW chitosan nanoparticles in ob/ob mice - (A) body weight and (B) fasting glucose tracking .......... 103 Figure 24.  Intraduodenal administration of human leptin plasmid-containing LMW chitosan nanoparticles in ob/ob mice - (A) body weight and (B) fasting glucose tracking ......................................................................................... 105 Figure 25.  Intraperitoneal (i.p.) administration of human leptin plasmid-containing LMW chitosan nanoparticles in ob/ob mice - (A) body weight and (B) fasting glucose tracking ......................................................................................... 107 Figure 26.  Intraperitoneal (i.p.) administration of human leptin plasmid-containing HMW chitosan nanoparticles in ob/ob mice - (A - F) body weight and fasting glucose tracking, (G) Percent weight and (H) blood glucose drop ............................................ 109 Figure 27.  Plasma human leptin levels post i.p. CMV-Lep-containing HMW chitosan nanoparticles in ob/ob mice ......................................................................................... 112 Figure 28.  Effects of oral administration of human leptin plasmid-containing HMW chitosan nanoparticles in ob/ob mice on (A) body weight and (B) fasting glucose ...... 113 Figure 29.  Plasma human leptin levels post oral CMV-Lep-containing HMW chitosan nanopartcles in ob/ob mice .......................................................................................... 114        ix LIST OF ABBREVIAIONS Abbreviation                     Description attB    attachment B BSA                                    bovine serum albumin CHO    Chinese hamster ovarian CMV    cytomegalovirus DDA    degree of deacetylation EF1α    elongation factor 1 alpha ELISA              enzyme-linked immunosorbent assay FBS    fetal bovine serum HEK    human embryonic kidney HG-DMEM  high glucose Dulbecco’s modified Eagle’s medium HMW                                  high molecular weight Int                                       ФC31 integrase i.p.                              intraperitoneal LB lysogeny broth LEP                                    leptin LMO2    LIM domain only 2 LMW                                   low molecular weight MER                                    minimal enhancer region MLV                                    murine leukemia virus NHPP    National Hormone and Peptide Program ObR                                    leptin receptor ORI    replication origin x PBS    phosphate buffered saline PCR                                    polymerase chain reaction PLAP    human placental alkaline phosphatase Poly A    polyadenylation signal P/S    penicillin/streptomycin RIA                                      radioimmunoassay RT-PCR                         reverse-transcriptase polymerase chain reaction SDS-PAGE                         sodium dodecyl sulfate polyacrylamide gel electrophoresis SEAP                                  human placental secreted alkaline phosphatase STAT signal transducers and activators of transcription TBS Tris buffered saline UTR                                  untranslated region WPRE woodchuck hepatitis B virus post-transcriptional regulatory                                            element         xi ACKNOWLEDGEMENTS  Dr. Timothy Kieffer has been an amazing supervisor in these past 2 years of my graduate career.  I will never forget the trauma after the first time he returned my thesis proposal to me with a short and powerful remark, “this needs a lot of work”, and the joy after receiving his comment on my thesis, “you did a good job writing”, 2 years later.  My 28-month graduate studies did not go by without unsuccessful experiments, struggles with lab techniques, mouse monitoring that had to take place during odd hrs (i.e. weekends, midnight…etc.), numerous sleepless nights after seeing “interesting” results, and last but not least intellectual challenges from dear labmates.  Tim’s guidance helped me through all these and, leading by example, he showed me the qualities of a passionate scientist.  Thank you Tim for the opportunity to work in your lab and the incomparable training you provided.  Of course, I truly thank everyone in the Kieffer Lab, including postdoctoral fellows, lab technicians, and fellow graduate students, and scientists at enGene Inc., who offered tremendous assistance in all aspects of my research project and, without whom, I would have been lost in the muddy path towards to my degree.  Travis, your training on cell culture and well-mannered tone of voice (most of the time), not to mention your well-known organization skills, will always be much appreciated.  Ali, thank you for always being patient and helpful and, most importantly, for taking me on as a volunteer before I joined the lab.  Majid, your persistent hard work will always inspire me and your humour will always be remembered.  Anthony and Eric, thank you for including me in the development process of your company’s technology and providing me with advice and directions throughout my time in graduate school.  I will always be grateful for the industrial experience acquired while working with enGene.  Carlos and Jun, your technical support and knowledge were of immense value to my project and I thank you for being helpful and approachable.  Gary, you have been a great support ever since I started working in the lab.  Thanks for all the help on data analysis, reports, and presentations, and equipping me with all the “grad student tips”.  Irene, Frank, Jasna, Blair, and Heather, you guys are incredible to work with.  I will never forget your encouragements, laughters, and “constructive” criticisms.  Finally, I would like to take this opportunity to thank my parents, who never failed to be there for me and pray for me.  Most significantly, I would like to thank my wife, Eva, for supporting and motivating me in everything I choose to do.  You’re always there to cheer me up when times are dishartening and share my joy when times are uplifting. Thank you for everything you do for me during my graduate school journey.    1 INTRODUCTION Gene Therapy  Gene transfer is a potential therapeutic option relying on the introduction of exogenous genetic materials into the body, where therapeutic gene products are expressed to supply a deficient protein or to offer therapeutic functions [1].  It was originally designed to correct or compensate for a phenotype associated with a particular genotype in cases such as cystic fibrosis, hemophilia, muscular dystrophy, metabolic diseases, and cancer [2].  However, in recent years, the scope of gene therapy has broadened to include manipulation of the immune system for DNA vaccination and treatment of inflammatory conditions and enhancement interventions for non-therapeutic purposes such as improving muscle strength and mass and controlling weight, height and hair growth [3, 4].  Turning genetic transferring strategy into a medicine, although simple in concept, has been met with great technical challenges and adverse outcomes, the most high-profile of which are the death of a patient and 4 accounts of oncogenic events occurred in human gene therapy clinical trials, resulting in the lack of any gene therapy product in the USA after over two decades of research and development [5]. Gene therapy clinical trials  In 1989, Rosenberg et al. conducted the first human gene therapy clinical trial at the Division of Cancer Treatment of the National Cancer Institute in Bethesda [6].  A retrovirus was utilized to introduce the gene encoding neomycin resistance into human tumor-infiltrating lymphocytes.  The transduced and modified lymphocytes were subsequently reinfused into five patients with metastatic melanoma.  The treatment was well-tolerated by all patients and no side effects due to the gene transduction were 2 reported.  Gene-modified cells were consistently found in the circulation of all five patients for three weeks and for as long as two months in two patients.  Cells remained retrievable from tumor deposits 64 days after cell infusion.  The procedure was safe according to all criteria, including the absence of infectious virus in tumor-infiltrating lymphocytes and in the patients.  This pioneering study demonstrated the feasibility of using retroviral gene transduction for human gene therapy, a novel technique that may be used to treat serious inherited diseases for which there is no conventional cure. Before long, in 1990, the first therapeutic human gene therapy clinical trial treating two children suffering from a form of severe combined immunodeficiency (SCID) resulting from adenosine deaminase (ADA) deficiency was approved.  In this trial, the adenosine deaminase (ADA) gene was transferred, using retroviral vectors, into the T cells of the two patients, who, after the termination of a 2-year treatment, maintained persistent ADA expression in the T cells [7].  Since that time, the number of trials initiated escalated rapidly reaching a peak of 113 approved trials for the year of 1999.  However, because of severe adverse events in 1999 and 2002, energy in the field of gene therapy dampened significantly as several regulatory agencies halted new or ongoing trials temporarily.  In 2003, only 53 new trials were approved worldwide, the lowest number since 1996 [8].  In 1999, 18-year-old Jesse Gelsinger, participating in a gene therapy trial for the treatment of ornithine transcarbamylase (OTC) deficiency, died after receiving a hepatic infusion of human adenovirus (type 5) containing human ornithine transcarbamylase (OTC) cDNA [9].  The investigators attributed the death to an overwhelming inflammatory reaction to the adenoviral vector.  Following this treatment-induced tragedy, this trial along with several others were discontinued by the US Food and Drug Administration (FDA) in January, 2000. 3  Adding to the gloomy field, in 2002, reports came describing the development of a leukemia-like condition in two of ten children treated for a rare form of X-linked severe combined immunodeficiency (SCID-X1) 2 years prior in France.  These two cases were subsequently shown to be related to retrovirus vector integration near the LMO2 (LIM domain only 2) proto-oncogene promoter, leading to aberrant transcription and expression of LMO2 [10].  These setbacks, while causing researchers to pause for reflection, did not stop gene therapy from progressing.  After the record low in 2003, the number of clinical trial approvals has rebounded, reaching 101, 112, 117, 89, and 108 approvals for the years 2004, 2005, 2006, 2007, and 2008, respectively [8].  To date, the majority (~63%) of these trials are conducted in the USA, while 29% of them are performed in Europe. Around two thirds of the trials are targeting cancer diseases, while cardiovascular, monogenic, infectious, and neurological diseases each cover 8% of the trials.  In terms of the choice of gene carriers, or vectors, investigators still focus mainly on using viruses, as ~66% of the trials involve the utilization of viral vectors (24% adenovirus, 20.9% retrovirus, 7.9% vaccine virus, 5.8% poxvirus, 4.3% adeno-associated virus, and 3.2% herpes simplex virus); however there is a trend towards using nonviral gene transfer techniques as ~25% trials avoid the use of viruses (18% naked DNA and 7% lipofection).  The types of therapeutic genes being transferred span a broad range from antigen (20%), cytokine (19%), tumour suppressor (11%), growth factor (8%), suicide genes (7%), receptor (5.4%), replication inhibitor (4.1%), and markers (3.3%).  In addition, 7.5% of the trials aim to correct protein deficiency.  So far, most trials are in the early phase of clinical trials (60% Phase 1) with some in Phase 2 (16.5%) and fewer in Phase 3 (3.4%).  It is clear from these numbers that gene therapy is making a slow 4 but steady progress towards the clinic.  Judging from this pace, it is surprising that only a limited number of gene therapy products are commercially available today.  With more than 5 years of clinical trials and $9.6 million (USD) plus government research grants in product development, in October, 2003, Shenzhen SiBiono GenTech (Shenzhen, China) commercialized with approval from the State Food and Drug Administration (SFDA; Beijing, China) the first gene therapy product, sold under the brand name Gendicine®, an adenoviral-based gene therapy for head and neck squamous cell carcinoma (HNSCC), which accounts for about 10% of the 2.5 million annual new cancer patients in China [11].  The therapy shows promosing efficacy as 64% of late-stage HNSCC tumors experienced complete regression and 32% experienced partial regression after 8 weeks of a joint treatment of radiotherapy and weekly gene therapy injections (1 x 1012 viral particles).  Following the launch of Gendicine®, China approved, in 2005, a second gene therapy product, Oncorine® (Shanghai Sunway Biotech; Shanghai, China), also indicated for head and neck cancer. Oncorine® is a type 5 adenovirus engineered to replicate selectively in tumour cells, within which viral propagation-associated cytotoxic effects are triggered, resulting in elimination of tumour cells while leaving healthy cells undamaged.  As shown in clinical studies, 72.7% head and neck cancer patients receiving chemotherapeutic agents in addition to Oncorine® underwent partial or complete tumour regression, compared to 40.4% in those treated with chemotherapy alone, demonstrating the synergistic effects of Oncorine® in combination with chemotherapy [12].  Currently, a potential third gene therapy product, co-developed by the San Diego- based Vical Inc. and the Japanese biopharmaceutical company, AnGes MG. Inc., is under final-stage review by the Japanese Ministry of Heath, Labor and Welfare for treatment of critical limb ischemia (CLI).  CLI is a severe obstruction of the arteries, 5 caused by arteriosclerosis and/or ischemic heart disease, which considerably reduces blood flow to the extremities (hands, feet and legs), resulting in severe pain and even skin ulcers or sores.  Without extensive treatment, such as revascularization and amputation, by a vascular surgeon, the condition will not improve on its own. Collategene®, the product candidate, utilizes a nonviral naked DNA transfer technology to deliver a gene expressing hepatocyte growth factor (HGF), a human protein that stimulates angiogenesis in areas of restricted blood flow, as a novel approach to address the important medical need.  A New Drug Application (NDA) was filed in March of 2008 for Collategene® in Japan following the announcement of positive interim results collected from the first 41 subjects completing a Phase 3 clinical trial.  Based on the primary efficacy endpoints, improvement of rest pain (Visual Analog Scale, or VAS) or ischemic ulcer size, at 12 weeks post-treatment, a statistically significant difference (p=0.014) was achieved between the placebo and treatment groups (30.8% vs. 70.4% improvement) with no major safety concerns related to treatment, leading to the recommendation of early trial termination by an Independent Data Monitoring Committee to prevent potential ethical issues against the placebo group subjects [13]. If the NDA is successful, Collategene® is expected to become the first commercialized gene therapy in Japan.  Outside of Japan, AnGes has already completed two Phase 2 clinical trials with Collategene® in the USA and has recently reached a broad agreement with the Food and Drug Administration (FDA; Silver Spring, MD) on the designs of their Phase 3 study, placing the nonviral gene therapy on its way towards regulatory and commercial acceptance.      6 Gene therapy vectors  In order for gene therapy to achieve acceptable safety and efficacy, genes must be delivered efficiently, tissue target-specifically, and safely.  Thus, the fundamental success of gene therapy relies on the development of suitable vectors.  Currently, viral and non-viral vectors are the two main categories of vectors used in gene delivery systems.  Viral-based vectors  As the predominant type of vector used in clinical trials, viral vectors, owing to viruses’ natural ability to infect cells and unload their genetic materials for survival, have the advantage of high in vivo transfection efficiency relative to their non-viral counterparts.  Examples of viral vectors include the RNA-based retroviruses and lentiviruses and the DNA-based adenoviruses, and adeno-associated viruses (AAVs) (see ref. [14] for virology review).  Retrovirus-based vectors, such as the murine leukemia virus (MLV), have a favourable feature of offering long term transgene expression due to their ability to stably integrate the genetic material into the host genome.  With their relatively large packaging capacity for DNA (up to 8 kb), retroviruses provide a versatile vehicle for the delivery of genes, making them the second most commonly used vector in gene therapy clinical trials [8].  However, one limitation for most retroviruses, with the exception of lentiviral vectors, is the requirement of target cell division for effective transduction, restricting their use to actively proliferating cells [15].  The use of integrative vector comes with several safety concerns, one of which is insertional mutagenesis, defined by the alteration in the expression levels of oncogenes or tumour suppressor genes as a result of transgene insertion.  This has been shown to be the cause of cancer in a few 7 patients receiving MLV-based gene therapy for X-linked severe combined immune deficiency (X-linked) [10].  However, under close monitoring and with further development, retroviral vectors continue to be a useful instrument for gene transfer in clinical trials [8].  Adenovirus is a non-enveloped, icosahedral virus of 60-90 nm in diameter with a linear, double-stranded DNA genome of 30-40 kb [16].  More than 50 different human serotypes have been identified [17].  The high nuclear transfer efficiency, the broad tissue tropism, and the low pathogenicity are all appealing properties of this virus. Despite efficient gene transfer, early adenoviral vectors were found to cause a direct toxicity and immunogenicity against the viral gene products in tranduced cells [18].  In fact, a patient undergoing gene therapy for treatment of orthithine transcarbamylase deficiency died from excessive immune reaction to the adenoviral vectors [9]. Conversely, the anitumoural effects resulting from the inherent cellular toxic and immunogenic nature of adenoviruses can be taken advantage of in cancer treatment, which is increasingly being pursued, making adenoviruses the most commonly used vectors in clinical trials today [8, 19].   Adeno-associated viruses (AAVs) are human parvoviruses that normally require a helper virus, such as adenovirus, to mediate a productive infection.  In the absence of a helper virus, AAVs establish a latent infection within the cell, either by site-specific integration into the host genome or by persisting in episomal forms [20].  As a small virus, AAV has a limited transgene packaging capacity (~5 kb) and it is difficult to produce large amounts of high-titer virus stocks.  There are 11 known human viral serotypes, the most extensively studied of which is serotype 2, and each serotype is believed to have different tissue tropism [21].  Advantages of AAV vectors include nonpathogenicity and nonimmunogenicity, extended duration of transgene expression 8 due to limited viral protein-induced cytotoxicity, a broad host and cell type tropism range, and the ability to transduce both dividing and nondividing cells in vitro and in vivo [22]. These characteristics have allowed AAV to be used in gene therapy clinical trials for the treatment of a variety of diseases including cystic fibrosis, hemophilia, and muscular dystrophy.  With the initial success in gene transfer and expression of human clotting factor IX in hemophilia B patients, more future clinical trials with AAV vectors are expected [23-25].  Despite their common use in gene therapy clinical trials, viral vectors have several unfavourable features, such as immunogenicity, insertional mutagenicity, toxicity, limited DNA carrying capacity, production and packaging challenges, recombination, and high cost, causing concerns over their use [26].  As a result, nonviral vectors, having reduced risks of the above limitations, are becoming an appealing alternative with simplicity of use, ease of large-scale production, large carrying capacity for genetic materials, and lack of specific immune response [27]. Nonviral-based vectors  In order to transfer DNA efficiently into the cell for expression, nonviral vectors must be designed to bring DNA from outside the cell to inside the nucleus by a multistep process consisting of cell uptake, endosomal escape, intracellular trafficking, and nuclear entry [26].  The progressive loss of DNA molecules at each step of this multistep journey to the nucleus underlies the low efficiency of nonviral-based DNA delivery.  However, with the goal to avoid the severe disadvantages of using viral-based systems, many efforts have been invested in devising nonviral-based techniques to augment DNA transfer efficiency. 9  Nonviral-based DNA transfer techniques are divided into two general groups: 1) naked DNA delivery via physical/mechanical and electrical methods, such as electroporation, bioballistic (gene gun), ultrasound, and hydrodynamic injection, and 2) delivery mediated by chemical carriers such as lipids and polymers.  Electroporation relies on the use of controlled electric fields to permeabilize the cell membrane, thereby enhancing DNA uptake into the cell following naked DNA injection [28].  This effective approach is especially suitable for the skin and muscle because of the ease of administration [29, 30].  In addition, electroporation has been shown to successfully mediate gene transfer following intratumoral and systemic injection of naked DNA, demonstrating the efficiency of DNA delivery via electric transfer [31, 32].  Gene gun, a method of direct gene transfer into tissues or cells by penetrating the cell membrane with DNA-coated gold particles, is used primarily for vaccination and immunotherapy [33, 34].  However, one drawback of this method is the shallow penetration of DNA into the tissue, limiting its flexibility for in vivo DNA delivery.  Ultrasound can be employed to facilitate gene transfer by increasing cellular permeability to DNA.  Irradiating the tissue with ultrasonic waves after DNA injection enhances gene expression [35, 36], which coupled with the safety and flexibility of ultrasound application, provides a great advantage for the potential clinical use of this method for gene transfer.  Hydrodynamic injection, first described in 1999 by Liu et al., has established itself in recent years to be the simplest method for gene transfer into small rodents [37].  The technique entails an injection in 5 – 8 sec. of 8 – 10% body weight in volume of isotonic DNA solution into the tail vein of a mouse, leading to elevation of pressure in the inferior vena cava, retrograde flow of DNA solution into the liver, enlargement of the fenestrae and generation of transient plasma membrane defects in hepatocytes, and gene transfer into hepatocytes [38].  These events, initiated by a seemingly harsh and yet well 10 tolerated method, collectively contribute to the highly elevated transgene expression in the liver.  More interestingly, Wolff and colleagues showed that hydrodynamic injection can be applied to deliver DNA into various skeletal muscles via a limb vein to achieve comparable efficiencies between small (mouse, rat) and large (rabbit, dog, monkey) animal models, supporting the potential advancement of this technique into clinical trials [39-41].  In addition to the physical methods described above, chemical carriers can be used to accomplish gene transfer by complexing with DNA and protecting it from extra- and intra-cellular nuclease degradation during the delivery process.  The chemical carriers are separated into two categories: liposomes and polymers.  Liposomes are microscopic phospholipid bubbles with a bilayered membrane structure that, upon mixing, form stable complexes with DNA.  Liposome : DNA ratio, lipid composition, complex size and shape, and preparation methods are believed to be major factors dictating the efficiency of gene transfer [42].  Introduced more than 20 year ago by Dr. Felgner in 1987, liposome-based delivery, more extensively reviewed by others [43, 44], still remains one of the most commonly used nonviral DNA transfer techniques today [45].  Despite the general use, liposome-based delivery suffers from a number of drawbacks, including the poorly understood structure of DNA – lipid complexes, variations resulting during fabrication, and formulation-dependent toxicity [46].  The second class of chemical carriers consists of an array of polymers, such as polyenthyleneimine (PEI), poly-L-lysine, poly(β-amino esters) (PAE), polyamidoamine (PAMAM) dendrimers, and chitosan.  PEI, having remarkable transfection efficiency based on advantageous properties of DNA protection, cell binding and uptake, endosomal escape, and carrier release, has been regarded as the gold standard of polymer-based gene transfer [47].  However, its utilization is hampered by the 11 significant cytotoxicity and limited polymer degradability observed both in vitro and in vivo following its use, at least for the high molecular weight and branched forms of the polymer, prohibiting repeated systemic administration of PEI and restricting it to a few localized applications [48, 49].  Similarly, while highly efficient at transfecting cells, the use of poly-L-lysine also suffers from substantial cytotoxicity caused by its dense cationic charges and immune responses induced by its protein-based nature [50].  In contrast, PAE, a highly efficient and easily synthesised biodegradable polymer by the conjugate addition of primary amines or bis-secondary amines to diacrylate compounds, possesses a faster degradation rate and exhibits more superior DNA delivery efficiencies with minimal toxicity in various cell lines, including COS-7, human hepatocellular carcinoma, and 3T3 fibroblasts, attracting much attention as a chemical DNA carrier [51-53].  PAMAM, or starburst dendrimers, are a unique class of polymer with highly definable and monodispersed morphology, characterized by dendritic branching of a core polymer with radial symmetry, that has also seen increasing use in gene transfer applications due to their high efficiency and negligible toxicity [54-56]. However, among the chemical carriers, chitosan is attracting increasing interest due to its extensively examined safety profile and versatility as a promising nonviral gene transfer vehicle.  This polymer will be thoroughly reviewed in the next section. Chitosan/ФC31 Gene Transfer Technology Chitosan Chitosan, an abundant polymer (D-glucosamine and N-acetyl-D- glucosamine linked by β-(1–4) glucosidic bonds) derived from alkaline N-deacetylation of chitin, found in the shells of marine crustaceans, exoskeletons of insects, and cell walls of fungi, is being actively explored as a nonviral gene carrier (Figure 1) [57]. 12        Figure 1.  (A) Schematic illustration of the structure of chitosan.  (B) Transmission electron micrographs of chitosan nanoparticles.  Schematics in (A) and (B) were obtained from [58] and [59].     n A. B. 13 Rather than a specific compound, chitosan is a general term referring to a family of linear, binary polysaccharides consisting of β-(1–4) linked acetylated and deacetylated units at varying ratios and of varying chain lengths.  With an apparent pKa value of 6.5 - 7 for the amino group on the deacetylated units, the polymer remains positively charged at neutral or acidic conditions [60]. Chitosan is biodegradable, biocompatible, non-toxic, and non-immunogenic.  It also possesses mucoadhesive properties, such as hydrogen bonding capacity, linear structure, sufficient chain flexibility, and cationic charges, to allow for its sustained interaction with the mucous-bearing epithelium, promoting more efficient uptake by epithelial cells [61-63].  The mucus-binding features of chitosan were evaluated by Ping and colleagues in turbimetric, adsorption, and rat intestine adhesion studies [62].  It was found that, while there was only modest interaction between the control polymer, poly(vinyl alcohol), and mucin, a strong force of contact existed between chitosan and mucin, indicated by the reduced amount of free mucin in the chitosan/mucin suspension compared to the control.  Furthermore, the binding between chitosan and mucin was positively correlated with the quantity of anionic charge-contributing sialic acid content in the mucin, as demonstrated in the adsorption studies, illustrating the importance of opposite charge attractions in mucoadhesion.  Moreover, the authors showed that the amount of chitosan adsorbed to the mucosal tissues in isolated rat intestine was directly influenced by the positive zeta potential of the polymer and that the anionic ethyl cellulose control could not be adsorbed, further suggesting that chitosan interacts with the mucosal surface via electrostatic forces.  Based on these observations, a strategy may be devised using chitosan to improve mucoadhesive properties of a delivery system.  In fact, by coating insulin-loaded liposomes with chitosan and orally administering the complexes to rats, Takeuchi et al. observed, compared to non-coated 14 liposomes, a faster and more sustained absorption of the loaded drug, attributable to chitosan’s mucoadhesiveness that enhances enteral absorption [64].  This is likely the result of cell binding and spontaneous endocytosis mediated by anionic cell surface proteoglycan [65, 66]. With its net positive charge, chitosan can easily complex with negatively charged DNA, based predominantly on electrostatic interaction, to form nanoparticles that facilitate gene transfer following luminal or oral delivery [67-71].  Lu’s group reported that oral administration of chitosan nanoparticles was effective at gene transfer as indicated by an over 25% increase in hematocrit levels in mice receiving chitosan- encapsulated DNA encoding mouse erythropoietin compared to those receiving naked DNA [72].  In addition to this, chitosan also has been successfully employed to transfer DNA via a variety of administration routes, such as intravenous [73], intratracheal [74], intrabiliary and intraportal [75], gastric and colonic [76], and ocular delivery [77]. The transfection efficiency of chitosan/DNA nanoparticles is governed by the properties of chitosan, including molecular weight (MW) and degree of deacetylation (DDA), and formulation variables, such as pH and N/P ratio, or the ratios of moles of the amine groups of cationic polymers to those of the phosphate groups of DNA.  These factors can greatly influence the physiochemical properties of the chitosan/DNA complexes, including size and polydispersity measured by dynamic light scattering and zeta potential assessed by laser Doppler electrophoresis [78-80].  The size, in diameter, of nanoparticles is determined based on how the nanoparticles diffuse within a fluid. Since this can be affected by the concentration and the type of ions in the fluid in which the complexes are in, the size is referred to as hydrodynamic.  As a measure of the distribution of nanoparticle size, polydispersity is calculated as the ratio of the standard deviation to the mean of the nanoparticle size.  Zeta potential is the charge of the layer 15 of ions between the surface of the nanopartilcles and the surrounding solution.  It is a practical indicator of the effects of formulation changes on the inter-particle stability of nanoparticles.  The molecular weight of chitosan is a direct function of the number of repeating units, or mers.  When joined as a co-polymer, each D-glucosamine unit is 161 g/mol and each N-acetyl-D-glucosamine unit is 203 g/mol.  Since chitosan consists of a mixture of D-glucosamine (C6H13NO5) and N-acetyl-D-glucosamine (C8H15NO6), a DDA of 98% denotes that the chitosan contains 98% D-glucosamine and 2% N-acetyl-D- glucosamine.  Therefore, the molecular weight of chitosan can be calculated based on the DDA and number of mers using the following formula: [203 x (1-DDA) ± 161 x (DDA)] x # mer.  For example, with a DDA of 98%, a 31-mer chitosan is calculated to have a molecular weight of 5,017 g/mol, or 5,017 Da (~5 kDa). Chitosan is typically commercially available in high MW with various DDA.  To decrease MW, chitosan polymer can be depolymerized with sodium nitrite, while the DDA can be increased by alkaline deacetylation or decreased by reacetylation with acetic anhydride [80, 81].  The MW of chitosan can be assessed indirectly through the intrinsic viscosity of the polymer using the Mark-Houwink equation or directly by gel permeation chromatography multi-angle laser light scattering (GPC-MaLLS), a technique that detects the number of molecules present in each molecular weight fraction, yielding information on the distribution of molecular weight [82, 83].  The DDA of chitosan can be measured by several techniques, such as nuclear magnetic resonance (NMR), linear potentiometric titration (LPT), ninhydrin test, and first derivative UV spectrophotometry [84].  Among these, first derivative UV spectrophotometry has been shown to be a fast, convenient, and dependable method, capable of detecting, at the wavelength of 203 nm, the percentage of acetylated units within a chitosan sample, from which the DDA of the polymer can be calculated. 16 When binding DNA to form nanoparticles, an N/P ratio of 6 was found to be sufficient to achieve > 90% encapsulation independent of the MW (10–213 kDa) and DDA (46–88%) of chitosan tested [80].  However, the stability of the nanoparticles is still largely determined by these properties of chitosan.  Huang et al. found that chitosan polymers of lower MW (10 kDa) or DDA (46%) were less capable of retaining the DNA upon incubation with PBS, an observation that was correlated with the inability of chitosan to protect the DNA from degradation by DNase and serum components. Similarly, uptake of the nanoparticles by A549 cells (a tumour-derived human alveolar basal epithelial cells) was also significantly hindered by lowering the MW or DDA of chitosan, as the reduction of the MW from 213 to 10 kDa led to a drop in nanoparticle uptake from 4.1 to 0.9 µg/mg protein and the lowering of the DDA from 88 to 46% resulted in a corresponding decrease from 4.1 and 0.4 µg/mg protein.  This restricted nanoparticle uptake consequently gave rise to poor transfection efficiency, which was related to the low zeta potential, or surface charge, of the nanoparticles synthesized with low MW or DDA chitosan.  Based on the tight correlation between transfection efficiency, cellular uptake, and zeta potential of the nanoparticle, it appears that cellular uptake, mediated by electrostatic interactions with the cell membrane, is a critical step leading to efficient transfection seen with high chitosan MW and DDA, highlighting the positive effects of either one of these two factors. Another variable to consider while formulating stable chitosan/DNA nanoparticles is the pH.  With an apparent pKa value of 6.5, the primary amines in chitosan become more positively charged from 50 to ~100% when pH decreases from 6.5 to 3.5 [60]. Since chitosan binds DNA electrostatically, with a higher number of positive charges on the chitosan chain, more stable nanoparticles, represented by a larger fraction of soluble globules, can be formed.  In fact, when compared to a lower fraction of globular 17 nanoparticles, nanoparticles that exhibited a higher proportion of globular and/or aggregated structures were more efficient at gene transfer in 293 cells [79].  In addition, strongly charged chitosan polymer chains are flexible and have a unique extended conformation, which is favourable in promoting the ionic interactions with the negatively charged phosphate groups in DNA.  As a result, a lower pH is beneficial in formulating more stable and efficient chitosan/DNA nanoparticles. The mechanisms behind chitosan-mediated tranfection appear to rely on the polymer’s ability to protect DNA from nuclease degradation, attach to the cell surface, transport DNA across the cell membrane, escape the endosome, and import into the nucleus (Figure 2).  Using DNase I as a model enzyme, the protective effect of chitosan was assessed by Mao et al., who found significant degradation of naked DNA in DNase I after 15 min., whereas chitosan-encapsulated DNA remained completely intact.  Even at a concentration of DNase I (250 U/ml), markedly surpassing physiological levels of nucleases, the nanoparticles still retained a significant portion of non-degraded DNA, demonstrating chitosan’s protective effect for the DNA [85].          18  Figure 2.  Transfection mechanisms of the nonviral gene transfer system consisting of chitosan DNA carrier and ФC31 integrase.  (A) Chitosan polymer condenses DNA to form cationic nanoparticles, which adhere to anionic heparan sulphate proteoglycan at the cell surface - the first step of chitosan-based DNA transfer.  (B) Endocytosis of chitosan nanoparticles is followed by endosomal escape.  Nuclear import of either the intact nanoparticles or the dissociated DNA subsequently takes place, after which ФC31 integrase is transcribed and translated.  The resulting ФC31 integrase mediates plasmid genomic integration between the plasmid attB site and one of the genomic pseudo attP sites. A. B. 19 To transport DNA into the cell, cationic chitosan nanoparticles first associate with the negatively charged plasma membrane, and through endocytosis, translocate into the cytoplasm and subsequently reside in the endosomes 1 hr post transfection.  This was visualized by separately labelling the DNA and endosome with two different fluorescent dyes, FITC and Texas Red-dextran, respectively.  Using the same labelling technique, this time labelling chitosan, instead of the endosome, with Texas Red, the colocalization of DNA and chitosan was observed in the nucleus 4 hr post transfection, indicating the endosomal escape and nuclear import of chitosan nanoparticles [57]. The process of endosomal escape is governed by at least one of the following theories: 1) endosomal buffering [86], and 2) polymer swelling.  Once inside the endosome, chitosan acts as a buffer for endosomal acidification by absorbing the protons that are being pumped into the organelle.  This leads to an influx of Cl- ions to prevent the build up of a charge gradient due to the influx of protons.  The influx of both ions then increases the osmolarity of the endosome and causes osmotic swelling of the organelle.  The swelling of chitosan may also occur as a result of charge repulsion between the proton-saturated polymer chains.  These two events may individually or collectively contribute to the destabilization of the endosome and the release of its content into the cytoplasm [87].  Finally, while colocalization of DNA and chitosan was observed in the nucleus, nuclear import of intact chitosan-DNA complexes remains controversial. While an effective gene transfer vehicle, chitosan, like all nonviral vectors, lacks a valuable feature which is the capacity for long term transgene expression.  To assemble a gene transfer system equipped with this property, chitosan is united with a well-characterized integrase, ФC31. 20 ФC31 integrase  ΦC31 integrase is a serine integrase from Streptomyces ΦC31 phage that, in nature, catalyzes recombination between a short sequence (~ 30 base pairs) within the phage genome, attP or attachment P site, and a short sequence within the bacterial genome, attB or attachment B site [88].  This unique quality of ΦC31 integrase can be applied to insert a therapeutic gene construct containing an attB site into the mammalian genome housing ~370 pseudo attP sites in an efficient, unidirectional, and site-specific manner (Figure 2B) [89-91].  Based on ~200 independent integration events from three human cells lines, Chalberg et al. studied the specificity of ФC31-mediated integration and found that all of the integration sites seemed to have a single copy of the integrated plasmid, and that there are ~20 sites of recurrent integration, accounting for almost 60% of the integration events [92].  Furthermore, all of these preferred integration sites show significant identity to a 28-bp pseudo attP consensus sequence, demonstrating that the integration reaction is sequence-driven.  From that study, it was also determined that the majority (61.3%) of the integration sites were intergenic, with some disproportionally high representation of sites in intronic (36.8%) and exonic (1.9%) locations, revealing a minor preference for integration into genes, as well as an inclination towards highly transcribed regions.  This is perhaps due to the integrase’s function limited to chromatin blockage-free regions of the chromosome, and may explain the above average expression levels of ФC31-integrated genes, compared to random integration, and the persistence of gene expression, favourable for gene therapy.  To assess safety, detailed molecular analysis has shown that none of the recurrent integration sites are 21 located near known cancer genes, and thus the threat of tumour formation due to insertional mutagenesis is greatly reduced.  When combined with a nonviral gene transfer technique, such as hydrodynamic tail vein injection, ФC31 was able to sustain the long term expression of a factor IX- expressing transgene in the mouse liver even after a two-thirds hepatectomy was performed to accelerate the loss of unintegrated DNA.  By contrast, in the absence of ФC31, the initial levels dropped steadily, reaching 40-fold lower levels a few weeks after injection and plunging to background levels after partial hepatectomy [89].  In another study carried out in a fumarylacetoacetate hydrolase (FAH)-deficient mouse model, a plasmid encompassing the FAH cDNA and an attB site was co-introduced into the mouse with the ФC31 integrase expression vector by hydrodynamic injection.  This led to complete cure of the disease and, more remarkably, serial transplantation of the corrected hepatocytes resulted in cure of secondary and even tertiary recipients, underlining the ability of ФC31 to mediate long term transgene expression, even through multiple rounds of cell divisions [90].  The effects of targeted plasmid integration using ФC31 integrase were also studied in a mouse model of Duchenne muscular dystrophy, the mdx mouse.  Bertoni et al.  injected the luciferase reporter or the dystrophin-expressing attB-carrying construct under the control of a muscle-specific promoter (CK6) with or without the ФC31 integrase expression vector (25 µg per construct) into the tibialis anterior muscle of anesthetized mice in a volume of 20 µl, following which electroporation of the muscle was performed with a two-needle electrode array at a setting of 5 pulses of 50 ms duration at a voltage of 360 V/cm [93].  It was found that the luciferase activity, measured on total extracts of muscles isolated at different times after injection, was 22 significantly higher in the leg receiving ФC31 integrase compared to the leg that did not starting at Day 15 until Day 540 post-treatment.  This was due to the persistence of the levels of plasmids over the course of 18 months in ФC31 injected muscles, as opposed to the significant decline observed in muscles without the integrase.  Similarly, at 5.5 months after intramuscular injection of the dystrophin-expressing construct, significantly more dystrophin-positive fibers (161 ± 41) were detected in the integrase-expressing than in the non integrase-expressing muscles (55 ± 31) by immunostaining. Furthermore, when Evans blue dye (EBD), a marker for degenerating fibers, was injected into the muscles as a functional test for dystrophin expression 6 months after the treatment, marked improvement in muscle health was documented in the muscles expressing the integrase, indicated by the significantly lower percentage of EBD- positive staining in the dystrophin-expressing cells than that seen in the muscles without the integrase (3.5 ± 3 vs. 28.2 ± 12%).  Based on these data, the authors concluded that higher levels of gene expression can be achieved in muscles in which plasmid integration was promoted with ФC31 integrase and those levels remained substantially higher over time (> 1 year) compared to those observed in muscles that did not receive the integrase.  The utility of ФC31 integrase in mediating long term transgene expression was examined additionally through subretinal DNA injections in Sprague-Dawley rats. Luciferase-expressing plasmid bearing an attB site was injected subretinally with or without the ФC31 integrase expression vector at a DNA dose of 2.5 µg per construct into one of the eyes by Chalberg and colleagues [94].  Immediately after injection, electroporation was performed by attaching an electrode to each cornea, with the negative electrode on the injected eye, and applying 5 pulses of 100 ms duration at a voltage of 10 V/mm.  Shortly after treatment, at Day 2, similar levels of luciferase activity, 23 averaging ~2.2 x 108 photons/s, > 3300-fold above background levels, were detected in all treated rats.  However, luciferase levels in the group that did not receive integrase decreased sharply, reaching near background levels by Day 18, while co-administration of the luciferase and integrase constructs led to an initial drop followed by a high level (~1 x 107 photons/s) of stable luciferase expression from Day 20 to 140.  The stable transgene expression observed with ФC31 integrase, after 4.5 months, was ~85-fold higher than that without integrase.  Furthermore, nested PCR analysis on genomic DNA samples prepared from the posterior eyecup of treated animals, using probes flanking one of the pre-determined integration hotspots in the rat genome, rps1, identified that plasmid genomic integration was indeed present only in the eye receiving integrase. Therefore, along with other previous reports, this study shows that ФC31 integrase is capable of conferring long term transgene expression by means of genomic integration. Moreover, the efficiency of ФC31 has been demonstrated in a collection of studies, including in vivo reporter gene transfer in rabbit joints [95], cytokine receptor γ chain gene transfer in human T cell line [96], and ex vivo collagen VII gene transfer in xenotranplantation models [97].  Safety remains to be a concern with any integrating system.  The most dangerous threat is the risk of perturbing protooncogenes, possibly contributing to oncogenesis, by inserting extra genetic materials into the host genome.  While current ФC31 integrase system does not have 100% integrating precision, it does provide a significant improvement over other means of transgene integration, such as through oncoretroviruses, lentiviruses, and Sleeping Beauty transposon, all of which have been shown to integrate with little sequence specificity [98, 99], likely accessing the ~300 documented cancer genes [100].  In constrast, due to its sequence-driven nature, ФC31-mediated integration events are predicted to occur in ~370 sites that are all 24 distant from cancer genes.  However, it should be noted that, at a frequency of approximately 10%, aberrant events, such as larger intrachromosomal deletions and apparent interchromosomal rearrangements, at least in cultured cells, have been found with ФC31 integrase-catalyzed integration [92].  These aberrant events occur largely at pseudo attP sites, and therefore, the associated risks for cancer gene dysregulation are similar to those with normal integration events.  Since none of the identified integration sites are in close proximity to cancer genes, the risk of oncogenesis is greatly reduced with ФC31 integrase, as opposed to random integrating systems.  However, chromosomal rearrangements outside of known human pseudo-attP sites have been reported.  Liu et al. identified highly abnormal karyotypes, comprising 14 chromosomal breakpoints, 9 of which locating beyond previously identified psudo-attP sites in human chromosomes, exclusively in primary human fibroblasts co-transfected with attB-bearing and ФC31-expressing plasmids and not in those transfected with attB-bearing plasmids alone [101].  Furthermore, an incident of transient displasic appearance of hepatocytes, although atypical, was documented following ФC31-mediated gene transfer in a mouse model of type 1 tyrosinemia, suggesting that the danger of ФC31-induced chromosomal aberrations is not unequivocally negligible [90].  Nevertheless, in most animal studies, tumour formation or other unfavourable events arising from use of the ФC31 integrase system in a variety of tissues, including the skin, muscle, eye, and joint, have not been discovered.  In addition, the consistent viability as well as the lack of increase in abnormal offspring following the use of ФC31 integrase in generating transgenic animals alleviate the safety concerns of ФC31 [102, 103].  Moreover, since ФC31 integrase is only required transiently for integration, by restricting its duration of 25 expression, long term effects of ФC31 and the possibility of immunogenicity against the enzyme can be minimized.  ФC31 integrase possesses site-specific advantages over other integrating systems, such as oncoretroviruses, lentivirus, and Sleeping Beauty transposon, which integrate with little sequence specificity [98, 99].  When coupled with chitosan, ФC31 creates a unique nonviral gene transfer system with the capacity for long term transgene expression.  Gene Transfer Delivery Routes Intrasplenic  Intrasplenic injection has been shown to be an effective route of gene transfer owing to the profound vasculature of the spleen and a direct connection via the circulation to another highly perfused organ, the liver.  By injecting adenoviral vector encoding human preproinsulin transgene intrasplenically to Wistar rats made diabetic by streptozotocin treatment, a significant reduction in elevated blood glucose levels was seen.  In addition, the expression of insulin transgene following this route of gene transfer stimulated the regeneration of hepatocytes post partial hepatectomy, indicating efficient gene transfer via intrasplenic injection [104].   Tissue distribution of the delivered DNA is one of the most critical aspects based on which the safety of gene transfer is determined.  By RT-PCR, Yamaguchi et al. reported that the expression of the transferred DNA was confined to the liver and spleen, and was not found in the pancreas, kidney, muscle, and lung, after gene transfer via intrasplenic injection, illustrating the utility of this mode of delivery [105].  Further highlighting the application of intrasplenic injection in gene transfer, many studies have 26 targeted the spleen, the most important lymphoid organ, for DNA vaccination via this administration route [106, 107].  Based on the potential to target a nearby organ (i.e. liver) through a relatively simple and safe procedure and the ease of gene transfer into the spleen, intrasplenic injection has established itself to be a useful route of DNA administration. Intraperitoneal  Intraperitoneal (i.p.) injection is another method of administration to be considered for the delivery of DNA because of 1) high accessibility of many major organs in the peritoneal cavity, 2) the presence of few biocomponents that reduce gene transfer activity, and 3) high capacity for the DNA suspension.  Previously, i.p. injection has been utilized by many investigators for the purpose of gene transfer.  It was demonstrated in a peritoneal tumor mouse model, using liposome- encapsulated chloramphenicol acetyltransferase (CAT) reporter gene, that ~18% of the injected dose (250 µg) of DNA was associated with the tumor and CAT expression was detected in the tumor at 2 and 24 hrs. post injection, respectively, suggesting effective gene delivery via i.p. injection.  Although gene expression was not directly assessed, the authors also found the plasmid DNA in the liver, pancreas, and spleen outside of the tumor [108].  In addition to these organs, Fellowes et al. detected plasmid DNA in the kidney and lung and mRNA in the kidney and spleen following an i.p. injection of a liposome-complexed anti-inflammatory cytokine gene, IL-10, into an arthritis mouse model.  Interestingly, by transport of the transfected cells in the peritoneal cavity, the plasmid was able to reach the paws, where inflammation was located and be expressed to cause therapeutic effects [109]. 27  Collectively, with careful analysis of the plasmid tissue distribution and expression, i.p. injection, due to the accessibility of a large number of cells simultaneously, may be an effective route of administration for gene transfer. Oral  Oral administration is the ideal route of drug delivery primarily due to convenience and patient compliance.  It is also considered the ‘holy grail’ of gene transfer as very little has been reported in the literature regarding this route of administration.  This challenging task encounters several obstacles, posed by the protective nature of the gastrointestinal tract, including the extreme acidity and the presence of protein-degrading enzymes such as pepsin in the stomach [110], secreted pancreatic enzymes in the intestinal lumen, and membrane-bound brush-border enzymes, all of which contribute to substantial loss of orally administered drug or nucleic acid [111].  By formulating DNA into nanoparticles with a polymer, such as chitosan, these barriers may potentially be overcome.  Bowman and colleagues recently showed that oral feeding of blood clotting Factor VIII DNA-containing chitosan nanoparticles achieved phenotypic correction in 13 out of 20 haemophilic mice in a bleeding challenge a month following treatment [67]. Complementary to this, perhaps the most well-known landmark in the field of oral gene transfer is the work published in Nature Medicine in 1999 by Roy et al. demonstrating oral chitosan-mediated gene transfer in a murine model of peanut allergy that effectively protected the mice from food allergen-induced hypersensitivity [71].  However, it should be noted that this route of gene transfer is especially complicated by the nature of the gastrointestinal (GI) tract programmed to guard the invasion of foreign substances. Previous successes, such as the ones cited above and a handful of other reporter gene 28 delivery studies (see ref. [68-70, 72]), might be dependent on the specific mouse model, transgene of interest, nanoparticle preparation and formulation, treatment regimen, and endpoint assessment that may or may not be easily translatable to other experimental settings.  Still, oral administration being the most acceptable route of drug delivery is continuously being explored as a method of gene transfer.  SEAP  Human placental secreted alkaline phosphatase (SEAP) is a commonly used reporter gene whose protein product is constitutively secreted by cells and can be detectable with high sensitivity by a chemiluminescent assay.  When delivered in vitro, the levels of the SEAP transgene expression can be readily determined by measuring the SEAP protein in the cell supernatant samples without the need to lyse the cells. Similarly, when delivered in vivo, both the levels and duration of transgene expression can be easily assessed by monitoring the amount of the SEAP protein in the circulation over time, and thus making SEAP a convenient reporter gene to be used for gene transfer studies.  Leptin  Leptin, a pleiotropic hormone expressed by and secreted mainly from white adipocytes, has been implicated in the regulation of food intake and glucose metabolism [112, 113].  The gene, localized to chromosomes 6 and 7q31.3 for mouse and human, respectively, encoding the 16 kDa hormone was first identified, through positional cloning, by Jeffrey Friedman’s group in 1994 [114].  The mutation at amino acid 105 of this gene, changing an arginine to a stop codon and giving rise to the synthesis of a truncated, inactive protein, was found to be the cause of the morbid obesity of a strain of mouse, ob/ob.  One year later, three papers simultaneously 29 demonstrated clearly that exogenous administration of the Ob protein, leptin, was able to eliminate the obesity of the ob/ob mice [112, 115, 116].  These findings supported the hypothesis, made by Coleman et al. more than 20 years ago, that a circulating factor, responsible for their obese phenotype, was missing in the ob/ob mice (see ref [117] for original experiments).  Leptin is a satiety factor expressed in adipose tissue in proportion to fat storage and adipocyte size [118, 119].  It has been shown in both human studies and in studies of several types of obese animal that the level of ob mRNA in white adipose tissue and the circulating leptin concentration are increased markedly in obesity [120-122].  In fact, a high correlation between body mass index (BMI) and circulating leptin can be found in human subjects [120].  Therefore, consistent with the lipostatic hypothesis, the greater the amount of adipose tissue, the higher the level of the adipocyte-derived signal; this interplay acts to report the status of body energy stores to the brain [123].  Furthermore, energy restriction or fasting can cause an even more rapid decrease in leptin levels, compared to the decrease in fat mass, in both rodents [121] and humans [124], suggesting that leptin may also serve as a sensor of short-term changes in energy stores.  Several splice variants of the leptin receptor gene exist (ObRa, ObRb, ObRc, ObRd, ObRe, ObRe), with ObRb, the long form of the receptor, being the only one that contains a 302 amino acid intracellular domain [125, 126].  ObRb is expressed in various regions of the brain and is believed to mediate leptin’s central actions.  Within the hypothalamus, the long form leptin receptor has been found in several hypothalamic nuclei, including the arcuate nucleus, ventromedial, dorsomedial and lateral hypothalamic nuclei, and the paraventricular nucleus [127, 128].  The murine and human leptin receptors are highly similar in amino acid sequences for both the 30 extracellular (78% identity) and intracellular domains (71% identity) [129].  Binding of leptin to ObRb leads to receptor homodimerization and the activation of JAK/STAT pathways.  Activated JAK2 subsequently phosphorylates tyrosine sites on the intracellular domain of the receptor, which provides a binding site for STAT3.  This activates STAT3 proteins that subsequently dimerize and translocate to the nucleus, where they bind DNA and activate transcription [130].  In addition to its roles in reducing food intake and body weight and raising energy expenditure, leptin appears to have independent actions on glucose and insulin homeostasis.  For example, leptin-deficient ob/ob mice exhibit profound diabetes that can be fully reversed by low doses of leptin that do not affect body weight and food intake [116].  In addition, administration of leptin intracerebroventricularly can acutely stimulate glucose uptake in skeletal muscle [131-134] and inhibit hepatic glucose production [113, 135].  Moreover, leptin improves insulin sensitivity dramatically in human lipodystrophy and in lipodystrophic mouse models, which are characterized by low serum leptin levels and by severe insulin resistance [136-138].  Together, leptin treatment in rodents mediates attenuated blood insulin and glucose levels and increased glucose metabolism in the peripheral targets, such as the muscle, liver, and pancreas.  Therefore, more than a reporter, the leptin gene encodes a protein product that exerts weight- and blood glucose-lowering effects in leptin-deficient mice, which are exquisitely sensitive to the presence of the protein.  The presence of leptin, even at levels that are barely detectable by regular assays, can lead to weight and glucose reduction in these mice.  As a result, the efficacy of leptin gene transfer can be conveniently evaluated based on apparent phenotypic changes of the treated mice without the requisite of inspecting the circulating transgene protein product.  31 Clinical Leptin Therapy  Genetically based leptin deficiency, although rare, has been reported in humans. In 1997, two related children of Pakistani origin were reported to possess a frame-shift mutation caused by the deletion of a single guanine nucleotide otherwise present in codon 133 of the leptin gene that leads to the production of a truncated protein, not targeted normally for secretion [139].  These children and the congenital leptin-deficient ob/ob mice share a number of similar phenotypes, such as a normal birth weight followed by the rapid development of severe obesity linked to hyperphagia and irregular satiety.  The older of these two children, a 9-year-old girl, was subsequently subjected to a 12-month leptin replacement therapy, consisting of daily (8:00 a.m.) subcutaneous injections of recombinant human methionyl leptin at a dose (0.028 mg/kg lean mass) calculated to achieve a peak serum leptin concentration equivalent to 10% of the child’s predicted normal serum leptin concentration (70 ng/ml) [140].  Remarkably, being well- tolerated, contributing to no reported adverse effects, the injection of leptin, as early as 2 weeks into the treatment, initiated body weight reduction, which continued over the 12-month treatment period at a rate of 1 – 2 kg/month that eventually resulted in a total of 16.4 kg weight loss, 95% of which was accounted for by the decrease in body fat. Leptin replacement also modified the patient’s eating behaviour, dropping the amount of food intake by 42%, to 930 kcal, at the first test meal after the start of treatment. Furthermore, long term treatment of this individual, along with two other congenital leptin-deficient patients, possessing the same mutation in the leptin gene, led to similar selective loss of fat mass, suppression of food intake, and restriction in caloric consumption during a test meal, confirming the critical roles of leptin in the regulation of human body weight, fat mass, and appetite and highlighting the sustained beneficial effects of leptin therapy seen in these subjects (over 4 years in one subject) [141].  In 32 that study, the effects of leptin therapy on the progression of appropriately timed puberty, gradual but sustained improvements in hyperinsulinemia and lipid profile as well as the acute increase and prolonged maintainence of circulating thyroid hormone (T4) concentrations were also documented. Unlike the ob/ob mice, different genetic mutations can underly leptin deficiency in humans.  Strobel et al. reported a homozygous missense mutation, harbouring a C  T substitution in the first base of codon 105 of the leptin gene that results in an Arg  Trp replacement in the mature protein, associated with hypogonadism and morbid obesity, in 4 pateints (1 adult male, 2 adult females, and 1 child) in a highly consanguineous extended Tukish family [142, 143].  This mutation does not cause the degradation of the protein, but instead, impairs the normal processing of leptin through the secretory pathway, leading to the synthesis of non-functional leptin protein.  The three adults (patients A, B, and C) underwent an 18- month leptin replacement therapy involving daily (18:00 – 20:00) subcutaneous injections of recombinant human methionyl leptin at doses in the range of 0.01 – 0.04 mg/kg calculated to achieve a normal leptin concentration based on body fat of 30% in females and 20% in males [144].  The evening administration was chosen to further model the circadian variation in endogenous leptin, characterized by a pulsatile circadian rhythm with marked nocturnal rise.  By the end of the 18-month treatment, the weight losses of patients A, B, and C were 76.2, 47.5, and 60.0 kg, corresponding to 53.8%, 43.5%, and 44.5% of their body weight, respectively.  Interestingly, without any type of structured dietary or physical activity interventions, the patients were able to lose as much as half of their body weight solely by leptin replacement.  Parallel to the decrease in body weight, physical activity during the day was increased progressively and linearly in all patients throughout the study.  Furthermore, for one of the patients, who had a clinical diagnosis of type 2 33 diabetes mellitus, 2-month leptin replacement decreased her fasting and postprandial glucose values and confined her hemoglobin A1c levels within the normal range at the time of reporting.  The other two patients, while maintaining normal fasting glucose levels, showed decreased insulin and C-peptide levels that were less than half of those measured originally, indicative of a marked decrease in insulin resistance.  Moreover, the authors documented, after leptin replacement, the reversal of clinical features of hypogonadism diagnosed before treatment, as indicated by increased facial hair, onset of facial acne, development of pubic and axillary hair, and growth of penis and testicles in the male patient and regular menstrual periods associated with serial midluteal phase progesterone measurements >10 ng/ml - signs of ovulation, in the two female patients. These results demonstrate that leptin replacement therapy is efficacious even in leptin- deficient adults with established severe obesity, providing beneficial outcomes, including profound weight loss, decreased energy intake, increased physical activity, and resolution of type 2 diabetes mellitus and hypogonadism. In addition to congenital leptin deficiency, leptin replacement therapy has also been applied to patients with low levels of leptin (< 3 ng/ml for male, < 4 ng/ml for female), such as those seen in severe lipodystrophy, caused by a deficiency or destruction of adipose cells.  These patients are characterized by insulin resistance, hyperglycemia, and hypertriglyceridemia.  By placing 9 female severe lipodystrophic patients on a 4-month leptin replacement therapy, consisting of subcutaneous administration of recombinant leptin with a dose of 0.03 – 0.04 mg/kg body weight/day every 12 hrs, Oral et al. reported a marked enhancement of metabolic control in these patients after the treatment [136].  The patients showed by an absolute reduction in the hemoglobin A1c value of 1.9%, a 60% decrease in fasting triglyceride levels, and an improvement in whole-body insulin sensitivity and oral glucose tolerance compared to 34 the baseline levels, suggesting that leptin deficiency contributes to the insulin resistance and other metabolic abnormalities, such as hypertriglyceridemia, associated with severe lipodystrophy and that these conditions can be treated by the replacement of the hormone. The potential of exogenous leptin to cause body weight and fat mass reduction in the presence of physiological levels of leptin has also been investigated in normal lean and obese adults.  Heymsfield and colleagues reported a study in which 54 lean (body mass index, 20.0 - 27.5 kg/m2; mean ± SD body weight, 72.0 ± 9.7 kg) and 73 overweight or obese (body mass index, 27.6 - 36.0 kg/m2; mean ± SD body weight, 89.8 ± 11.4 kg) healthy subjects received a 4- and 24-week intervention of daily subcutaneous recombinant methionyl human leptin injection, respectively [145]. Various doses (0.01, 0.03, 0.10, or 0.30 mg/kg/day) of leptin or placebo (sorbitol and sodium acetate, pH 4.0) were delivered through subcutaneous bolus injections.  All subjects were given the intervention for 4 weeks (part A) and the obese subjects were continued with the intervention for an additional 20 weeks (part B).  After 4 and 24 weeks of treatment, a statistically significant dose response for weight loss from baseline was found in both lean and obese subjects.  At 4 weeks, absolute weight reduction across the doses studied averaged between 0.4 and 1.9 kg with mean ± SD weight decrease of 0.4 ± 2.0 and 1.9 ± 1.6 kg for the placebo and 0.1 mg/kg dose groups, respectively.  In those who received 20 weeks of additional treatment, absolute weight reduction across the doses studied averaged between 0.7 and 7.1 kg with greatest average weight loss (7.1 ± 8.5 kg) in the highest dose cohort.  Likewise, a dose response for decrease in fat mass from baseline was also statistically significant at 4 and 24 weeks.  It was further shown that the loss in fat mass accounted for most of the loss in body weight (> 95% of the weight loss in the 2 highest-dose cohorts at 24 weeks). 35 However, energy intake did not change significantly with leptin dose at week 4 or 24.  In this study, while a dose-response relationship both after 4 weeks of exposure to recombinant leptin in lean and obese subjects and after 24 weeks of exposure in obese subjects was present, only patients in the highest dose groups (0.10 and 0.30 mg/kg/day) had significant weight loss compared with those taking placebo and considerable variability in the amount of weight lost by individual subjects was noted. As a result, the authors concluded that the therapeutic potential for recombinant leptin to treat obesity in subjects, already having leptin in their body, could not be resolved from the findings of this study.  Nevertheless, the fact that some patients, across a wide range of body weights, still responded to high doses of exogenous leptin administration argues against an absolute leptin resistance in obese individuals with elevated endogenous leptin levels and suggests that leptin therapy may contribute at least partially to weight loss in leptin-sufficient individuals. While less efficacious as a weight loss-inducing agent, exogenous leptin may be prescribed at a low dose as a weight loss-maintaining mediator.  Leibel’s group demonstrated that daily administration of recombinant leptin can effectively block the body’s natural tendency to regain lost weight by reversing the phenotypes of decreased energy expenditure, sympathetic nervous system (SNS) tone, circulating concentrations of thyroxine (T4) and triiodothyronine (T3) and increased skeletal muscle work efficiency that normally accompany body weight reduction [146].  In their study, 10 subjects (5 male, 5 female; 7 obese, 3 never-obese) went through 3 experimental stages: 1) initial body weight was measured and stabilized with a liquid formula diet, 2) the same liquid formula diet with adjusted calories was prescribed to cause and maintain a 10% weight loss, and 3) twice daily leptin injections in doses (0.019 – 0.121 mg/kg/d) calculated to maintain circulating leptin concentrations at levels detected prior to weight loss were 36 administered for a period of 5 weeks.  Interestingly, with the leptin treatment, the significant decline in 24-hr energy expenditure and energy expended in physical activity above resting associated with maintenance of a 10% body weight reduction were returned to pre-weight loss levels.  Furthermore, leptin injections fully restored the 23% increase in gross mechanical efficiency of skeletal muscle while bicycling to generate 10 W of power in patients with a 10% weight loss to that evaluated before weight reduction.  Moreover, in the same patients, circulating T4 and T3 were detected, respectively, at concentrations similar to and higher than those measured before weight loss and the decline in SNS activity (measured by heart rate analysis and by urinary epinephrine excretion) was brought back up to that observed at the initial weight stage after 5 weeks of leptin administration.  Collectively, the authors showed that leptin injections for 5 weeks, at doses that have previously been reported to have no significant effect on body composition or thyroid function in normal-weight subjects [145, 147], could effectively correct the weight reduction-associated phenotypes that would otherwise function coordinately to promote weight regain.  This emphasizes the difference between the induction of weight loss and the maintenance of reduced body weight; the latter may be more effectively targeted by pharmacologic and other strategies, such as leptin therapy.  Patients suffering from congenital leptin deficiency and lipodystrophy can be treated by long term replenishment of leptin, which may also contribute to weight loss in some leptin-sufficient subjects and provide maintenance of weight loss.  The supply of leptin by gene therapy may be beneficial for leptin replacement recipients who otherwise require repeated lifetime needle injections.  With the availability of a well- characterized leptin-deficient mouse model, ob/ob, leptin gene therapy studies may readily be undertaken. 37 Leptin Gene Therapy The idea of supplying the body with a gene for leptin as a means to provide a continuous source of the hormone is not unprecedented.  Previous literature has documented the effects of central leptin gene therapy in various rodent models.  Chen et al. observed that injection of recombinant adenoviruses encoding rat leptin gene into the carotid artery of normal Wistar rats led to sustained hyperleptinemia (8 ng/ml) accompanied by 30 – 50% reduction in food intake and significantly lowered plasma triglycerides and insulin compared to pair-fed controls [148].  Since blood glucose levels were similar in the two groups, improved insulin sensitivity was inferred in the hyperleptinemic rats.  Interestingly, this treatment also caused absence of body fat in the subcutaneous, visceral, retroperitoneal, or epididymal fat depots, an observation that was in sharp contrast to AdCMV-β-Gal-treated animals, saline-infused controls as well as to the pair-fed animals, suggestive of a specific lipoatrophic activity for leptin. Dhillon and colleagues also saw similar disappearance of body fat following a single i.c.v. injection of adeno-associated viral (AAV) vector carrying rat leptin gene into normal adult Sprague–Dawley rats.  In this long term study, suppression of body weight first began 2 weeks after treatment and persisted until 6 months after treatment.  During this period, a decrease in serum insulin and leptin concentrations was observed along with normoglycaemia [149].  The same protective effects, such as reduced food intake, body weight, adiposity, and blood glucose, after AAV-based central leptin gene therapy were also reported in rats fed with a high fat diet [150].  Furthermore, the same leptin gene transfer technique gave rise to similar metabolic improvements, such as lowered blood glucose, improved glucose tolerance and insulin sensitivity as well as elevated energy expenditure, in two separate mouse models: insulin-deficient Akita nonobese mice, and leptin-deficient obese ob/ob mice [151]. 38 Besides i.c.v. injection, other routes of administration have been explored for leptin gene therapy.  Dhillon et al. injected ob/ob mice i.v. with three different doses, 6×109, 6×1010 and 6×1011 particles, of rat leptin-encoding AAV vector via the tail vein. They found that while the lowest dose was ineffective, the middle dose (6×1010 particles) restricted body weight gain without affecting food consumption for 75 days of observation and the ten-fold higher dose (6×1011 particles) resulted in increased blood leptin levels and reduced both body weight and food consumption throughout the duration of the experiment (75 days) [152].  It should be noted that the low levels of circulating leptin detected in the range of 2 – 3 ng/ml at Day 54 and 75 post injection in the highest dosing group were apparently sufficient to decrease both body weight and food intake in the ob/ob mice, highlighting the extreme sensitivity to leptin in these mice. Finally, Morsy et al. demonstrated that compared to daily intraperitoneal (i.p.) recombinant leptin protein injection, gene therapy mediated by leptin-encoding adenoviral vector via a single i.p. injection was more efficacious at reversing the obesity of the ob/ob mice as it achieved greater percentage weight loss (~ 11 vs. 5 %) with a much lower peak (~ 30 vs. 300 ng/ml) circulating leptin one week after treatment [153]. The authors attributed this to the chronic secretion pattern of leptin provided by gene therapy versus the discontinuous bolus delivery by daily protein injection; the latter being subjected to rapid clearance.  These data support the development of a gene transfer methodology for the replacement of leptin.     39 THESIS INVESTIGATION Human gene therapy first took place in 1989.  Ever since then, the quest to find a safe and efficacious method for transferring exogenous genetic material into the cells within the body has been a steep slope.  Although researchers found ways to exploit the machineries of viruses to accomplish this task, the adverse, and sometimes fatal consequences emphasize the necessity to seek alternative options.  Polymers have shown promise as nonviral DNA vectors.  Previously, it has been demonstrated that chitosan, a biodegradable polymer, can effectively achieve gene transfer in vitro as well as in vivo.  To acquire long term expression with nonviral-based vectors, however, transgene genomic integration is necessary.  A bacteriphage integrase, ФC31, can insert the transgene plasmid into the host genome in a site-specific and unidirectional manner, minimizing insertional mutagenesis, a dangerous feature of integrating viruses. The research in this thesis investigates the potential to obtain long term gene transfer with a nonviral delivery system consisting of chitosan and ФC31 integrase.  The transfer of two transgenes, human placental secreted alkaline phosphatase (SEAP) and human leptin, were examined in two mouse models, C57BL/6j and the obese and diabetic ob/ob mice.  In addition, a variety of delivery routes, including intraduodenal, intrasplenic, i.p., and oral administration, were explored.  To begin, human leptin gene was first cloned into attB-bearing plasmid backbones downstream of a CMV or EF1α promoter.  Second, nanoparticles were synthesized and studied in vitro.  Third, long term in vivo SEAP gene transfer was conducted in C57B6/j mice.  Finally, in vivo leptin gene transfer was performed in C57BL6/j and ob/ob mice.   40 MATERIALS AND METHODS Cloning of Plasmid Constructs Human leptin-expressing plasmid constructs  The human leptin gene was used for all leptin gene transfer studies due to ease of distinction between the endogenous mouse leptin protein and the leptin protein expressed by the transgene.  Based on close sequence homology (84%) of the protein between mouse and human, no difference in protein function is anticipated [112, 154]. EF1α-Lep-WPRE-attB  The human leptin-expressing plasmid construct was cloned by inserting a human leptin cDNA (504 bp) downstream of a human constitutively active cellular promoter, EF1α (elongation factor 1 alpha), into pUC vector (generously provided by enGene Inc. (Vancouver, BC)), which also contains the WPRE (woodchuck hepatitis B virus post- transcriptional regulatory element), attB (attachment B) site, and the ampicillin resistance (β-lactamase) gene (Figure 3).       41   Figure 3.  The cloning procedures of the EF1α-Lep-WPRE-attB plasmid construct.  (A) The SEAP transgene in the parent pUC vector containing a SEAP cDNA, WPRE, and an attB site was excised by Pac I and Spe I restriction digests.  Pac I and Spe I-bearing human leptin cDNA, amplified from a template previous generated from human adipose tissue by RT-PCR, was ligated into the vector.  The ampicillin resistance (β-lactamase) gene was included in the vector as a selectable marker.  (B) Plasmid construct map for EF1α-Lep-WPRE-attB. A. B. 42  To do this, two restriction sites, Pac I and Spe I, were engineered at the 5’ and 3’ end of the human leptin cDNA, respectively, by PCR using a human leptin cDNA template, previously generated by RT-PCR from human adipose tissue, and the following primer set:  forward primer hLepFPS (5’ – GTG TAC TAG TTT AAT TAA ATG CAT TGG GGA ACC CTG TGC GGA TTC TT – 3’), where the red and bold font indicate Pac I site and start codon, respectively; reverse primer hLepRS (5’ – GTG TAC TAG TCA TCA GCA CCC AGG GCT GAG GTC CA – 3’), where the blue and bold font indicate Spe I site and stop codon, respectively, amplifying a product with a predicted size of 534 bp.  PCR conditions used were: 2 min. initial activation at 94°C, 15 sec. denaturing at 94°C, 20 sec. annealing at 60°C (1°C/sec. ramp), 1 min. extension at 72°C, 40 cycles, and 5 min. final extension at 72°C ending with 4°C permanent hold. AccuPrimeTM Taq DNA polymerase (Invitorgen; Carlsbad, CA) was used to ensure PCR fidelity.  The PCR product was purified and serially digested with Pac I and Spe I restriction enzymes to generate 5’ and 3’ sticky ends, respectively, for ligation with the EF1α vector backbone, which were also digested with Pac I and Spe I, removing a 1.7 kb fragment containing transgene for SEAP (Figure 3).  The final ligation product (EF1α- Lep; 6.8 kb) was used to transform competent E.coli. bacteria (DH5α).  Following transformation, bacteria were grown on ampicillin selection agar gel at 37°C overnight, picked from a single colony, and amplified in 3 ml LB (Luria-Bertani) broth for 10 hrs, after which plasmid DNA was extracted by miniprep (Qiagen, Germany), verified by diagnostic digests and DNA sequencing, and stored as glycerol stock at -80°C. CMV-Lep-WPRE-attB  A human leptin-expressing construct under the control of a CMV promoter was also cloned.  This was done by replacing the EF1α promoter, in the above cloned plasmid 43 construct, with a CMV promoter and a rabbit β-globin intron 1 excised from pSCORE3- CMV-MCS vector at Mlu I and Pac I sites (Figure 4).  The resulting vector retains the WPRE, attB site, and the ampicillin resistance gene.  Following diagnostic digests, the CMV-Lep-WPRE-attB plasmid construct was stored as glycerol stock at -80°C.   Figure 4.  Plasmid construct map of CMV-Lep-WPRE-attB.  The human CMV promoter and the rabbit β-globin intron were subcloned into the EF1α-Lep-WPRE-attB vector, replacing the EF1α promoter, at Mlu I and Pac I restriction sites.  WPRE, poly A, and attB are located 3’ to the human leptin cDNA.  The ampicillin resistance (β-lactamase) gene is used as the selectable marker.     44 gWIZTM-SEAP, EF1α-SEAP-WPRE-attB, CMV-SEAP-WPRE-attB, and CMV-Int  The gWIZTM-SEAP plasmid vector is a commercially available reporter plasmid that contains a proprietarily modified human CMV promoter followed by intron A from the CMV immediate early gene and a highly efficient rabbit β-globin transcription terminator, designed to produce high levels of gene expression in a broad range of mammalian cells and tissues (Figure 5) (Aldevron; Fargo, ND).     Figure 5.  Plasmid construct map of gWIZTM-SEAP.  gWIZTM-SEAP is a commercially available SEAP expression vector driven by the human CMV promoter/intron (Aldevron; Fargo, ND).      45  To examine the plasmid vector more closely, gWIZTM-SEAP was compared to the CMV-Lep-WPRE-attB plasmid construct in terms of promoter, intron, polyadenylation signal, origin of replication, and other elements within the vector (Table I).  It appears that, based on DNA sequence alignment and the consultation with Aldevron technical support, the CMV promoter per se in these two plasmids should not give rise to significantly different levels of gene expression; however, the combination of the various elements, including promoter, intron, polyadenylation signal, and the transgene, may play a role in altering the performance of the plasmids.  EF1α-SEAP-WPRE-attB, CMV-SEAP-WPRE-attB, and a CMV-driven ФC31 integrase expression vector (CMV-Int) were generously provided by enGene Inc. (Vancouver, BC).                           46               Table I.  Comparison of vector elements and their relative positions between gWIZTM- SEAP and CMV-Lep-WPRE-attB.  Shaded boxes denote differences between the two plasmid constructs.  gWIZTM-SEAP CMV-Lep-WPRE-attB Element Position Element Position Promoter CMV, human 245 – 900 CMV, human 27 – 723 Intron CMV IE intron A 900 – 1864 Rabbit β-globin intron 1 724 – 1341 Polyadenylation signal Rabbit β-globin transcript terminator 3432 – 3463 SV40 late 2496 – 2619 Origin of replication pUC 4252 – 4507 pUC 3163 – 3842 Selectable marker Kanamycin 5491 – 5746 Ampicillin 3992 – 4849 Transgene  SEAP 1849 – 3411 Leptin 1362 – 1865 Others – – WPRE, attB 1884 – 2904      47 Transfection of STC-1 and HEK 293T Cell Using LipofectamineTM 2000 Transfection of STC-1 cell line  STC-1 cells (a gut endocrine cell line, generously provided by Dr. Daniel Drucker; University of Toronto) were cultured in high-glucose Dulbecco' s modified Eagle's medium (4.5 g/l D-glucose, HG-DMEM) (Invitrogen; Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (GIBCO; Grand Island, New York) and 100 U/ml penicillin and streptomycin (1X P/S) (GIBCO; Grand Island, New York).  Cells were seeded in 6-well plates at a density of 1 x 106 cells per well one day prior to transfection. On the day of transfection, at ~40% confluency, cells were transfected with EF1α-Lep using LipofectamineTM 2000 transfection reagent (Invitrogen; Carlsbad, CA). LipofectamineTM/DNA complexes were prepared by first diluting 4 µg EF1α-Lep with 250 µl serum- and antibiotic-free HG-DMEM.  Then, 5 µl of LipofectamineTM 2000 (Invitrogen; Carlsbad, CA) were diluted with 250 µl serum- and antibiotic-free HG- DMEM, after which the two mixes were combined, vortexed, and incubated for 20 min. at room temperature.  Following incubation, 500 µl of the complexes were added to each well, which were continued in culture at 37°C for 5 hrs.  Media samples were changed to complete media (HG-DMEM, 10% FBS and 1X P/S) at the end of the 5-hr period and cells were kept at 37°C for another 48 hrs before cell supernatant samples were collected and spun down (15 min. @ 500 x g). The supernatant from the centrifuged samples were stored at -20°C until time of assay. Transfection of HEK 293T cell line  HEK 293T cells (a variation of the human embryonic kidney 293 cell line that contains the additional SV40 Large T-antigen obtained from the American Type Culture Collection, ATCC) were grown in HG-DMEM (10% FBS, 1X P/S).  For transfection, 4.4 48 x 104 cells per well were seeded into 6-well plates one day before the experiment.  Each well, 50% confluent, received 2 µg EF1α-Lep or CMV-Lep through LipofectamineTM/DNA complexes prepared by mixing 2 µg DNA in 50 µl HG-DMEM (serum- and antibiotic-free) with 2.5 µl LipofectamineTM in 50 µl HG-DMEM (serum- and antibiotic-free).  The complexes were incubated for 20 min. before use.  Cells were washed once with 1 ml HG-DMEM (serum- and antibiotic-free) and 2 ml of the same media were added back to each well.  LipofectamineTM/DNA complexes, 100 µl, were added to each well and cells were incubated at 37°C for 5 hrs.  Following incubation, media samples were changed to complete media (HG-DMEM, 10% FBS and 1X P/S) and cells were continued in culture for another 48 hrs before cell supernatant samples were collected and spun down (15 min. @ 500 x g).  The supernatant from the centrifuged samples were stored at -20°C until time of assay.  Testing of the Leptin Plasmid Constructs Using a Cell-Based Bioassay CHO-ObRb cell stimulation by leptin  CHO-ObRb cells (generously provided by Dr. Takashi Murakami; University of Tokushima), a CHO (Chinese hamster ovarian) cell line stably transfected with the gene for the long form of the mouse leptin receptor (ObRb) [155], overexpress the leptin receptor on the plasma membrane, a feature to be utilized in the leptin bioassay for assessment of the biological function of the leptin protein.  CHO-ObRb cells were cultured in Ham’s F-12 media (GIBCO; Grand Island, New York) (10% FBS, 1X P/S) and were seeded in 6-well plates at a density of 3.5 x 105 cells per well for the bioassay. It is expected that upon binding of the leptin protein to its receptor, a downstream 49 signalling pathway will be activated, leading to JAK2-induced phosphorylation of STAT3, which will serve as an indicator of leptin’s bioactivity (Figure 6).   Figure 6. Receptor-mediated leptin signalling pathway results in JAK2-induced STAT3 phosphorylation (pSTAT3), a marker used to assess the biological activity of the leptin protein expressed by EF1α-Lep and CMV-Lep.  ObRl: leptin receptor (long form), JAK2: Janus kinase 2, IRS-1/2: insulin receptor substrate-1/2, SOC3: suppressor of cytokine signaling 3, SHP2: Src homology 2-containing tyrosine phosphatase, Grb2: growth factor receptor-bound protein 2, PI3K: phosphoinositide 3 kinase, STAT3: signal transducer and activator of transcription 3, Akt: protein kinase B, ERK1/2: extracellular signal-regulated kinases 1/2, p38: p38 kinase, PLCγ: phospholipase C-gamma, PKC: protein kinase C, NO: nitric oxide, Rb: retinoblastoma tumor suppressor, NFκB: nuclear factor-κB, vegf: vascular endothelial growth factor.  Schematic was obtained from [156].  50  To test whether the leptin protein expressed by the CMV- and EF1α-Lep plasmid constructs is biologically functional, 300 µl cell supernatant samples, containing 60 and 144 ng of human leptin (based on the volume used and concentrations determined by leptin RIA), collected, respectively, from cells previously transfected with CMV- and EF1α-Lep were used to stimulate CHO-ObRb cells for 10 min. 1 day after seeding at 50% confluency following 2 x PBS- washes.  Recombinant murine leptin peptide, purchased from the National Hormone and Peptide Program (NHPP), was reconstituted in PBS at 250 ng/ml and used, 300 µl, as positive control.  Cell lysis and BCA Protein Assay  After stimulation, the cells were washed with ice cold PBS (2 x 2 ml) and lysed with 0.25 ml ice cold lysis buffer (1% w/v Triton X-100 (VWR), 20 mM Tris HCl pH 7.5 (Sigma-Aldrich; St. Louis, MO), 150 mM NaCl (Fisher), 5 mM EDTA (Fisher)). Immediately prior to use, 1X protease inhibitor cocktail (Sigma-Aldrich; St. Louis, MO) was added to the lysis buffer.  Cells were detached, by scraping with 1 ml pipette tips, and transferred on ice to 1.5 ml RNase- and DNase-containing microcentrifuge tubes to achieve a final concentration of 50 µg/ml for RNase and DNase (Sigma-Aldrich; St. Louis, MO), which were incubated on a rotating platform for 1 hr and spun (10,000 x g) for 10 min. at 4°C.  The supernatant was transferred to fresh microtubes after centrifugation.  Protein concentration was measured in a 96-well plate by BCATM (bicinchoninic acid) Protein Assay according to manufacturer’s Standard Microplate protocol (Thermo Scientific; Rockford, IL).  To obtain absorbance readings at 562 nm, the Infinite® M1000 plate reader was used to scan the plate (TECAN; Switzerland).  51 Western Blot Analysis of Phospho-STAT3 in Simulated CHO-ObRb Cells SDS-PAGE and protein transfer  Sample buffer, 4X, (30% glycerin (Fisher), 25 mM Tris-HCl pH 6.8, 8% SDS, 0.02% Bromophenol Blue (Sigma-Aldrich; St. Louis, MO)) was added to 8 µg protein to achieve 1X concentration of sample buffer.  The sample buffer-added protein was heated at 85°C for 10 min. and then centrifuged (15,000 x g) for 1 min.  The supernatant was loaded, 25 µl, on 8% polyacrylamide gel, which was ran by electrophoresis, in 1X running buffer (25 mM Tris base (Fisher), 192 mM glycine (EMD Chemicals; Gibbstown, NJ)), at 45 V initially until the dye front reached the top of the separating gel, at which point the voltage was increased to 105 V until the dye front reached ~0.5 cm from the bottom of the gel.  When electrophoresis was completed, the gel was equilibrated in transfer buffer (20 mM Tris base, 150 mM glycine (EMD Chemicals; Gibbstown, NJ), 20% methanol (Fisher), 0.038% SDS) for 15 min. at the end of which the transfer apparatus was assembled in the following order:  black plate, foam pad, 2 pieces of whatman paper, gel, PVDF (polyvinylidene fluoride) transfer membrane (Bio-Rad, Hercules, CA), 2 pieces of whatman paper (Bio-Rad, Hercules, CA), foam pad, clear plate.  The transfer was done in ice cold transfer buffer at 100 V for 60 min.  When the transfer was finished, the membrane was incubated in blocking solution (5% BSA in TBST (1X TBS, 0.1% v/v Tween20 (VWR), milli-Q water)) 30 min. at room temperature. Western blot  Two quick and then 3 x 5 min. TBST washes with rocking were done after the membrane was removed from the blocking solution.  The membrane was subsequently incubated with 12 ml 1:1000 rabbit α-pSTAT3 primary antibody (Cell Signalling 52 Technology; Danvers, MA) diluted in 1X TBST with 5% (w/v) BSA.  Then, the membrane was subjected to 2 x quick, 1 x 5 min., 1x 8 min., and 1 x 10 min. washes in TBST with rocking.  After the washes, the membrane was incubated with 12 ml 1:8000 donkey-α-rabbit HRP secondary antibody (GE Healthcare; Piscataway, NJ) diluted in 1X TBST with 5% (w/v) BSA.  Following secondary antibody incubation, the membrane was washed 3 x 10 min. in TBST with rocking and the ECLTM Western Blotting Detection Reagents (GE Healthcare; Piscataway, NJ) were added to cover the side of the membrane with the leptin protein.  The membrane was then wrapped in saran wrap and set inside a film cassette for film exposure.  The film exposure times were 10 sec., 1, 5, and 10 min. for pSTAT3 and 30 and 60 sec. for β-actin.  Synthesis of Chitosan Nanoparticles Preparation of chitosan solution  Chitosan (226 and 429 kDa) stock solutions (0.6% w/w, pH 4.8) were prepared by dissolving ultra pure chitosan powder, UltrasanTM (BioSyntech; Laval, QC), with 1N acetic acid (Figure 7).         53                  Figure 7.  Chitosan nanoparticles were synthesized through a series of production steps, including (A) chitosan solution preparation, (B) plasmid DNA extraction, and (C-D) chitosan/DNA mixing.  Following synthesis, the nanoparticles were subjected to quality control checkpoints, such as (E) gel motility test, (F) preliminary light scattering assessment, and (G) size, polydispersity index, and zeta potential measurement (Malvern Zetasizer).     F. B. C. D.  E. G. A. 54 For the dissolving process, 0.15 g (dry weight) chitosan was first wetted with 18 ml milli- Q water in a clean 100 ml flask holding a magnetic stir bar.  Acetic acid (1N) was then added slowly, 0.1 ml at a time, to a total of 1.0 and 1.25 ml for the 226 and 429 kDa chitosan, respectively, with gentle magnetic stirring at room temperature until chitosan was completely dissolved (1 - 2 hrs).  The pH of the solution was measured by a pH meter and residual chitosan on the pH probe was rinsed back into the solution with milli- Q water.  The solution was brought up at the end to a final volume of 25 ml with milli-Q water and stored at room temperature overnight before filtration.  In the biological safety cabinet, the chitosan solution was passed through 0.22 µm syringe filters into a borosilicate glass vial (Fisher).  Before and after filtration, the solution was diluted 5-fold in milli-Q water for absorbance measurement at 280 nm.  Chitosan stock solutions were stored at 4°C.  Low molecular weight chitosan solution (2.4% w/w, pH 4.8) was generously provided by enGene Inc. (Vancouver, BC). Plasmid DNA extraction  DNA was extracted using endotoxin-free plasmid Giga kits (Qiagen) (Figure 7). Bacteria from -80°C glycerol stock were grown in 3 ml LB (Luria-Bertani) supplemented with 200 mg/ml ampicillin sodium salt (amp±) (Fisher Scientific) starter culture for ~10 hrs.  Fresh LB (amp±), 5 x 3.5 ml, were inoculated with this starter culture, 0.55 ml each, and incubated at 37°C for ~13 - 15 hrs.  This was subsequently transferred, 3 ml each, to 5 x 500 ml fresh LB (amp±), which was incubated for 24 hrs before the culture was centrifuged (6,000 x g) for 15 min. at 4°C.  Following centrifugation, bacteria pellets were collected and frozen at -20°C overnight before use.  Plasmid DNA extraction was performed according to the manufacturer’s protocol.  The DNA pellets were 55 reconstituted in 0.3 - 1 ml endotoxin-free Buffer TE depending on pellet size and stored at 4°C overnight before absorbance measurement by UV spectrophotometry at the wavelengths of 260 and 280 nm. Chitosan nanoparticles  Chitosan nanoparticles were synthesized by mixing chitosan solution with plasmid DNA at a concentration of 50 or 250 µg/ml.  For the low and high molecular weight chitosan, amine to phosphate (N/P) ratios of 20 and 6 were used, respectively.  Single plasmid nanoparticles contained gWIZTM-SEAP, EF1α-SEAP, EF1α-Lep, or CMV-Lep, while double plasmid nanoparticles contained a mixture of CMV-Int and EF1α-SEAP, EF1α-Lep, or CMV-Lep at a 1:5 ratio (one part CMV-Int, 5 parts transgene).  DNA and chitosan solutions (pH 4.0 or 4.8) were diluted to desired concentrations with milli-Q water before loading into 10 and 20 ml syringes (BD) for the 9 and 25 ml batch size, respectively.  The concentrations of DNA were monitored by UV spectrophotometry and recorded during the sequential dilution steps.  To complex chitosan with DNA, infusion pumps, Harvard Pump 11 Plus (Harvard Apparatus; Holliston, MA), were utilized to combine the two solutions, after which nanoparticles were formed and collected in a 50 ml conical tube (Figure 7).  Immediately after nanoparticles collection, a preliminary light scattering assessment was performed by penetrating the solution with a conventional laser beam and observing the presence of light path through the solution. Gel Motility Test  Agarose gel electrophoresis was performed to assess DNA encapsulation efficiency by chitosan nanoparticles (Figure 7, 12).  EF1α-Lep and CMV-Int (1.5 µg) encapsulated by chitosan or in the form of naked DNA, in addition to 1 kb DNA ladder, were loaded on 0.7% SYBR® Safe-stained agarose gel (Invitrogen; Carlsbad, CA), which was run 56 for 2 hrs at 60 V.  After electrophoresis, the gel was imaged on a UV light box through a gel documentation (geldoc) system (BioRad Laboratories; Mississauga, ON).  Measurement of Size, Polydispersity, and Zeta Potential of Chitosan Nanoparticles  Following synthesis, nanoparticle size and polydispersity index, and zeta potential were measured by dynamic and electrophoretic light scattering, respectively, using Malvern Zetasizer, Nano ZS (Malvern Instruments; Worcestershire, UK) (Figure 7).  The nanoparticles were diluted 20-fold in filtered 10 mM NaCl before measurements, which were taken in triplicates for size and polydispersity index and in quadruplicates for zeta potential at 25 ºC.  Automated data acquisition and analysis were done by the DTS software based on pre-designed standard operating procedures.  Transfection of HEK 293T Cells Using Chitosan Nanoparticles  HEK 293T cells were transfected by various batches of chitosan nanoparticles encapsulating different plasmid DNA, including gWIZTM-SEAP, EF1α-SEAP, CMV- SEAP, EF1α-Lep, and CMV-Lep, synthesized with different molecular weight (5, 226, and 429 kDa) to determine the transfection efficiency of the synthesized nanoparticles. HEK 293T cells were cultured in HG-DMEM (10%FBS, 1X P/S) and seeded in 6-well plates one day prior to transfection at a density of 4.4 x 104 cells per well.  On the day of transfection, cells, 50% confluent, were washed once with 1 ml HG-DMEM (serum- and antibiotic-free, pH 6.0).  Nanoparticles containing 2 µg plasmid DNA were added to each well after the addition of 1 ml HG-DMEM (serum- and antibiotic-free, pH 6.0). Cells were incubated with the nanoparticles for 2 hrs at 37°C, following which the media were changed to 3 ml complete HG-DMEM (10% FBS, 1 x P/S, pH 7.4).  Cell 57 supernatant samples were collected 48 hrs post-transfection and spun down (15 min. @ 500 x g).  The supernatant from the centrifuged samples were stored at -20°C until time of assay. Long Term In Vitro Studies with ФC31 Integrase LipofectamineTM 2000  HEK 293T cells were grown in HG-DMEM (10% FBS, 1X P/S) and seeded at a density of 4.4 x 104 cells per well into 6-well plates one day before transfection.  Fifty percent confluent cells received either EF1α-SEAP and CMV-Int together or EF1α- SEAP alone through LipofectamineTM/DNA complexes prepared by mixing 2 µg DNA in 50 µl HG-DMEM (serum- and antibiotic-free) with 2.5 µl LipofectamineTM 2000 in 50 µl HG-DMEM (serum- and antibiotic-free).  The complexes were incubated for 20 min. before use.  For double plasmid transfection, EF1α-SEAP and CMV-Int were mixed at SEAP to Int plasmid ratios of 5:1, 1:1, or 1:5 while the total amount of DNA transfected was held constant at 2 µg.  Therefore, the 5:1 group received 1.67 µg EF1α-SEAP and 0.33 µg CMV-Int, the 1:1 group received 1 µg of EF1α-SEAP and CMV-Int each, and the 1:5 group received 0.33 µg EF1α-SEAP and 1.67 µg CMV-Int.  For single plasmid transfection, cells received either 2 or 1 µg EF1α-SEAP.  Cells were washed once with 1 ml HG-DMEM (serum- and antibiotic-free) and 2 ml of the same media were added back to each well.  One hundred microlitres of the LipofectamineTM/DNA complexes were added to each well and cells were incubated at 37°C for 5 hrs.  Following incubation, media samples were changed to complete media (HG-DMEM, 10% FBS and 1X P/S) and cells were continued in culture for another 48 hrs before cell supernatant samples were collected.  After sample collection, cells were passaged according to the following procedures: briefly, 2 ml 0.25% typsin-EDTA (GIBCO; Grand 58 Island, New York) were added to each well of the 6-well plate and an incubation period of 5 min. at 37°C was followed.  At the end of the incubation, 2 ml HG-DMEM (10%FBS, 1X P/S) were added to the wells containing typsinized cells and the entire suspension from a single well was transferred to a 15 ml Falcon tube.  The same was repeated for each well, keeping the cells from each well separated.  The cells were centrifuged (2 min. @ 200 x g) and the supernatant was discarded before the cells were re-suspended in 1 ml HG-DMEM (10%FBS, 1X P/S).  Fifty microlitres of this suspension were seeded into each well of the 6-well plate containing 3 ml HG-DMEM (10%FBS, 1X P/S), achieving a 1:20 split in cell density.  Cells were passaged every 6 days and cells were allowed to grow and secrete SEAP into the media for 3 days following each passaging before cell supernatant samples were collected.  After media sampling, cells were maintained in culture for an additional 3 days before the next cell splitting.  All cell supernatant samples were spun down (15 min. @ 500 x g) and the supernatant from the centrifuged samples were stored at -20°C until time of assay.  Cell supernatant samples collected at Day 2, 5, 11, 17, 23, and 29 post-transfection were analyzed for SEAP protein content. Chitosan nanoparticles  HEK 293T cells were transfected by chitosan (5 kDa) nanoparticles encapsulating EF1α-SEAP and CMV-Int at a SEAP to Int plasmid ratio of 5:1.  HEK 293T cells were cultured in HG-DMEM (10%FBS, 1X P/S) and seeded in 6-well plates one day prior to transfection at a density of 4.4 x 104 cells per well.  On the day of transfection, 50% confluent cells were washed once with 1 ml HG-DMEM (serum- and antibiotic-free, pH 6.0).  Nanoparticles containing 2 µg of total plasmid DNA were added to each well after the addition of 1 ml HG-DMEM (serum- and antibiotic-free, pH 6.0).  Cells were 59 incubated with the nanoparticles for 2 hrs at 37°C, following which the media were changed to 3 ml complete HG-DMEM (10% FBS, 1 x P/S, pH 7.4).  Cell supernatant samples were collected 48 hrs post-transfection.  After sample collection, the cells were passaged according to the procedures described in the previous section.  Cell passaging was repeated every 6 days and cells were allowed to grow and secrete SEAP into the media for 3 days following each passaging before cell supernatant samples were collected.  After media sampling, cells were maintained in culture for an additional 3 days before the next cell splitting.  All cell supernatant samples were spun down (15 min. @ 500 x g) and the supernatant from the centrifuged samples were stored at -20°C until time of assay.  Cell supernatant samples collected at Day 2, 5, 11, 17, 23, and 29 post-transfection were analyzed for SEAP protein content.  In Vivo Delivery of Nanoparticles (Intrasplenic and Intraperitoneal) – Preliminary Studies Intrasplenic injection  Male C57BL/6j mice (Jackson Laboratories; Bar Harbor, MO) were anaesthetized using 2.5 % isoflurane.  The fur from the left side of the mice was shaved and the skin swabbed with alcohol pads.  A skin incision of 0.5 -1 cm was made with a scalpel and the muscle layer was incised.  The spleen was exteriorized with forceps and a needle (27 gauge ½’’) was inserted deeply into the organ.  Nanoparticles or saline, 100 µl, were then injected into the spleen while the needle was being withdrawn.  After the injection, the spleen was returned to the peritoneal cavity, after which the muscle and the skin were closed with sutures and wound clips.  Two mice received chitosan nanoparticles (5 kDa) containing 25 µg gWIZTM-SEAP and one received saline as negative control. 60 Mice were overnight-fasted before the procedure and allowed free access to food and water after the procedure.  Blood samples were collected, from the saphenous vein, into heparinized capillary tubes 1 day pre- and post-treatment for SEAP assay.  Immediately after collection, blood was transferred into 1.5 ml microtubes containing sodium heparin (50 units) and kept on ice until centrifugation; blood samples were centrifuged at 7,000 x g for 9 min., after which plasma was transferred to clean 1.5 ml microtubes and stored at -20 °C until time of assay.  The same procedures were followed for all plasma collection. Intraperitoneal (i.p.) injection  Male C57BL/6j mice were administered i.p. with nanoparticles (5 kDa) delivering various doses of DNA.  For i.p. injection, the mice were given isoflurane inhalable anaesthetic inside the induction chamber briefly before treatment.  With the mouse being held by the skin of the back and scruff, nanoparticles or saline were injected using a 23 gauge ½’’ needle, attached to a 1 ml syringe, into the lower left quadrant of the abdomen, avoiding vital organs.  The mice were then placed back into a clean cage immediately after the treatment.  In the first study, a high DNA dose, 625 µg, was given to two mice, while in the second study lower DNA doses, 250 and 50 µg, were each given to one mouse.  Negative control received i.p. saline injection.  Mice were overnight-fasted before the procedure and allowed free access to food and water after the procedure.    Plasma samples were collected 1 day pre- and post-treatment for SEAP assay.   61 SEAP Reporter Gene Transfer and Long Term Circulating SEAP Tracking Intraperitoneal (i.p.) injection  EF1α-SEAP and gWIZTM-SEAP were delivered by chitosan nanoparticles (5 kDa) into male C57BL/6j mice.  In the gWIZTM-SEAP experiment, treatment mice received 50, 25, or 12.5 µg gWIZTM-SEAP-encapsulating nanoparticles.  Negative control received saline or 50 µg gWIZTM-SEAP in the form of naked DNA (n < 5).  In the EF1α-SEAP experiment, mice were administered nanoparticles containing 50 or 25 µg EF1α-SEAP and CMV-Int as treatment and 50 µg EF1α-SEAP naked DNA or saline as negative control.  Mice were overnight-fasted before the procedure and allowed free access to food and water after the procedure.  Mouse plasma samples in both experiments were collected from fed mice pre- and at Day 1, 3, 5, 7, 77, 89, 109, and 122 post-treatment for SEAP assay.  A follow-up study was conducted in which C57BL/6j mice were divided into three groups (n = 5).  The first two groups were administered with chitosan nanoparticles (5 kDa); one group receiving EF1α-SEAP with CMV-Int and the other receiving EF1α-SEAP without CMV-Int.  The last group was given EF1α-SEAP and CMV-Int naked DNA.  Plasma samples were collected 2, 3, 6, 8, and 11 days pre- and 1, 3, 5, 7, 14, 28, and 56 days post-treatment for SEAP assay Oral administration  The oral route of administration was used to deliver chitosan nanoparticles (5 kDa) containing gWIZTM-SEAP to male C57BL/6j mice (n = 4).  Negative controls received acetate buffer, pH 4.8, in the same manner (n = 3).  One mouse was untreated to serve as procedure control.  Mice were fasted overnight and water-restricted for 2 hrs. before treatment.  Food and water were returned to mice 2 hrs. after treatment.  For the gavage procedure, the mouse was placed on its stomach on a hard surface with its tail 62 facing towards the experimenter.  The base of the tail was grabbed with one hand and the scruff of the neck with the other hand.  The tail was wrapped and securely held between the fourth and fifth fingers of the neck-scruff hand.  With the mouse’s head extended backwards by the index finger, the gavage needle (22 gauge, round-tip), which was attached to a 1 ml syringe containing the nanoparticles or acetate buffer, was inserted gently into the mouse's mouth.  The gavage needle was lightly pressed against the roof of the mouth, advanced and slipped down the oesophagus.  The materials were then quickly injected and the gavage needle removed from the mouse. A volume of 300 µl was administered.  Mouse plasma samples were collected through the saphenous vein pre- and at Day 1, 3, 7, and 14 post-treatment for SEAP assay.  SEAP Chemiluminescent Assay Cell supernatant samples  Cell supernatant samples collected from transfection experiments were assayed for the SEAP protein using a SEAP chemiluminescent assay (Roche Applied Science; Indianapolis, IN).  Samples were diluted 20-fold in HG-DMEM (10% FBS, 1X P/S), the same media that were used to culture transfected cells.  Assay standards (0.1, 1, 10, 100, 1,000, 5,000, and 10,000 ng/ml) were prepared by diluting human placental alkaline phosphatase (PLAP) (Sigma-Aldrich; St. Louis, MO) serially in HG-DMEM (10% FBS, 1 x P/S).  Both the diluted samples and standards were added, 50 µl, in 150 µl of assay buffer, after which they were incubated at 65°C in a water bath for 30 min.  These were then cooled on ice and centrifuged at 21,000 x g for 2 min.  The supernatant, 175 µl, was transferred to fresh microtubes and equilibrated at room temperature for 5 min., following which the assay was continued according to the manufacturer’s protocol.  To 63 obtain luminescence readings for the assay, the Infinite® M1000 plate reader was used to scan the plate at 20 and 30 min. post the addition of the assay substrate (TECAN, Switzerland). Mouse plasma samples  To measure the SEAP protein in the plasma by SEAP chemiluminescent assay (Roche Applied Science; Indianapolis, IN), mouse plasma samples, 25 µl, were first diluted in 175 µl assay buffer.  The same volume of assay standards (0.2, 0.5, 1, 2, 5, 10, 20, and 50 ng/ml), reconstituted by serial dilution of PLAP, were also added to 175 µl assay buffer.  The diluted samples and standards were incubated at 65°C in a water bath for 2 hrs. for heat-inactivation of endogenous serum alkaline phosphatase.  Then, the samples/standards were cooled on ice and centrifuged (21,000 x g) for 2 min., after which the supernatant, 175 µl, was transferred to fresh microtubes and equilibrated at room temperature for 5 min.  The assay was continued according to the manufacturer’s protocol from here onwards.  The luminescence readings for the assay were measured using the Infinite® M1000 plate reader at 30 and 45 min. post the addition of the assay substrate (TECAN, Switzerland).  Human Leptin Gene Transfer and Body Weight and Blood Glucose Monitoring Low molecular weight (LMW) chitosan nanoparticles - oral administration  LMW (5 kDa) chitosan nanoparticles encapsulating EF1α-Lep with or without CMV- Int were administered orally to 8-week-old male ob/ob mice (Jackson Laboratories; Bar Harbor, MO), which were divided into 4 groups receiving: 1) 90 µg EF1α-Lep (n = 2), 2) 90 µg EF1α-Lep and CMV-Int (n= 4), 3) 450 µg EF1α-Lep (n = 4), and 4) 450 µg EF1α- Lep and CMV-Int (n = 3).  The total DNA doses were delivered through 6 oral gavage 64 treatments (15 or 75 µg DNA per treatment), carried out once every 2 days across a period of 2 weeks to overnight-fasted mice.  In addition, water gavage treatments were administered with the same frequency and duration after the end of the nanoparticle treatment.  The delivery volume of each nanoparticle or water gavage treatment was 300 µl.  Mice were overnight-fasted before each treatment and allowed free access to food and water 2 hrs. after each treatment.  Body weight and blood glucose in 4-hr fasted mice were monitored after the first nanoparticle treatment at least twice a week for 19 weeks.  Blood glucose monitoring was performed using a One Touch Ultra glucometer (LifeScan Inc.; Milpitus, CA). LMW chitosan nanoparticles - intraduodenal injection  Nine-week old male ob/ob mice (Jackson Laboratories; Bar Harbor, MO) were used in this experiment.  The surgical procedures of intraduodenal injection were as follows: anaesthesia of overnight-fasted mice was performed on heating pads using 2.5% isoflurane.  Mice were given subcutaneous injections of 50 µl ketoprofen (2 mg/ml) (Merial Animal Health; UK) for pain management and 0.5 ml saline (Baxter Healthcare Corp.; Deerfield, IL) for maintenance of body fluid 25 min. before surgery.  The duodenum of an anaesthetized mouse was isolated temporarily through an abdominal incision and hung by a glass hook, made from a Pasteur pipette, held in place by a burette clamp on a burette stand ~1 cm above the mouse abdomen.  A double-knot ligature was tied snugly at the pyloric sphincter to prevent backflow of nanoparticles into the stomach.  LMW (5 kDa) chitosan nanoparticles at a volume of 200 µl were injected into the lumen of a segment of the duodenum and incubated for 1 hr.  The mice were injected subcutaneously with 50 µl atropine sulphate (0.5 mg/ml) (Bimeda-MTC Animal Health; Cambridge, ON) to slow down GI tract motility 15 min. before the end of the 65 incubation [157].  After 1 hr incubation, the ligature was released and the duodenum was returned to the peritoneal cavity, filled with 1 ml ampicillin (5 mg/ml) solution (Fisher Scientific).  The mice were given 1 ml Lactated Ringer Solution (Hospira; Lake Forest, IL) subcutaneously immediately after suturing for body fluid replenishment.  Four groups of 9-week-old ob/ob mice were treated receiving: 1) 10 µg EF1α-Lep and CMV-Int (n = 2), 2) 50 µg EF1α-Lep (n = 1), 3) 50 µg EF1α-Lep and CMV-Int (n = 2), and 4) 50 µg gWIZTM-SEAP (n =3); the fourth group being negative control.  Body weight and blood glucose of these mice after a 4-hr fast were monitored after the treatment at least twice a week for 14 weeks. LMW chitosan nanoparticles - intraperitoneal (i.p.) injection  LMW (5 kDa) chitosan nanoparticles, 300 µl, containing 75 µg EF1α-Lep and CMV- Int (n = 2) or gWIZTM-SEAP (n =1) were administered via a single i.p. injection to 9- week-old male ob/ob mice.  Body weight and blood glucose of the treated mice after a 4-hr fast were tracked for 10 weeks post-treatment. High molecular weight (HMW) chitosan nanoparticles - intraperitoneal (i.p.) injection  HMW chitosan nanoparticles were administered to 7-week-old male C57BL/6j mice through two i.p. injections for the delivery of CMV-Lep and CMV-Int.  Mice received 200 µl per injection of the CMV-Lep and CMV-Int-containing nanoparticles synthesized using 226 or 429 kDa chitosan.  Negative controls received LMW (5 kDa) chitosan nanoparticles encapsulating CMV-Lep and CMV-Int or HMW (429 kDa) chitosan nanoparticles encapsulating CMV-SEAP and CMV-Int.  All groups contained 5 mice each.  A total of two injections, a month apart delivering 50 µg DNA each, were given to the mice, whose body weight and blood glucose were monitored after the first injection 66 for 10 weeks.  Plasma samples were collected 1 day before and 1 and 3 days after each injection treatment for circulating human leptin measurement. HMW chitosan nanoparticles - oral administration  The oral route of administration was also utilized to deliver HMW chitosan nanoparticles encapsulating CMV-Lep and CMV-Int to 8-week-old male C57BL/6j mice, which were divided into 4 groups: 1) 226 kDa chitosan, CMV-Lep and CMV-Int, 2) 429 kDa, CMV-Lep and CMV-Int, 3) 5 kDa, CMV-Lep and CMV-Int, and 4) 429 kDa, CMV- SEAP and CMV-Int; the fourth group being the negative control.  The oral gavage regimen constituted a total of 6 administrations of nanoparticles (300 µl per treatment) once every 2 days across a period of two weeks.  Mice were overnight-fasted before treatment and water restricted for 2 hrs before and after treatment.  In total, 450 µg DNA was delivered equally through the 6 administrations.  Mouse body weight and blood glucose were monitored after the treatment for 6 weeks.  Plasma samples from the saphenous vein were collected 1 day after the 6th oral gavage treatment (Day 14 of experiment) for human leptin measurement.  Human Leptin Radioimmunoassay and Enzyme-Linked Immunosorbent Assay  The human leptin protein was measured using the Alpco Human Leptin (ultrasensitive) RIA (22-LEP-R40, Alpco Diagnostics, NH), or Invitrogen Human Leptin ELISA (KAC2281, Invitrogen; Carlsbad, CA).  The claimed sensitivities of the RIA and ELISA are 0.25 ng/ml and 15.6 pg/ml, respectively, both requiring a sample volume of 100 µl.  The RIA and ELISA cover an assay range of 0.25 ~ 16 ng/ml and 15.6 ~ 10,000 pg/ml, respectively.  Both assays were reported to have 100% cross-reactivity with human leptin and no cross-reactivity with mouse leptin (Table II).  With the larger 67 number of samples (i.e. 100 samples) and a relatively low sensitivity, the RIA was used for detection of human leptin in cell supernatant samples, which usually carried high levels of the protein, while the detection of human leptin in mouse plasma, which were expected to contain much less of the protein, was done by the ELISA owing to its higher sensitivity and broader assay range.  Cell supernatant samples were diluted 20- or 50- fold and mouse plasma samples were diluted 10-fold in assay buffer for the measurement of human leptin by RIA and ELISA, respectively.  As controls for the plasma human leptin assay (ELISA), a set of naïve C57BL/6j plasma samples were spiked with known concentrations of recombinant human leptin (46.2, 69.3, 104, 156, 234, 467, 1,400 pg/ml).  Taking into account the 10-fold dilution of these standards in assay buffer, the concentrations of the standards were expected to be 4.6, 6.9, 10.4, 15.6, 23.4, 46.7, and 140 pg/ml.  It was observed that, despite small differences from the expected concentrations based on single measurements, the assay exhibited a sensitivity close to that reported by the vendor, as it was able to detect human leptin at a concentration as low as ~20 pg/ml (Figure 8), supporting the use of this ELISA assay for detection of very low levels of plasma human leptin.  As negative controls, naïve ob/ob plasma samples collected from 3 individual mice were used.         68         Table II.  Human leptin assay comparison between Alpco Human Leptin (Ultrasensitive) RIA and Invitrogen Human Leptin ELISA.  Alpco Human Leptin RIA Invitrogen Human Leptin ELISA Cat No. 22-LEP-R40 KAC2281 Sensitivity 0.25 ng/ml 15.6 pg/ml Standards 0.25, 0.5, 1, 2, 4, 8, 16 ng/ml 15.6, 31.2, 62.5, 125, 250, 500, 1,000, 10,000 pg/ml Cross-reactivity 100% human, 0% mouse 100% human, 0% mouse Sample volume 100 µl 100 µl Sample number 100 samples 74 samples          69      Figure 8.  Human leptin ELISA positive controls prepared by spiking naïve C57BL/6j plasma samples with known concentrations of recombinant human leptin.  Each standard was assayed in singlet.  The expected concentrations for standards A to G were 140, 46.7, 23.4, 15.6, 10.4, 6.9, 4.6 pg/ml, respectively.  Naïve ob/ob plasma samples were collected from from 3 individual mice and measured as negative control. Red dotted line denotes the limit of detection for the assay (~ 20 pg/ml).  Invitrogen Human Leptin ELISA (KAC2281, Invitrogen; Carlsbad, CA) was used.      70 DATA ANALYSIS Data are presented as mean ± standard error of the mean (SEM) or mean + SEM.  Statistical significance was assessed using a Student’s t-test (unpaired, two- tailed) or analysis of variance (ANOVA).  Two-Way ANOVA was followed by the Bonferroni post-tests to further evaluate the significance between groups.  Statistical significance was set at the 5% confidence interval where *P < 0.05, **P < 0.01, and ***P < 0.001.  Data analysis was performed using the Prism software package (GraphPad Software Inc.; La Jolla, CA).                 71 RESULTS Study 1: Cloning and In Vitro Characterization of Plasmid Constructs 1.1 DNA Sequencing of Leptin cDNA Since PCR was used to engineer two additional restriction sites flanking the human leptin cDNA and to amplify the DNA during the cloning procedure of the EF1α-Lep plasmid construct, it was critical to assess the DNA sequences of the human leptin PCR product before any subsequent use of the construct.  Sequencing results showed that there was a 100% alignment between the human leptin cDNA (504 bp) in the EF1α-Lep construct and the Homo sapiens leptin coding sequence deposited in the Genbank database (accession: NM_00230; version: NM_00230.1, GI: 4557714).  1.2  Human Leptin Protein Secretion From Transfected STC-1 and HEK 293T Cells Transfection of STC-1 cell line Following the verification of the leptin cDNA sequence in the EF1α-Lep construct, in vitro transfection experiments were conducted in two different cell lines, STC-1 and HEK 293T, to test the functionality of the plasmid construct.  The presence of human leptin protein in the cell supernatant samples collected from transfected cells can confirm the expression of the leptin transgene as well as the proper translation and secretion of the leptin protein.  By ELISA, human leptin protein was detected at 10.5 ± 1 ng/ml in cell supernatant samples collected from STC-1 cells transfected with EF1α-Lep, whereas no human leptin protein was detectable in samples collected from those transfected with CMV-EGFP (enhanced green fluorescent protein gene under the control of a CMV promoter) as negative control (Figure 9). 72    Figure 9.  Human leptin protein was detected in cell supernatant samples collected from cells transfected with human leptin-expressing plasmid constructs. LipofecamineTM 2000 was used to transfect (A) EF1α-Lep into STC-1 cells and (B) CMV- or EF1α-Lep into HEK 293T cells.  In (A), cell supernatant samples were collected 48 hrs post transfection and assayed neat for human leptin by ELISA (Linco Research Inc., MO).  In (B), cell supernatant samples were collected 48 hrs post transfection and diluted 1:50 with assay buffer for human leptin measurement by RIA (Alpco Diagnostics, NH).  Each transfection condition was performed in triplicates. Values are expressed as mean ± SEM.  Statistical difference is denoted as **P < 0.01 by Student’s t-test (unpaired, 2-tailed).  A. B. 73 Transfection of HEK 293T cell line In another cell line, HEK 293T, human leptin protein was detected at 200 ± 29 and 450 ± 40 ng/ml in supernatant samples of cells transfected with CMV-Lep and EF1α- Lep, respectively, while no human leptin protein was detectable in supernatant samples collected from cells devoid of transfection (Figure 9).   Since the use of a ubiquitous cellular promoter such as EF1α minimizes the possibility of differential promoter activation between cell lines, the difference between the levels of human leptin detected in transfected STC-1 and HEK 293T cells could be attributed to variations in cell density and health at the time of transfection, the species origin of the cell lines, and, more critically, in the transfection efficiency between these two cell lines.  Nevertheless, the presence of the human leptin protein in cell supernatant samples post transfection with EF1α-Lep in STC-1 cell line and with CMV- or EF1α-Lep in HEK 293T cells indicates that 1) the leptin plasmid constructs are compatible with cellular transcriptional and translational machinery and 2) the resulting protein product is able to be secreted by cells uptaking the human leptin constructs.  1.3  Functional Leptin Protein Detection by Cell-Based Bioassay Immunoreactivity of the human leptin protein, shown by the ELISA and RIA results above, while an important indicator of the protein’s presence, does not provide any definitive evidence for its biological function, which depends largely on complete processing and proper folding of the protein.  In order to determine if the human leptin protein secreted by cells transfected with leptin-expressing constructs was biologically functional, a cell-based bioassay was employed to assess the ability of the protein found in the cell supernatant samples, collected from the above transfection experiments, to activate leptin signalling pathway.  If the human leptin protein, 74 expressed by either the CMV- or EF1α-Lep plasmid construct, was processed and folded correctly, it is expected that it will bind to the long form of the leptin receptor, ObRb, and activate the leptin signalling pathway, one of the most critical steps of which is the phosphorylation of Signal Tranducer and Activator of Transcription 3 (STAT3) (Figure 5).  Indeed, it was found that by incubating ObRb-overexpressing CHO cells with cell supernatant samples collected from HEK 293T cells transfected with either the CMV- or EF1α-Lep plasmid construct, leptin signalling was successfully activated as indicated by the phosphorylation of STAT3 (pSTAT3) shown by western blot, similar to that found with CHO-ObRb cells stimulated by recombinant murine leptin peptide (rmLeptin) as positive control.  In contrast, media samples collected from untransfected HEK 293T cells did not result in the same STAT3 phosphorylation (Figure 10).           75   Stimulated by rmLep NC CMV EF1α CMV EF1α rmLep NC   Figure 10.  The human leptin protein expressed by either the CMV- or EF1α-Lep plasmid construct activates the leptin signalling pathway as indicated by the phosphorylation of STAT3 in CHO-ObRb cell line.  CHO-ObRb cells were stimulated (10 min) by 300 µl murine leptin peptide (rmLeptin; 250 ng/ml), cell supernatant samples collected from cells without transfection (NC; Negative control) or from those transfected with the CMV-Lep (CMV) or the EF1α-Lep plasmid construct (EF1α). Primary antibody - 1:1000 rabbit α-pSTAT3 primary antibody (Cell Signalling Technology; Danvers, MA); secondary antibody - 1:8000 donkey-α-rabbit HRP secondary antibody (GE Healthcare; Piscataway, NJ).  Each condition was performed in duplicate.  β-actin was used as the loading control.  The film exposure times for pSTAT3 and β-actin were 10 min. and 90 sec., respectively.    pSTAT3 β-actin 76 These results provide a solid basis for utilizing the leptin-expressing plasmid constructs in further in vitro and in vivo experiments.  Moreover, it is worthy of note that, in line with the high amino acid sequence homology (84%) between murine and human leptin and previous literature on leptin signalling activation [112, 114], the human leptin protein expressed by either the CMV- or EF1α-Lep plasmid constructs in our experiment was able to bind to the murine version of the leptin receptor in CHO-ObRb cells and activate downstream leptin signalling pathway, further warranting the use of these plasmid constructs in subsequent experimentation in mice.  1.4  SEAP Protein Secretion from Transfected HEK 293T Cells SEAP-expressing plasmid constructs under the control of different promoters, gWIZTM, CMV, and EF1α, were tested in vitro and the strengths of the promoters were compared.  HEK 293T cells transfected separately with the three plasmid constructs by LipofectamineTM 2000 secreted similar levels of SEAP into the cell supernatant (162166 ± 21612, 189027 ± 15190, and 169133 ± 15360 ng/ml for gWIZTM-, CMV-, and EF1α- SEAP, respectively), while no SEAP was detected in cell supernatant samples collected from non-transfected cells, demonstrating that all three plasmids can be expressed by the cells and suggesting that the three promoters are approximately equal in their strength to drive the expression of the SEAP transgene in vitro (Figure 11).       77       Figure 11.  Similar levels of the SEAP protein were detected in cell supernatant samples collected from HEK 293T cells transfected with SEAP-expressing plasmid constructs under the control of the gWIZTM, CMV, and EF1α promoter.  Cell supernatant samples were collected 48 hrs post transfection and diluted 1:20 with HG-DMEM for SEAP measurement by SEAP Chemiluminescent Assay (Roche Applied Science; Indianapolis, IN).  Each transfection condition was performed in triplicate.  Values are expressed as mean ± SEM.  NS (no statistical significant difference): P > 0.05 by Student’s t-test (unpaired, 2-tailed).       78 1.5  Ф31-mediated long term SEAP gene expression in LipofectamineTM- transfected cells  The capacity of ФC31 in providing sustained transgene expression over time was examined in vitro.  EF1α-SEAP containing the attB site was transfected into HEK 293T cells with or without the addition of the expression vector for ФC31 integrase (CMV-Int) using LipofectamineTM 2000.  Transfected cells were passaged every 6 days and continued in culture for a period of ~1 month.  For the co-transfection groups, various ratios (5:1, 1:1, and 1:5) of EF1α-SEAP to CMV-Int plasmids were used, while the total amounts of DNA transfected were held constant at 2 µg.  The single transfection groups received either 2 or 1 µg EF1α-SEAP alone, matching the molar concentrations of EF1α-SEAP given for the 5:1 and 1:1 ratio co-transfection groups, respectively.  Two days after transfection, cell secreted SEAP protein that achieved 91230 ± 4478, 57787 ± 5708, 22505 ± 1228 ng/ml in the 5:1, 1:1, and 1:5 groups, respectively, while at the same time, cells transfected with 2 and 1 µg EF1α-SEAP alone secreted slightly higher levels of SEAP at 109474 ± 8032 and 100416 ± 9639 ng/ml, respectively (Figure 12A).  79  Figure 12.  Long term SEAP gene expression mediated by ФC31 integrase.  (A) Significantly higher levels of SEAP were detected in the cell supernatant of HEK 293T cells co-transfected with the attB-bearing EF1α-SEAP and CMV-Int plasmids than those transfected with EF1α-SEAP alone on (B) Day 11, (C) 17, (D) 23, and (E) 29 post- transfection.  Transfection was performed using LipofectamineTM 2000.  Different SEAP to ФC31 (Int) ratios were tested for the co-transfection groups.  In all cases, the total of DNA transfected was held constant at 2 µg.  Values are expressed as mean ± SEM. Statistical differences between Int and Int-minus groups with equal molar amounts of the SEAP plasmid are denoted as *P < 0.05 and **P < 0.01 by Student’s t-test (unpaired, 2-tailed).  Leptin-expression DNA (EF1α-Lep) was transfected as negative control (NC). A. B.  C. D.  E. 80 At Day 5 post-transfection, all groups, co- or singly-transfected, secreted approximately equal amounts of SEAP at ~10000 ng/ml.  Interestingly, by Day 11, a clear separation in SEAP levels was noticed between cells receiving ФC31 and those that did not.  At this time point, while SEAP secretion from co-transfected cells was maintained at levels that were 7.6 and 18.7% (5:1 and 1:1 groups, respectively) of those measured at Day 2, it was detected at only ~3% of the Day 2 values for the singly-transfected cells (Figure 12B).  Furthermore, at all later time points on Days 17, 23, and 29, significant differences were found between ФC31-plus and ФC31-minus groups at equal molar concentrations of EF1α-SEAP (Figure 12C-D).  At the end of the experiment at Day 29, an average 3-fold elevation in SEAP secretion level was preserved in the ФC31- transfected groups (5:1 and 1:1) compared to groups (2 and 1 µg) that were never exposed to ФC31 (~753 vs. 247 ng/ml for ФC-plus and ФC-minus groups, respectively), demonstrating the advantage of the integrase in mediating long term transgene expression.  Study 2: Synthesis and Quality Control of Chitosan Nanoparticles 2.1 Low Molecular Weight Chitosan Nanoparticles for SEAP Reporter Gene Transfer  Low molecular weight chitosan (5 kDa) was used to synthesize three batches of nanoparticles encapsulating various plasmid constructs for in vivo SEAP reporter gene transfer.  The first batch (batch no. 112-18) of nanoparticles, containing the gWIZ-SEAP plasmid construct, was formulated at pH 4.8, while the second and the third batches (batch no. 112-66c and 112-66d) of nanoparticles, containing the EF1α-SEAP plasmid construct, were formulated at pH 4.8 and 4.0, respectively.  Following synthesis, the 81 nanoparticles were subjected to a series of quality control procedures, first of which was a gel motility test.  DNA encapsulated by chitosan was retained at the loading well while naked DNA moved freely in the agarose gel by electrophoresis (Figure 13).    Figure 13.  Complete encapsulation of EF1α-Lep/ CMV-Int, shown by DNA motility retardation in agarose gel (lane 4), was achieved by complexing the DNA with chitosan. Gel motility test was conducted by running nanoparticles on a 0.7 % agarose gel by electrophoresis.  Unbound DNA was used as negative control.  The same amount (1.5 µg) of DNA was loaded across lanes 2 - 4.  Lane 1: 1 kb ladder.  Lane 2: EF1α-Lep naked DNA.  Lane 3: CMV-Int naked DNA.  Lane 4: chitosan nanoparticles containing EF1α-Lep and CMV-Int.   82 This retardation of DNA motility seen with nanoparticles was an indicator of DNA encapsulation by chitosan.  Next, the size, polydispersity, and zeta potential of the nanoparticles were measured.  It was observed that these nanoparticles averaged 107 ± 1, 137 ± 1, and 133 ± 1 nm in diameter, contained a polydispersity index of 0.14 ± 0.01, 0.17 ± 0.01, and 0.16 ± 0.01, and carried a zeta potential of 30.4 ± 0.1, 33.7 ± 0.6, and 34.9 ± 0.6 mV in batches 112-18, 112-66c, 112-66d, respectively (Figure 14A).                     83    Figure 14.  Size, polydispersity index, zeta potential, in vitro testing results of synthesized nanoparticles.  (A) Three batches of nanoparticles were produced and characterized by size, polydispersity, through dynamic light scattering (DLS), and zeta potential, through electrophoretic light scattering (ELS), using a Malvern Zetasizer (Malvern Instruments, MA).  Nanoparticles were diluted 1:20 with 10 mM filtered NaCl before measurements, which were taken in triplicates for size and polydispersity and in quadruplicates for zeta potential at 25 ºC.  Values are expressed as mean ± SEM.  (B) Nanoparticles (pH 4.8) encapsulating 2 µg SEAP-expressing plasmid constructs under the control of either the gWIZ or the EF1α promoter were used to transfected HEK 293T cells.  Nanoparticles (pH 4.0) encapsulating the same amount of EF1α-SEAP were also used in the transfection experiment.  Cell supernatant samples were collected 48 hrs post transfection and diluted 1:20 with HG-DMEM for SEAP measurement by SEAP Chemiluminescent Assay (Roche Applied Science; Indianapolis, IN).  Water and EF1α- Lep plasmid were used as negative controls (NC).  Each transfection condition was performed in triplicate and media samples were assayed in duplicate.  Values are expressed as mean ± SEM.  Statistical differences are denoted as *P < 0.05 by Student’s t-test (unpaired, 2-tailed). A. B. 84 When used to transfect HEK 293T cells, the nanoparticles were able to transport the plasmid DNA into the nucleus for gene expression, resulting in the secretion of the SEAP protein into the cell supernatant.  Significantly higher levels of SEAP were measured in cell supernatant samples collected from cells transfected by gWIZTM- SEAP-containing nanoparticles than those transfected by EF1α-SEAP-contaning nanoparticles.  However, no difference in SEAP levels was found between supernatant samples collected from cells transfected by EF1α-SEAP-contaning nanoparticles synthesized at pH 4.8 and by those synthesized at pH 4.0, suggesting that the two pHs used for nanoparticle formulation here did not contribute to significant changes in transfection efficiency in vitro (Figure 14B).  Additionally, for a separate follow-up experiment investigating the effect of ФC31 integrase on long term transgene expression, two other batches of nanoparticles were also synthesized.  One of these batches (Nano 9) encapsulated both the EF1α-SEAP and the CMV-Int plasmid constructs and the other batch (Nano 8) encapsulated only EF1α-SEAP without CMV-Int.  The nanoparticles, all synthesized at pH 4.8, averaged 144 ± 2 and 149 ± 1 nm in diameter, contained a polydispersity index of 0.24 ± 0.01 and 0.20 ± 0.01, and carried a zeta potential of 34.7 ± 0.5 and 34.0 ± 0.7 mV in batches Nano 8 and 9, respectively, possessing comparable physiochemical properties to previous batch of nanoparticles (122-66c) formulated at the same pH (Figure 15).       85        Figure 15.  Nanoparticles synthesized for follow-up SEAP reporter gene transfer study possess comparable physiochemical properties to those of the nanoparticles used in the first i.p. SEAP gene transfer study.  Nanoparticles were produced and characterized by size, polydispersity, through dynamic light scattering (DLS), and zeta potential, through electrophoretic light scattering (ELS), using a Malvern Zetasizer (Malvern Instruments, MA).  Nanoparticles were diluted 1:20 with 10 mM filtered NaCl before measurements, which were taken in triplicate for size and polydispersity and in quadruplicate for zeta potential at 25 °C.  Values are expressed as mean ± SEM.    86 2.2 Low and High Molecular Weight Chitosan Nanoparticles for Leptin Transgene Transfer  High molecular weight (HMW) chitosan has been shown to form more stable nanoparticles with DNA due to its long polymer chain and excessive amount of positive charges, offering improved protection of DNA for in vivo delivery [80].  To study the effect of HMW chitosan on nanoparticle transfection efficiency, two types of HMW chitosan, 226 (Nano 11) and 429 kDa (Nano 12), were utilized to synthesize nanoparticles encapsulating CMV-Lep and CMV-Int.  The HMW chitosan nanoparticles averaged 1052 ± 45 and 1043 ± 11 nm in diameter, contained a polydispersity index of 0.79 ± 0.08 and 0.50 ± 0.02, and carried a zeta potential of 40.8 ± 0.6 and 40.0 ± 0.8 mV in batches Nano 11 and 12, respectively.  The diameter, polydispersity index, and zeta potential for the negative control 429 kDa chitosan nanoparticles (Nano 13) containing CMV-SEAP and CMV-Int were 1136 ± 59 nm, 0.78 ± 0.10, and 45.2 ± 0.9 mV.  Not surprisingly, based on their longer polymer chain, it was found that these HMW chitosan nanoparticles were larger in size, more polydispersed, and more positively charged compared to their low molecular weight (5 kDa) counterparts, which measured 126 ± 1 nm in diameter, contained a polydispersity index of 0.22 ± 0.01, and carried a zeta potential of 33.8 ± 0.1 mV in batch Nano 10 (Figure 16).        87    Figure 16.  High molecular weight (HMW) chitosan nanoparticles are larger, more polydispersed, and more positively charged than low molecular weight chitosan nanoparticles.  (A) Nanoparticles were synthesized by complexing low molecular weight (5 kDa) or HMW chitosan (226 and 429 kDa) with CMV-Int and CMV-Lep-WPRE-attB (red) or CMV-SEAP-WPRE-attB (blue) plasmid DNA at a concentration of 250 µg/ml. Size and polydispersity of nanoparticles were measured by Dynamic Light Scattering (DLS) and zeta potential was measured by Electrophoretic Light Scattering (ELS), both using Malvern Zetasizer (Malvern Instruments, MA).  Values are expressed as mean ± SEM.  (B) Nanoparticles, synthesized with different chitosan MW, encapsulating 2 µg CMV-Lep/CMV-Int were used to transfect HEK 293T cells.  Nanoparticles (429 kDa) containing CMV-SEAP/CMV-Int were used as negative control.  Cell supernatant samples were collected 48 hrs post transfection and diluted 1:50 with assay buffer for human leptin measurement by RIA (Alpco Diagnostics, NH).  Each transfection condition was performed in triplicate.  Values are expressed as mean ± SEM. Statistical differences are denoted as ** P < 0.01, *** P < 0.001 by Student’s t-test (unpaired, 2-tailed). A. B. 88 Interestingly, the nanoparticles performed quite differently in terms of in vitro transfection efficiency.  HEK 293T cells secreted the highest level of SEAP into the cell supernatant when transfected by the 5 kDa chitosan nanoparticles, followed by those transfected by the 429 kDa chitosan nanoparticles, with those transfected by the 226 kDa chitosan nanoparticles secreting the lowest level of SEAP.  This may suggest that low molecular (i.e. 5 kDa) chitosan nanoparticles are more efficient at transporting DNA in vitro due to more rapid DNA release from the weakly assembled chitosan-DNA complexes formed by low molecular weight (LMW) chitosan.  2.3 Ф31-mediated long term SEAP gene expression in nanoparticle- transfected cells  To evaluate ФC31’s ability to support long transgene expression, the expression vector for the integrase (CMV-Int) was co-packaged into chitosan nanoparticles along with the attB site-containing EF1α-SEAP at a SEAP to Int ratio of 5:1.  HEK 293T cells were transfected with nanoparticles containing either double plasmids (SEAP and Int) or single plasmid (SEAP).  The total amounts of transfected DNA were 2 µg for both groups.  Transfected cells were maintained in culture for a period of ~1 month, during which cell passaging took place every 6 days.  Transfected cells secreted similar levels of SEAP into the supernatant between the ФC-plus (14267 ± 980 and 2703 ± 230 ng/ml) and ФC-minus (12251 ± 1137 and 2167 ± 92 ng/ml) groups at Day 2 and 5 post- transfection (Figure 17).     89        Figure 17.  Transfection with nanoparticles co-packaging attB-bearing EF1α-SEAP and CMV-Int plasmids resulted in long term expression of the SEAP reporter gene in HEK 293T cells.  Values are expressed as mean ± SEM.  Statistical difference is denoted as *** P < 0.001 by Student’s t-test (unpaired, 2-tailed).         90 However, at Day 11, while cells transfected with EF1α-SEAP alone secreted levels of SEAP that were 97% less than the measurement taken at Day 5, cells that were exposed to ФC31 produced amounts of SEAP that were still more than 40% of the Day 5 values.  Remarkably, whereas SEAP secretion was completely abolished in the ФC- minus group, it was continued to be measured in the ФC-plus group at Day 23 and 29 post-transfection (380 ± 30 and 178 ± 80 ng/ml), suggesting that the effect of long term transgene expression attributable to ФC31 integrase can be achieved by chitosan nanoparticle transfection.  Study 3: In Vivo Gene Transfer of SEAP Reporter Gene  3.1 Delivery Route and Dosing – Preliminary Studies  The route of delivery is a critical factor determining the efficacy of gene transfer. With limited restriction on the target cell type, chitosan can be applied to transfer exogenous genetic materials in vivo via a variety of delivery routes, including intrasplenic (i.s.) and intraperitoneal (i.p.) injections.  A preliminary study was conducted to test the efficiency of gene transfer via these two delivery routes, through which nanoparticles encapsulating the gWIZ-SEAP plasmid construct were administered to C57BL/6j mice.  Maximum volumes, 100 µl and 2.5 ml for i.s. and i.p. injection, respectively, of nanoparticles with a DNA concentration of 250 µg/ml were chosen for the study.  In the two treated mice, measurements of SEAP in the circulation 24 hrs post-treatment revealed that, with a DNA dose of 25 µg, i.s. nanoparticle injection achieved 4.2 and 15.6 ng/ml SEAP in mouse i.s. #1 and 2, respectively, while at the same time, no SEAP was detectable in the mouse (i.s. #3) receiving saline.  Mice receiving 625 µg of DNA through i.p. injection achieved circulating SEAP levels of 23 91 and 48 ng/ml in mouse i.p. #1 and 2, respectively, 24 hrs post-treatment, while no SEAP was detectable in the mouse (i.p. #3) receiving saline via the same administration route (Figure 18A).     `                   92   Figure 18.  Intrasplenic (i.s.) and intraperitoneal (i.p.) administration of SEAP plasmid- containing nanoparticles in C57BL/6j mice led to in vivo transgene expression as indicated by an increase in plasma SEAP activity post-treatment.  (A) The highest level of plasma SEAP activity post i.p. administration of nanoparticles was 3 times higher than that post i.s. administration (48 (i.p. #2) vs. 15.6 (i.s. #2) ng/ml).  Maximum nanoparticle delivery volumes were administered for each method.  (B) Similar levels of plasma SEAP activity, 353 and 371 ng/ml, were observed post i.p. administration of 50 and 250 µg DNA, respectively.  Increasing the DNA dose to 625 µg led to a dramatic decrease in plasma SEAP activity (23 and 48  n < 2.  Nanoparticle concentration of 250 µg/ml was used in all treatment groups.  NC: Negative control; saline injection. A. B. 93 These results suggest that, when compared at the maximal nanoparticle delivery volume for each delivery route, i.p. injection appeared to be more efficient at achieving higher transgene expression levels, and based on this along with the relatively non- invasive nature of the procedure, i.p. injection was chosen for the subsequent dosing study. In the dosing study, two C57BL/6j mice were treated, one receiving 200 µl and the other receiving 1 ml of nanoparticles containing gWIZTM-SEAP DNA doses of 50 µg and 250 µg, respectively, through i.p. injection.  Remarkably, it was found that, by reducing the DNA dose from 625 to 250 µg, a higher level of circulating SEAP was achieved 24 hrs post-treatment at 371 ng/ml, compared to the 23 and 48 ng/ml SEAP achieved with the 625 µg DNA dose.  More interestingly, a similarly high level of circulating SEAP, 353 ng/ml, could be achieved with an even lower DNA dose of 50 µg, suggesting that while a DNA dose of 625 µg may have a negative effect, a DNA dose as low as 50 µg may be sufficient to reach maximal gene transfer (Figure 18B). Collectively, these findings demonstrate the efficacy of in vivo gene transfer by chitosan nanoparticles via both i.s. and i.p. administration, between which the i.p. route gave rise to much higher transgene expression, providing a baseline for further experimentation of gene transfer through i.p. nanoparticle administration.  3.2 Transgene Expression Profile – gWIZTM Promoter Intraperitoneal (i.p.) administration Preliminary studies showed that chitosan-mediated gene transfer can be effectively accomplished via the i.p. route of administration.  Based on this, a new study was conducted aiming to document the transgene expression profile over time following 94 a single i.p. injection of the gWIZTM-SEAP-containing nanoparticles into C57BL/6j mice. A total of three DNA doses, 50, 25, and 12.5 µg, were tested.  Additionally, 50 µg of naked gWIZTM-SEAP plasmid DNA and saline were delivered in the same manner as negative controls.  Consistent with that shown in preliminary studies, transgene expression was observed 24 hrs post-treatment, indicated by an increase in circulating SEAP detected at 343 ± 60, 219 ± 72, and 125 ± 33 ng/ml in mice receiving 50, 25, and 12.5 µg DNA, respectively, highly elevated from the pre-treatment values of 0.05 ± 0.02, 0.04 ± 0.02, and 0.06 ± 0.02 ng/ml (Figure 19).  Figure 19.  Intraperitoneal (i.p.) administration of nanoparticles containing gWIZTM- SEAP mediated successful gene transfer as demonstrated by an increase in circulating SEAP post-treatment.  Nanoparticles encapsulating the gWIZTM-SEAP plasmid construct were administered i.p. to C57B6/J mice at DNA doses of 50, 25, and 12.5 µg. High levels of circulating SEAP were detected (* denotes significant difference between the 50 and 12.5 µg groups) 24 hrs post-treatment, after which a decrease in SEAP was observed.  Nanoparticle concentration of 250 µg/ml was used in the study.  Values are expressed as mean ± SEM.  Statistical differences are denoted as *P < 0.05 by two way ANOVA.           .  95 Although not statistically significant, the relative levels of SEAP seen in the different treatment groups suggest a dose-response to the amount of DNA administered. The circulating SEAP levels quickly diminished thereafter and became completely undetectable by Day 9 post-treatment.  Furthermore, long term tracking of circulating SEAP at Day 77, 89, 109, and 122 did not show any subsequent increase in SEAP levels in addition to the initial phase, suggesting short term transgene expression as a result of the transient activity of the gWIZTM (commercial CMV) promoter.  Alternatively, without transgene integration, the lack of persistent transgene expression could also result from the loss of the delivered plasmid over time.  Oral administration  In addition to i.p. injection, oral feeding is another attractive route of gene transfer owing to its minimized invasiveness and the capacity for easy repeat administration. Nanoparticles containing 75 µg of the gWIZTM-SEAP plasmid construct or acetate buffer were orally fed to C57BL/6j mice, whose blood samples were collected pre- and at Day 1, 3, 7, and 14 post-treatment for SEAP measurement.  The SEAP protein was detected in the circulation of 1 out of 4 mice, following oral nanoparticle administration, at concentrations of 76.7, 30.8, 3.9, and 2.2 ng/ml at Day 1, 3, 7, and 14 respectively, suggesting the possibility of SEAP gene transfer via the oral route of administration (Figure 20).      96      `  Figure 20.  Oral administration of the nanoparticles containing gWIZTM-SEAP mediated successful gene transfer in 1 out of 4 treated mice.  Nanoparticles containing gWIZTM - SEAP were administered at a DNA dose of 75 µg to overnight fasted C57BL/6j mice through an oral feeding needle.  Plasma samples were collected pre- and Day 1, 3, 7, and 14 post-treatment, and diluted 1:4 (Day 1) or 1:2 with assay buffer for SEAP measurement by SEAP Chemiluminescent Assay (Roche Applied Science; Indianapolis, IN).  Nanoparticle concentration of 250 µg/ml was used in the study.  Individual mouse data are shown in the graph.  NC: negative control; acetate buffer (pH 4.8) gavage.     97 Although the success of this mode of treatment appeared to be inconsistent, the presence of a similar pattern of transgene expression, characterized by an initial increase following a sharp decrease in SEAP levels, also observed in mice receiving the nanoparticles through i.p. injection, supports the likelihood of gene transfer in the one positive responder.  Nevertheless, more studies are required to better assess the efficacy of gene transfer via oral administration.  3.3 Transgene Expression Profile – EF1α Promoter Intraperitoneal (i.p.) administration  The gWIZTM promoter, a commercially available CMV promoter, is one of the strongest promoters able to give rise to high levels of transgene expression.  However, due to the possibility of CMV promoter silencing in vivo, a constitutively active cellular promoter, EF1α, was used to examine the potential to obtain long term transgene expression with the current gene transfer system.  EF1α-driven SEAP plasmid construct bearing an attB site for genomic integration was encapsulated into chitosan nanoparticles with the ФC31 integrase expression vector (CMV-Int) and administered via a single i.p. injection to C57BL/6j mice at DNA doses of 50 and 25 µg.  EF1α-SEAP and CMV-Int naked DNA (50 µg) and saline were administered as negative controls. Plasma samples were collected pre- and at Day 1, 3, 5, 7, 77, 89, 109, and 122 post- treatment for SEAP measurement.  In contrast to the initial high levels found with gWIZTM-SEAP gene transfer, circulating SEAP was detected at near background levels during the first 7 days post-treatment, but was later observed to increase and eventually peak (5.1 ± 0.8 and 5.5 ± 2.4 ng/ml) at Day 89 post-treatment in mice receiving 50 and 25 µg DNA, respectively (Figure 21). 98       Figure 21.  Intraperitoneal (i.p.) administration of nanoparticles containing EF1α-SEAP and CMV-Int directed potential long term transgene expression, as indicated by peak circulating SEAP at Day 89 post-treatment.  C57BL/6j mice received 50 or 25 µg DNA through a single i.p. nanoparticle injection, after which plasma samples were collected at Day 1, 3, 5, and 7 for short term and at Day 77, 89, 109, and 122 for long term circulating SEAP monitoring.  Peak SEAP levels, 5.1 ± 0.8 and 5.5 ± 2.4 ng/ml, were measured at Day 89 post-treatment in mice receiving 50 and 25 µg DNA, respectively. By the end of the study at Day 122, circulating SEAP remained at 4.1 ± 0.5 and 4.2 ± 1.3 ng/ml in the high and low DNA dosing groups, respectively.  Nanoparticle concentration of 250 µg/ml was used in the study.  Values are expressed as mean ± SEM.  Statistical differences between 50 µg DNA treatment and saline control are denoted as *P < 0.05 and ***P<0.001 by two way ANOVA.     99 Furthermore, by the end of the study at Day 122 post-treatment, circulating SEAP was found to remain at 4.1 ± 0.5 and 4.2 ± 1.3 ng/ml in the 50 and 25 µg DNA dosing groups, respectively, suggesting long-term transgene expression potentially as a result of ΦC31 integrase-mediated plasmid integration into the host genome.  However, in order to firmly attribute long term circulating SEAP to transgene genomic integration, a follow-up study was conducted to address the issues of assay background noise and to include a negative control group for ФC31 integrase.  Intraperitoneal (i.p.) administration – follow-up study In a follow-up study, a total of 75 plasma samples were collected from 15 mice on 5 separate days, 2, 3, 6, 8, and 11 days before treatment, for pre-treatment SEAP measurement.  The values were averaged (1.44 ± 0.03 ng/ml) to establish an assay background baseline to which post-treatment SEAP levels were compared.  Following the treatment, plasma samples were collected at Day 1, 3, 5, 7, 14, 28, and 56 for SEAP protein monitoring, which revealed low levels of SEAP in the blood of all treated mice (Figure 22).           100        Figure 22.  Long term monitoring of circulating SEAP in C57BL/6j mice following an intraperitoneal (i.p.) injection of chitosan nanoparticles carrying EF1α-SEAP with or without CMV-Int.  Mice received a DNA dose of 50 µg either in the form of nanoparticles or naked DNA.  Plasma samples were collected 2, 3, 6, 8, and 11 days pre- and 1, 3, 5, 7, 14, 28, and 56 days post-treatment for SEAP assay, for which the background level was determined to be 1.44 ng/ml based on the average of 75 pre-treatment values. Most post-treatment SEAP levels, with the exception of those at Day 7, were above this background level.  Nanoparticle concentration of 250 µg/ml was used in the study. Values are expressed as mean ± SEM.      101 While most circulating SEAP levels were above background levels (1.44 ± 0.03 ng/ml), some (Day 1: EF1α-SEAP ± CMV-Int, Day 7: all groups) were detected below this level.  With these low levels of transgene expression, it is difficult to determine whether or not gene transfer was effective.  However, similar levels of plasma SEAP, < 2.5 ng/ml, were also observed during the early time points (Day 1- 7) of the previous experiment (Figure 21), in which circulating SEAP (5.1 ± 0.8 ng/ml) did not peak until much later at Day 89 post-treatment.  Based on this and the upward trending of SEAP observed at the end of the current follow-up experiment (Day 56), it is possible that circulating SEAP could have gradually increased to levels matching that observed in the previous experiment at later time points.  However, since there was no difference in SEAP levels between the integrase and integrase-minus groups, long term (56 days) transgene expression observed in this study, if true, may have been due to extrachromosomal persistence of the plasmid DNA and the lasting activity of the EF1α promoter, rather than transgene genomic integration.  Study 4: In Vivo Gene Transfer of Human Leptin Transgene 4.1 Gene Transfer with Low Molecular Weight Chitosan Nanoparticles in ob/ob Mice Through Oral, Intraduodenal, and Intraperitoneal Administration Oral administration  The ob/ob mouse model presents a unique system for gene transfer studies as its obese and diabetic phenotypes, caused by a single mutation in the leptin gene, can be cured upon the restoration of plasma leptin, an advantage for the assessment of the efficacy of leptin gene transfer.  The human leptin plasmid construct under the control of 102 the EF1α promoter was encapsulated into chitosan nanoparticles with or without the expression vector for ФC31 integrase at plasmid concentrations of 50 and 250 µg/ml. These nanoparticles were orally fed to overnight fasted ob/ob mice for a total of 6 times across a period of two weeks.  Subsequently, water was administered to the same mice for the same frequency and duration similar to the nanoparticle treatment as negative control.  Four groups of ob/ob mice, 90 or 450 µg DNA dosing with or without integrase, at the age of 8 weeks were treated and monitored in terms of body weight and fasting blood glucose throughout the study.  During the initial treatment period, all mice showed an equal attenuation of weight gain, and therefore long term body weight comparison between mice receiving the integrase and those that did not was necessary to judge whether or not this observation was due to treatment success (Figure 23).           103   Figure 23.  Ob/ob mice receiving 6 oral administrations of human leptin plasmid- containing nanoparticles showed no significant differences in body weight and fasting blood glucose between the various treatment groups.  Body weight (A) and blood glucose (B) of treated mice were tracked for 112 days following treatment.  Mice were 8 weeks old at the initiation of the treatment.  Black arrows indicate oral nanoparticle gavage treatment, while gray arrows indicate oral saline gavage treatment.  Single DNA doses of 15 and 75 µg were administered 6 times throughout the nanoparticle treatment. Values are expressed as mean ± SEM. A. B. 104 It was anticipated that mice receiving nanoparticles containing EF1α-Lep plus CMV-Int would have the leptin gene integrated into the genome of gut cells, resulting in the retention of the leptin transgene and long term weight reduction in these mice. However, since this was not seen, the initial attenuation of weight gain was likely the result of the vigorous oral gavage regimen and not the success of leptin gene transfer. Further supporting this notion, no significant difference in fasting blood glucose was observed between the treatment groups receiving various amounts of DNA with or without integrase.  The failure to achieve gene transfer may be attributed to the harsh gastric environment and the long distance away from the intestinal cells for the nanoparticles by oral administration.  Thus, with the hope to increase gene transfer efficiency, in another experiment, nanoparticles were directly exposed to the cells in the upper intestine by intraduodenal injection, bypassing the stomach and shortening the distance from the target cells.  Intraduodenal administration A total of four groups of 9-week-old ob/ob mice were each subjected to intraduodenal injections of chitosan nanoparticles containing 10 µg of EF1α-Lep plus CMV-Int, 50 µg EF1α-Lep, 50 µg EF1α-Lep plus CMV-Int, or 50 µg gWIZTM-SEAP. Post-treatment body weight and fasting blood glucose of mice receiving the leptin transgene were compared to negative controls receiving the SEAP reporter gene. While a brief attenuation of weight gain and mild reduction in fasting blood glucose were observed, the occurrence of these events in both the treatment and negative control groups at the same time indicates that the changes in body weight and fasting blood glucose were attributable to surgical recovery and the end of the well-documented 105 transient hyperglycaemia of the ob/ob mice, respectively, independent of the efficacy of leptin gene transfer (Figure 24).   Figure 24.  Ob/ob mice receiving a single intraduodenal administration of human leptin plasmid-containing nanoparticles showed no significant differences in body weight and fasting blood glucose between the treatment and control groups.  Body weight (A) and blood glucose (B) of treated mice were tracked for 56 days following treatment.  Mice were treated at the age of 9 weeks indicated by the black arrow.  Nanoparticles containing gWIZTM-SEAP was administered as negative control (NC).  Values are expressed as mean ± SEM.  A. B. 106 It is possible that the failure to transfer the leptin gene into the target cells in the intestine is due to the hostile intestinal environment, constituted by a suite of digestive enzymes, preventing sufficient protection of the plasmid DNA by chitosan.  To avoid contact with the digestive enzymes, the i.p. route of chitosan nanoparticles delivery was examined for the efficiency of gene transfer next. Intraperitoneal (i.p.) administration Nanoparticles encapsulating either EF1α-Lep and CMV-Int or gWIZTM-SEAP were administered to two and one ob/ob mice, respectively, through a single i.p. injection, transferring 75 µg DNA.  The delivery of gWIZTM-SEAP was used as negative control. Body weight and fasting blood glucose were tracked for 40 days after treatment.  During this period, reduced body weight and lowered fasting blood glucose, which would be expected with the presence of plasma leptin, were not seen in mice receiving EF1α-Lep plus CMV-Int (Figure 25).         107    Figure 25.  Ob/ob mice receiving a single i.p. administration of human leptin plasmid- containing nanoparticles did not exhibit reduced body weight or lowered fasting blood glucose compared to negative control.  Body weight (A) and blood glucose (B) of treated mice were tracked for 40 days following treatment.  Mice were treated at the age of 9 weeks indicated by the black arrow.  Nanoparticles containing gWIZTM-SEAP were administered as negative control (NC).  A DNA dose of 75 µg was used.  Values are expressed as mean ± SEM.  A. B. 108 With the small number of mice tested, it is not easy to draw a conclusion from the results, but it appears that, in this case, either the leptin gene was not transferred effectively, or that the transgene expression level was not sufficient to contribute to phenotypic changes, the latter of which should be further addressed with more experiments. 4.2 Gene Transfer with High Molecular Weight Chitosan Nanoparticles in C57BL/6j Mice Through Intraperitoneal and Oral Administration Intraperitoneal (i.p.) administration  HMW chitosan has been shown to bind DNA with stronger electrostatic forces producing nanoparticles with greater stability [80].  To examine whether this enhanced stability offered by high molecular weight chitosan may improve gene transfer efficiency and whether the CMV promoter may provide more robust transgene expression than the EF1α promoter, HMW chitosan nanoparticles encapsulating the human leptin- expressing plasmid construct under the control of a CMV promoter (CMV-Lep) and CMV-Int were synthesized with 226 or 429 kDa chitosan and were administered intraperitoneally to C57BL/6j mice.  LMW (5 kDa) chitosan nanoparticles containing CMV-Lep/ Int and HMW (429 kDa) chitosan nanoparticles encapsulating CMV-SEAP/Int were administered as negative controls.  A total of two injections, a month apart each delivering 50 µg DNA, were given to all mice whose body weight and fasting blood glucose were monitored throughout the study.  In addition, blood samples were collected for plasma human leptin protein assessment.  Interestingly, despite observing a decrease in body weight and fasting blood glucose in all groups of mice 24 hrs after the first injection, a higher percent drop in body weight was seen in the 5 kDa group compared to negative control (7.3 ± 1 vs. 3.7 ± 1.5%) (Figure 26G). 109              A.  B. C.  D. 110      Figure 26.  Body weight and blood glucose tracking in C57BL/6j mice following i.p. administration of HMW chitosan nanoparticles containing leptin-expressing DNA.  (A and B) Body weight and blood glucose tracking are presented as data from all groups or (C and D) from only the 5 kDa treatment group and negative control, NC or (E and F) as individual mouse data from the 5 kDa treatment group and NC.  (G and H) Percent drop in body weight and blood glucose following the first treatment (Day 0) in the 5 kDa treatment group and NC.  Black arrows indicate injection treatments, each of which is a DNA dose of 50 µg.  n = 5 for all groups.  Mice were 7 weeks old at the start of treatment.  Measurements of body weight and blood glucose were taken after a 6 hr fast.  The percent drop in body weight and blood glucose is calculated by taking the difference between the mean values 24 hr pre-treatment and those 24 hr post-treatment, dividing it by the mean values 24 hr  pre-treatment, and multiplying by 100%.  Values are expressed as mean ± SEM.  A-D: Statistical differences between 5 kDa treatment and NC are denoted as *P < 0.05 by two way ANOVA.  G and H: Statistical differences are denoted as *P < 0.05 by Student’s t-test (unpaired, 2-tailed).   E.  F. G.  H. 111 Moreover, there was a concurrent drop in fasting blood glucose in the 5 kDa group significantly higher than that in negative control (34.4 ± 10.1 vs. 5.0 ± 7.6 %), suggesting that leptin may be elevated in the circulation of treated mice above physiological levels (2-3 ng/ml) at this time point as a result of gene transfer (Figure 26H).  However, the reduced body weight of the mice in the 5 kDa group as well as those in the other groups quickly returned to pre-treatment levels and began to incline steadily by Day 3 post-treatment.  Similarly, there was no difference between the 5 kDa group and negative control in fasting blood glucose after the initial separation at Day 1 post-treatment.  These observations of the brief reductions in body weight and fasting blood glucose are consistent with previous study in which circulating SEAP levels following gWIZTM-SEAP delivery was only transiently detected (Figure 19).  Curiously, the same responses were not seen after the second injection taking place a month after the first injection (Figure 26A-F).  Although this could potentially be explained by the differences in age (7 vs. 11 weeks) and growth rate of mice, it may also be that the initial responses following the first treatment were simply experimental artefact due to handling stress.  To address this issue, plasma samples before and after the injections were assayed for human leptin to generate a profile of the circulating transgene product. Plasma human leptin was detected at 7.3 ± 0.4, 7.5 ± 0.4, and 9.3 ± 0.9 pg/ml at Day 1 and 9.0 ± 0.8, 9.0 ± 0.7, and 10.3 ± 1.1 pg/ml at Day 3 post-treatment for the 5, 226 and 429 kDa treatment groups, respectively. However, these values were all below the assay background (~20 pg/ml) and were no different than those detected in negative controls at Day 1 and 3 (14.8 ± 5.6 and 9.6 ± 0.8 ng/ml, respectively) (Figure 27), supporting the hypothesis that the transient drop in body weight and fasting blood glucose were experimental artefacts.  112         Figure 27.  Plasma human leptin levels in mice receiving i.p. administration of nanoparticles encapsulating human leptin-expressing DNA (CMV-Lep).  Blood samples were collected at Day 1 pre- and Day 1 and 3 post-treatment in mice administered with nanoparticles i.p. for human leptin assay.  No human leptin was detectable above the assay background (~20 pg/ml).  NC: negative control.  Values are expressed as mean ± SEM.      113 Oral administration  In addition to the i.p. route, HMW chitosan nanoparticles were also administered orally to C57BL/6j mice to study the potential to achieve oral gene transfer using HMW chitosan.  For the oral route, mice were divided into 4 groups identical to the i.p. HMW chitosan gene transfer experiment but were administered with 75 µg DNA per treatment for a total of six times through oral feeding.  Mice were overnight fasted before treatment and water restricted for 2 hrs before and after treatment.  After oral nanoparticle feeding, tracking of body weight and fasting blood glucose revealed no differences between the treatment groups and negative control (Figure 28).     Figure 28.  Body weight and blood glucose tracking in C57B6 mice following oral administration of chitosan nanoparticles containing leptin-expressing DNA.  Black arrows indicate oral administrations, each of which delivered a DNA dose of 75 µg. Mice were 8 weeks old at the start of treatment. Measurements of body weight and blood glucose were taken after a 6 hr fast.  NC: negative control.   114 For this reason, plasma human leptin levels were required to assess whether the treatments were inefficient (i.e. leptin slightly above physiological levels, 1-2 ng/ml, but not enough to cause changes) or ineffective (i.e. no leptin above physiological levels). Plasma samples 1 day following the last of the 6 oral treatments were assayed for the 5 kDa treatment group and negative control, showing human leptin levels of 15.6 ± 1.4 and 14.3 ± 0.2 pg/ml, respectively.  Falling below the assay background, these values, similar to those found with the i.p. route, suggest ineffective oral gene transfer (Figure 29).   Figure 29.  Plasma human leptin levels in mice receiving oral administration of nanoparticles encapsulating human leptin-expressing DNA (CMV-Lep).  Blood samples from the 5 kDa group and negative control were collected 1 day after the last of the 6 oral nanoparticle treatments for human leptin assay, which showed 156.2 ± 14.3 and 143.0 ± 2.2 pg/ml for the 5 kDa group and negative control, respectively.  No human leptin was detectable above the assay background (~20 pg/ml).  NC: negative control. Values are expressed as mean ± SEM.  NC: negative control.  Values are expressed as mean ± SEM.  115 DISCUSSION  Promoter Selection for Transgene Expression In Vivo  In many gene therapy applications, long term expression of the transgene is desired to improve therapeutic effects and minimize repeat administrations.  However, most gene transfer results in short-lived transgene expression.  Previous studies indicated that this is not due to the loss of plasmid DNA itself but primarily a function of the expression vector capable of remaining active with time [158].  Herweijer et al. observed that, by Day 7 after intraportal injection of a luciferase reporter gene driven by the CMV promoter, the luciferase levels dropped to 0.02% of the initial robust luciferase gene expression (3500 ng luciferase per liver) measured at Day 1.  On the other hand, despite the low initial expression levels (10 ng luciferase per liver), as much as 64% of the Day 1 luciferase activity was preserved at Day 7 when the liver-specific albumin promoter was used to drive the transgene expression, suggesting the influence of promoters on transgene expression [158].  Viral promoters, such as the CMV promoter, generally exhibit a characteristic expression profile, illustrated by very high levels of transgene expression 1 day after treatment, followed by a drastic diminishment within the first week of treatment.  This is in sharp contrast to transgene expression patterns derived from nonviral promoters, such as the albumin promoter, which tends to sustain low levels of expression [158].  Our data collected from the in vivo i.p. SEAP gene transfer studies using the CMV promoter (gWIZTM)-driven SEAP plasmid construct correspond well to these expression patterns.  In this case, we demonstrated that i.p. injection was able to achieve effective SEAP gene transfer, which resulted in high circulating SEAP (343 ± 60 ng/ml) 1 day post injection, after which it rapidly declined to 2% of this level by Day 7 and eventually became undetectable by Day 9 (Figure 19). 116  The mechanism of promoter inactivation remains poorly defined.  However, one possible reason to explain CMV-promoter silencing is the activation of the innate immune response by the unmethylated CpG motifs in the viral promoter, leading to the induction of cytokines, such as interferon γ, which inhibits CMV promoter activity [159, 160].  The use of constitutive cellular promoter might circumvent the viral promoter attenuation problem and therefore we explored the use of a human cellular promoter, EF1α, to obtain sustained transgene expression.  In vitro, we found that the EF1α promoter directed significantly higher human leptin gene expression than the CMV promoter (Figure 9B), but gave rise to similar levels of SEAP gene expression compared to the CMV and gWIZTM promoters in the HEK 293T cell line (Figure 11). Cell line-dependent activity of the EF1α promoter has been previously reported [161]. Our data suggest that the activity of this promoter may also be transgene-dependent. In vivo, in contrast to the strong short term activity of the gWIZTM promoter, following i.p. administration of chitosan-bound EF1α-SEAP, only background levels (~1 – 2 ng/ml) of the transgene protein were detected in the first week of the experiment.  This lack of prediction of in vivo expression performance based on in vitro observation is well established in the field of gene transfer [158, 162], stressing the necessity to test the expression vectors in vivo.  To select an expression vector capable of providing high and sustained levels of transgene expression, one may consider a number of previously described options of various combinations of promoter, intron, and 5’ and 3’ UTR.  For instance, by using the thyroid hormone-binding globulin promoter, two copies of the α1-microglobulin/bikunin enhancer, and a 71 bp leader sequence, factor VIII expression following adeno- associated virus gene transfer into the liver was sustained past 5 months after treatment [163].  In addition, Miao et al. described a vector, containing the α1-antitrypsin 117 promoter and the hepatic locus control region from the apolipoprotein E gene, that gave rise to therapeutic levels (0.5 – 2 µg/ml) of factor IX for over one and a half years following nonviral gene transfer [164].  Furthermore, long term (1 year) high levels of SEAP reporter gene expression, comparable to those obtained from the CMV promoter on Day 1 post-treatment, were recently demonstrated by using an optimal expression vector constructed with the albumin promoter, an α-fetoprotein MERII enhancer, 5’ intron from the factor IX gene, and the 3’UTR from the albumin gene including intron 14 [165].  In addition, use of the human polyubiquitin C (UbC) promoter successfully avoided transcriptional silencing following intranasal instillation of lipid/DNA complexes and resulted in sustained luciferase gene expression in the mouse lung for at least 8 weeks post-treatment [166].  Taken together, by choosing appropriate regulatory elements, the activity of a promoter can be tailored to direct persistent long term transgene expression.  Evaluation of Potential Long Term Transgene Expression Conferred by Nonviral Gene Transfer  In the current studies, we investigated a ФC31-based transgene genomic integration approach to obtain long term transgene expression.  Calos’ group has performed comprehensive molecular analysis on the phage ФC31 integrase and showed that it is capable of integrating the transgene plasmid into the host genome at specific sites, resulting in stable transgene expression [89, 90, 92].  Our in vitro data demonstrated that co-transfection of the attB site-bearing EF1α-SEAP plasmid and the expression vector for ФC31 integrase (CMV-Int) into HEK 293T cells using LipofectamineTM 2000 resulted in prolonged expression of the SEAP reporter gene for a period of ~1 month, indicated by the significantly higher SEAP contents detected in the 118 supernatant compared to cells receiving EF1α-SEAP alone over time (Figure 12).  We subsequently co-packaged EF1α-SEAP and CMV-Int into chitosan nanoparticles and evaluated the nanoparticles in a long term transfection experiment, which showed that while SEAP expression was entirely eliminated in cells receiving nanoparticles containing EF1α-SEAP alone by Day 17, it was sustained until the end of the experiment nearly 1 month post-transfection in cells given EF1α-SEAP in addition to the ФC31 expression vector by chitosan nanoparticles (Figure 17).  The success of ФC31 in mediating long term transgene expression in culture prompted us to further examine the CMV-Int-containing nanoparticles in mice.  For the in vivo study, nanoparticles co- encapsulating EF1α-SEAP and CMV-Int were administered to C57BL/6j mice via i.p. injection.  Transgene expression was measured at the protein level by tracking the SEAP protein in the circulation for up to 122 days post treatment.  Minimal SEAP levels (1 – 2 ng/ml) were detected during the first week, but eventually peaked (5.5 ± 2.4 ng/ml) at Day 89 post-treatment, suggestive of long term transgene expression (Figure 21).  To evaluate if this was due to ФC31, a follow-up study was conducted treating mice with or without ФC31.  Consistent with the previous study, low levels SEAP expression were measured during the first week post-treatment.  Long term monitoring, however, revealed an elevating trend of SEAP by the end of the study at Day 56 (Figure 22). Nevertheless, unlike the observation in vitro, a clear distinction in transgene expression levels as a result of the inclusion of ФC31 was unable to be discerned.  This might be related to inadequate levels of plasmid integration efficiency that is partially dependent on the transgene-to-integrase plasmid ratio.  When co-delivering attB-containing plasmids expressiong fumarylacetoacetate hydrolase (FAH) with ФC31 expression vectors at a 1:1 ratio, Held et al. measured significant FAH transgene integration frequency, based on the number of FAH-positive nodules in liver sections following 119 gene transfer, while integration frequencies were almost undetectable when the transgene-to-integrase ratios were modified to 5:1, 10:1, and 20:1 [90].  Furthermore, long term factor IX and dystrophin expressions in the liver and muscles, respectively, were also achieved with a transgene-to-integrase ratio of 1:1, suggesting a potential role of the balance of the amount of the delivered plasmids in mediating sustained transgene expression in vivo [89, 93].  Thus, the 5:1 SEAP-to-integrase ratio used in our study might not have been optimal to impart sufficient integration efficiency necessary for long term transgene expression.  On the basis of this hypothesis, the SEAP expression detected almost 2 months after treatment at levels similar between mice receiving ФC31 and those that did not is possibly due the persistent activity of the EF1α promoter rather than transgene genomic integration by ФC31.  This is a likely scenario as plasmid DNA can endure for long periods of time following gene transfer into the hepatocytes [158], which turn over with a half-life of approximately 120 days [167].  With i.p. injection, despite the lack of targeting specificity, plasmid DNA can certainly be transferred into the liver [108].  In terms of expression kinetics, it is unclear why the EF1α promoter gave rise to a delayed increase in gene expression for a period of time before the SEAP levels peaked (Figure 21, 22), but other promoters, such as the albumin promoter, have also been observed to direct a gradual increase of transgene expression after gene delivery [158].  This may be related to the cellular roles of the proteins whose expression these promoters are responsible for endogenously. EF1α, or polypeptide elongation factor 1 α, is a ubiquitously expressed protein that plays a key role in the elongation cycle during translation.  It is also involved in cytoskeleton reorganization, proliferation, actin bundling, and microtubule severing and is associated with the centrosome and mitotic machinery [168].  In short, EF1α regulates cellular functions that are both dependent on and independent of translational 120 controls.  Therefore, EF1α mRNA levels usually increase in rapidly growing cells [169], while decreased EF1α mRNA levels are found during cell differentiation [170].  In our studies, since the delivered plasmids presumably ended up mainly in post-mitotic cells, such as the hepatocytes, based on the endogenous functions of the EF1α protein, perhaps the EF1α promoter was only partially active while driving the expression of the SEAP transgene.  This led to the overall reduced levels of gene expression relative to those observed with the gWIZTM (CMV) promoter.  The slow increase in SEAP levels may then be explained by the turning over of the cells over time as cell division helps to raise the activity of the EF1α promoter.  During cell division, a fraction of the non- integrated DNA would be lost; however, those that remain may still contribute to transgene expression, which, with time, would eventually deline to background levels. In any case, to be therapeutically significant, sufficient transgene product is required, and the low levels of SEAP obtained with the EF1α vector in our studies speak to the importance of having a well-designed plasmid vector that is able to provide both high and sustained levels of transgene expression. It has been demonstrated that the dissociation of the transgene expression cassette from the plasmid bacterial backbone DNA, consisting of the origin of replication and antibiotic resistance gene, can confer sustained and high level expression of the transgene product [171].  Kay’s group undertook a study in which they cleaved a closed circular plasmid into two DNA fragments, liberating the human α1-antitrypsin (human AAT) expression cassette from the bacterial backbone DNA, and delivered the DNA into mice by tail vein injection.  The DNA was delivered in the form of intact circular DNA, 2- fragment DNA (expression cassette and bacterial backbone), or purified 1-fragment expression cassette.  They found that mice injected with the uncut circular plasmid expressed a high level of serum human AAT that declined by more than 3 logs within 4 121 weeks after treatment.  In contrast, mice receiving equal molar amounts of 2-fragment and 1-fragment DNA expressed 28- and 40-fold higher serum human AAT, respectively, over the same period, suggesting that the bacterial backbone has an inhibitory effect on transgene expression.  Since it was observed that the introduction of the 2-fragment DNA into the mice resulted in the formation of random concatemers, further experiments were conducted to differentiate whether the concatemers or the covalent linkage of the bacterial and expression cassette sequences were accountable for the decline in gene expression.  It was found, 3 weeks after DNA injection, that serum human AAT levels in mice receiving 2-fragment DNA were 20-fold higher than those in mice receiving single-cut linearized DNA, composed of the expression cassette covalently attached to the bacterial DNA, confirming that a covalent attachment of the bacterial and expression cassette DNA is required for the silencing effect to occur.  The authors further showed that this expression inhibitory effect remained regardless of what promoter/enhancer or bacterial origin of replication and antibiotic gene were used. With the development of a high yield, simple purification method of the shortened circular plasmid DNA, consisting only of the transgene expression cassette (i.e. minicricle DNA), from bacterial culture, minicircle DNA can now be applied to satisfy the need for high level and stable long term transgene expression in many gene transfer applications [172].  Nonviral Gene Transfer: Delivery Routes and Barriers to Overcome  As for conventional protein drugs, the delivery route for nucleic acid-based therapy is a crucial aspect for determining treatment success.  In order to evaluate the chitosan/ФC31 gene transfer system thoroughly, a number of delivery routes, including oral, intraduodenal, intrasplenic, and intraperitoneal administration, were explored. 122  For the oral route, chitosan nanoparticles encapsulating EF1α-Lep with or without ФC31 integrase were delivered to ob/ob mice through a total of 6 oral gavage treatments across 2 weeks.  To confirm the biological activity of the leptin protein expressed by EF1α-Lep, the construct was tested in vitro by a cell-based bioassay.  It was illustrated that the leptin protein was able to bind the leptin receptor and activate downstream signalling pathway, represented by the phosphorylation of STAT3, indicating that the protein was indeed biologically active (Figure 10).  This assay provided a basis for utilizing the human leptin-expressing construct in vivo as the DNA was able to direct the expression of leptin protein that is expected to exert biological functions should gene transfer be completed.  With leptin’s body weight and blood glucose lowering effects on the ob/ob mouse model, successful gene transfer would entail reductions in body weight and fasting glucose of the treated mice.  Our data did not show any decrease in body weight and fasting glucose in either the low (90 µg) or high (450 µg) DNA dosing groups, indicating ineffective leptin gene transfer (Figure 23). However, in another study where 75 µg gWIZTM-SEAP was delivered to C57BL/6j mice via a single oral administration of chitosan nanoparticles, successful SEAP reporter gene transfer, as indicated by an increase in circulating SEAP 1 day post-treatment, was achieved in 1 out of 4 treated mice (Figure 20).  Interestingly, consistent with the previously observed expression kinetics conferred by the gWIZTM promoter, the circulating SEAP declined drastically following the increase at Day 1 and became background levels 1 week after treatment in the positive responder.  This supports that, although an occasional finding, effective gene transfer via the oral route indeed occurred.  Nevertheless, the rare event of successful oral gene transfer highlights the considerable barriers encountered with this route of delivery. 123  The two main barriers posed by the gastrointestinal (GI) tract are: 1) the enzymatic barrier, caused by luminal secreted and luminal membrane-bound brush- border enzymes, and bile [110, 111, 173] and 2) the diffusion barrier, caused by the mucus layer covering GI epithelia [63].  It has been well-established that, without protection, naked DNA delivered through the oral route quickly becomes sheared by the nucleases abundant throughout the GI tract.  Although, in vitro, Chitosan-bound DNA is protected from DNase I degradation [85], the simplistic modeling of the GI lumen with a single enzyme is inadequate to reflect the true environment through which chitosan nanoparticles must travel.  It order to predict the stability of chitosan nanoparticles in the GI tract following oral administration, one must understand the biodegradation of chitosan.  Biodegradation is a process by which organic substances are disintegrated by enzymes produced by living organisms.  In the body, it is believed that chitosan is predominantly degraded by lysozyme (EC, a glycoside hydrolase found in many tissues and secretions such as tears, saliva, blood, and mucus, responsible for catalyzing the hydrolysis of the β-(1,4) glycosidic bonds within the chitosan polymer [174].  The rate of degradation is inversely proportional to the degree of deacetylation as lysozyme selectively targets N-acetylated chitosan.  In addition, chitosan depolymerization can also occur by acid hydrolysis.  Since the chitosan used in all our oral gene transfer studies was highly deacetylated (80 - 98%), acid hydrolysis may be more important in destabilizing chitosan nanoparticles than lysozyme-mediated degradation.  The pH-sensitive stability of chitosan/DNA nanoparticles may be further compromised by another hindrance in the duodenum - bile.  With constituents of cholesterol, bile pigment, bile acids, and phospholipids, bile can considerably damage the integrity of the nanoparticles.  Specifically, this bicarbonate ion-rich fluid, normally secreted upon trigger by acidic chyme, efficiently increases the pH of the nanoparticle 124 surrounding, thereby decreasing the forces of interaction between chitosan and DNA and weakening the nanoparticle stability.  Dai et al. found significant aggregation of chitosan/DNA nanoparticles with 10% rat bile exposure, expanding the size of nanoparticles by ~15-fold to 3.6 µm, a phenomenon that was attributable to the surface adsorption of negatively charged protein present in the bile in addition to bile’s pH buffering property, reducing the surface charge of the nanoparticles and thus decreasing inter-particle separation.  Further, a combination of bile-induced nanoparticle aggregation and degradation of particles and DNA led to a dramatic abolishment of in vitro transfection efficiency, indicated by a ~1000-fold decrease in luciferase reporter gene expression compared to transfection in the absence of bile [173].  As a result, the nanoparticle stability-undermining effects of bile should not be overlooked. The second GI barrier is the diffusion barrier, caused by the mucus layer covering GI epithelia.  Mucoadhesion of chitosan [62] can be a favourable feature because the restricted mobility of DNA carrying nanoparticles at the mucosal surface would result in: 1) a prolonged residence time at the site of absorption, 2) a placement of the delivery system at a given target site, 3) an increase in concentration gradient of the nanoparticles due to particle build up at the mucosal surface, and 4) a direct contact with intestinal cells which is the first step before nanoparticle uptake [63].  However, the duration of mucoadhesion is limited by the frequent renewal at the mucosal surface by a turnover process and the slow diffusion of nanoparticles across the mucus layer may hinder the nanoparticles from reaching the intestinal cells in time.  In this situation, while prolonging residence time, mucoadhesion may possess undesirable effects of entrapping particles, preventing them from arriving at cell surfaces, and causing them to be swept from the intestine.  On top of this, in the slightly alkaline conditions of the 125 intestine, modest amounts of chitosan actually remain positively charged, minimizing the proportion of intact nanoparticles and their capacity for mucoadhesion.  Given the many barriers faced with oral delivery, we hypothesized that direct regional administration and incubation of nanoparticles in a defined segment of the intestine may partially circumvent the oral delivery issues, mainly those created by gastric acidity, and be more effective at gene transfer.  Chitosan nanoparticles encapsulating EF1α-Lep with or without ФC31 integrase were surgically injected into ob/ob mice regionally inside the lumen of the duodenum, in which a 1 hr incubation period was allowed for nanoparticle uptake by intestinal cells.  After recovery, body weight and fasting blood glucose of the treated mice were assessed.  No difference in body weight and blood glucose was measured between mice receiving various doses (10 and 50 µg) of the leptin-expressing DNA and those receiving the high dose (50 µg) of the control vector, gWIZTM-SEAP (Figure 24).  Clearly, these results indicate that there may be additional factors other than gastric acidity and the distance between the oral cavity and target intestinal cells for the nanoparticles that hampered effective gene transfer.  In particular, the consequences of the dilution and mixing of the particle suspension with the GI tract fluid are worth much attention.  It has been suggested that the adsorption of lipases onto the surfaces of chitosan nanoparticles can lead to precipitation and rapid clearing of the particles [175].  In general, due to the defensive characteristics of the GI tract against foreign substances, gene transfer via the oral or intraduodenal route of administration remains challenging.  Despite the difficulty with oral and intraduodenal gene transfer, we were successful in demonstrating gene transfer via the intrasplenic and i.p. route of administration.  For the intrasplenic route, nanoparticles encapsulating 25 µg gWIZTM- 126 SEAP were injected into the spleen tissue of two C57BL/6j mice, whose circulating SEAP levels were measured at 4.2 and 15.6 ng/ml 1 day post-treatment, while SEAP was not detectable in saline-injected mice (Figure 18A).  Although with some variations in SEAP expression levels, which may be explained by the unintended loss of nanoparticles during the procedure, the presence of circulating SEAP in both treated mice post-injection validates the usefulness of intrasplenic injection in delivering chitosan-protected DNA.  Further studies are required to investigate the tissue distribution of the delivered DNA and the cell types from which the transgene product is secreted.  Based on reports by others, however, it is expected that the expression of DNA is restricted to the liver and spleen, providing safety for the intrasplenic route of DNA transfer [105].  For the i.p. route, a very high DNA dose (625 µg), corresponding to the maximum volume of nanoparticle delivery, was initially given via a single i.p. injection to two C57BL/6j mice.  SEAP expression levels, higher than those obtained with intrasplenic injection, were detected at 23 and 48 ng/ml 1 day post-injection in the treated mice (Figure 18A).  A follow-up dosing study, to our surprise, further revealed that a 60% decrease in the DNA dose (from 625 to 250 µg) led to an at least a ~7-fold increase in circulating SEAP, which was detected at 371 ng/ml (Figure 18B).  More interestingly, a similar level of SEAP (353 ng/ml) was measured with an even lower dose of DNA (50 µg), suggesting that while the highest DNA dose, delivered by a large volume of nanoparticles, is less effective, a dose as low as 50 µg is sufficient to maximize transgene expression.  Based on the doses tested here, a full-scale study, discussed earlier (Figure 19), was undertaken to examine short and long term transgene expression kinetics following the delivery of a total of 3 DNA doses (50, 25, 12.5 µg) via i.p. injection.  Together, these results demonstrate that 1) i.p injection is an effective 127 method of chitosan/DNA nanoparticle delivery and 2) maximal transgene expression can be achieved with a relatively low DNA dose (i.e. 50 µg) via i.p. administration.  It is worthy of note that this is the first time, to our knowledge, that successful i.p. gene transfer has been shown using a chitosan-based gene transfer system.  The i.p. route of delivery has the advantage of exposing the nanoparticles to many organs, including the liver, pancreas, spleen, and kidney, in the peritoneal cavity, potentially contributing to the high efficiency of DNA uptake and remarkable levels of transgene expression [108, 109].  However, while providing enhanced transgene expression, the lack of targeting specificity and broad tissue distribution of the delivered DNA are also major limitations that require careful consideration should the i.p. administration route be chosen for gene transfer.  A tissue specific promoter (e.g. the albumin and α1- antitrypsin promoters) might be utilized to restrict transgene expression in designated tissues. Optimization of the Chitosan/ФC31 Gene Transfer Technology Nanoparticles produced by complexation of chitosan and DNA proved to be an effective tool of gene transfer in several of our studies (see above); however, a number of shortcomings exist.  For instance, long term transgene expression, conferred by the well-designed chitosan/ФC31 technology, was never shown conclusively and successful oral and intraduodenal gene delivery was never achieved fully.  Therefore, in attempt to acquire complete efficacy and wide-ranging applicability, further optimization of the gene transfer system was investigated. To optimize the system, the first step was to modify the nanoparticle formulation in order to strengthen the stability of the nanoparticles.  As a critical parameter, pH has been shown to influence the force of DNA-chitosan interaction within nanoparticles [176, 128 177].   Using atomic force microscopy, it was demonstrated that adhesive forces between DNA and chitosan are strong at acidic pH (4.1, 6.1), while negligible at neutral or basic pH [178].  This is mainly due to protonation of more amine groups in the chitosan molecules at acidic pH, increasing the charge density necessary for strong chitosan/DNA interaction.  We formulated chitosan nanoparticles, containing EF1α- SEAP and CMV-Int, at pH 4.0 and compared them to previously synthesized nanoparticles formulated at pH 4.8.  As expected, the pH 4.0 nanoparticles were slightly larger and more positively charged because of the increase in protonation providing more cationic charges, which also created repulsive forces between the polymer chains and expanded the size of the nanoparticles (Figure 14A).  Along with the pH 4.8 nanoparticles, these nanoparticles were subsequently evaluated for transfection efficiency in vitro.  It was found that HEK 293T cells transfected with the pH 4.0 nanoparticles secreted levels of SEAP that were not significantly different than those secreted from pH 4.8 nanoparticle-transfected cells, suggesting that the pH of the formulation did not play a significant role in nanoparticle transfection ability at least in culture dishes (Figure 14B).  It is likely that the pH of the small amount (8 µl) of nanoparticles added to the culture media (1 ml) during transfection was effectively equilibrated to the pH of the media (i.e. pH 6.0) regardless of the starting pH, and thus the effects of the two pH formulations were diminished.  This might be an important observation since the nanoparticles would face the same situation in vivo, where the pH of the nanoparticles is largely determined by the physiological pH.  In fact, when delivered to mice, the pH 4.0 nanoparticles gave rise to levels of transgene expression that were essentially the same to those observed with the pH 4.8 nanoparticles (data not shown), lending support to the in vitro findings. 129 Given that the pH of nanoparticle formulation could be influenced by external factors (i.e. physiological pH), we set out to investigate another parameter of the formulation that was intrinsic to chitosan - the molecular weight (MW).  The MW of chitosan has profound influences on DNA retention and high MW chitosan has been shown to better entangle DNA with its long polymer chain, offering improved protection of DNA inside nanoparticles [80].  By measuring cumulative DNA release from chitosan/DNA nanoparticles in PBS over the course of 14 days, Huang et al. observed that, at a high degree of deacetylation (i.e. 88%), only 10% DNA release was measured with the high molecular weight chitosan (213 kDa), whereas as much as 50% DNA release was found with the 10 kDa chitosan 1 day post-incubation.  Furthermore, by the end of 14 days, while the 213 kDa chitosan still retained almost 40% DNA, the 10 kDa chitosan could only retain half of that amount.  Moreover, among the various chitosan molecular weights (10, 17, 48, 98, 213 kDa) tested, the 213 kDa chitosan (88% deacetylated) exhibited the highest zeta potential (±23 mV), cellular uptake (4.1 µg/mg protein), and transfection efficiency (12.1%), suggesting that high MW chitosan may be useful for improving transfection efficiency.  Corresponding to this, the limited reports on effective oral gene transfer have all been demonstrated using high MW chitosan (300- 1400 kDa), and therefore we hypothesized that high molecular weight (HMW) chitosan may enhance the transfection efficiency of chitosan/DNA nanoparticles [67, 68, 70-72]. New batches of nanoparticles were produced to encapsulate CMV-Lep/CMV-Int or CMV-SEAP/CMV-Int using chitosan with a molecular weight of 5, 226, or 429 kDa. Chitosan nanoparticles synthesized using HMW (226, 429 kDa) chitosan were larger, more polydispersed, and more positively charged than those synthesized using LMW (5 kDa) chitosan (Figure 16A).  When used to transfect HEK 293T cells, increasing levels of transfection efficiency, as shown by the amounts of human leptin secreted by 130 transfected cell into the cell supernatant, was obtained with 226, 429, and 5 kDa chitosan nanoparticles (Figure 16B).  This is consistent with the expectation that the readily dissociable LMW chitosan nanoparticles, being able to provide more free DNA for transcription, are more likely to confer higher in vitro transfection efficiency. Conversely, the tightly bound DNA inside HMW chitosan nanoparticles may be more efficient in vivo, where greater protection is needed due to more vicious DNA degradation. To evaluate their in vivo gene transfer efficiency, these nanoparticles were delivered to C57BL/6j mice via two delivery routes: i.p. and oral.  For the i.p. route, a single injection of the 5, 226, and 429 kDa chitosan nanoparticles containing 50 µg CMV-Lep/CMV-Int were given, after which body weight and fasting glucose were monitored.  One day post-injection, mice receiving the 5 kDa chitosan nanoparticles showed a percent body weight drop that was nearly twice as much as that seen in negative controls receiving CMV-SEAP/CMV via the 429 kDa chitosan nanoparticles (Figure 26).  In addition, the percent drop in fasting glucose in the 5 kDa group was significantly higher than that observed in negative controls, suggesting that circulating leptin, as a result of i.p. leptin gene transfer, may be present to cause the phenotypic changes.  However, analysis of the blood samples collected from these mice before and after treatment revealed that no human leptin was detectable above assay background in all groups of mice (Figure 27).  The lack of transgene product in the circulation confirms that the observations of body weight and fasting blood glucose reduction were pure experimental artefacts and that leptin gene transfer was not effective.  Similarly, following oral administration of the low and high MW chitosan nanoparticles, differences in body weight and fasting blood glucose were undetectable between the treatment groups and negative controls (Figure 28).  Furthermore, it was determined that human 131 leptin was absent in the blood sample collected from the 5 kDa treatment and 429 kDa control mice 1 day after the oral treatment regimen (Figure 29).  Collectively, these findings point towards the inability of enhancing in vivo gene transfer efficacy through chitosan MW modification. While the lack of oral gene transfer efficacy re-emphasizes the difficulty in transporting DNA across the numerous barriers inherent to this route of delivery, it was somewhat surprising that leptin gene transfer via the i.p. route was equally unproductive, particularly for the 5 kDa chitosan nanoparticle group.  Since the route of administration and the chitosan used (i.e. 5 kDa) were unchanged from the previous experiment in which, i.p. SEAP gene transfer was successful, one place left to be examined was the design of the plasmid constructs.  The DNA sequences of the gWIZTM-SEAP and CMV- Lep plasmid constructs were aligned and annotated carefully (Table I).  Although both constructs contain the human version of the CMV promoter and the pUC origin of replication, they also consist of a number of regulatory elements, including intron, polyadenylation signal, selectable marker, and transgene expression cassette, that were different from each other.  It is unknown if these differences, especially in intron, polyadenylation signal, and transgene expression cassette, outside of the promoter could contribute to differential transgene expression that may be responsible for the variations in gene transfer results.  Future i.p. gene transfer studies employing the leptin expression cassette fused to the gWIZTM vector backbone will serve to validate the efficiency of the gWIZTM construct in driving the expression of a different transgene and further evaluate the efficacy of gene transfer via the i.p. route. While i.p. gene transfer might be useful, the oral route of DNA delivery will continue to be most attractive for many researchers owing to its considerable advantages.  We have shown that chitosan MW modification alone was insufficient to 132 confer effective oral gene transfer and additional changes to the chitosan/ФC31 gene transfer system are necessary.  Since lipases are mainly present in the small and large intestines, strategic formulation entrapping chitosan nanoparticles within a lipid shell may be attractive as it provides protection of the orally administered payload during transit from the stomach, and, upon lipid digestion, releases chitosan nanoparticles in the intestines, where high concentrations of intact nanoparticles will be exposed to intestinal cells. A similar formulation encapsulating gelatine/DNA nanoparticles in a lipase- sensitive polyester, poly(ε-caprolactone), has been developed by Bhavsar and colleagues for successful delivery of the IL-10 transgene to a mouse model of acute colitis through oral administration [179, 180].  Additionally, several studies have explored the encapsulation of chitosan nanoparticles inside phospholipids, such as DSPC (distearoylphosphatidylcholine), DPPS (dipalmitoylphosphatidylserine), PC (phosphatidylcholine), DPPC (Dipalmitoylphosphatidylcholine), DMPG (dimyristoylphosphatidyl glycerol), and cholesterol, as a new class of colloidal delivery system for mucus-lining organ systems, including respiratory and intestinal epithelia [181, 182].  Jain et al. described a preparation method of the lipid/particulate delivery system that involves adding pre-formed chitosan nanoparticles to a lipid suspension constituted of soy lecithin and sorbitan monostearate.  Compared to unmodified chitosan nanoparticles, those further protected in a lipid shell retained a significantly higher fraction of the loaded protein antigen in simulated gastric fluid (pH 1.2), and, following oral administration to Wistar albino rats, more efficiently elicited both systemic and mucosal immune responses, suggesting the effectiveness of the lipid shell in shielding chitosan nanoparticles against the low gastric pH [182].  Equipped with the 133 advantages of lipids and polymeric particles, a composite DNA carrier consisting of an external lipid coating and an internal chitosan nanoparticle core may be a potential strategic modification for the chitosan/ФC31 gene transfer system to overcome the hurdles of oral gene transfer.                134 CONCLUSIONS  Gene transfer has been shown to be highly efficient with viral-based systems.  It can also be achieved with a few chemical DNA carriers, such as liposomes and PEI. However, unfavourable effects, including immunogenicity and virus-dependent insertional mutagenesis for viral-based systems and low efficiency and toxicity for chemical carriers, accentuate the necessity for developing new gene transfer systems. Chitosan, a natural, biodegradable polymer, when coupled with ФC31, a site-specific integrase, creates a unique and versatile gene transfer platform, potentially capable of offering long term gene transfer.  Before using the system for in vivo gene delivery, we validated the expression of the newly cloned human leptin-expressing constructs in vitro and showed that the leptin protein expressed by the constructs was biologically active. Our data also indicated that complete encapsulation of DNA was attained by complexing chitosan with DNA.  The transfection ability of the chitosan/DNA nanoparticles in vitro was established.  We also provided evidence that it is possible, via the i.p. route of nanoparticle administration, to achieve SEAP gene transfer, illustrated by high circulating SEAP levels 1 day post-treatment, after which transgene expression rapidly diminished and became background levels after 1 week post-treatment.  Our long term studies suggest that prolonged SEAP expression may be possible with the chitosan/ФC31 technology.  We were unable to show effective leptin gene transfer in C57BL/6j via the i.p. and oral or ob/ob mice via the i.p., oral, and intraduodenal routes of administration, although occasional success with oral SEAP gene transfer was observed.  Finally, our findings revealed that tansgene vector design may have a determining role on gene transfer efficacy.  135 REFERENCES 1. Glover, D.J., H.J. Lipps, and D.A. Jans, Towards safe, non-viral therapeutic gene expression in humans. Nat Rev Genet, 2005. 6(4): p. 299-310. 2. O'Connor, T.P. and R.G. Crystal, Genetic medicines: treatment strategies for hereditary disorders. Nat Rev Genet, 2006. 7(4): p. 261-76. 3. Kiuru, M. and R.G. Crystal, Progress and prospects: gene therapy for performance and appearance enhancement. Gene Ther, 2008. 15(5): p. 329-37. 4. Garmory, H.S., K.A. Brown, and R.W. Titball, DNA vaccines: improving expression of antigens. Genet Vaccines Ther, 2003. 1(1): p. 2. 5. Branca, M.A., Gene therapy: cursed or inching towards credibility? Nat Biotechnol, 2005. 23(5): p. 519-21. 6. Rosenberg, S.A., et al., Gene transfer into humans--immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction. N Engl J Med, 1990. 323(9): p. 570-8. 7. Blaese, R.M., et al., T lymphocyte-directed gene therapy for ADA- SCID: initial trial results after 4 years. Science, 1995. 270(5235): p. 475-80. 8. John Wiley and Sons, L. Gene therapy clinical trials worldwide. Gene Clinical Trials Worldwide  2009  [cited 2009 July 14]; Available from: 9. Raper, S.E., et al., Fatal systemic inflammatory response syndrome in a ornithine transcarbamylase deficient patient following adenoviral gene transfer. Mol Genet Metab, 2003. 80(1-2): p. 148-58. 10. Hacein-Bey-Abina, S., et al., LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science, 2003. 302(5644): p. 415-9. 11. Pearson, S., H. Jia, and K. Kandachi, China approves first gene therapy. Nat Biotechnol, 2004. 22(1): p. 3-4. 12. Ma, G., et al., Gene medicine for cancer treatment: Commercially available medicine and accumulated clinical data in China. Drug Design, Development and Therapy, 2008. 2: p. 115-122. 13. Vical, I. Vical Licensee AnGes MG Files NDA in Japan for Collategene Angiogenesis Product.  2008  [cited 2009 August 24]; Available from: 14. Kay, M.A., J.C. Glorioso, and L. Naldini, Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics. Nat Med, 2001. 7(1): p. 33-40. 15. Miller, D.G., M.A. Adam, and A.D. Miller, Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol Cell Biol, 1990. 10(8): p. 4239-42. 16. Stewart, P., Adenoviral Vectors for Gene Therapy. Adenovirus structure, ed. D. Curiel and J. Douglas. 2002, San Diego: Academic Press. 17. Horwitz, M., Virology. Adenoviruses, ed. B. Fields, D. Knipe, and P. Howley. Vol. 2. 1996, Philadelphia: Lippincott-Raven Publishers. 18. Yang, Y., et al., Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy. Proc Natl Acad Sci U S A, 1994. 91(10): p. 4407- 11. 19. Heise, C. and D.H. Kirn, Replication-selective adenoviruses as oncolytic agents. J Clin Invest, 2000. 105(7): p. 847-51. 136 20. Berns, K.I. and R.M. Linden, The cryptic life style of adeno-associated virus. Bioessays, 1995. 17(3): p. 237-45. 21. Wu, Z., A. Asokan, and R.J. Samulski, Adeno-associated virus serotypes: vector toolkit for human gene therapy. Mol Ther, 2006. 14(3): p. 316-27. 22. Tal, J., Adeno-associated virus-based vectors in gene therapy. J Biomed Sci, 2000. 7(4): p. 279-91. 23. Stedman, H., et al., Phase I clinical trial utilizing gene therapy for limb girdle muscular dystrophy: alpha-, beta-, gamma-, or delta-sarcoglycan gene delivered with intramuscular instillations of adeno-associated vectors. Hum Gene Ther, 2000. 11(5): p. 777-90. 24. Wagner, J.A., et al., Safety and biological efficacy of an adeno-associated virus vector-cystic fibrosis transmembrane regulator (AAV-CFTR) in the cystic fibrosis maxillary sinus. Laryngoscope, 1999. 109(2 Pt 1): p. 266-74. 25. Kay, M.A., et al., Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector. Nat Genet, 2000. 24(3): p. 257-61. 26. Luo, D. and W.M. Saltzman, Synthetic DNA delivery systems. Nat Biotechnol, 2000. 18(1): p. 33-7. 27. Dincer, S., M. Turk, and E. Piskin, Intelligent polymers as nonviral vectors. Gene Ther, 2005. 12 Suppl 1: p. S139-45. 28. Somiari, S., et al., Theory and in vivo application of electroporative gene delivery. Mol Ther, 2000. 2(3): p. 178-87. 29. Maruyama, H., et al., Skin-targeted gene transfer using in vivo electroporation. Gene Ther, 2001. 8(23): p. 1808-12. 30. Widera, G., et al., Increased DNA vaccine delivery and immunogenicity by electroporation in vivo. J Immunol, 2000. 164(9): p. 4635-40. 31. Heller, L., et al., Electrically mediated plasmid DNA delivery to hepatocellular carcinomas in vivo. Gene Ther, 2000. 7(10): p. 826-9. 32. Liu, F. and L. Huang, Electric gene transfer to the liver following systemic administration of plasmid DNA. Gene Ther, 2002. 9(16): p. 1116-9. 33. Lin, M.T., et al., The gene gun: current applications in cutaneous gene therapy. Int J Dermatol, 2000. 39(3): p. 161-70. 34. Muangmoonchai, R., et al., Transfection of liver in vivo by biolistic particle delivery: its use in the investigation of cytochrome P450 gene regulation. Mol Biotechnol, 2002. 20(2): p. 145-51. 35. Newman, C.M., et al., Ultrasound gene therapy: on the road from concept to reality. Echocardiography, 2001. 18(4): p. 339-47. 36. Lu, Q.L., et al., Microbubble ultrasound improves the efficiency of gene transduction in skeletal muscle in vivo with reduced tissue damage. Gene Ther, 2003. 10(5): p. 396-405. 37. Liu, F., Y. Song, and D. Liu, Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Ther, 1999. 6(7): p. 1258-66. 38. Zhang, G., et al., Hydroporation as the mechanism of hydrodynamic delivery. Gene Ther, 2004. 11(8): p. 675-82. 39. Zhang, G., et al., Efficient expression of naked dna delivered intraarterially to limb muscles of nonhuman primates. Hum Gene Ther, 2001. 12(4): p. 427-38. 40. Hagstrom, J.E., et al., A facile nonviral method for delivering genes and siRNAs to skeletal muscle of mammalian limbs. Mol Ther, 2004. 10(2): p. 386-98. 137 41. Wells, D.J., Opening the floodgates: clinically applicable hydrodynamic delivery of plasmid DNA to skeletal muscle. Mol Ther, 2004. 10(2): p. 207-8. 42. Templeton, N.S. and D.D. Lasic, New directions in liposome gene delivery. Mol Biotechnol, 1999. 11(2): p. 175-80. 43. Templeton, N.S., Cationic liposome-mediated gene delivery in vivo. Biosci Rep, 2002. 22(2): p. 283-95. 44. Audouy, S.A., et al., In vivo characteristics of cationic liposomes as delivery vectors for gene therapy. Pharm Res, 2002. 19(11): p. 1599-605. 45. Felgner, P.L., et al., Lipofection: a highly efficient, lipid-mediated DNA- transfection procedure. Proc Natl Acad Sci U S A, 1987. 84(21): p. 7413-7. 46. Filion, M.C. and N.C. Phillips, Major limitations in the use of cationic liposomes for DNA delivery. 1998. International Journal of Pharmaceutics(162): p. 1-2. 47. Gebhart, C.L. and A.V. Kabanov, Evaluation of polyplexes as gene transfer agents. J Control Release, 2001. 73(2-3): p. 401-16. 48. Hong, S., et al., Interaction of polycationic polymers with supported lipid bilayers and cells: nanoscale hole formation and enhanced membrane permeability. Bioconjug Chem, 2006. 17(3): p. 728-34. 49. Breunig, M., et al., Breaking up the correlation between efficacy and toxicity for nonviral gene delivery. Proc Natl Acad Sci U S A, 2007. 104(36): p. 14454-9. 50. Hudecz, F., et al., Carrier design: cytotoxicity and immunogenicity of synthetic branched polypeptides with poly(L-lysine) backbone Journal of Controlled Release, 1992. 19(1-3): p. 231-243. 51. Akinc, A., et al., Synthesis of poly(beta-amino ester)s optimized for highly effective gene delivery. Bioconjug Chem, 2003. 14(5): p. 979-88. 52. Zugates, G.T., et al., Synthesis of poly(beta-amino ester)s with thiol-reactive side chains for DNA delivery. J Am Chem Soc, 2006. 128(39): p. 12726-34. 53. Lynn, D.M. and R. Langer, Degradable Poly(β-amino esters):  Synthesis, Characterization, and Self-Assembly with Plasmid DNA. Journal of the American Chemical Society, 2000. 122(44): p. 10761–10768. 54. Bielinska, A.U., J.F. Kukowska-Latallo, and J.R. Baker, Jr., The interaction of plasmid DNA with polyamidoamine dendrimers: mechanism of complex formation and analysis of alterations induced in nuclease sensitivity and transcriptional activity of the complexed DNA. Biochim Biophys Acta, 1997. 1353(2): p. 180-90. 55. Kukowska-Latallo, J.F., et al., Efficient transfer of genetic material into mammalian cells using Starburst polyamidoamine dendrimers. Proc Natl Acad Sci U S A, 1996. 93(10): p. 4897-902. 56. Hill, I.R., et al., In vitro cytotoxicity of poly(amidoamine)s: relevance to DNA delivery. Biochim Biophys Acta, 1999. 1427(2): p. 161-74. 57. Ishii, T., Y. Okahata, and T. Sato, Mechanism of cell transfection with plasmid/chitosan complexes. Biochim Biophys Acta, 2001. 1514(1): p. 51-64. 58. Kjøniksen, A., et al., Effect of surfactant concentration, pH, and shear rate on the rheological properties of aqueous systems of a hydrophobically modifed chitosan and its unmodified analogue Polymer Bulletin, 1997. 38(1): p. 71-79. 59. Martien, R., B. Loretz, and A.B. Schnurch, Oral gene delivery: design of polymeric carrier systems shielding toward intestinal enzymatic attack. Biopolymers, 2006. 83(4): p. 327-36. 138 60. Anthonsen, M.W. and O. Smidsrød, Hydrogen ion titration of chitosans with varying degrees of N-acetylation by monitoring induced 1H-NMR chemical shifts Carbohydrate Polymers, 1995. 26(4): p. 303-305. 61. Rao, S.B. and C.P. Sharma, Use of chitosan as a biomaterial: studies on its safety and hemostatic potential. J Biomed Mater Res, 1997. 34(1): p. 21-8. 62. Ping, H. and S.S. Davis, In vitro evaluation of the mucoadhesive properties of chitosan microspheres. International Journal of Pharmaceutics, 1998. 166(1): p. 75-88. 63. Ponchel, G. and J. Irache, Specific and non-specific bioadhesive particulate systems for oral delivery to the gastrointestinal tract. Adv Drug Deliv Rev, 1998. 34(2-3): p. 191-219. 64. Takeuchi, H., et al., Enteral absorption of insulin in rats from mucoadhesive chitosan-coated liposomes. Pharm Res, 1996. 13(6): p. 896-901. 65. Mislick, K.A. and J.D. Baldeschwieler, Evidence for the role of proteoglycans in cation-mediated gene transfer. Proc Natl Acad Sci U S A, 1996. 93(22): p. 12349-54. 66. Labat-Moleur, F., et al., An electron microscopy study into the mechanism of gene transfer with lipopolyamines. Gene Ther, 1996. 3(11): p. 1010-7. 67. Bowman, K., et al., Gene transfer to hemophilia A mice via oral delivery of FVIII- chitosan nanoparticles. J Control Release, 2008. 132(3): p. 252-9. 68. Guliyeva, U., et al., Chitosan microparticles containing plasmid DNA as potential oral gene delivery system. Eur J Pharm Biopharm, 2006. 62(1): p. 17-25. 69. Zheng, F., et al., Chitosan nanoparticle as gene therapy vector via gastrointestinal mucosa administration: results of an in vitro and in vivo study. Life Sci, 2007. 80(4): p. 388-96. 70. Kai, E. and T. Ochiya, A method for oral DNA delivery with N-acetylated chitosan. Pharm Res, 2004. 21(5): p. 838-43. 71. Roy, K., et al., Oral gene delivery with chitosan--DNA nanoparticles generates immunologic protection in a murine model of peanut allergy. Nat Med, 1999. 5(4): p. 387-91. 72. Chen, J., et al., Transfection of mEpo gene to intestinal epithelium in vivo mediated by oral delivery of chitosan-DNA nanoparticles. World J Gastroenterol, 2004. 10(1): p. 112-6. 73. Fernandes, J.C., et al., Bone-protective effects of nonviral gene therapy with folate-chitosan DNA nanoparticle containing interleukin-1 receptor antagonist gene in rats with adjuvant-induced arthritis. Mol Ther, 2008. 16(7): p. 1243-51. 74. Koping-Hoggard, M., et al., Improved chitosan-mediated gene delivery based on easily dissociated chitosan polyplexes of highly defined chitosan oligomers. Gene Ther, 2004. 11(19): p. 1441-52. 75. Jiang, X., et al., Chitosan-g-PEG/DNA complexes deliver gene to the rat liver via intrabiliary and intraportal infusions. J Gene Med, 2006. 8(4): p. 477-87. 76. Niu, L., et al., Gene therapy for type 1 diabetes mellitus in rats by gastrointestinal administration of chitosan nanoparticles containing human insulin gene. World J Gastroenterol, 2008. 14(26): p. 4209-15. 77. de la Fuente, M., B. Seijo, and M.J. Alonso, Bioadhesive hyaluronan-chitosan nanoparticles can transport genes across the ocular mucosa and transfect ocular tissue. Gene Ther, 2008. 15(9): p. 668-76. 139 78. Malvern, I. Polymer size and zeta potential measurements.  2009  [cited 2009 August 3]; Available from: 79. Koping-Hoggard, M., et al., Relationship between the physical shape and the efficiency of oligomeric chitosan as a gene delivery system in vitro and in vivo. J Gene Med, 2003. 5(2): p. 130-41. 80. Huang, M., et al., Transfection efficiency of chitosan vectors: effect of polymer molecular weight and degree of deacetylation. J Control Release, 2005. 106(3): p. 391-406. 81. MacLaughlin, F.C., et al., Chitosan and depolymerized chitosan oligomers as condensing carriers for in vivo plasmid delivery. J Control Release, 1998. 56(1-3): p. 259-72. 82. Cherkasova, E.I., L.A. Smirnova, and V.F. Smirnov, Measurement of molecular mass of chitosan oligomers. Polymer Science, 2006. 48(3): p. 557-60. 83. Wang, T. and J.A. Lucey, Use of multi-angle laser light scattering and size- exclusion chromatography to characterize the molecular weight and types of aggregates present in commercial whey protein products. J Dairy Sci, 2003. 86(10): p. 3090-101. 84. Tan, S.C., et al., The degree of deacetylation of chitosan: advocating the first derivative UV-spectrophotometry method of determination. Talanta, 1998. 45(4): p. 713-9. 85. Mao, H.Q., et al., Chitosan-DNA nanoparticles as gene carriers: synthesis, characterization and transfection efficiency. J Control Release, 2001. 70(3): p. 399-421. 86. Boussif, O., et al., A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci U S A, 1995. 92(16): p. 7297-301. 87. Cho, Y.W., J.D. Kim, and K. Park, Polycation gene delivery systems: escape from endosomes to cytosol. J Pharm Pharmacol, 2003. 55(6): p. 721-34. 88. Groth, A.C. and M.P. Calos, Phage integrases: biology and applications. J Mol Biol, 2004. 335(3): p. 667-78. 89. Olivares, E.C., et al., Site-specific genomic integration produces therapeutic Factor IX levels in mice. Nat Biotechnol, 2002. 20(11): p. 1124-8. 90. Held, P.K., et al., In vivo correction of murine hereditary tyrosinemia type I by phiC31 integrase-mediated gene delivery. Mol Ther, 2005. 11(3): p. 399-408. 91. Ehrhardt, A., et al., A direct comparison of two nonviral gene therapy vectors for somatic integration: in vivo evaluation of the bacteriophage integrase phiC31 and the Sleeping Beauty transposase. Mol Ther, 2005. 11(5): p. 695-706. 92. Chalberg, T.W., et al., Integration specificity of phage phiC31 integrase in the human genome. J Mol Biol, 2006. 357(1): p. 28-48. 93. Bertoni, C., et al., Enhancement of plasmid-mediated gene therapy for muscular dystrophy by directed plasmid integration. Proc Natl Acad Sci U S A, 2006. 103(2): p. 419-24. 94. Chalberg, T.W., et al., phiC31 integrase confers genomic integration and long- term transgene expression in rat retina. Invest Ophthalmol Vis Sci, 2005. 46(6): p. 2140-6. 95. Keravala, A., et al., PhiC31 integrase mediates integration in cultured synovial cells and enhances gene expression in rabbit joints. J Gene Med, 2006. 8(8): p. 1008-17. 140 96. Ishikawa, Y., et al., Phage phiC31 integrase-mediated genomic integration of the common cytokine receptor gamma chain in human T-cell lines. J Gene Med, 2006. 8(5): p. 646-53. 97. Ortiz-Urda, S., et al., Stable nonviral genetic correction of inherited human skin disease. Nat Med, 2002. 8(10): p. 1166-70. 98. Bushman, F.D., Targeting survival: integration site selection by retroviruses and LTR-retrotransposons. Cell, 2003. 115(2): p. 135-8. 99. Yant, S.R., et al., High-resolution genome-wide mapping of transposon integration in mammals. Mol Cell Biol, 2005. 25(6): p. 2085-94. 100. Futreal, P.A., et al., A census of human cancer genes. Nat Rev Cancer, 2004. 4(3): p. 177-83. 101. Liu, J., et al., Phi c31 integrase induces chromosomal aberrations in primary human fibroblasts. Gene Ther, 2006. 13(15): p. 1188-90. 102. Allen, B.G. and D.L. Weeks, Transgenic Xenopus laevis embryos can be generated using phiC31 integrase. Nat Methods, 2005. 2(12): p. 975-9. 103. Groth, A.C., et al., Construction of transgenic Drosophila by using the site- specific integrase from phage phiC31. Genetics, 2004. 166(4): p. 1775-82. 104. Matsumoto, T., et al., Insulin gene transfer with adenovirus vector via the spleen safely and effectively improves posthepatectomized conditions in diabetic rats. J Surg Res, 2003. 110(1): p. 228-34. 105. Yamaguchi, M., et al., Adenovirus-mediated insulin gene transfer improves nutritional and post-hepatectomized conditions in diabetic rats. Surgery, 2000. 127(6): p. 670-8. 106. Kasinrerk, W., S. Moonsom, and K. Chawansuntati, Production of antibodies by single DNA immunization: comparison of various immunization routes. Hybrid Hybridomics, 2002. 21(4): p. 287-93. 107. Ho, S.H., et al., Intrasplenic electro-transfer of IL-4 encoding plasmid DNA efficiently inhibits rat experimental allergic encephalomyelitis. Biochem Biophys Res Commun, 2006. 343(3): p. 816-24. 108. Reimer, D.L., et al., Liposomal lipid and plasmid DNA delivery to B16/BL6 tumors after intraperitoneal administration of cationic liposome DNA aggregates. J Pharmacol Exp Ther, 1999. 289(2): p. 807-15. 109. Fellowes, R., et al., Amelioration of established collagen induced arthritis by systemic IL-10 gene delivery. Gene Ther, 2000. 7(11): p. 967-77. 110. Allemann, E., J. Leroux, and R. Gurny, Polymeric nano- and microparticles for the oral delivery of peptides and peptidomimetics. Adv Drug Deliv Rev, 1998. 34(2-3): p. 171-189. 111. Bernkop-Schnurch, A. and M.E. Krajicek, Mucoadhesive polymers as platforms for peroral peptide delivery and absorption: synthesis and evaluation of different chitosan-EDTA conjugates. J Control Release, 1998. 50(1-3): p. 215-23. 112. Halaas, J.L., et al., Weight-reducing effects of the plasma protein encoded by the obese gene. Science, 1995. 269(5223): p. 543-6. 113. van den Hoek, A.M., et al., Leptin deficiency per se dictates body composition and insulin action in ob/ob mice. J Neuroendocrinol, 2008. 20(1): p. 120-7. 114. Zhang, Y., et al., Positional cloning of the mouse obese gene and its human homologue. Nature, 1994. 372(6505): p. 425-32. 115. Campfield, L.A., et al., Recombinant mouse OB protein: evidence for a peripheral signal linking adiposity and central neural networks. Science, 1995. 269(5223): p. 546-9. 141 116. Pelleymounter, M.A., et al., Effects of the obese gene product on body weight regulation in ob/ob mice. Science, 1995. 269(5223): p. 540-3. 117. Coleman, D.L., Effects of parabiosis of obese with diabetes and normal mice. Diabetologia, 1973. 9(4): p. 294-8. 118. Hamilton, B.S., et al., Increased obese mRNA expression in omental fat cells from massively obese humans. Nat Med, 1995. 1(9): p. 953-6. 119. Harris, R.B., et al., Early and late stimulation of ob mRNA expression in meal-fed and overfed rats. J Clin Invest, 1996. 97(9): p. 2020-6. 120. Considine, R.V., et al., Serum immunoreactive-leptin concentrations in normal- weight and obese humans. N Engl J Med, 1996. 334(5): p. 292-5. 121. Frederich, R.C., et al., Expression of ob mRNA and its encoded protein in rodents. Impact of nutrition and obesity. J Clin Invest, 1995. 96(3): p. 1658-63. 122. Maffei, M., et al., Increased expression in adipocytes of ob RNA in mice with lesions of the hypothalamus and with mutations at the db locus. Proc Natl Acad Sci U S A, 1995. 92(15): p. 6957-60. 123. Kennedy, G.C., The role of depot fat in the hypothalamic control of food intake in the rat. Proc R Soc Lond B Biol Sci, 1953. 140(901): p. 578-96. 124. Boden, G., et al., Effect of fasting on serum leptin in normal human subjects. J Clin Endocrinol Metab, 1996. 81(9): p. 3419-23. 125. Tartaglia, L.A., et al., Identification and expression cloning of a leptin receptor, OB-R. Cell, 1995. 83(7): p. 1263-71. 126. Lee, G.H., et al., Abnormal splicing of the leptin receptor in diabetic mice. Nature, 1996. 379(6566): p. 632-5. 127. Mercer, J.G., et al., Localization of leptin receptor mRNA and the long form splice variant (Ob-Rb) in mouse hypothalamus and adjacent brain regions by in situ hybridization. FEBS Lett, 1996. 387(2-3): p. 113-6. 128. Fei, H., et al., Anatomic localization of alternatively spliced leptin receptors (Ob-R) in mouse brain and other tissues. Proc Natl Acad Sci U S A, 1997. 94(13): p. 7001-5. 129. Tartaglia, L.A., The leptin receptor. J Biol Chem, 1997. 272(10): p. 6093-6. 130. Vaisse, C., et al., Leptin activation of Stat3 in the hypothalamus of wild-type and ob/ob mice but not db/db mice. Nat Genet, 1996. 14(1): p. 95-7. 131. Cusin, I., et al., Chronic central leptin infusion enhances insulin-stimulated glucose metabolism and favors the expression of uncoupling proteins. Diabetes, 1998. 47(7): p. 1014-9. 132. Haque, M.S., et al., Role of the sympathetic nervous system and insulin in enhancing glucose uptake in peripheral tissues after intrahypothalamic injection of leptin in rats. Diabetes, 1999. 48(9): p. 1706-12. 133. Kamohara, S., et al., Acute stimulation of glucose metabolism in mice by leptin treatment. Nature, 1997. 389(6649): p. 374-7. 134. Minokoshi, Y., M.S. Haque, and T. Shimazu, Microinjection of leptin into the ventromedial hypothalamus increases glucose uptake in peripheral tissues in rats. Diabetes, 1999. 48(2): p. 287-91. 135. Pocai, A., et al., Central leptin acutely reverses diet-induced hepatic insulin resistance. Diabetes, 2005. 54(11): p. 3182-9. 136. Oral, E.A., et al., Leptin-replacement therapy for lipodystrophy. N Engl J Med, 2002. 346(8): p. 570-8. 137. Petersen, K.F., et al., Leptin reverses insulin resistance and hepatic steatosis in patients with severe lipodystrophy. J Clin Invest, 2002. 109(10): p. 1345-50. 142 138. Shimomura, I., et al., Leptin reverses insulin resistance and diabetes mellitus in mice with congenital lipodystrophy. Nature, 1999. 401(6748): p. 73-6. 139. Montague, C., et al., Congenital leptin deficiency is associated with severe early onset obesity in humans. Nature, 1997. 387: p. 903–908. 140. Farooqi, I.S., et al., Effects of recombinant leptin therapy in a child with congenital leptin deficiency. N Engl J Med, 1999. 341(12): p. 879-84. 141. Farooqi, I.S., et al., Beneficial effects of leptin on obesity, T cell hyporesponsiveness, and neuroendocrine/metabolic dysfunction of human congenital leptin deficiency. J Clin Invest, 2002. 110(8): p. 1093-103. 142. Strobel, A., et al., A leptin missense mutation associated with hypogonadism and morbid obesity. Nat Genet, 1998. 18(3): p. 213-5. 143. Ozata, M., I.C. Ozdemir, and J. Licinio, Human leptin deficiency caused by a missense mutation: multiple endocrine defects, decreased sympathetic tone, and immune system dysfunction indicate new targets for leptin action, greater central than peripheral resistance to the effects of leptin, and spontaneous correction of leptin-mediated defects. J Clin Endocrinol Metab, 1999. 84(10): p. 3686-95. 144. Licinio, J., et al., Phenotypic effects of leptin replacement on morbid obesity, diabetes mellitus, hypogonadism, and behavior in leptin-deficient adults. Proc Natl Acad Sci U S A, 2004. 101(13): p. 4531-6. 145. Heymsfield, S.B., et al., Recombinant leptin for weight loss in obese and lean adults: a randomized, controlled, dose-escalation trial. JAMA, 1999. 282(16): p. 1568-75. 146. Rosenbaum, M., et al., Low-dose leptin reverses skeletal muscle, autonomic, and neuroendocrine adaptations to maintenance of reduced weight. J Clin Invest, 2005. 115(12): p. 3579-86. 147. Welt, C.K., et al., Recombinant human leptin in women with hypothalamic amenorrhea. N Engl J Med, 2004. 351(10): p. 987-97. 148. Chen, G., et al., Disappearance of body fat in normal rats induced by adenovirus- mediated leptin gene therapy. Proc Natl Acad Sci U S A, 1996. 93(25): p. 14795- 9. 149. Dhillon, H., et al., Central leptin gene therapy suppresses body weight gain, adiposity and serum insulin without affecting food consumption in normal rats: a long-term study. Regul Pept, 2001. 99(2-3): p. 69-77. 150. Dube, M.G., et al., Central leptin gene therapy blocks high-fat diet-induced weight gain, hyperleptinemia, and hyperinsulinemia: increase in serum ghrelin levels. Diabetes, 2002. 51(6): p. 1729-36. 151. Ueno, N., et al., Leptin transgene expression in the hypothalamus enforces euglycemia in diabetic, insulin-deficient nonobese Akita mice and leptin-deficient obese ob/ob mice. Peptides, 2006. 27(9): p. 2332-42. 152. Dhillon, H., et al., Long-term differential modulation of genes encoding orexigenic and anorexigenic peptides by leptin delivered by rAAV vector in ob/ob mice. Relationship with body weight change. Regul Pept, 2000. 92(1-3): p. 97-105. 153. Morsy, M.A., et al., Leptin gene therapy and daily protein administration: a comparative study in the ob/ob mouse. Gene Ther, 1998. 5(1): p. 8-18. 154. Glaum, S.R., et al., Leptin, the obese gene product, rapidly modulates synaptic transmission in the hypothalamus. Mol Pharmacol, 1996. 50(2): p. 230-5. 155. Uotani, S., et al., Functional properties of leptin receptor isoforms: internalization and degradation of leptin and ligand-induced receptor downregulation. Diabetes, 1999. 48(2): p. 279-86. 143 156. Garofalo, C. and E. Surmacz, Leptin and cancer. J Cell Physiol, 2006. 207(1): p. 12-22. 157. Smith, T.K. and W.J. Robertson, Synchronous movements of the longitudinal and circular muscle during peristalsis in the isolated guinea-pig distal colon. J Physiol, 1998. 506 ( Pt 2): p. 563-77. 158. Herweijer, H., et al., Time course of gene expression after plasmid DNA gene transfer to the liver. J Gene Med, 2001. 3(3): p. 280-91. 159. Harms, J.S. and G.A. Splitter, Interferon-gamma inhibits transgene expression driven by SV40 or CMV promoters but augments expression driven by the mammalian MHC I promoter. Hum Gene Ther, 1995. 6(10): p. 1291-7. 160. Krieg, A.M., Direct immunologic activities of CpG DNA and implications for gene therapy. J Gene Med, 1999. 1(1): p. 56-63. 161. Kim, S.Y., et al., The human elongation factor 1 alpha (EF-1 alpha) first intron highly enhances expression of foreign genes from the murine cytomegalovirus promoter. J Biotechnol, 2002. 93(2): p. 183-7. 162. Donoviel, D.B., et al., Analysis of muscle creatine kinase gene regulatory elements in skeletal and cardiac muscles of transgenic mice. Mol Cell Biol, 1996. 16(4): p. 1649-58. 163. Wang, L., et al., Sustained correction of bleeding disorder in hemophilia B mice by gene therapy. Proc Natl Acad Sci U S A, 1999. 96(7): p. 3906-10. 164. Miao, C.H., et al., Long-term and therapeutic-level hepatic gene expression of human factor IX after naked plasmid transfer in vivo. Mol Ther, 2001. 3(6): p. 947-57. 165. Wooddell, C.I., et al., Sustained liver-specific transgene expression from the albumin promoter in mice following hydrodynamic plasmid DNA delivery. J Gene Med, 2008. 10(5): p. 551-63. 166. Gill, D.R., et al., Increased persistence of lung gene expression using plasmids containing the ubiquitin C or elongation factor 1alpha promoter. Gene Ther, 2001. 8(20): p. 1539-46. 167. Magami, Y., et al., Cell proliferation and renewal of normal hepatocytes and bile duct cells in adult mouse liver. Liver, 2002. 22(5): p. 419-25. 168. Okazaki, K. and S. Yumura, Differential association of three actin-bundling proteins with microfilaments in Dictyostelium amoebae. Eur J Cell Biol, 1995. 66(1): p. 75-81. 169. Sanders, J., J.A. Maassen, and W. Moller, Elongation factor-1 messenger-RNA levels in cultured cells are high compared to tissue and are not drastically affected further by oncogenic transformation. Nucleic Acids Res, 1992. 20(22): p. 5907-10. 170. Roth, W.W., et al., Expression of a gene for mouse eucaryotic elongation factor Tu during murine erythroleukemic cell differentiation. Mol Cell Biol, 1987. 7(11): p. 3929-36. 171. Chen, Z.Y., et al., Silencing of episomal transgene expression by plasmid bacterial DNA elements in vivo. Gene Ther, 2004. 11(10): p. 856-64. 172. Chen, Z.Y., C.Y. He, and M.A. Kay, Improved production and purification of minicircle DNA vector free of plasmid bacterial sequences and capable of persistent transgene expression in vivo. Hum Gene Ther, 2005. 16(1): p. 126-31. 173. Dai, H., et al., Chitosan-DNA nanoparticles delivered by intrabiliary infusion enhance liver-targeted gene delivery. Int J Nanomedicine, 2006. 1(4): p. 507-22. 144 174. Varum, K.M., et al., In vitro degradation rates of partially N-acetylated chitosans in human serum. Carbohydr Res, 1997. 299(1-2): p. 99-101. 175. Wydro, P., B. Krajewska, and K. Hac-Wydro, Chitosan as a lipid binder: a langmuir monolayer study of chitosan-lipid interactions. Biomacromolecules, 2007. 8(8): p. 2611-7. 176. Strand, S.P., et al., Influence of chitosan structure on the formation and stability of DNA-chitosan polyelectrolyte complexes. Biomacromolecules, 2005. 6(6): p. 3357-66. 177. Liu, W., et al., An investigation on the physicochemical properties of chitosan/DNA polyelectrolyte complexes. Biomaterials, 2005. 26(15): p. 2705-11. 178. Xu, S., et al., Direct force measurements between siRNA and chitosan molecules using force spectroscopy. Biophys J, 2007. 93(3): p. 952-9. 179. Bhavsar, M.D., S.B. Tiwari, and M.M. Amiji, Formulation optimization for the nanoparticles-in-microsphere hybrid oral delivery system using factorial design. J Control Release, 2006. 110(2): p. 422-30. 180. Bhavsar, M.D. and M.M. Amiji, Oral IL-10 gene delivery in a microsphere-based formulation for local transfection and therapeutic efficacy in inflammatory bowel disease. Gene Ther, 2008. 15(17): p. 1200-9. 181. Grenha, A., et al., Microspheres containing lipid/chitosan nanoparticles complexes for pulmonary delivery of therapeutic proteins. Eur J Pharm Biopharm, 2008. 69(1): p. 83-93. 182. Jain, S., R.K. Sharma, and S.P. Vyas, Chitosan nanoparticles encapsulated vesicular systems for oral immunization: preparation, in-vitro and in-vivo characterization. J Pharm Pharmacol, 2006. 58(3): p. 303-10.  


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