Nanoparticle Mediated Gene Therapy Diabetic Retinopathy Biology Essay

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A. SPECIFIC AIMS: Diabetic retinopathy (DR), brought on as a result of complications attributed to diabetes, is the leading cause of preventable blindness among adults aged 20-74 years in the industrialized world. Approximately half of all patients with diabetes will, at one time or another, have some degree of retinopathy and with the prevalence of diabetes expected to rise steadily in the foreseeable future DR will continue to manifest as a significant complication in patients with diabetes. Although pan-retinal photocoagulation (PRP) continues to be the standard treatment for DR, its clinical limitations and adverse effects have led to an exploration of alternative strategies for treatment [x-Jardeleza]. One holding great promise is gene therapy utilizing novel DNA nanoparticles (NPs). For some time ocular gene therapy using viral vectors have shown limited success (e.g., AAV2 phase I clinical trials for Leber Congenital Ameroses (LCA)), with notable limitations which include size of expression cassette delivered, cellular tropism, and issues of safety. Along with others we have successfully created and used novel self-compacted DNA nanoparticles capable of efficient long-lasting expression within 1 hour of administration. The technology used to construct these DNA nanoparticles is able to compact a DNA vector of up to 20 kb in size into a nanoparticle having a final diameter of only 8 nm. Our own proof-of-principle studies have demonstrated that these NPs are safe, effective, non-toxic and efficient as non-viral gene transfer machinery for use in the retina [4-11]. The clinical viability for the use NPs as a therapeutic strategy in the treatment of DR is favorable given data supporting its efficacy with other forms of retinal disease [x]. In this grant application, we plan to utilize our optimized NP-mediated gene transfer strategy, alone or combined with siRNA targeting system, for the treatment of DR. The DNA NPs will be delivered intravitreally into the Ins2Akita, an established mouse model for the study of the molecular basis involved in the early progression of DR [x-2005 Barber]. The Ins2Akita mice were inherited as an autosomal dominant, which resulted in a single amino acid substitution in the insulin 2 gene that causes misfolding of the insulin protein [12]. Heterozygous males for this mutation have significant hyperglycemia, hypoinsulinemia, and early signs of DR that made it as an excellent type 1 diabetic mouse model for studying DR [12, 13]. Since the high levels of VEGF and PEDF are present in vitreous fluid and inhibitors of angiogenesis are generally abundant in the vitreous fluid [14, 15], we will inject the animal intravitreally. Our immediate goal is to develop a safe and an efficient non-viral therapeutic to combat visual loss in a model of DR.

Aim 1. Anti-VEGF targeting in Ins2Akita mice of DR using NP vectors. We will purchase four pre-defined small interfering RNAs (siRNAs), (Dharmacon, Inc.) that target sequences within the VEGF principal receptors on the endothelial cell plasma membrane following NP-mediated introduction into the eye. The short hairpin RNA (shRNA) expression vectors driven by the RNA polymerase III H1 promoter will be cloned first, and their efficiency and specificity will be tested prior to NP construction and intravitreal delivery to the diabetic mouse model of DR.

Aim 2. Evaluation of optimal NP-mediated PEDF gene transfer into ocular cells following intravitreal injection in a diabetic mouse model of DR. The ultimate treatment for vision-threatening DR may require multiple modalities. In this aim, NPs carrying the PEDF gene of interest driven by strong universal CBA and/or CMV early enhancer/chicken β-actin (CAG) promoters will be injected into the intravitreal space of Ins2Akita males at postnatal (P) day 60 before the onset of DR. Outcome measurements of function will be obtained by electroretinography (ERG) and psychophysical vision testing by OptoMotry; measurements of structure will be obtained by histological analysis of retinal vessel formation at the light and EM levels and by immunohistochemistry (IHC); and measurements of biochemical activity will be obtained by qRT-PCR and quantitative Western blotting analysis to evaluate PEDF expression in order to assay multiple aspects of the disease process.

B. BACKGROUND AND SIGNIFICANCE: Diabetic retinopathy (DR) is a significant visual complication of diabetes and is the leading cause of blindness among working-aged adults. Globally, the incidence of diabetes is expected to rise steadily as a consequence of lifestyle attributes that lead to obesity. The quality of life of patients is severely affected by the partial or complete loss of vision, therefore there is a great need for developing novel therapeutics that are safe and effective for preventing and treating DR.

(1) Model of DR: The Ins2Akita mouse, first identified in 1997[12], is an autosomal dominant mutant diabetic type 1 model without insulitis or obesity[12] and has been the most extensively studied model of this kind [12, 13, 16-18]. These non-obese mice were noted to be hyperglycemic and hypoinsulinemic by 4-6 weeks of age[12] and develop diabetic complications including early onset of DR[13], neuropathy[19], and nephropathy[18]. The Ins2Akita mouse encodes for a single base pair substitution at the insulin 2 gene on chromosome 7 in a C57BL6 mouse background resulting in replacement of cysteine with tyrosine at position 96 (C96Y) and is transmitted as an autosomal dominant trait. The substitution disrupts the majority of circulating insulin in the C57BL/6 wild type because of improper folding of the insulin 2 protein, resulting in severely hyperglycemic and hypoinsulinemic syndrome. The phenotype of the syndrome is more pronounced in Akita male than in the female. The Ins2Akita mouse has an advantage over other mouse strains for the investigation of DR because: (i) it's a very common genetic diabetic mouse model; (ii) it shows early age of diabetes onset (4-6 weeks) [12, 13]and early signs of DR (12 weeks) [13] when compared with others models, such as diabetic type II db/db mouse (early signs of DR ~20 weeks)[20]; (iii) it is known to produce intraocular neovascularization which mimics the vascular proliferative phase of DR; (iv) it is easily bred and fertilized; (v) its diabetic status mimics human insulin-dependent diabetes mellitus (IDDM) which can be maintained in a state without exogenous insulin[13]; and (vi) the DR manifested is more common in those with type I diabetes[21]. Therefore, it is a suitable model for testing NP-mediated non-viral treatment hypotheses for DR.

(2) The role of VEGF in DR: To prevent the onset or progression of DR, key factors involved in both the vascular and neuronal abnormalities which lead to DR must be addressed. VEGF is a critical component in the tissue growth and organ repair processes of angiogenesis and vasculogenesis. It is vital for promoting the formation of collateral vessels after ischemic events and plays a key role in wound healing. However, this very function of VEGF also plays a key role in the development of both proliferative DR and diabetic macular edema. In diabetic rats, VEGF was overexpressed in diabetic retinas, indicating that VEGF seems to play an important role in the breakdown of the blood-retinal barrier in simple DR. In recent years, the use of anti-VEGF agents has emerged as a new approach to the treatment of these devastating diabetic complications. Inhibition of VEGF can prevent the diabetes-induced permeability, indicating a direct role for VEGF in this pathology. Intraocular delivery of anti-VEGF therapies is now widely used to treat AMD, and clinical trials for treatment of DR are currently being evaluated. All members of the VEGF family stimulate cellular responses by binding to cell surface tyrosine kinase receptors (the VEGFRs). VEGF receptor-1 (VEGFR-1), also known as Flt-1 (fms-like tyrosine kinase receptor-1), is found primarily in endothelial cells and are essential for vasculogenesis and vascular maintenance. VEGF receptor-2 (VEGFR-2), also known as the kinase domain region (KDR), is found primarily in endothelial and hematopoietic cells and their precursors. VEGFR-2 mediates almost all endothelial cell responses to VEGF and is important for vascular and hematopoietic development. VEGFR-3 (fit-4) is restricted primarily to the adult lymphatic endothelium. VEGFR-3 preferentially binds to VEGF-C and VEGF-D and may be involved in the control of lymphangiogenesis. VEGF and its receptors are good targets for therapeutic intervention of DR because VEGF receptors are highly specific and are expressed in increased numbers during pathological vascular growth. Down-regulation of VEGF inhibits retinal angiogenesis when exogenously administered. Therefore, our plan is to downregulate the expression of VEGF via specifically designed RNAi.

(3) The role of PEDF in DR: PEDF, on the other hand, acts as an anti-angiogenic factor in the retina by inhibiting the migration of endothelial cells [22]. PEDF is a 50-kDa protein that is endogenously expressed in ocular tissues and other neuronal and non-neuronal tissues. It has neuronal differentiating and survival activities and behaves as an inhibitor of angiogenesis; The PEDF gene shares structural and sequence homology with members of the serpin gene family[23]. Yet, unlike many serpins, PEDF does not inhibit serine proteases[24]. Instead, it exhibits potent antiangiogenic, neurotrophic and neuroprotective activities. Numerous studies support the role of PEDF in neuronal development, differentiation, and survival[25-27]. More recently, PEDF has been demonstrated to be a major component of the ocular vitreous and aqueous humor where it has been implicated as a potent inhibitor of angiogenesis[22]. Therefore, PEDF is best candidate for prevention the increased vascular permeability and to block the neuronal cell death in DR disease. The second option we plan to take is to over-express PEDF in the DR model to inhibit DR-associated angiogenesis. Studies also show that VEGF and PEDF behave in a balancing fashion and the ratio of VEGF/PEDF corresponds with retinal neovascularization [28]. Regarding these considerations, a combination of transgenes that act on different aspects of angiogenesis may increase the efficacy of gene therapy for DR prevention.

(4) The roles of CBA/CAG promoter: Main studies of DR have been focused on retinopathy associated with the vascular network known as microangiopathy, which is characterized by damage to endothelial cells lining retinal blood vessels and pericytes. Retinal neovascularization in DR occurs away from the RPE and toward the vitreous space. In our experimental plan, we propose to use the CBA and/or CAG universal promoters that are known to enhance vector expression without inducing humoral and cellular immune responses[29, 30] (such as the conventional CMV does) to drive therapeutic genes in the vitreal space of DR mouse model (please also see our preliminary data, Fig. 1).

(5) Gene therapy for ocular diseases: Ocular diseases can be generally divided into three categories which are: 1) ocular disorders that are a known to be caused by a specific type of genetic mutation; 2) ocular diseases where the specific genetic component is unknown or unclear; and 3) ocular diseases that are not genetically based. To date, over 150 retinal disease-causing genes have been identified. Disorders in the first two categories cause a large variety of debilitating retinal degenerations for which there are no curative treatments. Gene therapy in eye can be simply split into two categories, gene replacement and gene regulation (such as a degree to which a gene is knockdown, turned on or off). Gene therapy needs a vector to deliver the therapeutic gene to the target cells. The most challenge facing gene therapy is to deliver quantitatively precise expression of genes in the right cell type and at the right time without host immunity caused. Thus far, delivery methods can be broadly divided into viral and non-viral approaches. Historically, viral vectors have been the most efficient (in terms of cell delivery) and the most popular delivery choice in ocular gene therapy, but they can be limited by cell tropism, size of the expression cassette to be transferred, and host immunity to repeated infections. Recent advances in the formulation of non-viral vectors have greatly improved their efficiency and non-viral gene therapy research has been growing in popularity.

(6) Ocular nano-gene therapy: A NP is defined as any molecular particle that is less than 100 nanometers in size and behaves as a whole unit in terms of its transport and properties. Nanotechnology is a multidisciplinary interactions between the interdisciplinary convergence of basic fields (such as chemistry, physics, mathematics and biology) and applied fields (such as materials science and the various areas of engineering), which contributes to the functional outcomes of the technology. Although multiple different types of NPs have been used to deliver therapeutic genes, a currently popular and successful formulation involves the compaction of a single molecule of plasmid DNA with a PEG substituted lysine 30-mer[8, 10, 31, 32]. The resulting particles have a very small size (8-20nm in diameter) and are either rod or ellipsoidal in shape (depending on the counterion present at the time of compaction).This technology can be used to compact any type of nucleic acid, linear or circular, single or double stranded. Self-compacted DNA NPs as a system for non-viral gene transfer have no theoretical limitation on plasmid size and have been tested with plasmids up to 20kb, do not provoke an immune response in the systems tested (lung and eye), and can be highly concentrated [6-8]. The compacted particle is efficiently taken up into dividing and non-dividing cells and remains episomal. These NPs have been shown to be safe and effective in a human clinical trial for cystic fibrosis and are currently being employed in multiple different organ systems[8, 10, 31-34].The efficiency of CK30PEG-compacted DNA NPs as a system for non-viral gene transfer to ocular tissues have shown no theoretical limitation on plasmid size, do not provoke immune response, and can be highly concentrated [4-8, 11, 32, 35].

Significance: The loss of one's vision is indisputably distressing. DR, which is caused by diabetes, is regarded as "the silent killer of eye". Because of the relative mild early symptoms and the progressive, irreversible pathological developing process, most patients can have long time diabetes before they are diagnosed. Nonetheless, the conventional treatment is limited. The cost for the patient and the society are burden. The impact on patient's quality of life is significant. To explore the next-generation of therapeutics for DR is essentially needed. Recently, gene therapy for diabetes and its complications are spreading out on every corner. Given the dangers inherent in the use of viral vectors, our strategy will enable us to access the favorable aspects of viral vectors while providing the safety and pharmaceutical qualities inherent in non-viral gene delivery systems. In a long run, patients will accept only non-viral vectors as the ways of treatment. In this study, we will generate strategies focus on microvascular and neuropathy to block the vascular and neuronal complications associated with the development and progression in a DR mouse model through non-viral NP delivery. This will be an important step for generate a clinical relevant therapeutic strategy for treatment and prevention of the onset of DR. Therefore, the outcome of this application will contribute significantly to the future application of gene therapy for ocular disorders in general and DR in particular.

C. PRELIMINARY STUDIES: To determine the utility expression of CK30PEG NP in ocular gene therapy, we conducted a set of experiments using nanocompacted DNA to express eGFP driven by the mouse rod opsin promoter (MOP 500) and a ubiquitous CBA promoter. Balb/c mice were injected subretinally on P30 and the retinas were cyosectioned at post-injection (PI) 15 days. Analysis of eGFP fluorescence demonstrated that substantial expression of eGFP was detected in all layers of the retina after injection with nano-CBA-eGFP, whereas, nano-MOP-eGFP generated transgene expression only in photoreceptor cells.

Fig. 1 In vivo testing of nano-MOP-eGFP and nano-CBA-eGFP. Compacted DNA in water (4µg, 1 µl) was subretinally injected into the eyes of adult Balb/c mice. (A) nano-CBA-eGFP, (B) nano-MOP-eGFP, (C) Saline injected control. At PI-15 the retinas were cyosectioned and native eGFP expression was examined by confocal fluorescence microscopy. OS: Outer Segment; ONL: Outer Nuclear Layer; INL: Inner Nuclear Layer; GCL: Ganglion Cell Layer. Scale bar: 10 µm.


Aim 1. Four pre-defined siRNAs will be constructed as shRNA driven by the H1 RNA promoter and cloned into our recently developed mammalian expression vector. The most efficient vector will be compacted into NPs and delivered into the vitreal space of DR mice. A control vector will be designed to house the inactive shRNA sequence directed by the H1 promoter as well. Following injection, the levels of VEGF mRNA will be quantified using RNase protection assays and protein levels will be measured using Western blot analysis.

Aim 2. In this section, we will 1) generate compacted PEG-NP-eGFP and PEG-NP-PEDF under control of CBA and/or CAG promoters and 2) evaluate eGFP and PEDF expression and localization following intravetreal at PI-7, PI-30 and PI-90. Rescue will be assessed structurally, functionally, and biochemically. We anticipate these experiments will produce functional and structural recovery in DR mice model. Thus, the potential scientific and clinical benefits of these proof-of-principles experiments are substantial.

Timetable for completion of the grant: Given our experience with surgical procedures, anatomical methods, and molecular biology, it is anticipated that data gathering for this project will be completed within one year. We do not anticipate any problems with these two parts of the specific aims since the principle investigator (PI) is very familiar with proposed methods and has a substantial publication record in the field of gene therapy. The studies proposed in this application will contribute significantly to the future application of NP gene therapy for ocular diseases. Pending the successful demonstration of effective non-viral NP-mediated induction in ocular cells in mice, and completion of additional toxicology studies along with our group's previous study on NP-gene therapy for ocular disorders, we plan to use these data from the Dr. William "Bill" W. Talley II Research Award to apply for a R01 grant of NP-mediated gene therapy with DR diseases.

E. PROPOSAL AS IT RELATES TO HHODC GOALS AND DIABETES RESEARCH: The proposed grant application fits well with goals of Harold Hamm Oklahoma Diabetes Center (HHODC); the results acquired from this work will help prevent the development of diabetes and its complications in those at risk, particularly in Oklahoma, the most severely affected state in America. This aspect is very important in regions concerned with gene therapy for genetic ocular disorders, especially the working class people. Knowledge gained from these studies will find direct application in the DR with diabetes. The payoffs are both theoretical and applied. At the theoretical level we are working toward a better understanding of the applicability of bio-science models to human populations. On the applied level our results from the proposed application will directly affect quality of life and could be of potential benefit for treating human DR with diabetes by providing the new high technology options.

F. PROPOSAL AS IT RELATES TO CAREER GOALS FOR PI: PI has been active in research for the past 14 years, culminating in a very unique combination of knowledge and experience. He first worked as a medical doctor specializing in neurology. He later expanded his professional interests to include biomedical research, earning his Ph.D. in Neurology in 2002 and since that time has worked on AAV for gene therapy for neurological and genetic mitochondrial disorders. During his post-doctoral training, he worked on developing and applying safe and effective gene delivery strategies for genetic and metabolic disorders involving pre-clinical experiments of gene therapy through AAV vectors, as well as with the human parvovirus vectors. After finishing his post-doctoral training at the University of Florida, he joined the University of Oklahoma Health Sciences Center (OUHSC) as a junior faculty member in the Department of Cell Biology to pursue the research work in gene therapy for neuro-visual diseases using both non-viral nanoparticles and AAV vectors. He has received first hand training in vision research since he joined OUHSC. PI has been active in research for the past 14 years. He has been highly productive as shown by the record of his publications. All the needed resources for the PI to proceed with his proposed research are available at OUHSC and within the Department of Cell Biology. Funding secured from Dr. William "Bill" W. Talley II Research Award would be a significant stepping stone for PI as he works towards his goal of becoming a successful independent investigator and a leading expert in genetic visual diabetic research.

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