Non-Viral Gene Therapy for Retinal Degeneration

视网膜变性的非病毒基因疗法

• 批准号: 8160322
• 负责人: RAJENDRA KUMAR-SINGH
• 金额: $ 41.25万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8160322
• 关键词: Acute; Address; Adenoviruses; Adult; American; Animal Model; Binding; Blindness; Cell Culture Techniques; Cell Nucleus; Cell surface; Cells; Chemicals; Chemistry; Clinical; Clinical Trials; Cloning; Complex; Cytosol; DNA; DNA Integration; DNA delivery; Development; Disadvantaged; Disease; Electroretinography; Elements; Endosomes; Eye Part; Gene Expression; Gene Transfer; Genes; Genetic; Genetic Heterogeneity; Glial Fibrillary Acidic Protein; Goals; Histology; Human; Immune; Immune response; In Situ Nick-End Labeling; Individual; Insertional Mutagenesis; Integrase; Investigational Drugs; Laboratories; LacZ Genes; Light; Longevity; Luciferases; Lysosomes; Malignant Neoplasms; Matrix Attachment Regions; Measures; Mediating; Mitosis; Mitotic; Modeling; Modification; Monophenol Monooxygenase; Muller' s cell; Mus; Neonatal; Non-Viral Vector; Nuclear; Nucleic Acids; Nucleosomes; Outcome Study; Patients; Peptides; Phase I Clinical Trials; Photoreceptors; Process; Production; Proteins; Public Opinion Poll; Publishing; Quantum Dots; Recombinant Proteins; Recombinants; Research; Retina; Retinal; Retinal Degeneration; Retinal Diseases; Retinitis Pigmentosa; Serious Adverse Event; Source; Staining method; Stains; Structure of retinal pigment epithelium; Surface; System; Technology; Testing; Therapeutic; Time; Tissues; Toxic effect; Toxicology; Transgenes; Transgenic Animals; Translating; Viral Vector; Virus; Vision research; Work; body system; clinical application; conditioned fear; design; fluorophore; gene delivery system; gene therapy; gene therapy clinical trial; gene transfer vector; glial cell-line derived neurotrophic factor; glutamylalanine; immunogenicity; improved; in vivo; knockout animal; large scale production; leucyl-alanine; nanoparticle; nanoscale; neurotrophic factor; non-viral gene delivery; non-viral gene therapy; novel; nucleolin; photoreceptor degeneration; plasmid DNA; pre-clinical; recombinant viral vector; recombinant virus; retinal apoptosis; scaffold; small molecule; subretinal injection; trafficking; transgene expression; uptake; vector

DESCRIPTION (provided by applicant): Retinal degeneration is one of the most genetically heterogeneous groups of disorders known, involving over 184 loci. Several ocular gene therapy clinical trials have remarkably demonstrated that gene therapy is a valid approach to treat retinal diseases. Each of these clinical trials and almost every preclinical gene therapy study thus far have utilized viruses as the gene transfer vector. Viruses have significant advantages as gene transfer vectors- primarily their ability to efficiently deliver genes to post-mitotic retinal cells in vivo. However, viruses also have some disadvantages, including induction of host immune responses, a limited transgene capacity, insertional mutagenesis and difficulty in production. Despite these disadvantages, viruses are the current vector of choice in almost all ocular gene therapy studies because of a lack of alternatives. If the above disadvantages could be resolved by the development of non-viral gene transfer vectors that could deliver genes to post-mitotic tissues such as adult retina, it would have substantial impact on the field of preclinical and clinical ocular gene therapy. Unfortunately, non-viral vectors only work efficiently in cell culture or in neonatal retina where mitosis is ongoing. Hence, unlike viruses, non-viral vectors generally fail to rescue animal models of retinal degeneration unless applied in neonatal murine retina - results from which cannot be directly translated to post-mitotic human retina. Recently, we developed a 3.5 Kd peptide (POD) that can form nanoparticles resembling viruses in size (136nm) when complexed with DNA and enable transgene expression in post-mitotic retina. Although gene transfer with POD nanoparticles was not as efficient as with viruses, it was sufficient to enable a short-term delay in retinal degeneration in vivo. This is only one of two studies thus far demonstrating a delay in retinal degeneration in an adult mouse using a non-viral vector. The major limitation of our study was that of short-term transgene expression from POD nanoparticles. The primary objective of this study is to prolong transgene expression from POD nanoparticles by use of nuclear DNA integration or DNA retention elements. The second objective of this study is to improve the efficiency of gene transfer of POD such that it could be more potent and the third objective is to validate the improvements in POD in two relevant animal models of retinal degeneration. The high level of genetic heterogeneity observed in retinal degeneration hampers the timely availability of therapies for patients as each gene and virus combination needs to be developed through a lengthy process. Such approaches are not economically feasible for the >184 loci. Hence, we propose to use POD nanoparticles not to deliver individual genes but instead, genes encoding neurotrophic factors such as to develop a non-viral, non gene-specific approach to treat retinal degeneration. Upon completion of these studies we will have a novel non-viral vector ready for use in clinical trials pending toxicology studies. If successful, these studies would be a paradigm shift in ocular gene therapy. PUBLIC HEALTH RELEVANCE: According to public opinion polls, blindness is the second most feared condition amongst Americans after cancer. Gene therapy for blindness requires the application of viruses that can cause serious adverse events in patients. The objective of this proposal is to develop a non-viral approach to gene therapy. Upon completion, this study will be ready to advance a non-viral gene therapy approach to clinical trials, pending standard toxicology studies as required by the FDA.

描述（由申请人提供）：视网膜变性是已知最具遗传异质性的疾病组之一，涉及超过 184 个基因座。多项眼部基因治疗临床试验已显着证明基因治疗是治疗视网膜疾病的有效方法。迄今为止，每一项临床试验和几乎每一项临床前基因治疗研究都利用病毒作为基因转移载体。病毒作为基因转移载体具有显着的优势——主要是它们能够有效地将基因传递到体内有丝分裂后视网膜细胞。然而，病毒也有一些缺点，包括诱导宿主免疫反应、转基因能力有限、插入突变和生产困难。尽管存在这些缺点，但由于缺乏替代品，病毒仍然是目前几乎所有眼部基因治疗研究中选择的载体。如果能够通过开发能够将基因传递到有丝分裂后组织（例如成人视网膜）的非病毒基因转移载体来解决上述缺点，这将对临床前和临床眼部基因治疗领域产生重大影响。不幸的是，非病毒载体只能在细胞培养物或正在进行有丝分裂的新生儿视网膜中有效地发挥作用。因此，与病毒不同，非病毒载体通常无法挽救视网膜变性的动物模型，除非应用于新生小鼠视网膜，其结果不能直接转化为有丝分裂后的人类视网膜。最近，我们开发了一种 3.5 Kd 肽 (POD)，当与 DNA 复合时，它可以形成类似病毒大小 (136 nm) 的纳米颗粒，并能够在有丝分裂后视网膜中表达转基因。尽管 POD 纳米粒子的基因转移不如病毒有效，但足以在体内短期延迟视网膜变性。这只是迄今为止两项证明使用非病毒载体延迟成年小鼠视网膜变性的研究之一。我们研究的主要限制是 POD 纳米颗粒的短期转基因表达。本研究的主要目的是通过使用核 DNA 整合或 DNA 保留元件来延长 POD 纳米粒子的转基因表达。本研究的第二个目标是提高 POD 基因转移的效率，使其更加有效，第三个目标是在两种相关的视网膜变性动物模型中验证 POD 的改善。在视网膜变性中观察到的高度遗传异质性阻碍了患者及时获得治疗，因为每种基因和病毒组合都需要经过漫长的过程来开发。对于 >184 个基因座，此类方法在经济上不可行。因此，我们建议使用 POD 纳米颗粒不是传递单个基因，而是传递编码神经营养因子的基因，例如开发一种非病毒、非基因特异性方法来治疗视网膜变性。这些研究完成后，我们将拥有一种新型非病毒载体，可用于等待毒理学研究的临床试验。如果成功，这些研究将成为眼部基因治疗的范式转变。 公共健康相关性：根据民意调查，失明是美国人中仅次于癌症的第二大最可怕的疾病。失明的基因治疗需要使用病毒，这可能会给患者带来严重的不良事件。该提案的目的是开发一种非病毒基因治疗方法。完成后，这项研究将准备好将非病毒基因治疗方法推进临床试验，等待 FDA 要求的标准毒理学研究。