Non-Viral Gene Therapy for Retinal Degeneration

描述（由申请人提供）：视网膜变性是已知的最具遗传异质性的疾病之一，涉及超过184个位点。几项眼部基因治疗临床试验显著地表明，基因治疗是治疗视网膜疾病的有效方法。到目前为止，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著的优势-主要是它们能够有效地将基因传递到体内有丝分裂后的视网膜细胞。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、插入诱变和生产困难。尽管存在这些缺点，但由于缺乏替代方法，病毒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成人视网膜等有丝分裂后组织的非病毒基因转移载体来解决上述缺点，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，非病毒载体仅在细胞培养或正在进行有丝分裂的新生儿视网膜中有效工作。因此，与病毒不同，非病毒载体通常不能拯救视网膜变性的动物模型，除非将其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的肽（POD），当与DNA络合时，可以形成大小类似病毒的纳米颗粒（136nm），并在有丝分裂后的视网膜中实现转基因表达。虽然用POD纳米颗粒进行基因转移不如用病毒有效，但它足以使体内视网膜变性的短期延迟。这只是迄今为止两项研究中的一项，证明了使用非病毒载体延缓成年小鼠的视网膜变性。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利用核DNA整合或DNA保留元件延长POD纳米颗粒的转基因表达。本研究的第二个目的是提高POD的基因转移效率，使其更有效；第三个目的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需要经过一个漫长的过程来开发。这种方法在经济上对bbbb184位点是不可行的。因此，我们建议使用POD纳米颗粒不是传递单个基因，而是编码神经营养因子的基因，例如开发一种非病毒，非基因特异性的方法来治疗视网膜变性。在完成这些研究后，我们将有一个新的非病毒载体准备用于临床试验，等待毒理学研究。如果成功，这些研究将是眼部基因治疗的一个范式转变。

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.