Non-Viral Gene Therapy for Retinal Degeneration

描述（由申请人提供）：视网膜变性是已知遗传异质性最大的疾病之一，涉及超过184个基因座。多项眼部基因治疗临床试验已证明基因治疗是治疗视网膜疾病的有效方法。迄今为止，这些临床试验和几乎所有临床前基因治疗研究都使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-主要是它们能够在体内有效地将基因递送到有丝分裂后的视网膜细胞。然而，病毒也有一些缺点，包括诱导宿主免疫应答、有限的转基因能力、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，病毒是目前几乎所有眼部基因治疗研究中的首选载体。如果能够通过开发非病毒基因转移载体将基因转移到有丝分裂后的组织如成人视网膜来解决上述缺点，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，非病毒载体仅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工作。因此，与病毒不同，非病毒载体通常不能挽救视网膜变性的动物模型，除非应用于新生鼠视网膜-其结果不能直接翻译为有丝分裂后的人视网膜。最近，我们开发了一种3.5 Kd肽（POD），当与DNA复合时，可以形成类似病毒大小（136 nm）的纳米颗粒，并使转基因在有丝分裂后的视网膜中表达。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短期延迟。这只是迄今为止证明使用非病毒载体在成年小鼠中延迟视网膜变性的两项研究之一。我们研究的主要局限性是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保留元件来延长转基因从POD纳米颗粒的表达。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水平遗传异质性阻碍了患者及时获得治疗，因为每个基因和病毒组合需要通过漫长的过程来开发。这些方法对于>184个基因座在经济上是不可行的。因此，我们建议使用POD纳米颗粒不递送单个基因，而是递送编码神经营养因子的基因，以开发非病毒、非基因特异性的方法来治疗视网膜变性。这些研究完成后，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，这些研究将成为眼部基因治疗的范式转变。 公共卫生相关性：根据民意调查，失明是美国人仅次于癌症的第二大恐惧症。失明的基因治疗需要应用可能导致患者严重不良事件的病毒。该提案的目的是开发一种非病毒的基因治疗方法。完成后，这项研究将准备推进非病毒基因治疗方法的临床试验，等待FDA要求的标准毒理学研究。

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.