Non-Viral Gene Therapy for Retinal Degeneration

项目描述 视网膜变性是已知的最具遗传异质性的疾病组之一，涉及超过100种视网膜病变。 184个位点。一些眼部基因治疗的临床试验已经显著地证明了基因治疗是一种有效的治疗方法。 治疗视网膜疾病的方法。这些临床试验和几乎所有的临床前基因治疗研究 迄今为止，已经利用病毒作为基因转移载体。病毒作为基因转移有着显著的优势 载体-主要是它们在体内有效地将基因递送到有丝分裂后视网膜细胞的能力。然而，病毒 也具有一些缺点，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。尽管有这些缺点，病毒是目前 由于缺乏替代品，几乎所有眼部基因治疗研究都选择了载体。如果上述 这些缺点可以通过开发非病毒基因转移载体来解决， 基因的有丝分裂后组织，如成人视网膜，它将有实质性的影响领域的临床前 和临床眼部基因治疗。不幸的是，非病毒载体仅在细胞培养物中有效地起作用，或者在细胞培养物中有效地起作用。 正在进行有丝分裂的新生视网膜。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，除非应用于新生鼠视网膜-其结果不能 直接翻译到有丝分裂后的人类视网膜。最近，我们开发了一种3.5Kd的肽（POD）， 当与DNA复合时，纳米颗粒的大小类似于病毒（136 nm），并使转基因表达成为可能 在有丝分裂后的视网膜中。虽然用POD纳米颗粒进行基因转移不如用病毒有效，但它是有效的。 足以使体内视网膜变性短期延迟。这只是迄今为止的两项研究之一 证明了使用非病毒载体在成年小鼠中视网膜变性的延迟。主要限制 我们的研究的重点是从POD纳米颗粒的短期转基因表达。的主要目的 本研究是通过使用核DNA整合或 DNA保留元件。本研究的第二个目的是提高转基因效率， 第三个目标是在两个方面验证POD的改进， 视网膜变性的相关动物模型。在视网膜病变中观察到的高水平的遗传异质性 退化阻碍了患者及时获得治疗，因为每个基因和病毒组合 需要经过一个漫长的过程。这种方法在经济上是不可行的， >184个位点。因此，我们建议使用POD纳米颗粒不递送单个基因，而是递送基因， 编码神经营养因子，如开发一种非病毒，非基因特异性的方法来治疗视网膜病变。 退化这些研究完成后，我们将有一个新的非病毒载体准备用于临床 正在进行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。

Project Description 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.