Non-Viral Gene Therapy for Retinal Degeneration

项目说明 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 184个基因座。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 治疗视网膜疾病的方法。每一项临床试验和几乎每项临床前基因治疗研究 到目前为止，已经利用病毒作为基因转移载体。病毒在基因转移方面有显著的优势 载体-主要是它们有效地将基因运送到体内有丝分裂后视网膜细胞的能力。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。尽管有这些缺点，但病毒仍是当前的 由于缺乏替代品，几乎所有眼科基因治疗研究都选择载体。如果出现上述情况 缺点可以通过开发非病毒基因转移载体来解决，这种载体可以 基因转移到成年视网膜等有丝分裂后组织，将对临床前领域产生实质性影响 和临床眼部基因治疗。不幸的是，非病毒载体只有在细胞培养或 新生儿视网膜，其中有丝分裂正在进行。因此，与病毒不同的是，非病毒载体通常无法挽救 视网膜变性的动物模型，除非应用于新生的小鼠视网膜-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3.5kD的多肽(POD)，它可以形成 当纳米粒子与DNA络合时与病毒大小相似(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进行基因转移，但它 足以在体内实现视网膜退化的短期延迟。这只是迄今为止两项研究中的一项 展示了使用非病毒载体的成年小鼠视网膜退化的延迟。主要的限制 我们的研究是通过POD纳米颗粒的短期转基因表达。的主要目标是 本研究旨在利用核DNA整合技术延长POD纳米颗粒的转基因表达。 DNA保留元素。本研究的第二个目标是提高基因转移的效率。 第三个目标是验证POD在两个方面的改进 视网膜变性相关动物模型的建立。视网膜中观察到的高度遗传异质性 退化阻碍了患者及时获得治疗，因为每一个基因和病毒组合 需要通过一个漫长的过程来开发。这样的方法在经济上是不可行的 &gt；184个座位。因此，我们建议使用POD纳米颗粒而不是单个基因，而是基因 编码神经营养因子，例如开发一种非病毒、非基因特异性的方法来治疗视网膜 退化。这些研究完成后，我们将拥有一种新的非病毒载体，可用于临床 等待毒理学研究的试验。如果成功，这些研究将是眼科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.