Vector engineering for non-viral delivery of large genomic DNA to the RPE

摘要 近年来，大量的遗传性致盲疾病使眼部基因治疗成为一个活跃的研究领域 几年，最终在广为人知的临床试验中达到顶峰。然而，对于这两个人来说，有效的治疗方法仍然难以捉摸。 原因：基因表达在持续时间和水平上往往仍然不足，而且包装能力有限 标准载体的使用禁止将疾病基因及其顺式调控元件包括在内。克服困难 这些障碍对该领域的发展和临床的广泛应用至关重要。现行基因 递送策略使用基于cdna的载体，这种载体缺乏基因组DNA中的非编码元件和顺式元件。 来调节基因的表达。这些基因组序列可以保持转录本的稳定性，提高 翻译并产生生理上相关的表达水平。在此应用程序中，我们检验假设 提供完整的纠正基因，包括其真正的启动子、增强子、内含子和未翻译的 区域(UTRs)改善RPE65相关小鼠模型中转基因表达的水平和持续时间 Leber先天性黑色素沉着症(LCA)，一种影响视网膜色素上皮(RPE)的疾病。这种病 表现在儿童早期，导致逐渐的视力丧失，常常导致失明。由于高企 RPE65相关LCA的患病率，已经进行了几项临床试验，使用腺相关 携带人RPE65基因的病毒(AAV)。然而，几乎所有的尝试都未能阻止正在进行的视觉 损失。因此，我们的目标是通过传递完整的人类RPE65基因来建立一种有效的治疗方法 利用其5‘/3’调控元件、外显子和内含子治疗LCA小鼠模型(RPE65-/-)。我们的目标是实现 以细胞特异性的方式全面调控和长期表达，以确保拯救LCA疾病 表型。我们已经将人RPE65基因组片段克隆到表达载体中，并将开发出 利用裸露的DNA或以纳米粒子(NPs)形式与多赖氨酸复合的DNA的有效传递平台 聚乙二醇偶联肽(CK30PEG)。我们将测试寿命和基因表达水平 在使用这些平台交付RPE65之后，并评估它们在 RPE65-/-小鼠。在AIM 1中，我们将设计能够在RPE中达到治疗性表达水平的载体 在目标2中，我们将评估LCA模型在RPE中的长期救援和转导效率 和发病后的疾病表型。总之，这项申请的结果将有助于 RPE相关疾病的非病毒基因治疗进展。

Summary The vast number of inherited blinding disorders has made ocular gene therapy an active research field in recent years, culminating in well-publicized clinical trials. However, efficacious therapies are still elusive for two reasons: gene expression often remains inadequate in duration and levels, and the limited packaging capacity of standard vectors prohibits the inclusion of disease genes with their cis-regulatory elements. Overcoming these barriers is critical for the advancement of the field and widespread clinical application. Current gene delivery strategies use cDNA-based vectors that lack the non-coding and cis-elements found in genomic DNA that regulate gene expression. These genomic sequences can preserve the stability of the transcript, improve translation and produce physiologically relevant levels of expression. In this application, we test the hypothesis that providing the entire corrective gene including its authentic promoter, enhancer, introns and untranslated regions (UTRs) improves the levels and duration of transgene expression in mouse model of RPE65-associated Leber Congenital Amaurosis (LCA), a disease that affects the retinal pigment epithelium (RPE). This disease manifests in early childhood leading to a gradual vision loss often resulting in blindness. Due to the high prevalence of RPE65-associated LCA, several clinical trials have been conducted using adeno-associated viruses (AAV) to deliver human RPE65 cDNA. However, almost all attempts failed to halt the ongoing visual loss. We thus aim to establish an effective therapeutic approach by delivering the whole human RPE65 gene with its 5’/3’ regulatory elements, exons and introns to treat a mouse model of LCA (RPE65-/-). We aim to achieve full regulation and long-term of expression in a cell-specific manner to ensure rescue of the LCA disease phenotype. We have cloned the human RPE65 genomic fragment into an expression vector and will develop an effective delivery platform utilizing naked DNA or DNA formulated as nanoparticles (NPs) with polylysine peptides conjugated to polyethylene glycol (CK30PEG). We will test the longevity and levels of gene expression after RPE65 delivery using these platforms, and evaluate their ability to mediate full phenotypic rescue in the RPE65-/- mice. In aim1, we will engineer vectors that can achieve therapeutic levels of expression in the RPE and in aim 2 we will evaluate long-term rescue and transduction efficiency in the RPE of the LCA model before and after the onset of the disease phenotype. In summary, results from this application will facilitate the advancement of non-viral gene therapy for RPE-associated diseases.