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

用于将大基因组 DNA 非病毒传递至 RPE 的载体工程

• 批准号: 10667049
• 负责人: Muna I. Naash
• 金额: $ 23.25万
• 依托单位: UNIVERSITY OF HOUSTON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10667049
• 关键词: Affect; Biochemical; Blindness; Cells; Clinical; Clinical Trials; Cloning; Cone; Cytomegalovirus Infections; DNA; Dependovirus; Disease; Distal; Early treatment; Elements; Engineering; Enhancers; Ensure; Equilibrium; Equipment; Exons; Eye; Gene Delivery; Gene Expression; Gene Expression Regulation; Gene Transduction Agent; Genes; Genetic; Genomic DNA; Genomics; Goals; Hereditary Disease; High Prevalence; Housing; Human; Human Cloning; Inherited; Injections; Introns; Leber' s amaurosis; Longevity; Matrix Attachment Regions; Mediating; Methodology; Methods; Modeling; Molecular Biology; Mus; Onset of illness; Outcome; Outcome Measure; Papio; Parents; Peptides; Phenotype; Physiological; Polyethylene Glycols; Polylysine; Positioning Attribute; Quantitative Reverse Transcriptase PCR; RPE65 protein; Regulation; Regulatory Element; Research; Resources; Retina; Retinal Diseases; Retinal gene therapy; Retinitis Pigmentosa; Rod; Structure; Structure of retinal pigment epithelium; Testing; Therapeutic; Toxic effect; Transcript; Translations; Treatment Efficacy; Untranslated RNA; Untranslated Regions; Viral; Vision; Visual; Western Blotting; adeno-associated viral vector; cis acting element; clinical application; cytotoxic; delivery vehicle; design; disease phenotype; early childhood; efficacious treatment; efficacy evaluation; experience; experimental study; expression vector; extracellular; gene therapy; improved; in vivo; innovation; mouse model; nanoparticle; non-viral gene therapy; novel; preservation; promoter; scaffold; targeted treatment; therapeutic gene; therapeutically effective; therapy development; tool; transduction efficiency; transgene expression; translational impact; uptake; vector

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.

概括 大量的遗传性盲目疾病使眼基因疗法成为了最近的活跃研究领域 几年，最终在盛大的临床试验中。但是，有效的疗法仍然难以捉摸两个 原因：基因表达通常在持续时间和水平上不足，包装能力有限 标准载体禁止将疾病基因纳入其顺式调节元件。克服 这些障碍对于该领域的发展和广泛临床应用至关重要。当前基因 输送策略使用基于cDNA的矢量，这些向量缺乏基因组DNA中发现的非编码和顺式元素 调节基因表达。这些基因组序列可以保留转录本的稳定性，改善 翻译并产生与身体相关的表达水平。在此应用中，我们检验假设 提供整个纠正基因，包括其正宗启动子，增强子，内含子和未翻译 区域（UTRS）改善了与RPE65相关的小鼠模型中转化表达的水平和持续时间 Leber先天性弹药（LCA），一种影响视网膜色素上皮（RPE）的疾病。这种疾病 在幼儿时期的表现会导致年级视力损失通常会导致失明。由于高 与RPE65相关的LCA的患病率，使用腺相关进行了几项临床试验 病毒（AAV）输送人RPE65 cDNA。但是，几乎所有尝试都无法阻止正在进行的视觉 损失。因此，我们旨在通过传递整个人RPE65基因来建立有效的治疗方法 具有5'/3'的调节元件，外显子和内含子来处理LCA的小鼠模型（RPE65 - / - ）。我们旨在实现 以细胞特异性的方式进行全面调节和长期表达，以确保拯救LCA疾病 表型。我们将人RPE65基因组片段克隆到表达载体中，并将​​发展 利用裸体DNA或DNA的有效递送平台，该平台用聚赖氨酸配制为纳米颗粒（NP） 与聚乙烯乙二醇（CK30PEG）结合的肽。我们将测试基因表达的寿命和水平 RPE65使用这些平台交付后，并评估了它们调解完整表型救援的能力 RPE65 - / - 小鼠。在AIM1中，我们将设计可以在RPE中实现热表达的向量 在AIM 2中，我们将在LCA模型的RPE中评估长期的救援和转移效率 在疾病表型发作之后。总而言之，此应用程序的结果将有助于 针对RPE相关疾病的非病毒基因疗法的进步。