Hybrid synthetic targeted lenti-VLP nanoparticle for gene delivery

描述（由申请人提供）：对更好的基因递送的需求阻碍了生物医学研究的潜在进步，最关键的是阻碍了在基因功能水平上操作的更好的治疗方法的开发。这个问题对于非分裂细胞最为严重，而非分裂细胞实际上是主要的细胞类型。因此，对基因治疗的相当大的兴趣已经大大缩小到主要开发离体方法和使用具有几种重要的独特能力（包括递送到非分裂细胞）的慢病毒载体的努力。适用于所有开发的慢病毒载体的主要限制是缺乏肠胃外给药的适用性，这主要是由于它们依赖于挑剔的包膜蛋白，该包膜蛋白提供细胞结合的关键功能，然后是细胞膜融合和细胞内递送。尽管进行了许多努力，包括高通量基因工程（例如“定向进化”），多年来用于假型逆转录病毒载体的来自VSV的天然包膜G蛋白仍然是慢病毒载体的最有效包膜之一。这个问题已经对多功能靶向慢病毒载体的开发构成了基本上不可逾越的障碍。有趣的是，我们开发的用于非病毒基因递送的靶向纳米颗粒已经证明了使用配体-聚合物缀合的多功能配体介导的组织靶向，但对非分裂细胞的活性仍然是一个主要障碍。因此，我们正在开发一种混合物，将我们成功的合成组织选择性靶向与适合非分裂细胞的慢病毒颗粒相结合。我们正在开发一种配体靶向的聚合物涂层，以取代慢病毒包膜蛋白功能缺失的慢病毒样颗粒（慢病毒-VLP）没有包膜蛋白生产。由于这种蛋白质是慢病毒载体不稳定性的主要来源之一，因此预期大大增强的稳定性是额外的益处。我们的初步结果表明，简单的均聚物阳离子多肽可以取代慢病毒载体包膜蛋白的功能，但效力需要大大提高。为了确定在该I期研究计划中杂合合成配体介导的靶向lenti-VLP的可行性，我们首先将构建和筛选许多具有确定结构的阳离子多肽，以鉴定用于结合lenti-VLP和替换包膜蛋白功能但不引起lenti-VLP不稳定性的SAR。然后，我们将确定用于将靶向配体偶联到所选多肽而不对细胞递送功能产生不利影响的方法，并优化细胞培养物中的细胞选择性配体介导的基因递送和动物模型中的新生血管组织。 公共卫生相关性：对更好的基因递送的需要是开发在基因功能水平上操作的急需的治疗性治疗选择的主要障碍。许多努力集中在具有许多独特优点的慢病毒载体的离体使用上，但主要限制是由于依赖于提供细胞结合和细胞内递送的挑剔的包膜蛋白而不能肠胃外施用。相反，我们的核酸靶向纳米颗粒已经证明了配体介导的组织靶向，但效率，特别是对于非分裂细胞的效率仍然是一个主要障碍。计划的研究是确定缺乏包膜蛋白的杂交合成配体靶向慢病毒样颗粒的可行性，并在动物模型中优化细胞选择性配体介导的基因递送用于新生血管组织。

DESCRIPTION (provided by applicant): A need for better gene delivery has blocked the potential advancement in biomedical research and most critically development of better therapeutics that operate at the gene function level. This problem is most acute for non-dividing cells, which in fact are the predominant cell type. Thus the considerable interest in gene therapy has narrowed considerably to efforts largely developing ex-vivo approaches and use of lentiviral vectors with several important unique capabilities that include delivery to non-dividing cells. A major limitation that applies to all the lentiviral vectors developed is a lack of applicability for parenteral administration, largely due to their dependence on a finicky envelope protein that provides the critical functions of cell binding and then cell membrane fusion and intracellular delivery. Despite many efforts, including high throughput genetic engineering (e.g. "directed evolution"), the natural envelope G protein from VSV used to pseudotype retroviral vectors for many years remains one of the most effective envelope for lentiviral vectors. This problem has posed an essentially insurmountable barrier to the development of versatile targeted lentiviral vectors. Interestingly, our development of targeted nanoparticles for non-viral gene delivery has demonstrated versatile ligand-mediated tissue targeting using ligand-polymer conjugation but activity on non-dividing cells remains a major hurdle. Thus we are developing a hybrid combining our successful synthetic tissue selective targeting with lentiviral particles adept for non-dividing cells. We are developing a ligand-targeted polymer coating to replace the lentiviral envelope protein function missing in lenti-viral like particles (lenti-VLP) produced without envelope protein. Since this protein is one of the major sources of lentiviral vector instability, greatly enhanced stability is expected to be an added benefit. Our preliminary results indicate that simple homopolymer cationic polypeptides can replace lentiviral vector envelope protein function but the potency needs to be enhanced substantially. To determine feasibility of a hybrid synthetic ligand- medated targeted lenti-VLP in this Phase I research plan, we first will construct and screen a number of cationic polypeptides with defined structures to identify an SAR for binding lenti-VLP and replacing envelope protein function but without causing instability of the lenti-VLP. We then will identify methods for coupling targeting ligands to selected polypeptides without adversely affecting the cell delivery function and optimize cell selective ligand-mediated gene deliver in cell culture and for neovasculature tissue in an animal model. Public Health Relevance: A need for better gene delivery is a major barrier to development of critically needed therapeutic treatment options that operate at the gene function level. Many efforts are focused on ex-vivo uses of lentiviral vectors with many unique advantages but a major limitation is an inability for parenteral administration, due to dependence on a finicky envelope protein that provides cell binding and intracellular delivery. Conversely, our targeted nanoparticles for nucleic acids have demonstrated ligand- mediated tissue targeting but efficiency, especially for non-dividing cells remains a major hurdle. The planned study is to determine feasibility of a hybrid synthetic ligand-targeted lenti-virus like particle lacking envelope protein and optimization of cell selective ligand-mediated gene delivery for neovasculature tissue in an animal model.