Hybrid synthetic targeted lenti-VLP nanoparticle for gene delivery

描述(由申请人提供)：对更好的基因传递的需求阻碍了生物医学研究的潜在进步，最关键的是，在基因功能水平上操作的更好的治疗方法的开发。这个问题对于未分裂的细胞最为严重，事实上，这些细胞是主要的细胞类型。因此，人们对基因治疗的兴趣大大缩小，主要集中在开发体外方法和使用具有几种重要的独特能力的慢病毒载体，其中包括向未分裂的细胞输送。适用于所有已开发的慢病毒载体的一个主要限制是缺乏对非肠道给药的适用性，这主要是因为它们依赖于一种挑剔的包膜蛋白，该包膜蛋白提供细胞结合、细胞膜融合和细胞内递送的关键功能。尽管包括高通量基因工程(如定向进化)在内的许多努力，来自VSV的天然包膜G蛋白多年来一直用于伪型逆转录病毒载体，但仍然是慢病毒载体最有效的包膜之一。这一问题对开发多功能靶向慢病毒载体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传递的靶向纳米颗粒已经展示了使用配体-聚合物结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，我们正在开发一种将我们成功的合成组织选择性靶向与适合于非分裂细胞的慢病毒颗粒相结合的杂交技术。我们正在开发一种配体靶向的聚合物涂层，以取代在没有包膜蛋白的情况下产生的Lenti-Virus样颗粒(Lenti-VLP)中缺失的慢病毒包膜蛋白功能。由于该蛋白是慢病毒载体不稳定的主要来源之一，预计极大地增强稳定性将是一个额外的好处。我们的初步结果表明，简单的均聚阳离子多肽可以取代慢病毒载体包膜蛋白的功能，但其效力需要大幅提高。在这一阶段的研究计划中，为了确定杂交合成配体标记的靶向Lenti-VLP的可行性，我们首先将构建和筛选一些具有明确结构的阳离子多肽，以确定一种能够与Lenti-VLP结合并取代包膜蛋白功能但不会导致Lenti-VLP不稳定的SAR。然后，我们将确定将靶向配体偶联到选定的多肽而不会对细胞递送功能产生不利影响的方法，并在动物模型中优化细胞选择性配体介导的细胞选择性配体介导的基因递送。 公共卫生相关性：对更好的基因传递的需求是开发在基因功能水平上运作的迫切需要的治疗选择的主要障碍。许多努力都集中在慢病毒载体的体外应用上，这些载体具有许多独特的优点，但一个主要的限制是不能静脉给药，因为依赖于提供细胞结合和细胞内递送的挑剔的包膜蛋白。相反，我们的核酸靶向纳米粒子已经证明了配体介导的组织靶向，但效率，特别是对非分裂细胞的效率仍然是一个主要障碍。这项计划中的研究将在动物模型中确定缺乏包膜蛋白的混合合成配体靶向Lenti病毒样颗粒的可行性，以及细胞选择性配体介导的新生血管组织基因传递的优化。

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.