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运作的更好的治疗方法的发展。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，对基因治疗的极大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独特能力的慢病毒载体，包括递送到非分裂细胞。所有慢病毒载体的一个主要限制是缺乏肠外给药的适用性，这主要是由于它们依赖于一种精细的包膜蛋白，这种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。尽管做出了许多努力，包括高通量基因工程(例如；“定向进化”)，多年来用于伪逆转录病毒载体的VSV天然包膜G蛋白仍然是慢病毒载体最有效的包膜之一。这一问题对多用途靶向慢病毒载体的开发构成了本质上不可逾越的障碍。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证明了利用配体-聚合物偶联的多用途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合成的组织选择性靶向与适合非分裂细胞的慢病毒颗粒相结合的杂交体。我们正在开发一种配体靶向聚合物涂层，以取代在没有包膜蛋白的慢病毒样颗粒（lentivlp）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性的主要来源之一，因此大大增强的稳定性预计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，但效力还需要大幅度提高。为了确定在I期研究计划中混合合成配体介导的靶向lentivlp的可行性，我们首先将构建和筛选一些具有确定结构的阳离子多肽，以确定结合lentivlp并取代包膜蛋白功能的SAR，但不会引起lentivlp的不稳定性。然后，我们将确定靶向配体与选定多肽偶联的方法，而不会对细胞传递功能产生不利影响，并在细胞培养和动物模型中优化细胞选择性配体介导的基因传递。

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.