A better gene therapy envelope for transducing G0 CD4+ T cells

描述(由申请人提供)： 基因治疗中的一个挑战是载体转导G0静止的CD4+T细胞和G0干细胞的能力有限。开发这样的载体很重要，因为与激活细胞相比，转导静止细胞更具生理性，成本更低，劳动强度更低，可能更安全，而且带有基因标记的细胞可能在体内存活更长时间。VSV-G蛋白是一种包膜蛋白，通常用于转导活化的CD4+T细胞的伪型慢病毒载体，然而，VSV-G假型慢病毒载体不能有效地转导G0型CD4+T细胞。我们实验室的一个长期目标是设计一种更好的包膜来转导G0静息的CD4+T细胞。因此，我们通过测量静息细胞中的结合、融合、逆转录和整合来研究为什么VSV-G假型不能转导静息的CD4+T细胞。使用这种分析HIV感染的各个步骤的方法，我们以前证明了，与教条相反，HIV转导静止的CD4+T细胞。当我们比较慢病毒载体与VSV-G或HIV包膜(Env)的假型时，我们发现HIV Env伪型与静止的CD4+T细胞的融合效率是VSV-G假型的100倍。对这一发现的进一步研究表明，HIV Env与静止细胞的融合效率要高得多，因为HIV Env与辅助受体的结合触发了信号，从而诱导了细胞变化，从而促进了病毒的融合。为了设计更好的转导G0静止的CD4+T细胞的包膜，我们将测试其他包膜，单独和与HIV Env结合的能力，与静止的T细胞融合的能力，并探索HIV Env与静止的CD4+T细胞有效融合的机制。 公共卫生相关性： 基因疗法正在发展成为治疗许多疾病的一种可行的替代方案，包括免疫系统紊乱、某些癌症，甚至艾滋病毒感染。在基因治疗中，感兴趣的基因被输送到靶细胞，在某些情况下，靶细胞是静止的细胞；然而，为了获得有效的基因转移，通常需要激活静止的细胞进入细胞周期。一个重要但难以实现的目标是，在不激活某些静止细胞的情况下，将感兴趣的基因传递给它们，因为细胞周期不仅改变了静止细胞的功能，而且可能存在安全风险。在这项资助中，我们建议确定允许将基因输送到静止的CD4+T细胞的方法，作为实现在不激活细胞的情况下实现基因输送目标的第一步。

DESCRIPTION (provided by applicant): A challenge in gene therapy is the limited ability of vectors to transduce G0 resting CD4+ T cells and G0 stem cells. It is important to develop such vectors because transducing resting cells, as compared to activated cells, is more physiological, less expensive, less labor-intensive, possibly safer, and the gene-marked cells may be longer lasting in vivo. VSV-G protein is an envelope that is commonly used to pseudotype lentiviral vectors for transducing activated CD4+ T cells; however, lentiviral vectors pseudotyped with VSV-G do not transduce G0 CD4+ T cells efficiently. A long-term goal of our lab is to design a better envelope for transducing G0 resting CD4+ T cells. Therefore, we investigated why VSV-G pseudotypes do not transduce resting CD4+ T cells by measuring binding, fusion, reverse transcription, and integration in resting cells. Using this approach of assaying individual steps of HIV infection, we previously showed, contrary to dogma, that HIV transduces resting CD4+ T cells. When we compared lentiviral vectors pseudotyped with VSV-G or HIV envelope (Env), we found that HIV Env pseudotypes fused to resting CD4+ T cells 100 times more efficiently than VSV-G pseudotypes. Further investigation of this finding suggested that HIV Env fuses to resting cells much more efficiently because binding of HIV Env to co-receptor triggers signaling that induces cellular changes that enhance viral fusion. Toward the goal of designing a better envelope for transducing G0 resting CD4+ T cells, we will test the ability of other envelopes, singly and in combination with HIV Env, to fuse to resting T cells, and explore the mechanism underlying the ability of HIV Env to efficiently fuse to resting CD4+ T cells. PUBLIC HEALTH RELEVANCE: Gene therapy is developing into a viable alternative for the treatment of many diseases including immune system disorders, certain cancers, and even HIV infection. In gene therapy, a gene of interest is delivered to a target cell, which in some cases is a quiescent cell; however, to obtain efficient gene transfer it is often necessary to activate a quiescent cell to enter the cell cycle. An important but elusive goal is delivery of the gene of interest to certain quiescent cells without activating them, because cell cycling not only alters the function of the quiescent cell, but may also be a safety risk. In this grant we propose to identify methods that allow delivery of a gene to quiescent CD4+ T cells as the first step toward achieving the goal of gene delivery without cellular activation.