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

描述（由申请人提供）： 基因治疗的一个挑战是载体抑制G 0静息CD 4 + T细胞和G 0干细胞的能力有限。开发这样的载体是重要的，因为与活化细胞相比，转导静息细胞更符合生理，更便宜，劳动强度更低，可能更安全，并且基因标记的细胞在体内可能更持久。VSV-G蛋白是通常用于假型化慢病毒载体以转导活化的CD 4 + T细胞的包膜;然而，用VSV-G假型化的慢病毒载体不能有效地活化G 0 CD 4 + T细胞。 我们实验室的一个长期目标是设计一个更好的包膜来转导G 0静息CD 4 + 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，研究了VSV-G假型为什么不抑制静息CD 4 + T细胞。使用这种检测HIV感染的各个步骤的方法，我们以前表明，与教条相反，HIV转导静息的CD 4 + T细胞。当我们比较用VSV-G或HIV包膜（Env）假型化的慢病毒载体时，我们发现HIV Env假型与静息CD 4 + T细胞融合的效率比VSV-G假型高100倍。对这一发现的进一步研究表明，HIV Env与静息细胞融合的效率要高得多，因为HIV Env与辅助受体的结合触发了诱导细胞变化的信号传导，从而增强了病毒融合。 为了设计一种更好的转导G 0静息CD 4 + T细胞的包膜，我们将测试其他包膜单独和与HIV Env组合与静息T细胞融合的能力，并探索HIV Env有效融合静息CD 4 + T细胞的机制。 公共卫生相关性： 基因治疗正在发展成为治疗许多疾病的可行替代方案，包括免疫系统疾病，某些癌症，甚至HIV感染。在基因治疗中，感兴趣的基因被递送到靶细胞，在某些情况下靶细胞是静止细胞;然而，为了获得有效的基因转移，通常需要激活静止细胞以进入细胞周期。一个重要但难以捉摸的目标是将感兴趣的基因递送到某些静止细胞而不激活它们，因为细胞周期不仅改变静止细胞的功能，而且可能是安全风险。在这项研究中，我们提出了确定方法，使基因传递到静止的CD 4 + T细胞作为第一步实现基因传递的目标，没有细胞活化。

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.