Anti-HIV Gene Delivery to Human Hematopoietic Cells

描述(由申请人提供)：这项建议的目标是研究慢病毒载体传递的、经证实的基于基因的HIV-1干扰方式在人类造血干细胞(HSCs)、髓系细胞和CD4+T细胞中的毒性和功能。据估计，在接受高效抗逆转录病毒疗法(HAART)的艾滋病患者中，有30-45%的患者存在某种形式的耐药性。因此，抗药性HIV-1s的不断出现使得开发具有新的作用机制的治疗方法势在必行。我们假设，通过采用经过验证的、基于基因的HIV-1破坏方式的组合，以及使用慢病毒载体将其输送到CD34+和T细胞，抗HIV-1基因阳性细胞可能对感染无效，不能有效复制病毒，并且受保护的细胞将随着时间的推移而丰富。我们的长期目标是提供病毒和细胞基因破坏策略的基本研究结果，这些策略可能会用于已经或即将失败的HAART患者的自体T和CD34+细胞或同种异体CD34+细胞移植来源。类似于用于HAART的联合药物策略，我们正在完成一种慢病毒递送载体，该载体包含多个针对细胞和HIV信息和蛋白质的基因，从而扰乱病毒的进入、整合、病毒和细胞功能。此外，已经选择了目标位置，如果出现突变，所产生的病毒应该不太适合。 我们的目标将通过成功完成四个高度互动的特定目标来实现：1)包含多个HIV-1干扰基因的完整慢病毒载体。2)抗HIV慢病毒载体在不破坏正常细胞功能的同时是否提供了对HIV-1的保护？3)抗HIV基因的表达是否改变了人类的造血和胸腺生成？4)含有抗HIV基因的HSC是否产生了保护其免受HIV-1攻击的髓系细胞和CD4+T细胞？ 这些研究完成后，将提供含有针对HIV-1及其细胞途径的多个抗病毒基因的慢病毒载体是否会破坏造血功能的信息。我们的发现还将深入了解，在存在HIV-1感染的情况下，抗病毒基因的选定组合是否具有细胞保护和选择性优势。最后，预计在我们的研究中开发和验证的许多基因中断和慢病毒载体传递策略将适用于其他基础研究和临床应用。 项目简介：据估计，在接受高效抗逆转录病毒疗法(HAART)的艾滋病患者中，有30-45%的患者存在某种形式的耐药性。因此，抗药性HIV-1病毒的不断出现需要新的治疗方法。我们的长期目标是提供有关病毒和细胞抗HIV-1策略的基因传递的基本研究成果，使血细胞对HIV-1感染具有抵抗力，并减少HIV-1的生长。这些新疗法可能用于已经或即将失败的HAART患者的T细胞和CD34+细胞移植来源。

DESCRIPTION (provided by applicant): The goals for this proposal are to investigate toxicity and function of lentiviral vector delivered, proven gene- based HIV-1 disruption modalities in human hematopoietic stem cells (HSCs), myeloid and CD4+ T cells. It is estimated that 30-45% of AIDS patients on Highly Active Anti-Retroviral Therapy (HAART) having demonstrable virus have some form of drug resistance. Thus, the continuing emergence of drug resistant HIV-1s makes it imperative to develop therapeutics with novel mechanisms of action. We posit that by employing a combination of proven, gene-based HIV-1 disruption modalities, and the use of lentiviral vectors for delivery to CD34+ and T cells, anti-HIV-1 gene positive cells may be refractory to infection, not replicate virus efficiently, and protected cells will enrich over time. Our long-term goals are to provide basic research findings on viral and cellular gene disruption strategies that may find use for autologous T and CD34+ cell or allogenic CD34+ cell transplant sources for patients that have or are going to fail HAART. Analogous to combination drug strategies used for HAART, we are completing a lentiviral delivery vector containing multiple genes that target cellular and HIV messages and proteins, thereby disrupting viral entry, integration, viral and cellular function. Moreover, target sites have been selected that if mutations arise the resulting virus should be less fit. Our goals will be achieved by the successful completion of four highly interactive Specific Aims: 1) Complete lentiviral vectors containing multiple HIV-1 disruption genes. 2) Do anti-HIV lentiviral vectors provide protection from HIV-1 while not disrupting normal cellular function? 3) Does expression of anti-HIV genes alter human hematopoiesis and thymopoiesis? 4) Do HSCs containing anti-HIV genes give rise to myeloid and CD4+ T cells that are protected from HIV-1 challenge? When completed these studies will provide information on whether lentiviral vectors containing multiple anti- viral genes targeting HIV-1 and its cellular pathways disrupt hematopoietic function. Our findings will also provide insight into whether selected combinations of anti-viral genes confer a cell protective and selective advantage in the presence of HIV-1-infection. Lastly, it is anticipated that many of the gene disruption and lentiviral vector delivery strategies developed and validated during our studies will be applicable for other basic research and clinical uses. Project Narrative: It is estimated that 30-45% of AIDS patients on Highly Active Anti-Retroviral Therapy (HAART) having demonstrable virus have some form of drug resistance. Thus, the continuing emergence of drug resistant HIV-1s requires new therapies for treatment. Our long-term goals are to provide basic research findings on gene delivery of viral and cellular anti-HIV-1 strategies that make blood cells resistant to HIV-1 infection and reduce growth of HIV-1. These new therapies may be utilized for T cell and CD34+ cell transplant sources for patients that have or are going to fail HAART.