HIV vectors for stem cell and intrakine gene delivery

描述(申请人提供)：人类免疫缺陷病毒1型(HIV) 是获得性免疫缺陷综合症(ADS)的病原体。谁 据估计，到今年年底，世界人口的0.7% 都会感染艾滋病毒。高效抗逆转录病毒疗法(HAART) 改善了许多艾滋病毒血清阳性者的生活质量 世界。大多数其他感染者无法访问HAART，而HAART 并不总是有效的。因为目前还没有疫苗，所以有 开发新疗法的令人信服的理由，包括引入 基因修饰的细胞，以防止感染艾滋病毒。 以小鼠白血病病毒(MLV)为基础的载体已被广泛用作基因 转运车辆。不幸的是，MLV的有效转导需要 有丝分裂。以艾滋病毒为基础的媒介在他们的 能够转导各种静止的人类细胞，包括CD34+ 被认为代表原始造血干细胞的亚群 (HSC)。这项建议集中在HSC亚群的转导和再繁殖 针对HIV的基因治疗载体的分析与发展 艾滋病毒本身。将在不同的HSC子集之间进行仔细的比较 (例如，CD34+、LIN-CD34-、CD34+KDR+、CD34+KDR-和‘侧群’细胞) 它们被艾滋病毒载体转导的能力。读数将包括 甲基纤维素。集落和LT-CIC检测，以及NOD/SCID小鼠的再繁殖 转导细胞。为了最大限度地减少可变性，HSC子集将单独 用编码不同转基因的HIV载体转导并移植到 同一只老鼠。在这些研究的同时，编码CCR5和CCR5的艾滋病毒载体 将构建CXCR4内激活子。使用‘基因的可诱导载体 由于CXCR4非常关键，SDF-KDEL INTERKING将使用Switch系统 用于干细胞归巢。这些载体最初将用于转导T细胞 和巨噬细胞，以评估对HTV感染的保护。一旦药效是 证明，细胞内载体将用于HSC转导和 SCID-HU THY/LIV模型中的差异。合成的转导胸腺细胞 将在体外接受H1V病毒的挑战，以确定体内是否有 起到了保护作用。 最后，将构建编码HSV胸苷激酶的自杀性HIV载体， 可在更昔洛韦存在的情况下在TAT和Rev.存在的情况下诱导， 也表达TAT和REV的转导细胞将经历细胞死亡。这 载体将在已建立的和原代T细胞中测试HIV的有效性 抑制力。这将与上面的动画内编码向量和一个 反义包膜构建，包括单独构建和组合构建。这些中间载体 比较应该有助于确定更好的转基因组合。如果 这些研究的成功可能会促进临床分级的发展 抗HIV慢病毒载体转导肝星状细胞。

DESCRIPTION (provided by applicant): Human immunodeficiency virus type 1 (HIV) is the causative agent of the acquired immunodeficiency syndrome, or ADS. WHO estimates that, by the end of this year, 0.7 percent of the world's population will be infected with HIV. Highly active anti-retroviral therapy (HAART) has improved the quality of life for many HIV seropositive individuals in the First World. Most other infected individuals have no access to HAART, and HAART is not always effective. Because a vaccine is not yet available, there are compelling reasons to develop novel therapeutics, including the introduction of gene-modified cells to protect against HIV. Vectors based upon murine leukemia virus (MLV) have been widely used as gene transfer vehicles. Unfortunately, efficient transduction by MLV requires mitosis. HIV-based vectors have demonstrated remarkable promise in their ability to transduce a variety of resting human cells, including CD34+ subpopulations that are thought to represent primitive hematopoietic stem cells (HSC). This proposal concentrates on HSC subset transduction and repopulation analyses and the development of HIV-based gene therapy vectors directed against HIV itself. Careful comparisons will be made between different HSC subsets (e.g., CD34+, lin-CD34-, CD34+KDR+, CD34+KDR-, and 'side-population' cells) in their ability to be transduced by HIV vectors. Readout will include methylcellulose. colony and LT-CIC assays, and repopulation of NOD/SCID mice by transduced cells. To minimize variability, the HSC subsets will be separately transduced with HIV vectors encoding different transgenes and engrafted in the same mouse. In parallel with those studies, HIV vectors encoding both CCR5 and CXCR4 intrakines will be constructed. An inducible vector employing the 'gene switch' system will be used for the SDF-KDEL intrakine since CXCR4 is critical for stem cell homing. These vectors will be initially used to transduce T cells and macrophages to evaluate protection against HTV infection. Once efficacy is demonstrated, the intrakine vectors will be used for HSC transduction and differentiation in the SCID-hu thy/liv model. Resultant transduced thymocytes will be challenged with H1V ex vivo to determine whether the intrakines have a protective effect. Lastly, a suicide HIV vector encoding HSV thymidine kinase will be constructed, inducible in the presence of both Tat and Rev. In the presence of ganciclovir, transduced cells which also express Tat and Rev will undergo cell death. This vector will be tested in established and primary T cells for efficacy of HIV inhibition. This will be compared to the intrakine-encoding vector above and an antisense envelope construct, both alone and in combination. These intervector comparisons should serve to identify the superior transgene combination. If successful, these studies may facilitate the development of clinical-grade anti-HIV lentiviral vectors for transduction of HSC.