Targeted in vivo gene transfer to hematopoietic stem cells

描述（由申请人提供）：向人造血干细胞（HSC）的有效基因转移具有纠正许多人类血液学和非血液学疾病的潜力。目前，基因转移到HSC中通常是离体实现的，包括分离干细胞，将校正基因转移到细胞中，并将细胞再输注到患者体内。为了实现治疗性基因转移的目的，可以在体内实现靶向基因转移至HSC的载体将具有很大的实用性。对于这个R21的建议，我们的目标是开发和测试体内慢病毒为基础的基因转移载体，将通过造血干细胞表面上表达的分子靶向感染。我们将（i）开发用于靶向感染HSC的慢病毒载体。这将通过将结合HCS上表达的细胞表面受体（包括CD 34和激酶受体c-kit和Flt 3）的蛋白质结构域融合到突变辛德毕斯病毒包膜上来实现，所述突变辛德毕斯病毒包膜结合其受体严重受损但能够进行膜融合。这些载体将使用体外细胞培养系统进行广泛表征。然后，我们将继续（ii）评估为靶向体内造血干细胞感染而产生的载体。我们将研究将慢病毒载体靶向HSC的两种递送途径：（a）注射入骨髓和（B）动员HSC从骨髓进入血液后的全身递送。这些实验将严格测试体内靶向感染HSC的可行性。体内靶向载体至细胞具有增加向细胞的基因转移同时降低病毒载量的潜在优势。它还避免了基因转移到可能阻碍基因治疗的细胞，即树突细胞。造血干细胞的靶向将为体内靶向提供原理证明，并支持其用作关键血液学疾病（如免疫缺陷、AIDS和范可尼贫血）的治疗方法。此外，从这些研究的结果将有显着的影响，一般使用逆转录病毒载体的靶向治疗基因转移。 公共卫生相关性：该拨款提案旨在开发和测试病毒载体，这些病毒载体被设计用于感染体内特定的细胞类型。在这里，我们建议开发这些针对干细胞的感染，这些干细胞负责制造血液中的所有细胞。这些病毒载体的开发将使通过将这些载体直接注射到体内来治疗血液疾病成为可能。我们的发现也可以应用于其他疾病。

DESCRIPTION (provided by applicant): Efficient gene transfer to human hematopoietic stem cells (HSC) has the potential to correct a number of human hematologic and non-hematologic diseases. At present, gene transfer into HSC's is typically achieved ex vivo, involving isolation of stem cells, transferring a corrective gene into the cells and re-infusion of cells into the patient. For the purpose of achieving therapeutic gene transfer, there would be great utility in a vector that could achieve targeted gene transfer in vivo to HSC. For this R21 proposal we aim to develop and test in vivo lentiviral based gene transfer vectors that will target infection through molecules that are expressed on the surface of hematopoietic stem cells. We will (i) develop lentivirus vectors for targeting infection of HSC. This will be achieved by fusing protein domains that bind cell surface receptors expressed on HCS (including CD34, and the kinase receptors c-kit and Flt3) onto a mutant Sindbis virus envelope that is severely impaired for binding its receptor but is competent for membrane fusion. These vectors will be extensively characterized using in vitro cell culture systems. We will then proceed to (ii) evaluate vectors generated for targeting infection of hematopoietic stem cells in vivo. We will investigate two routes of delivery for targeting lentiviral vectors to HSC: (a) injection into the bone marrow and (b) systemic delivery after mobilization of HSC out of the marrow into blood. These experiments will critically test the feasibility of targeting infection to HSC in vivo. Targeting vector to cells in vivo has the potential advantage of increasing gene transfer to cells while decreasing viral load. It also avoids gene transfer to cells that could impede gene therapy i.e. dendritic cells. The targeting of hematopoietic stem cells will provide proof of principle for in vivo targeting and support its use as a therapeutic approach for key hematologic disorders, such as immunodeficiencies, AIDS and Fanconi anemia. Furthermore, results from these studies will have significant implications for targeted therapeutic gene transfer using retroviral vectors in general. Public Health Relevance: This grant proposal aims to develop and test viral vectors that are engineered to infect a specific cell type in the body. Here we propose to develop these to target infection of stem cells that are responsible for making all the cells of the blood. The development of these viral vectors will enable treatment of blood disorders by direct injection of these vectors into the body. The discoveries we make can also be applied to other diseases.