Deleted adeno AAV vectors for stable gene transfer into hematopoietic stem cells

本研究的主要目的是建立一种新的腺- aav杂交载体，使其能够稳定高效地将基因转移到造血干细胞中，为β -链血红蛋白病的基因治疗奠定基础。对ad积分。不含所有病毒基因的AAV杂交载体结合了腺病毒载体的高滴度、高传染性、大容量等特点，具有AAV itr的整合能力。本项目的目标包括：i) delta - ad的机制。AAV集成将进一步表征d3eltaAD的优势。目前用于基因治疗的经典病毒载体的AAV载体将在红白血病细胞系的转导研究中得到证明。ii)通过将AV Rep函数纳入混合载体，可以提高集成频率，促进位点特异性集成。要做到这一点，deltaAD的独特结构。只有在293细胞的病毒复制后期，AAV才能在HCS特异性启动子的控制下使rep基因处于转录活性位置。作为替代方案，将评估功能性Rep蛋白分子是否可以共包装到deltaAD中。AAV衣壳。iii)基于Ad5的deltaAD的趋向性。将对AAV载体进行修饰，使其基于car或不依赖于alpha5beta3/5-整合素的细胞进入高效感染hsc。其他人类或动物腺病毒将被分析感染造血干细胞的能力。一个嵌合载体将被创建，其中包含来自那些成功感染造血干细胞的病毒的异源纤维。或者，针对Ad5纤维的双特异性抗体/内化HSC受体或具有HSC特异性肽配体的纤维基因修饰剂将用于重新靶向Ad5载体。它将被测试是否重新定向delta。AAV载体能稳定转导静止型HSC。iv)优化的delta。AAV载体将用于在靶细胞中表达γ -珠蛋白基因，并使其达到适当的水平和持久性。为此，将测试一系列珠蛋白表达盒，包括与完整γ -珠蛋白基因连接的6至8 kb长功能最佳的LCR盒。e靶向杂交载体的转导研究将在整个人骨髓或纯化的CD34+细胞中进行。deltaAD的能力。AAV载体转导造血干细胞将通过集落试验和SCID-NOD小鼠体内扩增实验进行测试。

The main objective of this proposal is to adapt a new hybrid adeno-AAV vector for stable and efficient gene transfer into hematopoietic stem cells (HSCs) creating a basis for gene therapy of beta-chain hemoglobinopathies. The integrating deltaAD.AAV hybrid vector devoid of all viral genes combines features of adenoviral vectors including high titer, high infectivity, and a large capacity with the integration capability of AAV ITRs. The goals of this project include: i) The mechanism of deltaAD.AAV integration will be further characterized and the advantages of d3eltaAD.AAV vectors over classical viral vectors currently used for gene therapy will be demonstrated in transduction studies on erythroleukemic cell lines. ii) The integration frequency will be improve and sites-specific integration will be stimulated by incorporating the AV Rep function into hybrid vectors. To do this, the unique structure of deltaAD.AAV will be applied to bring the rep gene in a transcriptionally active position under the control of a HCS specific promoter only at late stages of virus replication in 293 cells. As an alternative, it will be evaluated whether functional Rep protein molecules can be co-packaged into deltaAD.AAV capsids. iii) The tropism of Ad5 based deltaAD.AAV vectors will be modified for efficient infection of HSCs based on CAR-or alpha5beta3/5-integrin independent cell entry. Other human or animal adenoviruses will be analyzed for the ability to infect HSCs. A chimeric vector will be created containing the heterologous fiber from those viruses successful at infecting HSCs. Alternatively, bispecific antibodies to the Ad5 fiber knob/internalizing HSC receptors or fibers genetically modifier with HSC specific peptide ligands will be used to retarget Ad5 vectors. It will be tested whether re-targeted deltaAD.AAV vectors can stably transduce quiescent HSC. iv) Optimized deltaAD.AAV vectors will be used to express gamma-globin genes in target cells at adequate levels and persistence. For this end, a series of globin expression cassettes will be tested including 6 to 8 kb long optimally functioning LCR cassettes linked to the complete gamma-globin gene. Transduction studies with e-targeted hybrid vectors will be performed with whole human bone marrow or purified CD34+ cells. The ability of deltaAD.AAV vectors to transduce HSCs will be tested by colony assays and in vivo expansion experiments in SCID-NOD mice.