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

这项建议的主要目的是使一种新型的杂合腺病毒载体能够稳定高效地将基因转移到造血干细胞(HSCs)中，为β-链状血红蛋白病的基因治疗奠定基础。不含所有病毒基因的整合型deltaAD.AAV杂合载体结合了腺病毒载体的高滴度、高感染性和大容量的特点与AAV ITR的整合能力。该项目的目标包括：i)DeltaAD的机制。AAV整合的进一步特征和d3eltaAD的优势。AAV载体与目前用于基因治疗的经典病毒载体相比，将在红白血病细胞系的转导研究中得到证实。Ii)通过将AV Rep功能整合到杂交载体中，整合频率将提高，并将刺激特定部位的整合。为此，将应用deltaAD.AAV的独特结构，仅在293细胞中病毒复制的后期阶段，才将rep基因置于HCS特异性启动子控制下的转录活性位置。作为替代方案，将评估功能性Rep蛋白分子是否可以共包装成deltaAD.AAV衣壳。基于Ad5的deltaADAAV载体的趋向性将基于CAR或α5β3/5整合素独立的细胞进入而被修饰以有效感染HSCs。将分析其他人或动物的腺病毒感染HSCs的能力。将创建一个嵌合载体，其中包含那些成功感染HSCs的病毒的异源纤维。或者，针对Ad5纤维结节/内化HSC受体的双特异性抗体或带有HSC特异性多肽配体的纤维基因修饰剂将被用于重定向Ad5载体。将测试重定向的deltaAD.AAV载体是否能稳定地转导静止的HSC。Iv)优化的deltaAD.AAV载体将用于在目标细胞中以适当的水平和持久性表达伽马珠蛋白基因。为此，将测试一系列珠蛋白表达盒，包括与完整的伽马珠蛋白基因相连的6至8kb长的最佳功能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.AAV载体转导HSCs的能力将通过集落试验和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.