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

该提案的主要目的是使新的杂合腺-AAV载体适应于稳定和有效的基因转移到造血干细胞（HSC）中，为β-链血红蛋白病的基因治疗创造基础。缺乏所有病毒基因的整合性Δ AD.AAV杂合载体将腺病毒载体的特征（包括高滴度、高感染性和大容量）与AAV ITR的整合能力相结合。该项目的目标包括：i）将进一步表征deltaAD.AAV整合的机制，并将在红白血病细胞系的转导研究中证明d3 eltaAD. AAV载体相对于目前用于基因治疗的经典病毒载体的优势。ii）通过将AV Rep功能并入杂合载体中，将提高整合频率并刺激位点特异性整合。为此，将应用deltaAD.AAV的独特结构，仅在293细胞中病毒复制的晚期阶段，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到deltaAD.AAV衣壳中。iii）基于Ad 5的deltaAD.AAV载体的向性将被修饰用于基于CAR-或α 5 β 3/5-整联蛋白非依赖性细胞进入的HSC的有效感染。将分析其他人或动物腺病毒感染HSC的能力。将产生嵌合载体，其含有来自成功感染HSC的那些病毒的异源纤维。或者，针对Ad 5纤维结/内化HSC受体或具有HSC特异性肽配体的纤维遗传修饰剂的双特异性抗体将用于重新靶向Ad 5载体。将测试重新靶向的Δ AD.AAV载体是否可以稳定地抑制静止HSC。iv）优化的deltaAD.AAV载体将用于在靶细胞中以足够的水平和持久性表达γ-珠蛋白基因。为此，将检测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长的最佳功能LCR盒。将使用全人骨髓或纯化的CD 34+细胞进行e靶向杂交载体的转导研究。将通过集落测定和SCID-NOD小鼠中的体内扩增实验来测试deltaAD.AAV载体扩增HSC的能力。

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.