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.
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