Efficient Gene Transfer into Human CD34⁺ Peripheral Blood Progenitor Cells Using Pseudotyped Recombinant Adeno-Associated Viral Vectors

Due to their pluripotency, human CD34⁺ hematopoietic stem cells are targets of interest for the treatment of many acquired and inherited disorders of the hematopoietic system using gene therapeutic approaches. Human CD34⁺ peripheral blood progenitor cells (PBPC) can be readily mobilized into the blood and harvested by leukapheresis, providing an easily accessible source of hematopoietic progenitor cells. Unfortunately, for gene transfer into CD34⁺ PBPC, most current vector systems either lack sufficient transduction efficiency or an acceptable safety profile. Standard adeno-associated virus-based vectors have an advantageous safety profile, yet lack the required efficiency. Therefore a panel of pseudotyped recombinant adeno-associated viral (rAAV2/1 - rAAV2/6) vectors expressing the eGFP gene was screened on human G-CSF-mobilized CD34⁺ PBPC to determine their efficacy. In addition, the difference in transgene expression between conventional single-stranded rAAV and self complementary rAAV (scAAV) vectors was determined. For each vector n≥6 was performed and data are shown as mean ± SD. Of all screened conventional rAAV vectors, rAAV2/6 proved to be the most efficient (13.5% ± 9.8% GFP⁺ and CD34⁺ PBPC; p<0.001 vs other vectors) on human CD34⁺ PBPC, followed by rAAV2/2 (2.6% ± 2.0% GFP⁺ cells) and rAAV2/1 (1.4% ± 1.2% GFP⁺ cells). For rAAV2/3, rAAV2/4 and rAAV2/5 no relevant gene transfer efficiency (<1% GFP⁺ cells) was observed. Furthermore, the relevance of the single-to-double-strand conversion block in transduction of human PBPC could be shown using scAAV vectors. scAAV2/6 and scAAV2/2 (both p<0.001) showed significantly higher gene expression (38.4% ± 12.2% and 11.8% ± 5.7% GFP⁺ cells, respectively) compared to their conventional counterparts in this cell entity. Similar results were observed for scAAV2/1 vectors (2.8% ± 1.9% GFP⁺ cells), though the difference was not significant. Of note, as previously observed using AAV peptide library-derived rAAV vectors (Sellner et al., 2008, Exp Hematol. 36), also here inter-patient variances in CD34⁺ PBPC susceptibility were found. For the first time we were able to obtain clinically relevant gene transfer and expression levels (>10%) with expression rates up to 60% in human CD34⁺ PBPC using an AAV-based vector system, thereby providing an efficient alternative vector system for gene transfer into this clinically important target cell.