Novel Efficient Primary Human Peripheral Blood Progenitor Cell-Targeted Recombinant Adeno-Associated Viral Vectors.

Introduction: G-CSF mobilized peripheral blood progenitor cells (PBPC) are an excellent target for the cure of acquired and inherited disorders of the hematopoietic system. AAV-based vectors have an advantageous safety profile, due to their low immunogenicity, the lack of any associated human disease and its episomal localization. Unfortunately, currently standard rAAV2-based vectors lack the required efficiency for gene transfer into human blood progenitor cells. To increase the gene transfer efficiency on primary hematopoietic cells we applied an AAV random peptide library on primary human CD34+ PBPC.

Materials and Methods: The AAV random peptide library was applied on primary human CD34+ PBPC samples (3 selection rounds). Several mutant blood progenitor-targeted rAAV2 capsid clones were obtained and sequenced. Five new rAAV vectors with the most efficient clones were produced. Titers were verified using real time PCR-based titration. To determine efficiency and specificity, a panel of leukemia (CML and AML) and non-leukemia cell lines, as well as primary human CD34⁺ PBPC were transduced. GFP expression was determined against uninfected control cells.

Results: On a panel of leukemia cell lines at least one capsid mutant was significantly more efficient (%GFP⁺; p<0.01) than standard rAAV2 vectors. On solid tumor cell lines rAAV2 was more efficient, suggesting the increased specificity of our capsid mutants for hematopoietic cells. Significantly higher gene transfer efficiency (up to 16% GFP⁺ cells) could be obtained with the newly generated vectors compared to standard rAAV2 (up to 9-fold higher; p<0.01). Using a different approach, up to 25% gene transfer into CD34+ PBPC was observed.

Conclusion: These novel vectors may finally enable efficient (>20%) gene transfer using rAAV-based vectors into primary human PBPC and may offer a safe alternative to other vector systems which is applicable in vivo.