Generation and Application of a CML-Specific Recombinant Adeno-Associated Virus (rAAV) Vector.

Introduction: Chronic myelogenous leukemia can be controlled but in most patients not be cured by tyrosine kinase inhibition. Direct targeting using gene therapy vectors combined with vaccination strategies may allow to eradicate residual leukemic progenitors. Adeno-associated virus (AAV) vectors are stable DNA vectors which were proven to be effective in the clinical gene therapy for e.g. coagulation disorders. The various AAV serotypes lack specificity for BCR-ABL+ leukemia cells. Recently developed AAV-library techniques allow a retargeting of vectors. We generated a set of rAAV vectors specific for BCR-ABL+ cells.

Material and Methods: After four selection rounds on BCR-ABL+ cells, the peptide sequences of the persisting clones were cloned into an AAV helper plasmid. rAAV-GFP stocks (2 K562-specific, 1 random) of each of the mutants were produced. Titers were determined using real time PCR-based titration assay. Both, a panel of leukemic cell lines and CML primary material were transduced with these vectors and gene transfer was determined by FACS analysis. Specificity was tested in a competitive transduction assay using BCR-ABL⁺ and BCR-ABL⁻ leukemic cell lines. Transduction of primary CML cells was confirmed using FACS-sorted GFP⁺ cells and subsequent BCR-ABL-FISH.

Results: Using the CML-specific rAAV clone on a panel of BCR-ABL+ cell lines, gene transfer rates of >60% could be obtained (random clone: <1%; rAAV-2: <5%), whereas the BCR-ABL⁻ cell lines were not susceptible to these vectors (gene transfer < 1 %). Admixing BCR-ABL− to BCR-ABL+ cells did not result in a significant drop of the gene transfer rates in the BCR-ABL− cell lines, suggesting that vector particles were not blocked by unspecific binding. Using primary material, significant gene transfer was observed (>5%; 6x more efficient than rAAV-2). In those cells, the CML-genotype was confirmed by BCR-ABL-FISH.

Conclusion: In this study, we were able to generate and apply a CML-specific rAAV vector on CML cell lines and primary material. Efficient and selective gene transfer in these cells could be obtained compared to standard rAAV-2 vectors and randomly generated clones. These data hold promise for future developments.