677. Generation and Application of an Efficient Chronic Myelogenous Leukemia (CML)-Targeted Recombinant Adeno-Associated Virus (rAAV) Vector: New Therapeutic Options?

Abstract

Top of pageAbstract Introduction: Despite great advances in vector development, most standard rAAV-2 vectors still lack the target cell specificity required for an efficient in vivo application. In addition, the gene transfer efficiency in many promising target cells (e.g.: peripheral blood progenitor cells (PBPC)) is low. Thus, designing gene therapy protocols for diseases like chronic myelogenous leukemia (CML) remained difficult, as standard rAAV-2-based vectors lack both the required gene transfer efficiency and selectivity. A recent advancement in vector development (Muller et al., 2003) now allows the generation of rAAV capsid mutants that offers higher target cell specificity and transduction efficiency. This method was applied to obtain a CML-targeted rAAV vector and was tested on a panel of leukemic cell lines, as well on primary CML material. Material and Methods: Performing latter method on the CML cell line K562 (4 selection rounds), several mutant CML-targeted rAAV2 capsid clones were obtained, cloned into an rAAV2 helper plasmid and for each of the mutants rAAV2-eGFP vector stocks were produced. Titers were verified using our real time PCR-based titration assay. To determine efficiency and specificity, a panel of leukemic (CML and AML) and non-leukemic cell lines, as well primary human CML and PBPC cells were transduced with these vectors. Mock, normal rAAV2 and random mutant clone-transduced cells served as controls. Transduction of primary human CML and PBPC cells was confirmed using FACS-sorted GFP+ cells with subsequent bcr-abl-FISH or FACS analysis for GFP/CD34+ co- expression, respectively. Results: Transduction of a panel of six leukemic cell lines with the CML-targeted rAAV2 vectors (moi 100 iu) resulted in an over 10-fold increase of gene transfer efficiency compared to random mutant and standard rAAV2 vectors; gene transfer rates of >60% were obtained. In primary human cells, for both CML (n=4) and PBPC (n=2), an increase in gene transfer was observed (CML: AAV2: 2.4 |[plusmn]| 0.2 vs AAV2-cml: 5.1 |[plusmn]| 1.6 (p=0.04). CD34+: AAV2: 3.2 |[plusmn]| 0.9 vs AAV2-cml: 8.5 |[plusmn]| 4.3 (p>0.05)). All transduced cell were positive for either the BCR-ABL fusion gene (CML) or the PBPC marker CD34. In contrast, gene transfer of the mutant CML-targeted rAAV2 vectors into non-leukemic cell lines resulted in a reduction in gene transfer efficiency compared to standard rAAV2 vectors (<50%). Conclusion: In this study, we were able to generate a CML-targeted rAAV2 vector, which was able to transduce leukemic cell lines and primary human CML cells with significant higher efficiency and specificity than standard rAAV2 vectors. The development of these vectors holds promise for future clinical applications.