Generation of Highly Efficient Recombinant Adeno-Associated Virus (rAAV) Vectors Targeted to Chronic Myelogenous Leukemia (CML) - and CD34+ Progenitor Cells Obtained by the „AAV Random Peptide Library“ Method.
Authors: Sellner Leopold , Veldwijk Marlon , Stiefelhagen Marius , Kleinschmidt Juergen A. , Wenz Frederik , Zeller W. Jens , Fruehauf Stefan 
Published: October 2019

Abstract
Resistance of chronic myelogenous leukemia (CML) to imatinib is a serious clinical challenge. In vivo targeted gene therapy may be a strategy to overcome resistance. For gene therapy of CML standard rAAV2-based vectors lack the required gene transfer efficiency as well as the in vivo selectivity. An advancement in vector development (Muller et al., Nat. Biotech., 2003) now allows the generation of rAAV capsid mutants that offer higher target cell specificity and efficiency. We used the “AAV random peptide library” method to generate capsid mutants efficient and specific for CML cell lines, primary CML cells and primary human peripheral blood progenitor cells PBPC. After previously applying this method successfully on CML cell lines, an effective vector for CML cell lines could be obtained. On primary human CML and PBPC the new vector was more efficient than rAAV2 but still at an unsatisfying level. To increase the gene transfer on primary cells we applied the “AAV library” on primary human CML and PBPC. A total of 44 different mutant capsid clones were obtained, most of them at three or less copies. Four clones were observed >10x to 39x and were selected for further testing. To determine efficiency and specificity, a panel of leukemic (CML, imatinib-sensitive and -resistant, and AML) and other solid tumor cell lines were transduced (moi 100). On a panel of leukemic cell lines similar or higher gene transfer efficiency of the rAAV capsid mutants was observed compared to standard rAAV2 vectors. The higher transduction efficiency in imatinib-resistant cell line LAMA84-R than in their sensitive counterpart LAMA84-S was particularly striking, as the difference in susceptibility for the capsid mutants vs rAAV2 in LAMA84-S (table 1). On solid tumor cell lines rAAV2 was more efficient, confirming the higher specificity of our capsid mutants. Compared to our previous work (<1% GFP+ cells), the new capsid mutants now result in efficient gene transfer into the AML cell line KG1a, and to lesser extend the CML cell line EM3. For both cell lines gene transfer with the capsid mutants was readily more efficient than with standard rAAV2 vectors. Preliminary data on primary PBPC showed a uniformly higher transduction efficiency for all capsid mutants (%GFP_mut1-4: 13.6% ± 2.2% vs %GFP_AAV2: 5.3%). Experiments with primary human CML cells are ongoing. Using the “AAV random peptide library” method on primary cells, we were able to generate rAAV vectors with higher transduction efficiency on leukemic cell lines and primary PBPC than standard rAAV2 vectors. The development of these highly efficient AAV mutant capsid vectors holds promise for future development.
Gene transfer efficiency (%GFP+ cells) Cell line Capsid mutant Standard rAAV2 Data are given as mean ± SD LAMA84-S 78.2 ± 7.9 9.2 ± 2.5 LAMA84-R 97.8 ± 0.3 93.0 ± 0.2 HL-60 54.0 ± 5.4 57.6 ± 8.2 BV173 48.4 ± 7.4 30.6 ± 1.8 EM3 3.9 ± 0.7 0.2 ± 0.2 KG1a 24.7 ± 0.7 12.8 ± 5.0