Chapter 5 —— 117 —— Similarly to the bipartite donor AAV vectors co-targeting ATP1A1 and AAVS1 or LMNA, the lowest and highest stably transduced cell foldenrichment factors were associated with the highest and lowest AAVHRA1.IN17 dosages, respectively (Figure 5D and 5E). However, clearly, the in-linkage donor vector AAV-HRA1.IN17 achieved a more thorough and homogeneous positive selection of gene targeted cells than that obtained with the bipartite donor vectors AAV-HRS1.A1 and AAV-HRLMN.A1. In fact, the highest fold-enrichment for gene targeted cells was obtained in hMSC cultures co-transduced with AdVP.C9KARA and AAV-HRA1.IN17 at 0.25 TU cell-1 where ouabain selection resulted in a 40-fold increase in the frequency of gene knock-ins (i.e., 96±1.6% and 2.4±0.7% with and without selection, respectively) (Figure 5D). The purity levels for gene targeted cells in hMSC and HeLa cell cultures attained upon AdVP.C9KARA and AAV-HRA1.IN17 cotransductions and ouabain selection were consistently high, varying from a minimum of 94.6% to a maximum of 99.4% regardless of donor vector input amounts (Figure 5D and 5E). Finally, junction PCR analysis of genomic DNA from stably transduced cell populations confirmed strict ouabaindependent enrichment for ATP1A1-targeted cells (Figure 5F). Taken together, these data establish the selector AAV in-linkage design as a robust strategy for achieving a strict selection for gene targeted cells through precise HR and, simultaneously, the purging of random and/or off-target donor DNA insertions from engineered cell populations. Discussion AAV vectors are commonly used in genome editing protocols as sources of donor HR substrates. However, the recombinogenic character of AAV vector genomes, that bear these substrates, fosters their “capture” at on-target and off-target or random chromosomal breaks through non-homologous end (F) Genotyping of ATP1A1 in co-transduced cell populations. ATP1A1 gene targeting in HeLa cells co-transduced with the indicated vectors and incubated or not with ouabain was assessed through junction PCR. Mock-transduced cells provided for negative controls.
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