68. Evidence for Correction of Mutations in the c-Kit Gene by rAAV Mediated Gene Targeting in W/Wv Mice
Authors: Arthur W. Nienhuis, John T. Gray, Serena Vayda, Amanda Cline, Marguerite V. Evans-Galea, David M. Bodine
Journal: Molecular Therapy (2006)

Recombinant adeno-associated viral vectors (rAAV) correct genetic lesions by gene targeting via interactions between homologous sequences in the vector and a chromosomal gene at a frequency of up to 1% in cultured and primary cells (reviewed in Hendrie PC, Russell DW, Mol Ther. 12:9–17, 2005). Although gene addition with retroviral vectors has proven therapeutic for Severe Combined Immunodeficiency (SCID), integration carries the risk for insertional mutagenesis and proto-oncogene activation (Science 302: 415–419, 2003) so that alternative strategies are needed. Mice with mutations in both c-Kit genes (W locus) are sterile, have defects in pigmentation and have mild macrocytic anemia. Useful assays for gene correction are available since bone marrow cells from W/Wv mice do not form 14 day spleen colonies or achieve long-term engraftment of lethally irradiated mice. A single wild-type cell is able to reconstitute unirradiated W/Wv mice. The W mutation, a G to A substitution in the splice donor site at the exon 10 - intron 10 boundary, eliminates an AarI site and the Wv mutation, a C to T transition in the last codon of exon 13, creates a NsiI site. The targeting vector, rAAV-c- Kit, was derived by subcloning a 4.6 kb EcoR1-BglII fragment from the c-Kit locus, recovered from a BAC clone (RP23-2), into a plasmid containing rAAV2 ITRs. Using a self-complementary rAAV2 vector genome encoding Green Fluorescent Protein (GFP), we found that vector particles packaged with AAV serotype 1 capsid proteins were 10 fold more efficient at transducing lineage depleted, c-Kit+, ScaI+ murine bone marrow cells than particles packaged in serotype 2 capsid proteins. At a MOI of 105 serotype 1 vector particles/ target cell, more than 95% of the c-Kit+, ScaI+ cells were transduced. Using optimized conditions, we transduced 1.2 × 107 lineage depleted W/Wv bone marrow cells with 1.2 × 1012 rAAV-c-Kit, serotype 1 vector particles in serum-free medium containing 100 ng/ml of SCF, TPO, IL-6 and Flt-3L and 10 ng/ml of IL-3. Many spleen colonies were present in irradiated recipients 14 days after injection with transduced cells but none were found in controls which received mock transduced W/Wv cells. Southern blot analysis provided evidence that the AarI and NsiI sites reflecting the W and Wv mutations, respectively, had been completely or partially changed (AarI gained and NsiI lost) in the majority (4 of 5) colonies suggesting biallelic correction during colony development. In an initial experiment designed to evaluate the ability of rAAV-c-Kit to achieve gene correction in long-term repopulating cells, we found that 2 of 5 irradiated wildtype mice injected with transduced W/Wv lineage depleted cells survived at 6 weeks with normal hematopoiesis. Overall, our data suggest that rAAV vectors can be used to achieve gene correction in primitive, primary hematopoietic cells at a detectable frequency which may be useful for gene therapy of disorders such as SCID in which there is a positive selective advantage for gene corrected cells.