Lentiviral transduction of P140K MGMT into human CD34(+) hematopoietic progenitors at low multiplicity of infection confers significant resistance to BG/BCNU and allows selection in vitro.

摘要: Lentiviral vectors may improve hematopoietic stem cell (HSC) gene transfer because of their enhanced ability to transduce nondividing cells. However, many studies report efficient transduction only at high multiplicities infection (MOI). This study reports human CD34+ cells with a drug resistance allowing post-transduction selection using lentivirus under low-MOI conditions that did not require cytokine stimulation or viral concentration. We used the P140K methylguanine-DNA-methyltransferase mutant (P140K MGMT) as insert into second-generation lentiviral backbone and triple-plasmid transfection generate vesicular stomatitis virus (VSV)-G protein-pseudotyped virus. The MGMT product, O6-alkylguanine-DNA-alkyltransferase (AGT), provides protection from therapeutic combination 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) wild-type AGT inhibitor O6-benzylguanine (BG). Low-speed spinoculation more than addition Polybrene multiple exposures. Addition cytokines was required. Low-MOI (≤1) lin− resulted in an average 41% 89% rate assessed by PCR, respectively, concordant expression conferred substantial clonogenic survival advantage after BG/BCNU treatment. During vitro selection, 87% surviving cell-derived colony-forming units (CFU) were transduced. work shows potential utility for HSCs purpose vivo marrow protection. Because will enrich transduced progenitors, MOI can be avoided, improving safety profile transfer.