Brian M. Davis

Generation of a packaging cell line for prolonged large-scale production of high-titer HIV-1-based lentiviral vector.

An Erratum has been published for this article in Journal of Gene Medicine 7(6), 2005, 835.

Characterization of the P140K, PVP(138-140)MLK, and G156A O6-Methylguanine-DNA Methyltransferase Mutants: Implications for Drug Resistance Gene Therapy

The G156A O6-alkylguanine-DNA alkyltransferase (AGT) mutant protein, encoded by the G156A O6-methylguanine-DNA methyltransferase gene (MGMT), is resistant to O6-benzylguanine (BG) inactivation and, after transduction into hematopoietic progenitors, transmits remarkable resistance to BG and BCNU. As a result, a clinical trial, in which the MGMT gene is transduced into CD34+ cells of patients with cancer, has been approved. A newly identified AGT mutation, P140K, generates dramatically increased BG resistance relative to G156A, and suggests that gene transfer of P140K may confer improved hematopoietic cell protection. To address this hypothesis, we measured BG + BCNU and BG + TMZ resistance in G156A, P140K, or P138M/V139L/P140K (MLK) MGMT-transduced K562 cells. In addition, we performed a detailed characterization of individual properties including BG resistance, activity, and protein stability of these mutants in human hematopoetic K562 cells and E86 retroviral producer cells. In K562 cell extracts, the MLK and P140K mutants retained full activity at doses up to 1 mM BG, while G156A had a BG ED50 of 15 microM, compared with 0.1 microM for wtAGT. In the absence of BG, the G156A protein possessed a 56% reduction in specific O6-methyltransferase activity compared with wtAGT. MLK, P140K, and wtAGT all possessed similar specific activities, although the O6-methyl repair rate of all mutants was reduced 4- to 13-fold relative to wtAGT. The wtAGT, MLK, and P140K proteins were stable, with half-lives of greater than 18 hr. In contrast, only 20% of the G156A protein was stable after 12 hr in cycloheximide and, interestingly, the remaining protein appeared to retain most of the activity present in non-cycloheximide-treated cells. Differences in BG resistance, activity, and stability between P140K, MLK, and G156A suggest that P140K may be the optimal mutant for drug resistance gene transfer. However, hematopoietic K562 cells transduced with MFG-G156A, P140K, or MLK had similar degrees of BG and BCNU as well as BG and TMZ resistance when treated with concentrations of BG (< or =25 microM) achieved in clinical trials, suggesting similar efficacy in many in vivo applications.

Safe two-plasmid production for the first clinical lentivirus vector that achieves >99% transduction in primary cells using a one-step protocol.

We report the design of a unique two‐plasmid production system for the first lentiviral vector to be evaluated in humans, VRX496. VRX496 is an optimized VSV‐G pseudotyped vector derived from HIV‐1 that expresses antisense to the HIV envelope gene. We found that a two‐plasmid approach to production resulted in higher vector production titers when compared with a three‐plasmid approach, which is particularly important for vector production at the large scale. Therefore, we carefully designed a single packaging construct, VIRPAC, for safety by reducing its homology with VRX496 and by insertion of functionally validated genetic elements designed to reduce the risk of generation of a replication‐competent lentivirus (RCL). A native cis‐acting ribozyme is used to prevent read through into the envelope gene from the upstream gag‐pol genes in the packaging vector, thus preventing RNAs containing gag‐pol and env together for comparable safety to a three‐plasmid system. We demonstrate that there is no significant in vivo vector mobilization using a primary SCID‐hu mouse transplantation model, which correlates with the presence of an anti‐HIV payload and suggests that inclusion of antisense may be a useful tool to restrict mobilization in other vector constructs. Gene transfer is achieved using a one‐step transduction procedure that is simple and clinically translatable, which reaches stable transduction efficiencies of >99% in CD4+ T lymphocytes within 3 days of culture initiation. Copyright

Delta MGMT-Transduced Bone Marrow Infusion Increases Tolerance to O6-Benzylguanine and 1,3-Bis(2-chloroethyl)1-nitrosourea and Allows Intensive Therapy of 1,3-Bis(2-chloroethyl)-1-nitrosourea-Resistant Human Colon Cancer Xenografts

O6-Benzylguanine (BG) is a potent inhibitor of the DNA repair protein 06-alkylguanine DNA alkyltransferase (AGT), and sensitizes tumors to BCNU in vitro and in xenografts. The combination of BG and BCNU is now undergoing phase I clinical testing. The maximally tolerated dose of BCNU given after BG is expected to be lower then the doses tolerated as a single agent owing to BG sensitization of hematopoietic progenitors. We have previously shown that retroviral expression of G156A mutant MGMT (deltaMGMT) in mouse and human marrow cells results in significant BG and BCNU resistance. In this study we evaluated the effect of deltaMGMT-transduced marrow infusion on the therapeutic index of multiple BG and BCNU treatments in tumor-bearing nude (nu/nu athymic) mice. Prior to subcutaneous implantation of BCNU-resistant SW480 human colon cancer cells, cohorts of mice were given intraperitoneal injections of nonablative doses of BG (30 mg/kg) and BCNU (10 mg/kg, one-half of the LD10) and then infused with 1-2 x 10(6) isogeneic deltaMGMT (n = 29 mice) or lacZ-transduced (n = 20 mice) marrow cells. The xenograft-bearing mice were treated with multiple cycles of BG (30 mg/kg) and BCNU (10-25 mg/kg). After three cycles, deltaMGMT mouse bone marrow was repopulated with CFU containing the provirus, and demonstrated a 2.7-fold increase in AGT activity and a 5.5-fold increase in BCNU IC90 compared with LacZ mice. After five cycles, the BCNU IC90 of CFU cells increased nine-fold over control cells, indicating selective enrichment of CFU precursor cells expressing high levels of deltaMGMT. Starting with the third cycle of therapy, tolerance to BG and BCNU was significantly improved in deltaMGMT mice compared with LacZ mice, as evidenced by preserved peripheral blood counts, bone marrow cellularity, and CFU content 1 and 2 weeks posttreatment and a significantly higher survival rate. Xenograft growth was significantly delayed in mice tolerating multiple cycles and higher dose intensity of BG and BCNU as compared with mice receiving less intensive therapy. We conclude that deltaMGMT-transduced marrow cells can improve the therapeutic index of BG and BCNU by selectively repopulating the marrow and providing significant marrow tolerance to this combination, allowing intensive therapy of a BCNU-resistant tumor.

Current progress in the gene therapy of cancer

Gene transfer for the treatment of cancer is a rapidly expanding field. Recent studies can be divided into four main areas: 1) transfer of suicide genes that convert inactive prodrugs into cytotoxic compounds, 2) transfer of genes encoding cytokines and stimulatory markers to enhance immunogenicity against tumors, 3) transfer of tumor-suppressor genes to block tumor cell proliferation, and 4) transfer of drug resistance genes into hematopoietic stem cells to increase their resistance to myelo-suppressive chemotherapeutic agents. This review discusses recent advances in technique and knowledge and their application to the gene therapy of cancer.

Applied molecular evolution of O6-benzylguanine-resistant DNA alkyltransferases in human hematopoietic cells

Applied molecular evolution is a rapidly developing technology that can be used to create and identify novel enzymes that nature has not selected. An important application of this technology is the creation of highly drug-resistant enzymes for cancer gene therapy. Seventeen O6-alkylguanine-DNA alkyltransferase (AGT) mutants highly resistant to O6-benzylguanine (BG) were identified previously by screening 8 million variants, using genetic complementation in Escherichia coli. To examine the potential of these mutants for use in humans, the sublibrary of AGT clones was introduced to human hematopoietic cells and stringently selected for resistance to killing by the combination of BG and 1,3-bis(2-chloroethyl)-1-nitrosourea. This competitive analysis between the mutants in human cells revealed three AGT mutants that conferred remarkable resistance to the combination of BG and 1,3-bis(2-chloroethyl)-1-nitrosourea. Of these, one was recovered significantly more frequently than the others. Upon further analysis, this mutant displayed a level of BG resistance in human hematopoietic cells greater than that of any previously reported mutant.

Differential competitive resistance to methylating versus chloroethylating agents among five O6-alkylguanine DNA alkyltransferases in human hematopoietic cells

P140K-MGMT and G156A-MGMT genes encode two O6-benzylguanine–resistant O6-alkylguanine DNA alkyltransferase proteins that confer a high degree of O6-benzylguanine and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or O6-benzylguanine and temozolomide resistance to primary hematopoietic cells. In this study, we directly compared these and three other O6-benzylguanine–resistant MGMT genes for their ability to protect the human erythroleukemia cell line, K562, using a direct competitive selection strategy to identify the mutation that conferred the greatest degree of protection from O6-benzylguanine and either BCNU or temozolomide. MFG retroviral vector plasmids for each of these mutants [G156A-MGMT (ED50 for O6-benzylguanine, 60 μmol/L); and P140K-MGMT, MGMT-2 (S152H, A154G, Y158H, G160S, L162V), MGMT-3 (C150Y, A154G, Y158F, L162P, K165R), and MGMT-5 (N157T, Y158H, A170S; ED50 for benzylguanine, >1,000 μmol/L)] were mixed, and the virus produced from Phoenix cells was transduced into K562 cells. Stringent selection used high doses of O6-benzylguanine (800 μmol/L) and temozolomide (1,000 μmol/L) or BCNU (20 μmol/L) administered twice, and following regrowth, surviving clones were isolated, and the MGMT transgene was sequenced. None of the mutants was lost during selection. Using temozolomide, the enrichment factor was greatest for P140K-MGMT (1.7-fold). Using BCNU selection, the greatest enrichment was observed with MGMT-2 (1.5-fold). G156A-MGMT, which is the least O6-benzylguanine–resistant MGMT gene of the mutants tested, was not lost during selection but was selected against. The optimal mutant MGMT useful as a drug resistance gene may depend on whether a methylating or chloroethylating agent is used for drug selection. [Mol Cancer Ther 2006;5(1):121–8]

Drug Selection of Mutant Methylguanine Methyltransferase from Different Oncoretroviral Backbones Results in Multilineage Hematopoietic Transgene Expression in Primary and Secondary Recipients

Optimized hematopoietic gene therapy requires vectors with strong expression in the desired target cell population and the ability to select for the expressing transduced cells. In the context of drug resistance selection of repopulating hematopoietic stem cells in the mouse, we examined tissue expression after transduced marrow transplantation of the drug selection gene, G156A mutant O6-methylguanine-DNA methyltransferase (G156A MGMT). To gain more experience with the rigor of the impact of selection on tissue-specific gene expression, we also asked whether there are expression differences between three different onco-retroviral backbones--MPSV, SF, and MFG. MGMT expression was compared after O6-benzylguanine (BG) and 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) drug selection in vivo. After mice were transplanted with cells transduced with MPSV, MFG, or SF retroviral vectors expressing G156A MGMT and drug treated, nearly complete replacement by transduced progenitors was observed in the marrow. Each backbone supported MGMT expression in all four hematopoietic lineages in vivo indicating that MGMT-mediated selection is indeed robust. Expression in marrow, spleen, and thymus was very similar between the vectors and differences were most likely due to differences in gene copy number per selected cell. In primary and secondary recipients, the highest expression was observed in MFG and this was the vector that transduced at the greatest proviral copy number per cell. These data indicate that strong selection pressure using the MGMT gene to protect primary and secondary repopulating murine stem cells from the toxicity of BCNU. Regardless of the vector backbone used, multiorgan expression was observed without evidence of gene silencing. These data help establish mutant, BG-resistant MGMT as a potent selection gene for stem cell selection in vivo.