Live and let die: In vivo selection of gene-modified hematopoietic stem cells via MGMT-mediated chemoprotection

Abstract

Gene transfer into hematopoietic stem cells (HSC) provides a potential means of correcting monogenic defects and altering drug sensitivity of normal bone marrow to cytotoxic agents. These applications have significant therapeutic potential but the translation of successful murine studies into human therapies has been hindered by low gene transfer in large animals (including humans), and recent serious side effects in a human immunodeficiency trial related to insertional mutagenesis. The latter trial, along with other subsequent trials, while bringing into focus the potential risks of integrating vector systems, also clearly demonstrate the potential usefulness of in vivo selection as it relates to inefficient stem cell transduction. Developing from initial studies by our group and other investigators in which drug resistance was utilized to demonstrate the feasibility of using gene transfer to effect protection from myelotoxicity of chemotherapeutic agents, expression of mutant forms of O 6 -methyguanine-DNA-methytransferase (MGMT) coupled with the simultaneous use of pharmacologic inhibitors and chemotherapeutic agents has been shown to provide a powerful method to select HSC in vivo. While stem and progenitor cell protection and resulting selection in vivo has potential applications for the treatment of selected cancers (allowing dose escalation) andor correction of monogenic disease (allowing an iatrogenic survival advantage of transduced cells in vivo), such an in vivo selection may have untoward effects on stem cell behavior. These deleterious effects may include stem cell exhaustion; lineage skewing; accumulation of genotoxic lesions; and clonal dominance driven towards a pro-leukemic phenotype. Knowledge of the likelihood of such deleterious events occurring as well as their potential implications will be critical to future clinical applications and may also enhance our understanding of both normal stem cell behavior and the evolution of hematopoietic malignancies.

DNA Base Excision Repair: Evolving Biomarkers for Personalized Therapies in Cancer

“…A detailed discussion of individual pathways is beyond the scope of this chapter as several recent excellent reviews on DNA repair are available [1][2][3][4][5][6]. Briefly, direct repair is involved in the repair of alkylated bases (such as O 6 methyl guanine) by MGMT (O 6 methyl guanine DNA methyl transferases [7][8][9][10]. DNA mismatch repair (MMR) corrects base-base mismatches and insertion-deletion loops (IDLs) erroneously generated during DNA replication and by exogenous DNA damage [11][12][13].…”

Efficiency and Safety of O6-Methylguanine DNA Methyltransferase (MGMTP140K)-MediatedIn VivoSelection in a Humanized Mouse Model

“…Besides mutants of the dehydrofolate reductase enzyme or the multidrug resistance 1 (MDR1) gene, mutants of the O 6 -methylguanine DNA methyltransferase (MGMT) gene have been investigated with considerable success in this context. MGMT encodes an evolutionarily conserved DNA repair protein that removes highly toxic O 6 -guanine adducts from the cellular DNA and confers resistance to chemotherapeutic drugs with a high O 6 -methylating or -chloroethylating potential such as temozolomide (TMZ) and other triazene derivatives or chloroethylnitrosoureas such as 1,3-bis(2-chlorethyl)-1-nitrosourea (BCNU), respectively (Milsom and Williams, 2007). Transgenic overexpression of the O 6 -benzylguanine (BG)-resistant MGMT P140K point mutant has been demonstrated to allow for efficient enrichment of transduced hematopoietic cells by the combined application of the BG and BCNU or TMZ.…”

Lentiviruses: Vectors for Cancer Gene Therapy

“…The mutant form of MGMT (MGMT-P140K) has been shown to be effectively resistant to O 6 -benzylguanine (BG), an inhibitor to wild-type MGMT, and also alkylating agents, such as Temozolomide or 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) (Chinnasamy et al 2004;Zielske and Gerson 2002). By using a lentiviral vector to introduce MGMT-P140K into hematopoietic stem cells (HSC), hematopoiesis can be restored, and transduced bone marrow cells can be protected and selected in vivo in preclinical models (Liu and Gerson 2006;Milsom and Williams 2007). MGMT-P140K provides leverage to bone marrow cells over tumor cells expressing wild-type MGMT against BG and chemotherapeutic treatments (Fig.…”