Selectively weakened binding of methotrexate by human dihydrofolate reductase allows rapid ex vivo selection of mammalian cells

Abstract

Ex vivo selection of transduced hematopoietic stem cells (HSC) with drug-resistance genes offers the possibility to enrich transduced cells prior to engraftment, toward increased reconstitution in transplant recipients. We evaluated the potential of highly methotrexate (MTX)-resistant variants of human dihydrofolate reductase (hDHFR) for this application. Two subsets of hDHFR variants with reduced affinity for MTX that had been previously identified in a bacterial system were considered: those with substitutions at positions 31, 34, and/or 35, and those with substitutions at position 115. The variants were characterized for their resistance to pemetrexed (PMTX), an antifolate that is related to MTX. We observed a strong correlation between decreased binding to both antifolates, although the identity of specific sequence variations modulated the correlation. We chose a subset of hDHFR variants for tests of ex vivo MTX resistance, taking into consideration their residual specific activity and their decrease in affinity for the related antifolates. Murine myeloid progenitors and other differentiated hematopoietic cells were transduced and exposed to MTX in a nucleotide-free medium. Bone marrow (BM) cells including 15% cells infected with F31R/Q35E were enriched to 98% transduced cells within 6 days of ex vivo selection. hDHFR variant F31R/Q35E allowed a strong ex vivo enrichment upon a short exposure to MTX relative to a less resistant variant of hDHFR, L22Y. We have thus demonstrated that bacterial selection of highly antifolate-resistant hDHFR variants can provide selectable markers for rapid ex vivo enrichment of hematopoietic cells.

Efficient Enrichment of Gene-Modified Primary T Cells via CCR5-Targeted Integration of Mutant Dihydrofolate Reductase

“… 20 , 21 , 22 , 23 , 24 Subsequently, an L22F/F31S double mutant was developed that outperformed L22Y, maintaining catalytic activity while exhibiting a marked decrease in MTX-binding affinity. 25 Another variant, F31R/Q35E, could withstand up to 1 μM MTX; murine bone marrow cells transduced with this mutant were enriched within a 4-day culture. 25 Previous clinical trials have characterized serum concentrations of MTX in order to better guide the selection of a relevant dose for chemoselection studies: 100 nM to 1,000 nM serum concentrations of MTX have been achieved in patients who were on a low-dose (10–500 mg/m 2 ) regimen of the drug.…”

Engineering Human T Cells for Resistance to Methotrexate and Mycophenolate Mofetil as an In Vivo Cell Selection Strategy

“…While a number of drug-resistance enzymes have been employed for selection of gene modified cells, including O 6 -mehtylguanine-DNA-methyltransferease (MGMT), multidrug resistance associated protein 1 (MDR1), bacterial hygromycin resistance gene (Hy) and neomycin phosphotransferase ( neo ) variants [1], many of these selective transgenes have proven disadvantageous in the clinic. For example, transgenes of non-human origin used for in vitro selection (e.g., Hy, neo , and Herpes simplex thymidine kinase, HSV-tk), often lead to immunological rejection of gene-modified cells [2]–[6].…”

“…Additionally, when MDR1-transduced autologous CD34 + cells were transferred into cancer patients, no enrichment of transduced cells was detected following etoposide treatment [7], [8]. Previous clinical trials of MGMT-, neoR- and Hy- mediated selection have also been halted due to safety concerns with long-term administration of selection drugs, (i.e., with DNA-alkalizing agents, neomycin, and hygromycin respectively) [1], [9]. Thus, there is a need for alternative strategies that will enable drug selection of gene modified cells with a tolerable toxicity profile in human patients.…”

Site-Specific PEGylation of Therapeutic Proteins

“…They used mPDA to PEGylate eGFP by performing in-lysate prenylation followed by capture and release strategy from hydrazine beads as described above; the presence of the new catalyst substantially decreased the elution time in the PEGylation/release step. They used this same catalyst to PEGylate Dihydrofolate reductase (DHFR) conjugated to a ketone group incorporated by nonsense suppression with p -acetyl phenylalanine; DHFR is being investigated for a number of different applications in drug delivery [ 105 , 106 ]. Rashidian et al were able to increase the PEGylation rate of DHFR using the mPDA catalyst by 2.5-fold compared to aniline.…”