Efficient and stable MGMT-mediated selection of long-term repopulating stem cells in nonhuman primates

Abstract

HSC transplantation using genetically modified autologous cells is a promising therapeutic strategy for various genetic diseases, cancer, and HIV. However, for many of these conditions, the current efficiency of gene transfer to HSCs is not sufficient for clinical use. The ability to increase the percentage of gene-modified cells following transplantation is critical to overcoming this obstacle. In vivo selection with mutant methylguanine methyltransferase (MGMTP140K) has been proposed to overcome low gene transfer efficiency to HSCs. Previous studies have shown efficient in vivo selection in mice and dogs but only transient selection in primates. Here, we report efficient and stable MGMTP140K-mediated multilineage selection in both macaque and baboon nonhuman primate models. Treatment consisting of both O6-benzylguanine (O6BG) and N,N'-bis(2-chloroethyl)-N-nitroso-urea (BCNU) stably increased the percentage of transgene-expressing cells from a range of initial levels of engrafted genetically modified cells, with the longest follow-up after drug treatment occurring over 2.2 years. Drug treatment was well tolerated, and selection occurred in myeloid, lymphoid, and erythroid cells as well as platelets. Retrovirus integration site analysis before and after drug treatments confirmed the presence of multiple clones. These nonhuman primate studies closely model a clinical setting and should have broad applications for HSC gene therapy targeting human diseases of malignant, genetic, and infectious nature, including HIV.

Figure 1. Efficient MGMT^P140K-mediated in vivo selection and chemoprotection in the baboon (M00228).

(A) Gene marking in MGMT^P140K-GFP granulocytes (closed circles), MGMT^P140K-GFP lymphocytes (open circles), YFP granulocytes (closed squares), and YFP lymphocytes (open squares), before and after in vivo selection with either O⁶BG and TMZ or BCNU. (B) A truncated representation of the time following in vivo selection with O⁶BG and TMZ or BCNU. Granulocytes are the only subset represented for clarity of selection (MGMT^P140K-GFP [MGMT-GFP]) and elimination (YFP). (C) Graphs are the corresponding neutrophil and PLT counts of the same baboon during the drug treatment cycles. (D) Provirus copy number data of MGMT^P140K-GFP peripheral blood cells (closed circles) and YFP peripheral blood cells (open circles) of the flow cytometry data represented in A and B. (A–D) Dashed arrows represent in vivo selection with either O⁶BG and TMZ, and solid arrows represent in vivo selection with BCNU.

Figure 2. Multilineage MGMT^P140K-mediated in vivo selection.

Bar graph representation of flow cytometry data from baboon M00228 gated on peripheral blood cells positive for MGMT-GFP or YFP and a monoclonal antibody for the indicated subset. White bars represent MGMT-GFP cells before drug treatment, and black bars represent MGMT-GFP cells after drug treatment (selection). Light gray bars represent YFP cells before drug treatment, and gray bars represent YFP cells after drug treatment (elimination).

Figure 3. Efficient MGMT^P140K-mediated in vivo selection of cells harboring anti-HIV transgenes in the macaque (M05189).

Gene marking in granulocytes (closed circles) and lymphocytes (open circles), before and after in vivo selection of MGMT^P140K-GFP genetically modified cells in a macaque. Treatment with O⁶BG-2X and BCNU is denoted by solid arrows.

Figure 4. Efficient MGMT^P140K-mediated in vivo selection in macaques (J02370 and T04228).

Gene marking in granulocytes (closed circles) and lymphocytes (open circles), before and after in vivo selection of MGMT^P140K-GFP genetically modified cells in macaques. Treatment with O⁶BG-2X and BCNU is denoted by solid arrows.

Figure 5. Flow cytometric analysis of transgene-expressing rbc and PLTs following MGMT-mediated in vivo selection.

Gating on rbc and PLTs using forward scatter (F-SC) and side scatter (S-SC) and EGFP-expressing rbc (top row) and PLTs (bottom row) plotted by side scatter is shown for a control macaque and macaque J02370.

Figure 6. Efficient MGMT^P140K-mediated in vivo selection in the macaque following reduced-intensity conditioning (K05079).

Gene marking plotted is as provirus copy number from whole wbc, before and after in vivo selection of MGMT^P140K-GFP genetically modified cells in a macaque. Treatment with O⁶BG-2X and BCNU is denoted by a solid arrow.