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. 2009 May 21;113(21):5094-103.
doi: 10.1182/blood-2008-09-176412. Epub 2009 Mar 31.

Long-term polyclonal and multilineage engraftment of methylguanine methyltransferase P140K gene-modified dog hematopoietic cells in primary and secondary recipients

Brian C Beard  1 , Reeteka Sud, Kirsten A Keyser, Christina Ironside, Tobias Neff, Sabine Gerull, Grant D Trobridge, Hans-Peter Kiem
Affiliations

Long-term polyclonal and multilineage engraftment of methylguanine methyltransferase P140K gene-modified dog hematopoietic cells in primary and secondary recipients

Brian C Beard et al. Blood. .

Abstract

Overexpression of methylguanine methyltransferase P140K (MGMTP140K) has been successfully used for in vivo selection and chemoprotection in mouse and large animal studies, and has promise for autologous and allogeneic gene therapy. We examined the long-term safety of MGMTP140K selection in a clinically relevant dog model. Based on the association of provirus integration and proto-oncogene activation leading to leukemia in the X-linked immunodeficiency trial, we focused our analysis on the distribution of retrovirus integration sites (RIS) relative to proto-oncogene transcription start sites (TSS). We analyzed RIS near proto-oncogene TSS before (n = 157) and after (n = 129) chemotherapy in dogs that received MGMTP140K gene-modified cells and identified no overall increase of RIS near proto-oncogene TSS after chemotherapy. We also wanted to determine whether in vivo selected cells retained fundamental characteristics of hematopoietic stem cells. To that end, we performed secondary transplantation of MGMTP140K gene-modified cells after in vivo selection in dog leukocyte antigen (DLA)-matched dogs. Gene-modified cells achieved multilineage repopulation, and we identified the same gene-modified clone in both dogs more than 800 and 900 days after transplantation. These data suggest that MGMTP140K selection is well tolerated and should allow clinically for selection of gene-corrected cells in genetic or infectious diseases or chemoprotection for treatment of malignancy.

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Figures

Figure 1
Figure 1
Stable increase in gene marking after in vivo selection of MGMTP140K gene-modified cells. Gene marking in granulocytes (●), lymphocytes (○), and donor chimerism (----) before and after in vivo selection of MGMTP140K-GFP gene-marked cells in a dog. Treatment with O6BG and BCNU denoted by ↓.
Figure 2
Figure 2
Durable in vivo selection and chemoprotection. (A) Gene marking in granulocytes (●) and lymphocytes (○) before and after in vivo selection of MGMTP140K-GFP gene-marked cells in a dog. Treatment with O6BG and temozolomide denoted by black arrows. (B) The corresponding platelet count of the same dog during the chemotherapy treatment cycles.
Figure 3
Figure 3
Similar distribution of retroviral integrants relative to proto-oncogenes before and after chemotherapy. Representative gel of RIS amplified by LAM-PCR before (A) and after (B) chemotherapy with either O6BG and BCNU (G069 and G154) or O6BG and temozolomide (G179 and G197). (C) The positions of RIS mapped relative to the RefSeq gene TSS of proto-oncogenes as defined in either the Sanger Cancer Gene Census or the Retroviral Tagged Cancer Gene Database. *P < .001 (SD).
Figure 4
Figure 4
Engraftment and multilineage repopulation potential of MGMTP140K gene-modified cells in secondary recipients after in vivo selection. (A) Schematic representation of secondary transplantation in DLA-matched dogs. The white dog represents the initial recipient of the gene-marked hematopoietic cells from the black dog (1°). The 2° transfer of the MGMTP140K gene-modified cells back into the original donor (black dog). (B,C top panels) Percentage of donor-positive WBC (----) and donor gene-modified WBC (●) in primary recipients (white dogs) is shown as a function of time. Chemotherapy denoted by small black arrows above the graph. Large black arrow indicates the time point at which the bone marrow was transplanted back into the original donors (black dogs). (B,C bottom panels) Percentage of gene-modified WBC in the secondary recipients (black dogs). Bar graphs to the right of the gene-marking plots are an alternate representation of hematopoietic contribution from each dog (white and black).
Figure 5
Figure 5
Multiple clones and common clones contribute to hematopoiesis in primary and secondary recipients. (A,B) Representative gel of RIS amplified by LAM-PCR in primary (1°) and secondary (2°) recipients. (C) Detection of a common integration site in different hematopoietic lineages using LTR-specific (forward) and dog genomic (reverse) primers of the primary and secondary dogs pictured in panel A. “ND” is a different dog that received lentivirus-transduced cells.
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