Potential Genotoxicity from Integration Sites in CLAD Dogs Treated Successfully with Gammaretroviral Vector-Mediated Gene Therapy
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Gene Therapy (2008) 15, 1067–1071 & 2008 Nature Publishing Group All rights reserved 0969-7128/08 $30.00 www.nature.com/gt SHORT COMMUNICATION Potential genotoxicity from integration sites in CLAD dogs treated successfully with gammaretroviral vector-mediated gene therapy M Hai1,3, RL Adler1,3, TR Bauer Jr1,3, LM Tuschong1, Y-C Gu1,XWu2 and DD Hickstein1 1Experimental Transplantation and Immunology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA and 2Laboratory of Molecular Technology, Scientific Applications International Corporation-Frederick, National Cancer Institute-Frederick, Frederick, Maryland, USA Integration site analysis was performed on six dogs with in hematopoietic stem cells. Integrations clustered around canine leukocyte adhesion deficiency (CLAD) that survived common insertion sites more frequently than random. greater than 1 year after infusion of autologous CD34+ bone Despite potential genotoxicity from RIS, to date there has marrow cells transduced with a gammaretroviral vector been no progression to oligoclonal hematopoiesis and no expressing canine CD18. A total of 387 retroviral insertion evidence that vector integration sites influenced cell survival sites (RIS) were identified in the peripheral blood leukocytes or proliferation. Continued follow-up in disease-specific from the six dogs at 1 year postinfusion. A total of 129 RIS animal models such as CLAD will be required to provide an were identified in CD3+ T-lymphocytes and 102 RIS in accurate estimate of the genotoxicity using gammaretroviral neutrophils from two dogs at 3 years postinfusion. RIS vectors for hematopoietic stem cell gene therapy. occurred preferentially within 30 kb of transcription start Gene Therapy (2008) 15, 1067–1071; doi:10.1038/gt.2008.52; sites, including 40 near oncogenes and 52 near genes active published online 27 March 2008 Keywords: hematopoietic stem cells; transplantation; retrovirus; LAM–PCR; integration site Although the theoretical risk of retroviral-mediated (SCID-X1) and adenosine deaminase (ADA) in SCID- oncogene activation through insertional mutagenesis ADA.4–6 Initial excitement in the early reports from these had been considered to be low, this risk has been shown trials was subsequently tempered by serious adverse to be significant in recent human gene therapy clinical events which occurred in four of the eight children with trials using gammaretroviral vectors.1–3 The importance therapeutic levels of in vivo transgene expression in the of the individual variables responsible for genotoxicity French SCID-X1 trial2 and in one of 10 children in the UK in hematopoietic stem cell gene therapy trials using SCID-X1 trial.7 The selective advantage conferred by the gammaretroviral vectors remain uncertain. Human transgene was thought to not only contribute to the clinical trials, as well as small and large animal studies, observed clinical success but also to genotoxic clonal using gammaretroviral vectors have implicated the outgrowth.6 vector, transgene and retroviral insertion sites (RIS) in In the recent non-myeloablative gene therapy trial the development of serious adverse events. In each case, for chronic granulomatous disease (CGD), in which the gammaretroviral vector integration itself has been no selective advantage was provided by the CYBB thought to contribute to the genotoxicity observed, but (gp91phox) transgene, two patients developed clonal may not be the only factor responsible for clonal dominance of retrovirally marked cells, accompanied outgrowth. by multiple RV insertions in EVI1-MDS1, PRDM16 and Successful hematopoietic stem cell gene therapy in SETBP1.3 Thus, factors other than the transgene, such as humans using gammaretroviral vectors have generally RIS, appear to play a major role in clonal dominance. relied upon an in vivo selective growth advantage for the Previous animal studies, including mice, dogs and transduced cells provided by a therapeutic transgene, non-human primates, also point to genotoxic risk from such as the interleukin-2 receptor g chain (IL2Rg)in gammaretroviral vectors. Insertional activation of Evi-1 X-linked severe combined immunodeficiency disease by gammaretroviral vectors was first described in mice.8 A study of 14 non-human primates had integrations in Correspondence: Dr DD Hickstein, Experimental Transplantation EVI-1 after gammaretroviral gene transfer, with one and Immunology Branch, National Cancer Institute, National animal developing a granulocytic sarcoma.9 A recent Institutes of Health, 10 Center Drive, MSC1203, Bldg 10-CRC, study examining RIS in dogs marked with a gamma- Rm 3-3142, Bethesda, MD 20892-1203, USA. retroviral vector also revealed a bias of RIS occurring E-mail: [email protected] 10 3These authors contributed equally to this work. in and near proto-oncogenes. Received 21 December 2007; revised 18 February 2008; accepted 18 In the current study, we conducted integration site February 2008; published online 27 March 2008 analysis in six dogs with canine leukocyte adhesion Integration sites in RV vector-treated CLAD dogs M Hai et al 1068 deficiency (CLAD) that had been successfully treated M 6 12 18 24 -C M 6 12 18 -C M 6 12 -C with gammaretroviral vector-mediated ex vivo gene bp bp bp 11 500 500 500 therapy. CLAD is analogous to leukocyte adhesion 400 400 400 deficiency in children, and thus represents a preclinical 300 300 300 large animal model for leukocyte adhesion deficiency. 200 The characteristics of the six treated dogs with CLAD, 200 200 including age at infusion, conditioning regimen and transduced cell dose infused, are shown (Supplementary 100 100 100 Table 1). In this study, the therapeutic CD18 transgene A1 A2 A3 did not confer a strong selective growth advantage to the M 6 12 1824 30 36 -C M 6 12 1824 -C M 6 12 1824 30 -C transduced cells, and a clinically applicable non-myelo- bp bp bp 500 500 ablative conditioning regimen was used to facilitate 500 400 400 400 engraftment of the transduced cells. The percentages of 300 300 300 CD18+ leukocytes in the peripheral blood in the six dogs 200 ranged from 0.7 to 11.2% at 12–36 months follow-up and 200 200 represent therapeutic levels (Supplementary Table 1). To evaluate the diversity of clones contributing to 100 100 100 long-term hematopoiesis after autologous transplanta- B1 B2 C1 tion of CD34+ BM transduced with a gammaretroviral MNL-C MNL-C vector carrying canine CD18, we identified integration bp bp sites by linear amplification-mediated (LAM) PCR from 500 500 all six treated animals (Figure 1). LAM–PCR, performed 400 400 at 6 month intervals using DNA from peripheral blood 300 300 leukocytes demonstrated multiple banding patterns 200 200 indicative of polyclonal hematopoiesis in all six dogs (Figure 1a). Similar polyclonal hematopoiesis was evi- 100 100 dent for both myeloid and lymphoid lineages in dogs B1 B1 C1 and C1 examined at 36 and 31 months postinfusion, Figure 1 Evaluation of polyclonality by linear amplification- respectively (Figure 1b). mediated PCR (LAM-PCR). Genomic DNA was collected from six To confirm the polyclonal nature of the samples, CLAD dogs (A1, A2, A3, B1, B2 and C1) treated by gammaretroviral linker-mediated (LM)–PCR was used to search for vector PG13/Mscv-cCD1811 at designated months postinfusion of potentially over-represented clones or RIS within genes autologous transduced CD34+ cells. DNA was isolated from (a) peripheral blood leukocytes and (b) flow sorted CD3+ lymphocytes and gene regions. When LM–PCR amplicons were and neutrophils using either the Blood and Cell Culture DNA Kit sequenced, the integration sites in all six dogs were (Qiagen Inc., Valencia, CA, USA) or the Wizard Genomic DNA polyclonally derived (complete list of integration sites in purification Kit (Promega Corp., Madison, WI, USA). Cells were Supplementary Table 2) with no predominant clone(s) sorted by immunostaining for T-cells using an antibody to canine observed. We obtained a total of 618 RIS from the treated CD3 (CA17.2A12; AbD Serotec, Raleigh, NC, USA) conjugated to CLAD dogs by sequence analysis of LM–PCR amplicons: AlexaFluor 647 by using a Zenon labelling kit, and for neutrophils using an antibody to canine neutrophils (CADO48A; VMRD Inc., 387 RIS from peripheral blood leukocytes from all six Pullman, WA, USA) conjugated to phycoerythrin. Labelled cells dogs at 5–21 months postinfusion (36 to 88 RIS per dog), were sorted to purities of 91.6 and 87.2% for CD3+ cells in dogs B1 129 RIS from sorted CD3+ lymphocytes and 102 RIS from and C1, respectively, with o0.5% neutrophil contamination. sorted neutrophils from dogs B1 and C1 at 31–36 months Neutrophils were sorted to purities of 97.8 and 93.5% for B1 and postinfusion. We identified 535 unique RIS after adjust- C1, respectively, with 0.1% CD3+ cell contamination. Cells were ment of 83 RIS that overlapped between the cell types sorted using a FACSVantage SE with DiVa software (BD Bios- ciences, San Jose, CA, USA) equipped with 488 and 633 nm and time points examined (see Supplementary Table 2). excitation lasers. LAM–PCR was performed on extracted DNA, as RIS isolated separately from lymphocytes and neutro- previously described.11 LAM–PCR samples were electrophoresed phils confirmed polyclonal insertion sites in each lineage. on a high-resolution Spreadex gel (Elchrom Scientific AG, Cham, In addition, 28 RIS were shared between the sorted Switzerland) and photographed using a gel documentation system lymphocytes and neutrophils RIS, indicating that a (Biochemi