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Retroviral integration and human gene therapy
Frederic D. Bushman
Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.
Address correspondence to: Frederic D. Bushman, Department of Microbiology, University of Pennsylvania School of Medicine, 3610 Hamilton Walk, Philadelphia, Pennsylvania 19104-6076, USA. Phone: (215) 573-8372; Fax: (215) 573-4856; E-mail: bushman@mail.med.upenn.edu.
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Published August 1, 2007 - More info
J Clin Invest. 2007;117(8):2083–2086. https://doi.org/10.1172/JCI32949.
© 2007 The American Society for Clinical Investigation
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Abstract
Recent reports have challenged the notion that retroviruses and retroviral vectors integrate randomly into the host genome. These reports pointed to a strong bias toward integration in and near gene coding regions and, for gammaretroviral vectors, around transcription start sites. Here, we report the results obtained from a large-scale mapping of 572 retroviral integration sites (RISs) isolated from cells of 9 patients with X-linked SCID (SCID-X1) treated with a retrovirus-based gene therapy protocol. Our data showed that two-thirds of insertions occurred in or very near to genes, of which more than half were highly expressed in CD34+ progenitor cells. Strikingly, one-fourth of all integrations were clustered as common integration sites (CISs). The highly significant incidence of CISs in circulating T cells and the nature of their locations indicate that insertion in many gene loci has an influence on cell engraftment, survival, and proliferation. Beyond the observed cases of insertional mutagenesis in 3 patients, these data help to elucidate the relationship between vector insertion and long-term in vivo selection of transduced cells in human patients with SCID-X1.
Authors
Annette Deichmann, Salima Hacein-Bey-Abina, Manfred Schmidt, Alexandrine Garrigue, Martijn H. Brugman, Jingqiong Hu, Hanno Glimm, Gabor Gyapay, Bernard Prum, Christopher C. Fraser, Nicolas Fischer, Kerstin Schwarzwaelder, Maria-Luise Siegler, Dick de Ridder, Karin Pike-Overzet, Steven J. Howe, Adrian J. Thrasher, Gerard Wagemaker, Ulrich Abel, Frank J.T. Staal, Eric Delabesse, Jean-Luc Villeval, Bruce Aronow, Christophe Hue, Claudia Prinz, Manuela Wissler, Chuck Klanke, Jean Weissenbach, Ian Alexander, Alain Fischer, Christof von Kalle, Marina Cavazzana-Calvo
Abstract
We treated 10 children with X-linked SCID (SCID-X1) using gammaretrovirus-mediated gene transfer. Those with sufficient follow-up were found to have recovered substantial immunity in the absence of any serious adverse events up to 5 years after treatment. To determine the influence of vector integration on lymphoid reconstitution, we compared retroviral integration sites (RISs) from peripheral blood CD3+ T lymphocytes of 5 patients taken between 9 and 30 months after transplantation with transduced CD34+ progenitor cells derived from 1 further patient and 1 healthy donor. Integration occurred preferentially in gene regions on either side of transcription start sites, was clustered, and correlated with the expression level in CD34+ progenitors during transduction. In contrast to those in CD34+ cells, RISs recovered from engrafted CD3+ T cells were significantly overrepresented within or near genes encoding proteins with kinase or transferase activity or involved in phosphorus metabolism. Although gross patterns of gene expression were unchanged in transduced cells, the divergence of RIS target frequency between transduced progenitor cells and post-thymic T lymphocytes indicates that vector integration influences cell survival, engraftment, or proliferation.
Authors
Kerstin Schwarzwaelder, Steven J. Howe, Manfred Schmidt, Martijn H. Brugman, Annette Deichmann, Hanno Glimm, Sonja Schmidt, Claudia Prinz, Manuela Wissler, Douglas J.S. King, Fang Zhang, Kathryn L. Parsley, Kimberly C. Gilmour, Joanna Sinclair, Jinhua Bayford, Rachel Peraj, Karin Pike-Overzet, Frank J.T. Staal, Dick de Ridder, Christine Kinnon, Ulrich Abel, Gerard Wagemaker, H. Bobby Gaspar, Adrian J. Thrasher, Christof von Kalle
Abstract
Gene transfer into HSCs is an effective treatment for SCID, although potentially limited by the risk of insertional mutagenesis. We performed a genome-wide analysis of retroviral vector integrations in genetically corrected HSCs and their multilineage progeny before and up to 47 months after transplantation into 5 patients with adenosine deaminase–deficient SCID. Gene-dense regions, promoters, and transcriptionally active genes were preferred retroviral integrations sites (RISs) both in preinfusion transduced CD34+ cells and in vivo after gene therapy. The occurrence of insertion sites proximal to protooncogenes or genes controlling cell growth and self renewal, including LMO2, was not associated with clonal selection or expansion in vivo. Clonal analysis of long-term repopulating cell progeny in vivo revealed highly polyclonal T cell populations and shared RISs among multiple lineages, demonstrating the engraftment of multipotent HSCs. These data have important implications for the biology of retroviral vectors, the dynamics of genetically modified HSCs, and the safety of gene therapy.
Authors
Alessandro Aiuti, Barbara Cassani, Grazia Andolfi, Massimiliano Mirolo, Luca Biasco, Alessandra Recchia, Fabrizia Urbinati, Cristina Valacca, Samantha Scaramuzza, Memet Aker, Shimon Slavin, Matteo Cazzola, Daniela Sartori, Alessandro Ambrosi, Clelia Di Serio, Maria Grazia Roncarolo, Fulvio Mavilio, Claudio Bordignon
The first unambiguous success for human gene therapy was reported in 2000 by Marina Cavazzana-Calvo, Alain Fischer, and their coworkers (1). Two boys with X-linked SCID (SCID-X1) caused by a genetic defect in a cytokine receptor gene were successfully treated by restoring the missing coding region. Previous attempts to treat patients with adenosine deaminase–deficient SCID (ADA-SCID) had been widely reported, but how much the gene therapy treatments benefited the patients was never fully clarified (the patients remained on effective enzyme replacement therapy throughout) (2). The gene therapy treatments for SCID-X1, in contrast, were stunningly successful. Adding back the gene for the missing cytokine chain restored a proliferation signal, allowing the gene-modified cells to expand in number and repopulate the treated patients, resulting in clear-cut reconstitution of immune function. Ten patients were ultimately treated by the French team of Cavazzana-Calvo and Fischer (ref. 3 and references therein). Subsequently, another group treated a further ten SCID-X1 patients (ref. 4 and references therein), a third group successfully treated two patients with chronic granulomatous disease (CGD) (5), and a fourth group achieved unambiguous success treating five ADA-SCID patients (6). At last, gene therapy had arrived.
However, with these successes came the first serious adverse events in retrovirus-based gene therapy. Three of the SCID-X1 patients treated by the French team developed a leukemia-like lymphoproliferative disease (7, 8). In the two adverse events reported in detail to date, integration of the therapeutic vector, a gammaretrovirus derivative, activated transcription of the LIM domain only 2 (LMO2) protooncogene. Two additional factors appear to have contributed to transformation in addition to insertional activation of LMO2: an activation signal from the newly introduced cytokine chain and a third “hit” in the form of a chromosomal rearrangement. One patient died of the lymphoproliferative disease. The other two patients responded to chemotherapy and are doing well, and furthermore continue to benefit from the gene therapy.
In this issue of the JCI, we get an update on the two SCID-X1 trials and the successful ADA trials, together with a detailed look at the distribution of genomic sites targeted for integration by the retroviral vectors in patient cells. The analysis of integration site patterns is useful in several respects. From a basic science perspective, such data add to our understanding of the molecular mechanisms of retroviral integration. Clinically, following integration site distributions over time helps elucidate the effects of insertional activation on persistence of the selected surviving clones of modified cells. Potentially most important is the possibility, although not yet realized, that such information can help forecast incipient adverse events.
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Integration targeting in successful gene therapy trials
The articles in this issue of the JCIbegin by updating us on the status of gene therapy patients from the SCID-X1 and ADA-SCID trials. Schwarzwaelder et al. (4) report that the patients treated in the British SCID-X1 trial continue to do well, and Deichmann et al. (3) update the first and longer-running SCID-X1 trial. At present there is no explanation for why adverse events occurred in one SCID-X1 trial and not the other. Less time has elapsed since cell infusion for the patients from the second trial, so differences are still hard to evaluate. The numbers of patients are still very low, so low, in fact, that the differences between the two trials are not statistically significant. Even if the Cavazzana-Calvo and Fischer et al. (1) trial experiences a fourth adverse event, so that 4 of 10 patients suffer adverse events versus 0 of 10 in the Thrasher trial (4), the difference still is not statistically significant. In the third related study reported in this issue of the JCI, Aiuti et al. (15) report that the five ADA-SCID patients continue to do well up to 47 months after cell infusion and are free of adverse events.
The three studies (3, 4, 15) then go on to investigate the locations of the integrated vectors in patient cells (Figure 1). All three studies used gammaretrovirus-based vectors, and the global integration patterns were as expected based on previous work using cell culture models (10, 14). Integration sites were enriched near gene 5′ ends and associated CpG islands compared with expectation based on random placement of sites. Analysis of the activity of genes near integration sites using transcriptional profiling data from the cell types transduced (CD34+ progenitor cells) indicated that active genes were particularly favored for integration.
Figure 1Gene correction using retroviral vectors, and monitoring the placement of integration sites after evolution in patients. A population of cells is removed from the patient (i) and transduced ex vivo with a retroviral vector carrying the corrected gene (ii–iii). The gene-corrected cells are subsequently reinfused into the patient (iv). The population of vector integration sites is diverse prior to transplantation, with each cell harboring integrated proviral copies at different locations in the genome. Examination years later of the population of gene-corrected cells reveals that the population of provirus has changed because of evolution of cells in vivo (v). In some cases this can include proliferation of cells where integrated proviruses activated oncogenes, leading to an adverse event.
Integration of retroviral DNA marks the multipotent progenitor cells that then give rise to the mature cell types of the hematopoietic lineage. Thus it is possible to investigate what types of progenitors can contribute to each lineage by asking whether the same integration sites, presumably derived by infection of a single multipotent progenitor cell, appear in different lineages. Aiuti et al. (15) separated cells into myeloid (granulocytes) and lymphoid (B and T cells) lineages and found integration sites in common between the two lineages. Similar data has been previously reported for the SCID-X1 trial (16). A technical concern is that imperfect cell fractionation could have yielded the observed result, but the results were obtained for multiple integration sites in several samples. Thus the simplest interpretation is that the therapeutic vector, at least on occasion, integrated in a precursor cell capable of giving rise to multiple lineages.
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