Dotting the I's and Crossing the T's: Integration Analyses in Transduced Patient T Cells (2009)

In this issue of Molecular Therapy, Wang et al. report an investigation of the integration profile of a lentiviral vector in T lymphocytes isolated from patients undergoing a phase I/II clinical trial with the VIRxSYS anti-HIV vector VRX496.1Wang GP Levine BL Binder GK Berry CC Malani N McGarrity G et al.Analysis of lentiviral vector integration in HIV+ study subjects receiving autologous infusions of gene-modified CD4+ T cells.Mol Ther. 2009; 17: 844-850Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar These analyses have important implications for the future use of lentiviral vectors in hematopoietic cell gene therapies. Gene therapy received a serious setback several years ago when a hematopoietic stem cell gene therapy trial for X-linked inherited immunodeficiency resulted in the development of T-cell leukemia in several patients, although for the majority of the subjects the treatment was curative.2Gaspar HB Thrasher AJ Gene therapy for severe combined immunodeficiencies.Expert Opin Biol Ther. 2005; 5: 1175-1182Crossref PubMed Scopus (40) Google Scholar The leukemias have now been shown to be associated with the integration site of the gamma retroviral vector used in the studies, which integrated in close proximity to a proto-oncogene, resulting in increased expression of that gene and enhanced proliferation and clonal expansion of T lymphocytes containing the integration.3Hacein-Bey-Abina S Garrigue A Wang GP Soulier J Lim A Morillon E et al.Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1.J Clin Invest. 2008; 118: 3132-3142Crossref PubMed Scopus (1380) Google Scholar The preference for insertion of gamma retroviruses near active promoter regions prompted investigators to begin exploring the integration patterns of other vectors, including lentiviral vectors. Several experiments monitoring lentiviral integration sites have been carried out in various cell types.4Barr SD Ciuffi A Leipzig J Shinn P Ecker JR Bushman FD HIV integration site selection: targeting in macrophages and the effects of different routes of viral entry.Mol Ther. 2006; 14: 218-225Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar,5Ciuffi A Llano M Poeschla E Hoffmann C Leipzig J Shinn P et al.A role for LEDGF/p75 in targeting HIV DNA integration.Nat Med. 2005; 11: 1287-1289Crossref PubMed Scopus (485) Google Scholar,6Marshall HM Ronen K Berry C Llano M Sutherland H Saenz D et al.Role of PSIP1/LEDGF/p75 in lentiviral infectivity and integration targeting.PLoS ONE. 2007; 2: e1340Crossref PubMed Scopus (194) Google Scholar,7Wang GP Ciuffi A Leipzig J Berry CC Bushman FD HIV integration site selection: analysis by massively parallel pyrosequencing reveals association with epigenetic modifications.Genome Res. 2007; 17: 1186-1194Crossref PubMed Scopus (343) Google Scholar Results from these analyses demonstrated that HIV and HIV-based vectors have a strong preference for integration into actively transcribed chromatin regions but not adjacent to promoter regions. These studies suggested that lentiviral vectors possess a possible safety advantage over other types of retroviral vectors—in particular, the gamma retroviral vectors—but this would need to be verified by analyzing integration events in patient samples from clinical trials. The first US Food and Drug Administration–approved clinical trial using a lentiviral vector was initiated a few years ago by VIRxSYS; it involved T-lymphocyte-based gene therapy for the treatment of HIV-1 infection. The CD8+-depleted CD4+ T lymphocytes from HIV-infected individuals were isolated and then co-stimulated with CD3 and CD28. The stimulated CD4+ lymphocytes were then transduced with a lentiviral vector (VRX496) that encodes an antisense transcript complementary to the HIV envelope messenger RNA (mRNA) and a functional long-terminal repeat.8Levine BL Humeau LM Boyer J MacGregor RR Rebello T Lu X et al.Gene transfer in humans using a conditionally replicating lentiviral vector.Proc Natl Acad Sci USA. 2006; 103: 17372-17377Crossref PubMed Scopus (415) Google Scholar The transduced T cells were expanded ex vivo and infused into the patients. Recently published results from this study revealed long-term persistence of the gene-modified T lymphocytes and a transient reduction in viral load in one of the first five subjects enrolled.8Levine BL Humeau LM Boyer J MacGregor RR Rebello T Lu X et al.Gene transfer in humans using a conditionally replicating lentiviral vector.Proc Natl Acad Sci USA. 2006; 103: 17372-17377Crossref PubMed Scopus (415) Google Scholar Thus far there have been no adverse events, which is an important concern for future use of lentiviral vectors in gene therapy settings. The safety of the first trial has led to a follow-on phase I/II trial involving multiple infusions of transduced T lymphocytes in early-stage HIV-infected subjects with well-controlled HIV loads. Given the adverse events in the gamma retroviral vector–based trials, it is paramount to evaluate the integration profiles of lentiviral vectors in primary cell populations. Moreover, it is important to determine whether there is clonal expansion or selection for specific integration sites of the VIRxSYS vector in the T lymphocytes isolated from patients. In the studies conducted by Wang et al., the integration sites of VRX496 from three patients were analyzed in CD4+ T cells before infusion and in cells recovered from the patients at 6, 14, 28, or 32 weeks after infusion. The authors used 454 pyrosequencing to characterize 7,782 sites from the pre-infusion samples and 237 sites from three of the patients at different time points after infusion. Chromosomal DNAs were restriction digested, and a polymerase chain reaction (PCR)–specific linker was added to one end of the digested samples. The vector-derived DNAs were amplified using primers complementary to the linker and a vector-derived sequence. To analyze the vectors in the high-throughput 454 sequencing protocol, the investigators bar-coded the second-round PCR amplification primers,9Wang GP Garrigue A Ciuffi A Ronen K Leipzig J Berry C et al.DNA bar coding and pyrosequencing to analyze adverse events in therapeutic gene transfer.Nucleic Acids Res. 2008; 36: e49Crossref PubMed Scopus (92) Google Scholar allowing them to simultaneously sequence multiple samples. To determine whether the vector integration sites resembled those of HIV integration, the pre- and postinfusion samples were analyzed in comparison to 40,000 previously characterized HIV integration sites. These analyses showed that, like HIV, the vector favors integration into a weakly conserved primary sequence, with highly similar base preferences at each position of the integration site. Additional analyses revealed that the vectors integrated preferentially into the gene-rich chromosomes 16, 17, 19, and 22, whereas the gene-poor Y, 4, and 13 chromosomes were disfavored. Moreover, the integration events favored transcription units in chromosomal regions of high gene density. In similarity to the results observed with HIV integration analyses, the vectors preferentially integrated into regions of genes with active histone marks and did not integrate into regions with silencing histone marks. Two important outcomes of these analyses are the relationship between integration preferences and proto-oncogene 5′ ends. There was no enrichment for integrations near proto-oncogenes as was found for the gamma retroviruses. The authors did point out, though, that those integration events near the 5′ ends of proto-oncogenes were more frequent than would be predicted by chance alone. Nevertheless, for proto-oncogenes known to be important in lymphoid cell cancers, none of the 237 patient-derived integration events was within 50 kb of any of these genes. Clonal expansion of specific integration events would suggest the potential for enhanced cell proliferation, which of course would be a genotoxic warning signal. When these investigators sought to identify specific integration events that occurred at a specific locus in samples isolated at more than one of the time points, they found no overlap, strongly suggesting no selective expansion of any transduced T-lymphocyte population. Overall, the results of these analyses are highly encouraging, because they correlate well with the known HIV integration patterns, and T-cell malignancies have not been associated with HIV integration, despite the millions of infected individuals. The VRX496 vector contains an intact viral long-terminal repeat, which could be activated upon introduction of Tat from an HIV infection. Thus, the transgene—in this case an antisense transcript—will be transcribed in a fashion similar to HIV mRNAs. What is uncertain is whether other promoters introduced into the bodies of lentiviral vectors can have long-range enhancer effects or read-through transcription into adjacent host sequences. Several ongoing and planned clinical trials with lentiviral vectors are targeting hematopoietic progenitor cells. It is not known whether the patterns of integration in this population of cells will be different from that of mature T lymphocytes, and clearly this warrants careful investigation and scrutiny. One study comparing lentiviral vector with gamma retroviral vector integration in non-human primate hematopoietic stem cells showed that both vectors integrated within 100 kb of proto-oncogenes at a frequency higher than a random data set.10Beard BC Dickerson D Beebe K Gooch C Fletcher J Okbinoglu T et al.Comparison of HIV-derived lentiviral and MLV-based gammaretroviral vector integration sites in primate repopulating cells.Mol Ther. 2007; 15: 1356-1365Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar Clearly, more careful analyses of the biological consequences of these integration patterns are warranted in appropriate animal models.