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Background Recent observations of insertional mutagenesis in preclinical and clinical settings emphasize the relevance of investigating comprehensively the spectrum of integration sites targeted by specific vectors. Methods We followed the engraftment of lentivirally transduced human cord blood (CB) progenitor cells after transplantation into NOD/SCID mice using a self-inactivating HIV-1-derived vector expressing the enhanced green fluorescent protein (EGFP). Results The mean of transduction of CD34 + CB cells was 41%, as deduced from the percentage of EGFP + cells before transplantation. At 3 weeks posttransplantation, the average of EGFP + cells in the human cell population was 65 ± 8%, and increased to 75 ± 10% at 12 weeks post-transplantation. In order to determine the proviral integration sites in human NOD/SCID repopulating cells (SRCs) we used the ligation-mediated polymerase chain reaction (LM-PCR) technique. Sixty-eight percent of the integrations were found to be located in RefSeq genes, most of them in intron regions. Twenty percent of these integrations occurred within a distance of 10 kb from the transcription start site; a percentage that is significantly lower compared to that observed in cells transduced by gammaretroviral vectors. Sixty-two percent of integrations occurred in genes with a biological function in cell metabolism, and four integrations were located in genes with a role in tumorigenesis. Conclusions These investigations indicate that integration of lentiviral vectors in human repopulating cells capable of engrafting NOD/SCID mice preferentially occur in coding regions of the human genome. Nevertheless, the clustering of integrations at the transcriptional start is not as high as that observed for gammaretroviral vectors. Copyright  2006 John Wiley & Sons, Ltd.

Reports on insertional "genotoxicity" in patients have created intense interest in characterizing retroviral vector integrations on the genomic level. The retroviral vector SF91m3 was used for transduction of human peripheral blood progenitor cells (PBPC). These PBPC were transplanted into nonobese diabetic/severe combined immunodeficient mice. A total of 186 retroviral vector integration sites were isolated by ligation-mediated PCR from chimeric mouse bone marrow of five PBPC donors, sequenced, and blasted against the human genome. Preferred integration near the transcription start regions, within CpG islands, and within Alu regions was observed. Detailed analysis of targeted RefSeq genes showed a favored integration within the first intron. Integrations were most common in genes coding for signaling proteins, transcription factors, and kinases. In all genes targeted independently multiple times the respective orientation of the provirus within the gene was identical, indicating integration hot spot regions and similar steric determinants for integration sites. Possible explanations for these findings could be nonrandom vector integration, clonal selection due to gene expression interference, or engraftment issues related to gene insertion in signaling and cell cycle genes. The low frequency of integrations in exons may be reassuring as to the safety of retroviral gene therapy with normal human PBPC.

Methods to analyze the clonality of an adverse event in preclinical or clinical retroviral stem cell gene therapy protocols are needed. We analyzed the progeny of retrovirally transduced human peripheral blood progenitor cells (PBPCs) after transplantation and engraftment in immune-deficient mice. The integration site of the provirus serves as a unique tag of the individual transduced PBPC. A plasmid library of junctions between proviral and human genomic DNA was generated. We were able to detect individual transduced cell clones that amounted to 0.14%–0.0001% of chimeric bone marrow cells. This is the first report in which the contribution of individual marrow-repopulating cells to human hematopoiesis is directly quantified.

Genetically modified T-Lymphocytes (TLCs) have been used for adoptive immunotherapy in the context of allogeneic hematopoietic stem cell transplantation (SCT). Infused TLCs have been shown to be susceptible to elimination through exposure to ganciclovir in the event of graft-versus-host disease (GvHD). Yet, reports on insertional mutagenesis in a mouse gene marking study and a clinical gene therapy trial for X-chromosomal severe combined immunodeficiency (X-SCID) reminded us the actual risk of insertional oncogene activation and subsequent leukemia development. We investigated retroviral integration sites in vitro and in vivo. Therefore, donor TLCs transduced with the MoLV-based TK/neoR vector Mo3TIN for a clinical HSV-Tk study were examined. TLCs of four different donors as well as whole blood samples of two patients transplanted with donor TLCs were analyzed either using highly sensitive and specific ligation-mediated PCR (LM-PCR). A total of 114 retroviral integration sites were detected in vitro. 41.2% of all integrations appeared near the transcription start regions (+/−10kb) of genes. Further analysis showed that 57 (50%) of all integrations targeted RefSeq genes. 24 of those appeared in intron 1 (42% of all integrations into genes) while 18% (10/57) of all integrations into genes landed in exon sequences whereas 6 hit the first exon. 18 of the targeted genes (15.8% of all integrations) could be at last assigned to signal transduction pathways, whereas the transcription factor family was afflicted 13 times (11.4% of all integrations). Among the targeted genes we found integrations into the CD8, CD100, CD44, CX3CR1, HLA-DMP and IL10-receptor genes. Within at a range of 5kb up- and 5kb downstream of vector integrations 15 genes were located that were not hit. 5 are known as transcription factors, whereas two of those are involved in leukemia, namely the homo sapiens myeloid/lymphoid or mixed-lineage leukemia 5 gene (MLL5) and the homo sapiens ALL1 fused gene from 5q31 (AF5Q31). Current analyses are focusing at the in vivo pattern of retroviral integration in DNA of TLCs obtained from transplanted patient’s TLCs. Therefore we developed a new high sensitive PCR method (HS-PCR), an improved LM-PCR to even detect minimal quantities of transduced DNA.

It is of concern whether the introduction of a transgene into hematopoietic stem cells by retroviral vectors will lead to an alteration of the growth and engraftment characteristics. Earlier studies in mice indicated that retroviral multidrug-resistance 1 gene transfer may be associated with a myeloproliferative disorder. In human or primate cells this could not be reproduced in bulk cell populations. Analysis on the clonal level were lacking. One method to study the in vivo behaviour of repopulating progenitor and stem cells is marking the cells with replication-incompetent retroviral vectors that integrate into identifiable host DNA sequences, thus allowing the tracking of cell progeny based on unique proviral insertion sites. In this study CD34-enriched peripheral blood stem cells from 2 rhesus macaque monkeys were split into two aliquots and transduced either with a multidrug-resistance 1 gene-retroviral vector based on the Harvey murine sarcoma virus (HaMDR1-vector) or a NeoR-retroviral vector based on the Moloney murine leukaemia virus (G1Na-vector). After autologous retransplantation, DNA from blood and bone marrow was collected at different time points in a period of 4 years and the animals are still alive. By using a highly sensitive and specific ligation-mediated polymerase chain reaction (LM-PCR) followed by sequencing of vector integration sites, we found in animal M120 32 different contributing hematopoietic clones 8 weeks and 50 weeks after transplantation and in animal M038 17 clones 58 weeks after transplantation. Based on the difference between the sequences of the HaMDR1-LTR and the G1Na-LTR, the clones can be allocated definitely to one of the two vectors. Remarkably, 36 clones descend from the G1Na-vector, whereas only 13 clones descend from the HaMDR1-vector. We conclude that hematopoiesis in these monkeys is polyclonal for prolonged periods after transplantation and that MDR1 gene transfer does not confer a proliferative advantage over vector-control-transduced hematopoietic stem cells.

Increasing use of hematopoietic stem cells for retroviral vector-mediated gene therapy and recent reports on insertional mutagenesis in mice and humans have created intense interest to characterize vector integrations on the genomic level. We have studied retroviral integration sites in the genome of human marrow-repopulating cells. Therefore, we transduced peripheral blood progenitor cells (PBPC) from 5 healthy donors with retroviral supernatant of the SF-vector (based on the Friend mink cell-forming/murine embryonic stem-cell virus; transduction efficiency: 7%–41%) and transplanted them into immunodeficient NOD/SCID mice. After 8 weeks of engraftment chimeric mouse bone marrow DNA was analyzed with ligation-mediated PCR (LM-PCR) followed by blast analysis.

Increasing use of hematopoietic stem cells for retroviral vector-mediated gene therapy and recent reports on leukemogenesis in mice and humans have created intense interest to characterize vector integrations on the genomic level. As techniques to determine insertion sites are more commonly applied in gene therapy laboratories there is a need to systematically collect and analyze the data arising from such studies in a vector insertion database. This will allow determining factors responsible for preferential integration of various vector types in specific chromosomal regions, genes or gene sections. The information derived from a vector insertion data base will be useful to recognize more “dangerous” vector types and may provide useful information for vector design. We have set up an automatic sequence analysis tool (ensuring quality criteria e.g. verification of LTR- and adapter sequence, score >40, e-value >10e-40, hit RefSeq, next RefSeq etc.) which simplifies data input enormously while ensuring high quality standards. Our group is establishing the "collaborative RISC (retroviral insertion estimation into chromosome) -Score Database (CRSD)"- assessment project, based on the M-CHIPS (Multi-Conditional Hybridisation Intensity Processing System) microarray data warehouse and analysis software (K. Fellenberg et al. 2001, 2002). The data obtained from the sequence analysis tool were automatically fed in the data base. A total of 287 retroviral vector integration sites were isolated and sequence analysis was performed with the above describe analysis tool. In human bone marrow repopulating cells they occurred with significantly increased frequency into chromosomes 17 and 19 (n=189). Analysis of targeted RefSeq genes showed a favored integration (48%) within the first intron. In comparison, retroviral vector integrations in T-cells (n=98) showed an entirely different chromosomal distribution pattern while the percentage of the targeted RefSeq genes was similar (46%). Further, more than 1200 sequences were submitted to the data base, originating from different vectors (SF-MDR-, MoLV-based TK/neoR-Mo3TIN-, Moloney-MGMT-, Harvey-based Neo-, Harvey-based MDR-, and lentiviral GFP-SIN-vectors) and different transduced cells (mouse hematopoietic cells, mouse fibroblasts, rhesus hematopoietic cells, human hematopoietic cells, human T-cells). The set-up and internal structure of the data base will be presented. Collaborations have been forged to include further groups and vector types. Bioinformatical analysis will allow recognizing even complex vector integration patterns and will broaden our understanding for the determinants of vector integration into the genome. This in turn can lead to the construction of "favorable" vectors and help to reduce the genotoxicity of retroviral or lentiviral vector-mediated gene transfer.