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Graft-versus-host disease (GvHD) is a severe complication in the context of allogeneic stem cell transplantation and adoptive immunotherapy. The transfer of a suicide gene into donor T-lymphocytes (TLCs) allows selective elimination of GvHD-causing cells. As retroviral gene transfer into hematopoietic stem cells can induce leukaemia, there is an urgent need also to analyze retroviral integration sites in TLCs. We examined suicide gene-transduced TLCs in four grafts and from four transplanted patients. One-hundred and fifteen integration sites were detected in vitro. Of these 90 could be mapped to the human genome; 50% (45) were located in genes and 32% (29) were detected 10 kb upstream or downstream of transcription start sites. We found a significant overrepresentation of genes encoding for proteins with receptor activity, signal transducer activity, transcription regulator activity, nucleic acid binding activity and translation regulator activity. Similar data were obtained from patient samples. Our results point to preferred vector integration patterns, which are specific for the target cell population and probably independent of selection processes. Thus, future preclinical analysis of the integration repertoire with abundant amounts of transduced cells could allow a prediction also for the in vivo situation, where target cells are scarce.

Despite significant improvements of the survival of HIV-infected patients during HAART, treatment limitations such as the development of drug-resistant HIV strains and long-term toxicities call for innovative treatment strategies. In this study, a novel antiviral gene that effectively inhibits virus entry was tested in a phase I/II clinical trial. The antiviral gene is expressed from a retroviral vector (M87o) and encodes for the membrane-anchored antiviral peptide C46. C46 comprises 46 amino acids, is derived from the second heptad repeat of the HIV-1 envelope glycoprotein gp41 and effectively inhibits fusion of the viral and cellular membranes during virus entry. The membrane-anchored form (maC46) was shown to effectively block entry of a broad range of HIV isolates, including viruses resistant to the fusion inhibitory peptide C36 (T20). C36 (T20) corresponds to the 36 C-terminal amino acids of C46. The gene was introduced into autolous CD4 T lymphocytes from 10 patients with severe immunodeficiency and HAART failure. Cells were expanded by co-stimulation with anti-CD3 and anti-CD28 immobilized on beads. Between 2 and 15 |[times]| 10E9 cells were infused. No major toxicities were observed and neither cellular nor humoral immune responses to the transgene product were detected. While a significant rise in T helper cell counts was seen, viral load was not affected. Gene marking could be detected throughout the one-year follow-up, but the levels were too low to account for the marked rise in CD4 counts.

Previous studies have demonstrated leukemic complications in mice after high-copy retroviral gene transfer of the multidrug resistance 1 (MDR1) cDNA, encoding a membrane-located efflux pump expressed in hematopoietic stem cells. In contrast, no such complications or MDR1-associated alterations of hematopoiesis were observed in numerous other studies exploring MDR1 gene transfer into cell lines, mice, dogs, nonhuman primates, and human subjects. Here, we show that leukemias associated with retroviral expression of MDR1 depend on high vector dose, and involve the selection of clones with combinatorial insertional mutagenesis of proto-oncogenes or other signaling genes. Compared with insertion patterns in normal long-term repopulating hematopoietic cells, such hits were overrepresented in leukemic clones, pointing to a causal role. A similar constellation of insertion sites was also observed in a leukemia arising after high-copy retroviral gene transfer of a fluorescent protein. Spectral karyotyping demonstrated additional chromosomal translocations in a subset of cases, indicative of secondary genetic instability. We also show that insertional mutants can be amplified in vitro prior to transplantation. On the basis of these findings, we suggest the use of preclinical dose-escalation studies to define a therapeutic index for retroviral transgene delivery.

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.

Chronic Granulomatous Disease (CGD) is a primary immunodeficiency characterized by recurrent and life-threatening bacterial and fungal infections. CGD is caused by mutations in any one of four genes encoding for subunits of the phagocytic NADPH oxidase complex. Most patients contain mutations or deletions within the gene coding for gp91phox, the larger subunit of the complex. Based on our preclinical work, two X-CGD patients, 26 and 25 years old, were treated with gene modified cells. G-CSF mobilized CD34+ cells were transduced with a monocistronic gp91phox retroviral vector. The transduction efficiency was 45% for Pat.1 and 40% for Pat. 2. The number of CD34+ cells reinfused was 1.2|[times]|10e7 per kg for Pat.1 and 0.9|[times]|10e7 per kg for Pat. 2. Before reinfusion, patients were treated with liposomal busulphan given i.v. at a dose of 4 mg/kg at two consecutive days, starting at day -3. The treatment was well tolerated and no adverse effects have been observed. Neutrophil counts declined to less than 100 cells per |[mu]|l at day 15 post reinfusion for both patients and recovered to more than 500 cells per |[mu]|l at day 30 for Pat. 1 and day 20 for Pat. 2. A significant fraction of gene marked cells has been detected in peripheral blood of both patients since day +21. Similarly, therapeutic relevant levels of NADPH oxidase activity have been observed since day +21. Both patients are well and have been free of severe bacterial and fungal infections since transplantation. Our data suggests that gene therapy is an option for the treatment of CGD, despite the fact that gp91phox corrected cells will not have per se a proliferative advantage over non-transduced cells.

Gene transfer into hematopoietic stem cells has been envisaged as an alternative to stem cell transplantation for the treatment of many genetic diseases of the blood system. In 2004 we initiated a gene therapy trial aimed at the correction of Chronic Granulomatous Disease (CGD), a rare inherited immunodeficiency caused by a functional defect in the microbial killing activity of phagocytes. Gene marking and functional correction of phagocytes were high shortly after transplantation of gene modified cells, leading to the eradication of therapy resistant infections from which patients had suffered for many years. However, one of our patients died 27 months after treatment due to a severe sepsis with multiorgan dysfunction. Although gene marking was still high at this time point, expression of the therapeutic gene, gp91phox, was minimal. This down regulation of transgene expression was due to CpG methylation within the viral LTR. Similar effects were observed in a second patient treated more than 3 years ago. In both patients CpG methylation was restricted to the promoter region of the viral LTR, while CpG dinucleotides within the enhancer region of the viral LTRs were not methylated. As a consequence, gp91phox gene expression was suppressed but the capacity of the viral LTRs to transactivate nearby sequences is still intact. Indeed we were able to detect cellular transcripts at predominant retroviral integration sites leading to an unbalanced clonal distribution of gene marked cells in peripheral blood and bone marrow. A third patient, a 5 years old child was treated in a similar way in Zurich. In this case only low levels of engraftment and gene correction were achieved. Although the treatment has been beneficial for all treated patients, the side effects observed in the two adults demand modifications in vector design for sustained gene expression and long term correction of the disease without side effects.

Gene transfer into hematopoietic stem cells has been successfully used to correct immunodeficiencies affecting the lymphoid compartment. However, similar results have not been reported for diseases affecting myeloid cells, mainly due to low engraftment levels of gene-modified cells observed in unconditioned patients. Here we report on two adult patients (P1 and P2, follow up >24 months) and one child (P3, 6 years, follow up 15 months) who received gene-transduced hematopoietic stem cells in combination with nonmyeloablative bone marrow conditioning for the treatment of X-linked Chronic Granulomatous Disease (X-CGD), a primary immunodeficiency caused by a defect in the oxidative antimicrobial activity of phagocytes. Therapeutically significant gene marking was detected in neutrophils of both adult patients (P1 and P2) leading to large numbers (up to 60%) of functionally corrected phagocytes 24 months after gene therapy. This high correction resulted from an unexpected but temporarily restricted expansion of gene transduced myeloid cells in vivo. In contrast gene marking and functionally reconstitution levels in P3 have been low (1–2%). Both adult patients suffered from active infections prior to gene therapy (P1 of bacterial liver abscesses and P2 of lung aspergillosis) and were free of severe bacterial and fungal infections until 24 months after transplantation. P3 suffered from an Aspergillus infection of the spinal cord with paraparesis before transplantation and recovered after gene therapy despite low numbers of functionally corrected cells in the peripheral blood. Large-scale mapping of retroviral integration site distribution revealed that activating insertions in the zinc finger transcription factor homologs MDS1/EVI1, PRDM16, or in SETBP1 have expanded gene-corrected long term myelopoiesis 3- to 4-fold in both adults, providing direct evidence in humans that these genes may influence regulation of normal long-term hematopoiesis. The hematopoietic repopulation in P1 was polyclonal until 18 months after therapy. P1 died of a severe bacterial sepsis after colon perforation 27 months after gene therapy. No evidence of malignant transformation was found in peripheral blood or bone marrow aspirates from this patient. Gene marking at death was still 60%; however the function of gene transduced cells, the number of corrected cell clones and the activity of a predominant clone was greatly decreased. P2 has been free of infections since transplantation (last monitoring: month 26). Hematopoietic repopulation was polyclonal in P2 until day 560. In conclusion, gene therapy in combination with bone marrow conditioning has provided a transitory therapeutic benefit for all 3 patients. Further improvements in vector design and conditioning regimes are under investigation to provide a stable and long term correction of the disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency in which phagocytic cells of affected patients have impaired antimicrobial activity due to a defect in the production of reactive oxygen species (ROS). CGD is caused by mutations in any one of four genes encoding for the subunits of the NADPH oxidase complex. Although curable by HSC transplantation, this strategy is usually limited only to patients with HLA-matched sibling or unrelated donors, as mismatched transplantation is associated with high morbidity and mortality due to graft failure and slow immune reconstitution. A therapeutic alternative for CGD patients is the genetic modification of autologous HSC. In January 2004 we initiated a Phase I/II clinical trial for X-CGD patients including conditioning with busulfan (8 mg/kg/total dose) prior to infusion of genetically modified HSC. G-CSF mobilized CD34+ cells from 2 adult patients (25 and 26 years) were transduced ex-vivo with a monocistronic gp91phox retroviral vector. Therapeutically significant gene marking levels were detected in neutrophils of both patients with up to 60% functionally corrected phagocytes 14 months after gene therapy. This high correction resulted from an unexpected but temporarily restricted expansion of gene transduced myeloid cells in vivo. In contrast gene marking levels in B-cells has remained constant at a level of 20%, while gene marking in T-cells is below 5%. Gene marking in bone marrow was detected at levels between 30% and 40% one year after transplantation of gene modified cells. Killing assays in vitro have demonstrated antibacterial and antifungal activity in gene transduced phagocytes and both patients recovered of Staph. aureus and Aspergillus fumigatus infections after gene therapy. Our results suggest that gene therapy in combination with bone marrow conditioning is a therapeutic option for inherited diseases affecting the myeloid compartment and can be successfully used to treat CGD.

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.

High titer retroviral vector supernatant, produced in compliance with GMP, has always been a prerequisite for gene therapy purposes in clinical trials. But most vector production procedures have their limitations: the vector often leads only to low titer supernatant, only a few harvest cycles can be done and harvest volumes are often small.

Retroviral vectors encoding the herpes simplex thymidine kinase gene (HSV-Tk) have been employed to render T-lymphocytes (TLCs) sensitive to the prodrug ganciclovir. Such genetically modified T cells have been used for adoptive immunotherapy in the context of allogeneic hematopoietic stem cell transplantation (SCT). Infused T cells have been shown to be susceptible to elimination through exposure to ganciclovir in the event of graft-versus-host disease (GvHD). Recent reports on insertional “genotoxicity” 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. Here we investigated retroviral integration sites in donor TLCs transduced with the MoLV-based TK/neoR vector Mo3TIN of four donors in a clinical HSV-Tk study. A total of 114 retroviral integration sites were detected using highly sensitive and specific ligation-mediated PCR (LM-PCR). 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). The zinc ion binding group makes up 4 (resp. 6, minding that GTF2HII contains a C2H2 type and KIAA0427 a C-x8-C-x5-C-x3-H-type zinc finger) of them. 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). Hit genes are currently examined more systematically in terms of function, e.g. involvement in signal transduction and transcription promoting processes. RISC (Retroviral Insertion estimate of Chromosomal Integration) scores and integration specific data will be submitted to a data warehouse, the collaborative RISC score database (CRSD). Such a systematic data collection similar to the IBMTR or EBMT databases will allow to recognize factors contributing to the safety, optimal transgene expression and persistence of transduced cells in the setting of allogenic matched donor transplantation.