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Regulators and industry are actively seeking improvements and alternatives to current models and approaches to evaluate potential carcinogenicity of gene therapies (GTs). A meeting of invited experts was organized by NC3Rs/UKEMS (London, March 2023) to discuss this topic. This article describes the consensus reached among delegates on the definition of vector genotoxicity, sources of uncertainty, suitable toxicological endpoints for genotoxic assessment of GTs, and future research needs. The collected recommendations should inform the further development of regulatory guidelines for the nonclinical toxicological assessment of GT products.

The development of genome-editing technology could lead to breakthrough gene therapy. Genome editing has made it possible to easily knock out or modify a target gene, while current gene therapy using a virus vector or plasmid hampering modification with respect to gene replacement therapies. Clinical development using these genome-editing tools is progressing rapidly. However, it is also becoming clear that there is a possibility of unintended gene sequence modification or deletion, or the insertion of undesired genes, or the selection of cells with abnormalities in the cancer suppressor gene p53 ; these unwanted actions are not possible with current gene therapy. The Science Board of the Pharmaceuticals and Medical Devices Agency of Japan has compiled a report on the expected aspects of such genome-editing technology and the risks associated with it. This article summarizes the history of that discussion and compares the key concepts with information provided by other regulatory authorities.

The former definition of gene therapy was the infusion of genes or cells transduced with genes into humans for the treatment or prevention of a disease, i.e., gene therapy adds a functional gene into the patients' genome, whereas the mutated gene remains as it is. Because most of the immune immunodeficiency disorders (PID) are caused by single gene mutations, this therapeutic option may provide a clinical effect. However, the treatment has a severe problem of leukemogenesis caused by insertional mutagenesis; therefore, it is not applicable for diseases caused by the gain-of-function of mutated genes. To address this, gene therapy using gene correction techniques will come to the forefront of the mainstream research. Herein, I have focused on the present outline of gene therapy by gene addition and described the future prospects of gene therapy by gene correction for PID.

Congenital adrenal hyperplasia (CAH) due to steroid 21-hydroxylase (21-OH) deficiency (21-OHD) is an autosomal recessive disorder, in which CYP21A2 mutations or deletions result in underproduction of glucocorticoid and mineralocorticoid, and overproduction of androgens. Patients with CAH are treated with oral steroid supplementation, but optimal control of blood steroid levels remains difficult. Thus, new therapeutic approaches are still needed. Previously, adenovirus-mediated administration of human CYP21A2 to adrenal glands rescued the phenotype of a mouse model of 21-OHD. In this study, we examined whether transduction of murine Cyp21a1 in extra-adrenal tissues could rescue steroid metabolism in 21-OHD mice. We transduced primary fibroblasts obtained from 21-OHD mice with a retroviral vector containing Cyp21a1. In vitro assays demonstrated that Cyp21a1-expressing fibroblasts can uptake progesterone from the culture media, convert it to deoxycorticosterone (DOC), and subsequently release DOC back into the media. Autotransplantation of Cyp21a1-expressing fibroblasts into the subcutaneous tissues of the back resulted in a significant reduction in the serum progesterone/DOC ratio in four of six 21-OHD mice at 4 weeks after injection. We also directly injected an adeno-associated viral vector containing Cyp21a1 into the thigh muscles of 21-OHD mice. Serum progesterone/DOC ratios were markedly reduced in all four animals at 4 weeks after injection. These results indicate that extra-adrenal induction of Cyp21a1 ameliorates steroid metabolism in 21-OHD mice. This study suggests a novel therapeutic strategy for congenital adrenal hyperplasia, which warrants further investigations.

Objective: We here describe treatment outcomes in two adenosine deaminase (ADA)-deficiency patients (pt) who received stem cell gene therapy (SCGT) with no cytoreductive conditioning. As this protocol has features distinct from those of other clinical trials, its results provide insights into SCGT for ADA deficiency., Patients and Methods: Pt 1 was treated at age 4.7 years, whereas pt 2, who had previously received T-cell gene therapy, was treated at age 13 years. Bone marrow CD34(+) cells were harvested after enzyme replacement therapy (ERT) was withdrawn; following transduction of ADA cDNA by the γ-retroviral vector GCsapM-ADA, they were administered intravenously. No cytoreductive conditioning, at present considered critical for therapeutic benefit, was given before cell infusion. Hematological/immunological reconstitution kinetics, levels of systemic detoxification, gene-marking levels, and proviral insertion sites in hematopoietic cells were assessed., Results: Treatment was well tolerated, and no serious adverse events were observed. Engraftment of gene-modified repopulating cells was evidenced by the appearance and maintenance of peripheral lymphocytes expressing functional ADA. Systemic detoxification was moderately achieved, allowing temporary discontinuation of ERT for 6 and 10 years in pt 1 and pt 2, respectively. Recovery of immunity remained partial, with lymphocyte counts in pts 1 and 2, peaked at 408/mm(3) and 1248/mm(3), approximately 2 and 5 years after SCGT. Vector integration site analyses confirmed that hematopoiesis was reconstituted with a limited number of clones, some of which were shown to have myelo-lymphoid potential., Conclusions: Outcomes in SCGT for ADA-SCID are described in the context of a unique protocol, which used neither ERT nor cytoreductive conditioning. Although proven safe, immune reconstitution was partial and temporary. Our results reiterate the importance of cytoreductive conditioning to ensure greater benefits from SCGT.

Patients affected by primary immunodeficiency (PID) can be cured by allogeneic hematopoietic stem cell transplantation (HSCT). In the absence of HLA-matched donors, however, incidence of HSCT-related complications is observed. Therefore, gene therapy has been developed as a highly desirable alternative treatment for patients lacking suitable donors. Retrovirus-based gene therapy was begun in 1990 for the patients of adenosine deaminase deficiency, followed by X-linked severe combined immunodeficiency, Wiskott-Aldrich syndrome and chronic granulomatous disease. Although treated patients have had excellent immune reconstitution and resolution of ongoing infections, complications such as a lymphoproliferative syndrome and a disappearance of gene-modified cells were observed in some clinical trials. To overcome these, ongoing and upcoming clinical trials use some new strategies. The use of preconditioning chemotherapy makes space in the bone marrow for the gene-treated stem cells and allows engraftment of multi lineage stem/progenitor cells. Self-inactivating vectors in which strong enhancers of long terminal repeat are eliminated may reduce the risk of insertional activation of proto-oncogene resulting in leukemia. These modifications will surely increase the safety and efficacy of stem cell gene therapy for PID.

The CH-296 recombinant fragment of human fibronectin is essential for murine leukemia virus (MLV)-derived retroviral transduction of CD34(+) cells for the purpose of stem cell gene therapy. Although the major effect of CH-296 is colocalization of the MLV-derived retrovirus and target cells at specific adhesion domains of CH-296 mediated by integrins expressed on CD34(+) cells, the precise roles of the integrins are unclear. We examined the kinetics of integrin expression on CD34(+) cells during the course of MLV-derived retrovirus-mediated gene transduction with CH-296. Flow cytometry revealed that the levels of both very late activation protein (VLA)-4 and VLA-5 on CD34(+) cells freshly isolated from cord blood were insufficient for effective MLV-derived retroviral transduction. However, increases were achieved during culture for preinduction and MLV-derived retrovirus-mediated gene transduction in the presence of a cocktail of cytokines. In addition, we confirmed by using specific antibodies that inhibition of the cell adhesion mediated by the integrins significantly reduced transduction efficiency, indicating that integrin expression is indeed important for CH-296-based MLV-derived retroviral transduction. Only a few cytokines are capable of inducing integrin expression, and stem cell factor plus thrombopoietin was found to be the minimal combination that was sufficient for effective transduction of an MLV-derived retrovirus based on CH-296. Our findings should be useful for improving the culture conditions for CH-296-based MLV-derived retroviral transduction in stem cell gene therapy.

To control severe GvHD while maintaining strong GvL effects in the context of allo-stem cell transplantation (allo-SCT), a phase I/II clinical trial of infusions of donor lymphocytes transduced with the herpes simplex virus thymidine kinase (TK-DLI) started at the Tsukuba University Hospital. To date, five (2 AML, 2 ALL, and 1 MDS) out of eight patients enrolled in the trial received approximately 7x10(7) transduced cells per kilogram of body weight and four patients showed some clinical responses such inhibition of the leukemic cell proliferation or mitigation of lymph node swelling. Especially, one MDS patient achieved complete remission and has remained in CR for 2 years after the treatment. GvHD developed in two patients (1 acute and 1 chronic) and the acute (grade III) was successfully controlled by administration of ganciclovir without any immunosuppressive drugs. Since HSV-TK as a strong antigen induced CTLs against transduced cells in patients, however, TK-DLI is expected to provide a more effective adoptive immune cell therapy by performance just after allo-SCT where the patient's immune function is severely damaged.

Recently, a serious adverse effect of uncontrolled clonal T cell proliferation due to insertional mutagenesis of retroviral vector was reported in X-SCID gene therapy clinical trial. To offset the side effect, we have incorporated a suicide gene into therapeutic retroviral vector for selective elimination of transduced cells. In this study, B-cell lines from two X-SCID patients were transduced with bicistronic retroviral vector carrying human gamma c chain cDNA and Herpes simplex virus thymidine kinase gene. After confirmation of functional reconstitution of the gamma c chain, the cells were treated with ganciclovir (GCV). The gamma c chain positive cells were eliminated under low concentration without cytotoxicity on untransduced cells and have not reappeared at least for 5 months. Furthermore, the gamma c chain transduced cells were still sensitive to GCV after five months. These results demonstrated the efficacy of the suicide gene therapy although further in vivo studies are required to assess feasibility of this approach in clinical trial.

Background: Hybrid adeno-retroviral vector systems utilize the high efficiency of adenovirus transduction to direct the in situ production of retroviral progeny. In this study, we show that a single-step transduction of glioma cells with trans-complementing hybrid adeno-retroviral vectors effectively turns these cells into retrovirus vector-producing cells, which in turn facilitates the transduction of adjacent cells., Methods: We have adapted the adeno-retroviral hybrid viral vector system to enhance the ganciclovir (GCV) killing of glioma cells following transfer of the herpes simplex virus thymidine kinase (HSV-tk) gene. To assess the effect of the in situ production of retroviral vectors on the transduction efficiency of glioma cells, 9L cells were transduced with adeno-retroviral hybrid vectors that separately express a retroviral genome (AVC2.GCEGFP or AVC2.GCTK) and retroviral packaging proteins (AxTetGP and AxTetVSVG). The generation of an integrated HSV-tk provirus by trans-complementation of the adeno-retroviral vectors was verified by analysis of the flanking retroviral LTR sequences. Tumors established on nu/nu mice were injected with the viruses followed by intraperitoneal injections of either PBS or GCV. We also estimated the copy numbers of the HSV-tk transgene present in the tumors of the treated mice. To determine the expression pattern of the HSV-tk transcripts within a tumor, in situ hybridization analysis was performed using an RNA probe specific for HSV-tk., Results: The co-transduction of rat 9L glioma cells with AVC2.GCEGFP together with vectors expressing packaging proteins of retroviruses increased the transduction efficiency. Transduction with AVC2.GCTK together with packaging vectors increased the in vitro sensitivity of cells to the pro-drug GCV by one log compared with control cells that were incapable of generating retrovirus. In vivo, the injection of established subcutaneous 9L tumors on athymic mice with a combination of AVC2.GCTK and packaging vectors followed by GCV treatment resulted in complete tumor regression in 50% of tumors at day 22, while no tumor regression was observed in control animals. Retroviral sequences diagnostic of 3' LTR reduplication in vivo were detected in genomic DNA extracted from the transduced tumors, indicating pro-viral integration of the retroviral genome derived from the adeno-retroviral hybrid vector. Furthermore, the relative copy number of the HSV-tk gene in tumors treated with the adeno-retroviral vectors was up to approximately 250-fold higher than in control tumors. In situ hybridization suggested dispersion of the HSV-tk product across a wider area of the tumor than in control tumors, which indicates the spread of the in situ generated retroviruses., Conclusions: Although the efficacy of this system has to be evaluated in orthotopic models, our observations suggest that this hybrid adeno-retroviral vector system could improve the suicide gene therapy of tumors., (Copyright 2004 John Wiley & Sons, Ltd.)

Clinical gene therapy trials for adenosine deaminase (ADA) deficiency have shown limited success of corrective gene transfer into autologous T lymphocytes and CD34(+) cells. In these trials, the levels of gene transduction and expression in hematopoietic cells have been assessed by DNA- or RNA-based assays and measurement of ADA enzyme activity. Although informative, these methods are rarely applied to clonal analysis. The results of these assays therefore provide best estimates of transduction efficiency and gene expression in bulk populations based on the assumption that gene transfer and expression are uniformly distributed among transduced cells. As a useful additional tool for evaluation of ADA gene expression, we have developed a flow cytometry (fluorescence-activated cell sorting, FACS) assay capable of estimating the levels of intracellular ADA on a single-cell basis. We validated this technique with T cell lines and peripheral blood mononuclear cells (PBMCs) from ADA-deficient patients that showed severely reduced levels of ADA expression (ADA-dull) by FACS and Western blot analyses. After retrovirus-mediated ADA gene transfer, these cells showed clearly distinguishable populations exhibiting ADA expression (ADA-bright), thus allowing estimation of transduction efficiency. By mixing ADA-deficient and normal cells and using enzymatic amplification, we determined that our staining procedure could detect as little as 5% ADA-bright cells. This technique, therefore, will be useful to quickly assess the expression of ADA in hematopoietic cells of severe combined immunodeficient patients and represents an important tool for the follow-up of patients treated in clinical gene transfer protocols.