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The Sendai virus vector has received a lot of attention due to its broad tropism for mammalian cells. As a result of efforts for genetic studies based on a mutant virus, we can now express more than 10 genes of up to 13.5 kilo nucleotides in a single vector with high protein expression efficiency. To prove this benefit, we examined the efficacy of the novel ribonucleic acid (RNA) virus vector harboring the human iduronate‐2‐sulfatase (IDS) gene with 1,653 base pairs, a causative gene for mucopolysaccharidosis type II, also known as a disorder of lysosomal storage disorders. As expected, this novel RNA vector with the human IDS gene exhibited its marked expression as determined by the expression of enhanced green fluorescent protein and IDS enzyme activity. While these cells exhibited a normal growth rate, the BHK‐21 transformant cells stably expressing the human IDS gene persistently generated an active human IDS enzyme extracellularly. The human IDS protein produced failed to be incorporated into the lysosome when cells were pretreated with mannose‐6‐phosphate, demonstrating that this human IDS enzyme has potential for therapeutic use by cross‐correction. These results suggest that our novel RNA vector may be applicable for further clinical settings. [ABSTRACT FROM AUTHOR]

Antiangiogenic therapy using Semliki Forest virus (SFV) carrying Endostatin gene for malignant brain tumor was investigated to improve its therapeutic efficacy. The efficiency of SFV- mediated gene delivery was first evaluated for B16 cells and compared with the efficiency in cells of endothelial origin (HMVECs). HMVECs are more susceptible to SFV infection than B16 cells. For the in vivo treatment model, phosphate-buffered saline, SFV-LacZ, retrovirus vector GCsap-Endostatin, and SFV-Endostatin were injected to mice bearing B16 brain tumors. A very significant inhibition of tumor growth was observed in the group that had been treated with SFV-Endostatin. A marked reduction of intratumoral vascularization was seen in the tumor sections from the SFV-Endostatin group compared with tumor sections from the SFV-LacZ or GCsap-Endostatin groups. Moreover, at day 7 after intravenous administration of SFV-Endostatin, the serum level of endostatin was augmented more than 3-fold compared to that after intravenous administration of GCsap-Endostatin. The results indicated that treatment with SFV-Endostatin inhibited the angiogenesis with established tumors. Gene therapy with Endostatin delivered via SFV may be a candidate for the development of new therapy for brain tumors. [ABSTRACT FROM AUTHOR]

Severe combined immunodeficiency (SCID) caused by adenosine deaminase deficiency (ADA–) is the first genetic disorder to be treated with gene therapy. Since 1990 when the first trial started for 2 patients with ADA– SCID, five clinical trials enrolling 11 patients have been conducted with different clinical approaches and the results obtained from these trials have recently been reported. According to these reports, T cell-directed gene transfer was useful in the treatment of ADA– SCID whereas the retroviral-mediated gene transfer to hematopoietic stem cells was insufficient for achievement of clinical benefits. This chapter reviews several crucial problems inherent in the current retroviral technology based on the clinical data observed in these pioneering ADA gene therapy trials and presents our new retroviral vector system for the next stem cell gene therapy. [ABSTRACT FROM AUTHOR]

The common gamma chain (γ[sub c] ) of cytokine receptors is mutated in X-linked severe combined immunodeficiency, a lethal disorder characterized by the absence of both humoral and cellular immune defenses. Allogeneic bone marrow transplantation from HLA-identical siblings usually results in complete reconstitution of the immune system and is the current treatment of choice. Genetic correction and reinfusion of autologous hematopoietic stem cells represents an alternative therapeutic approach for those patients who lack suitable marrow donors. In this study, we show that retroviral-mediated transfer of the γ[sub c] gene results in efficient expression in CD34+ cells and high transduction rate of colony-forming progenitors. [ABSTRACT FROM AUTHOR]

Donor lymphocytes function as a double-edged sword in allogeneic stem cell transplantation (allo-SCT), that is, they attack leukemic cells (graft versus leukemia-GVL) as well as patient's normal tissues such as liver, intestines, and skin, (graft versus host disease-GVHD). The strong GVL reaction has made an infusion of donor lymphocytes (DLI) an effective treatment for relapsed leukemia after allo-SCT, especially for relapse of chronic myelogenous leukemia. However, the use of DLI is severely limited by the risk of a potential life- threatening complication, GVHD. A phase I/II clinical study of DLI using donor lymphocytes transduced with a retroviral vector carrying the herpes simplex virus thymidine kinase (HSV-TK) gene for patients affected by relapsed leukemia after allo-SCT started at the Tsukuba University Hospital in 2004 as the first clinical trial of leukemia gene therapy in Japan. This is aimed at control of severe GVDH by administration of ganciclovir (GCV) if it occurs while maintaining the maximum GVL effects. So far, we have done eight transduction procedures and infused the HSV-TK transduced donor lymphocytes into 4 patients (2 AML, 1 ALL, and 1 MDS). All patients received about 1×108 cells per kilogram of body weight. In the result, three out of four patients showed good responses such as inhibition of leukemic cell growth and lowering the values of molecular markers after infusions of the transduced cells. One AML patient showed complete remission and no relapse signs are observed on 6 months after the treatment. One patient developed grade III GVHD, and was successfully treated with ganciclovir, in the absence of immunesuppressive drugs. No adverse effects related to gene therapy are observed. Other trials for relapsed leukemia are under way. Details of the present study including additional cases will be presented.Molecular Therapy (2006) 13, S426–S426; doi: 10.1016/j.ymthe.2006.08.1212 [ABSTRACT FROM AUTHOR]

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder caused by steroidogenic enzymes containing monogenetic defects. Most steroidogenic enzymes are cytochrome P450 groups that can be categorized as microsomal P450s, including 21-hydroxylase and 17α-hydroxylase/17,20 lyase, and mitochondrial P450s, including 11β-hydroxylase. It has been shown that ectopic administration of Cyp21a1 ameliorates steroid metabolism in 21-hydroxylase-deficient mice. However, the effectiveness of this approach for mitochondrial P450 has not yet been evaluated. In this study, primary fibroblasts from patients with 21-hydroxylase deficiency (CYP21A2D) (n = 4), 17α-hydroxylase/17,20 lyase deficiency (CYP17A1D) (n = 1), and 11β-hydroxylase deficiency (CYP11B1D) (n = 1) were infected with adeno-associated virus type 2 (AAV2) vectors. Steroidogenic enzymatic activity was not detected in the AAV2-infected CYP11B1D fibroblasts. Induced pluripotent stem cells (iPSCs) of CYP11B1D were established and differentiated into adrenocortical cells by induction of the NR5A1 gene. Adrenocortical cells established from iPSCs of CYP11B1D (CYP11B1D-iPSCs) were infected with an AAV type 9 (AAV9) vector containing CYP11B1 and exhibited 11β-hydroxylase activity. For an in vivo evaluation, we knocked out Cyp11b1 in mice by using the CRISPR/Cas9 method. Direct injection of Cyp11b1-containing AAV9 vectors into the adrenal gland of Cyp11b1-deficient mice significantly reduced serum 11-deoxycorticosterone/corticosterone ratios at 4 weeks after injection and the effect was prolonged for up to 12 months. This study indicated that CYP11B1D could be ameliorated by gene induction in the adrenal glands, which suggests that a defective-enzyme-dependent therapeutic strategy for CAH would be required. Defects in microsomal P450, including CYP21A2D and CYP17A1D, can be treated with extra-adrenal gene induction. However, defects in mitochondrial P450, as represented by CYP11B1D, may require adrenal gene induction. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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 γc chain cDNA and Herpes simplex virus thymidine kinase gene. After confirmation of functional reconstitution of the γc chain, the cells were treated with ganciclovir (GCV). The γc chain positive cells were eliminated under low concentration without cytotoxicity on untransduced cells and have not reappeared at least for 5 months. Furthermore, the γc 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. [Copyright &y& Elsevier]

Objective: apolipoprotein E (apoE) is a key regulator in cholesterol-rich lipoprotein metabolism. Inherited deficiency of this protein results in type III hyperlipoproteinemia in humans. ApoE, especially that derived from macrophages, can efficiently protect against development of atherosclerotic lesion. To use stem cell gene therapy or mini-transplant in treating abnormal lipid metabolism and preventing atherosclerosis, a minimal level of bone marrow chimerism must be determined. Methods: lethally irradiated apoE deficient mice (12–16 weeks of age) fed on normal chow were transplanted with normal bone marrow cells (C57BL/6.Ly5.1) mixed with those of apoE deficient mice (C57BL/6.Ly5.2) at various ratios. Plasma cholesterol levels were determined every 3 weeks for up to 42 weeks. Areas of atherosclerotic lesion in the aortas were quantified 6 months post-transplant. Plasma apoE was measured by Western blot analysis. Results: bone marrow transplantation (BMT) in apoE (−/−) mice resulted in a detectable level of plasma apoE as determined by Western blot analysis. The plasma cholesterol levels in mice with ≧60% chimerism were normalized by 6 weeks post-transplant. Mice with ≦40% chimerism showed significant reductions, but not normalization, in the plasma cholesterol levels even at 42 weeks posttransplant. However, atherosclerotic areas observed in 10%-chimeric mice were significantly smaller than those in control mice (P<0.01). Immunohistochemical studies in 10%-chimeric mice revealed foam cells derived from donor marrow (apoE (+/+)) and expressed immunoreactive apoE in the atherosclerotic lesion. The positive signals by Western blot analysis were represented in the plasma of up to 8% of the chimeric mice. Conclusion: chimerism of 10%, the minimum level analyzed, was sufficient to reduce the severity of atherosclerosis, although the plasma cholesterol levels were not completely normalized. The results indicate that stem cell gene therapy and mini-transplant may provide possible therapeutic approaches to treat patients with abnormal lipid metabolism and atherosclerosis. [Copyright &y& Elsevier]

An abstract of the article "Generalized Detoxification Associated with Engraftment of Gene-Corrected Repopulating Cells Achieved in ADA-SCID Patients by Stem Cell Gene Therapy without Myelopreparative Pre-Conditioning," by Makoto Otsu, Satoru Nakajima and Tadashi Ariga is presented.

In the previous meeting, we reported the initial follow-up of the two patients with adenosine deaminase (ADA)-deficiency whom we had treated in a clinical trial of retroviral-mediated stem cell gene therapy (SCGT). Our protocol is unique in that patients do not receive any kinds of marrow-preparative conditioning prior to infusion of gene-corrected autologous bone marrow (BM) cells. Since the successful cases of ADA gene therapy reported from Italy used low-dose busulfan to enhance the engraftment of gene-transduced BM cells, it is intriguing to see how the lack of conditioning affects the hematological and immunological reconstitution in our patients.Two patients (pt 1, a 4 year-old girl; pt 2, a 13 year-old boy) were given i.v. BM CD34+ cells transduced with the retroviral vector GCsapM-ADA following the cessation of ADA-enzyme (PEG- ADA) replacement. The first evidence of clinical benefits emerged in a form of systemic detoxification as we previously reported. Steady decrease of toxic metabolites, once accumulated shortly after PEG-ADA discontinuation, is still ongoing at present, 2 years after SCGT. Slow, but gradual increase of lymphocyte counts has followed as the second, reaching the recent values that rather surpass the pre- GT levels. T cells have been the most abundant population among the emerging lymphocytes in both patients, exhibiting relatively rapid development. Most striking is the appearance of peripheral B cells in pt 1 after long latency; B cells had never appeared during the first year, but then emerged and has shown a steady increase thereafter. NK cells showed similar reconstitution kinetics, with the evidence of 100% positive for the presence of ADA-transgene. Consistent with these observations, the proviral copy numbers in peripheral blood lymphocytes (PBL) of pt 1 have now reached 0.7 copies/cell and the latest value of ADA enzyme activity in PBL was shown to be near normal. Although pt 2 has shown much slower kinetics of lymphocyte development, he maintains healthy status with the recent evidence of improvement in T cell function.No serious adverse events, including clonal expansion of certain cell types, have been observed. Although some prophylactic measures are still necessary, both patients have been completely off PEG-ADA for longer than 2 years and now are ready for their new lives; pt 1 entering an elementary school and pt 2 proceeding to a high school. These results suggest that SCGT for ADA-deficiency without pre-conditioning can lead to meaningful hematological reconstitution and be a safe and effective treatment option for the patient who needs SCGT, especially when the conditioning may not be tolerated.Molecular Therapy (2006) 13, S418–S418; doi: 10.1016/j.ymthe.2006.08.1190 [ABSTRACT FROM AUTHOR]

X-SCID is characterized by profound defects of humoral and cellular immunity due to mutations in the gene encoding the common gamma chain (γc). Recent clinical trials of X-SCID gene therapy demonstrated the successful immunological reconstitution in the patients. However, a serious adverse effect of uncontrolled clonal T cell proliferation due to insertional mutagenesis of retroviral vector was reported.To offset the side effect, we applied the suicide gene system to X-SCID gene therapy as a fail-safe measure in case uncontrolled proliferation of gene transduced lymphocytes occurred. For this purpose, we constructed a bicistronic retroviral vector, pD-γc/TK, carrying human γc cDNA and HSVtk suicide gene. In the present study, MSCV LTR-based vector was used to overcome gene silencing, because a long-term reduction of the γc expression had been observed under the control of MoMLV LTR-based vector in our previous study. First, we transduced B-cell lines from X-SCID patients with this vector. Expression of the γc was stable for more than six months. After confirmation of functional reconstitution of the γc, the cells were treated with various concentrations of ganciclovir (GCV). The γc positive cells were selectively eliminated under lower concentrations of GCV (<25μM), and have not reappeared at least for 6 months. Furthermore, the γc transduced cells were still sensitive to GCV after six months. These results demonstrate that pD-γc/TK maintains a long-term simultaneous expression of the γc and HSVtk genes.To test the efficacy and safety feature of the gene therapy in vivo, we utilized pD-γc/TK for transduction of X-SCID murine model. Bone marrow cells obtained from X-SCID mice were prestimulated with growth factors including mSCF, mIL-11 and mTPO for 48hr and then transduced with the retroviral supernatant. The transduced cells were harvested and injected into lethally irradiated (800 rad) X-SCID mice. Two months after transduction, peripheral blood cells from the transduced mice were analyzed. Before transduction, the X-SCID mice show a decrease in the absolute number of lymphocytes comprising of a reduced numbers of B cells and CD8+ T cells with a relative accumulation of CD4+ T cells. Transfer of the γc gene resulted in an increase in CD8+T cells and B cells with a substantially corrected CD4:CD8 T-cell ratio. In addition, the γc expression was detected on the populations of these restored lymphocytes. These restored lymphocytes could be eliminated by 50 mg/kg GCV when injected into peritoneal for seven consecutive days. However, this treatment caused acute toxicity to the injected mice. Currently, we are optimizing the concentration of GCV, which could selectively eliminates the gene transduced lymphocytes without obvious toxicity in vivo.Molecular Therapy (2006) 13, S254–S254; doi: 10.1016/j.ymthe.2006.08.736 [ABSTRACT FROM AUTHOR]