Hematopoietic Tumors in a Mouse Model of X-linked Chronic Granulomatous Disease after Lentiviral Vector-Mediated Gene Therapy

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Connexin 43-Enhanced Suicide Gene Therapy Using Herpesviral Vectors

Tumor cell transduction with the herpes simplex virus (HSV) thymidine kinase (tk) gene and treatment with ganciclovir (GCV) is a widely studied cancer gene therapy. Connexin (Cx)-dependent gap junctions between cells facilitate the intercellular spread of TK-activated GCV, thereby creating a bystander effect that improves tumor cell killing. However, tumor cells often have reduced connexin expression, thus thwarting bystander killing and the effectiveness of TK/GCV gene therapy. To improve the effectiveness of this therapy, we compared an HSV vector (TOCX) expressing Cx43 in addition to TK with an isogenic tk vector (TOZ.1) for their abilities to induce bystander killing of Cx-positive U-87 MG human glioblastoma cells and Cx-negative L929 fibrosarcoma cells in vitro and in vivo. The results showed that low-multiplicity infection of U-87 MG cells with TOCX only minimally increased GCV-mediated cell death compared with infection by TOZ.1, consistent with the endogenous level of Cx in these cells. In contrast, bystander killing of L929 cells was markedly enhanced by vector-mediated expression of Cx. In vivo experiments in which U-87 MG cells were preinfected at low multiplicity and injected into the flanks of nude mice showed complete cures of all animals in the TOCX group following GCV treatment, whereas untreated animals uniformly formed fatal tumors. TOCX injection into U-87 MG intradermal and intracranial tumors resulted in prolonged survival of the host animals in a GCV-dependent manner. Together, these results suggest that the combination of TK and Cx may be beneficial for the treatment of human glioblastoma.     

Lentiviral Gene Therapy Using Cellular Promoters Cures Type 1 Gaucher Disease in Mice

Gaucher disease is caused by an inherited deficiency of the enzyme glucosylceramidase. Due to the lack of a fully functional enzyme, there is progressive build-up of the lipid component glucosylceramide. Insufficient glucosylceramidase activity results in hepatosplenomegaly, cytopenias, and bone disease in patients. Gene therapy represents a future therapeutic option for patients unresponsive to enzyme replacement therapy and lacking a suitable bone marrow donor. By proof-of-principle experiments, we have previously demonstrated a reversal of symptoms in a murine disease model of type 1 Gaucher disease, using gammaretroviral vectors harboring strong viral promoters to drive glucosidase β-acid (GBA) gene expression. To investigate whether safer vectors can correct the enzyme deficiency, we utilized self-inactivating lentiviral vectors (SIN LVs) with the GBA gene under the control of human phosphoglycerate kinase (PGK) and CD68 promoter, respectively. Here, we report prevention of, as well as reversal of, manifest disease symptoms after lentiviral gene transfer. Glucosylceramidase activity above levels required for clearance of glucosylceramide from tissues resulted in reversal of splenomegaly, reduced Gaucher cell infiltration and a restoration of hematological parameters. These findings support the use of SIN-LVs with cellular promoters in future clinical gene therapy protocols for type 1 Gaucher disease.     

Long-Term Follow-up of Foamy Viral Vector-Mediated Gene Therapy for Canine Leukocyte Adhesion Deficiency

The development of leukemia following gammaretroviral vector-mediated gene therapy for X-linked severe combined immunodeficiency disease and chronic granulomatous disease (CGD) has emphasized the need for long-term follow-up in animals treated with hematopoietic stem cell gene therapy. In this study, we report the long-term follow-up (4–7 years) of four dogs with canine leukocyte adhesion deficiency (CLAD) treated with foamy viral (FV) vector-mediated gene therapy. All four CLAD dogs previously received nonmyeloablative conditioning with 200 cGy total body irradiation followed by infusion of autologous, CD34⁺ hematopoietic stem cells transduced by a FV vector expressing canine CD18 from an internal Murine Stem Cell Virus (MSCV) promoter. CD18⁺ leukocyte levels were >2% following infusion of vector-transduced cells leading to ongoing reversal of the CLAD phenotype for >4 years. There was no clinical development of lymphoid or myeloid leukemia in any of the four dogs and integration site analysis did not reveal insertional oncogenesis. These results showing disease correction/amelioration of disease in CLAD without significant adverse events provide support for the use of a FV vector to treat children with leukocyte adhesion deficiency type 1 (LAD-1) in a human gene therapy clinical trial.     

与放疗联合的自杀基因治疗的现状

1 自杀基因的概念 目前国际上已有200多个基因治疗项目应用于临床,其中恶性肿瘤的基因治疗占了2/3,而自杀基因治疗又占恶性肿瘤基因治疗的1/5以上。自杀基因治疗已由最早的用于治疗卵巢癌发展到对多种肿瘤和疾病的治疗,如:脑胶质瘤、恶性黑色素瘤、头颈部鳞癌、结直肠癌及骨髓移植等。自杀基因治疗     

Correction of Murine SCID-X1 by Lentiviral Gene Therapy Using a Codon-optimized IL2RG Gene and Minimal Pretransplant Conditioning

Clinical trials have demonstrated the potential of ex vivo hematopoietic stem cell gene therapy to treat X-linked severe combined immunodeficiency (SCID-X1) using γ-retroviral vectors, leading to immune system functionality in the majority of treated patients without pretransplant conditioning. The success was tempered by insertional oncogenesis in a proportion of the patients. To reduce the genotoxicity risk, a self-inactivating (SIN) lentiviral vector (LV) with improved expression of a codon optimized human interleukin-2 receptor γ gene (IL2RG) cDNA (coγc), regulated by its 1.1 kb promoter region (γcPr), was compared in efficacy to the viral spleen focus forming virus (SF) and the cellular phosphoglycerate kinase (PGK) promoters. Pretransplant conditioning of Il2rg^−/− mice resulted in long-term reconstitution of T and B lymphocytes, normalized natural antibody titers, humoral immune responses, ConA/IL-2 stimulated spleen cell proliferation, and polyclonal T-cell receptor gene rearrangements with a clear integration preference of the SF vector for proto-oncogenes, contrary to the PGK and γcPr vectors. We conclude that SIN lentiviral gene therapy using coγc driven by the γcPr or PGK promoter corrects the SCID phenotype, potentially with an improved safety profile, and that low-dose conditioning proved essential for immune competence, allowing for a reduced threshold of cell numbers required.     

Correction of Murine Rag2 Severe Combined Immunodeficiency by Lentiviral Gene Therapy Using a Codon-optimized RAG2 Therapeutic Transgene

Recombination activating gene 2 (RAG2) deficiency results in severe combined immunodeficiency (SCID) with complete lack of T and B lymphocytes. Initial gammaretroviral gene therapy trials for other types of SCID proved effective, but also revealed the necessity of safe vector design. We report the development of lentiviral vectors with the spleen focus forming virus (SF) promoter driving codon-optimized human RAG2 (RAG2co), which improved phenotype amelioration compared to native RAG2 in Rag2^−/− mice. With the RAG2co therapeutic transgene, T-cell receptor (TCR) and immunoglobulin repertoire, T-cell mitogen responses, plasma immunoglobulin levels and T-cell dependent and independent specific antibody responses were restored. However, the thymus double positive T-cell population remained subnormal, possibly due to the SF virus derived element being sensitive to methylation/silencing in the thymus, which was prevented by replacing the SF promoter by the previously reported silencing resistant element (ubiquitous chromatin opening element (UCOE)), and also improved B-cell reconstitution to eventually near normal levels. Weak cellular promoters were effective in T-cell reconstitution, but deficient in B-cell reconstitution. We conclude that immune functions are corrected in Rag2^−/− mice by genetic modification of stem cells using the UCOE driven codon-optimized RAG2, providing a valid optional vector for clinical implementation.     

慢病毒载体的改进及其在血液病基因治疗中的应用

慢病毒载体（lentiviral vector，LV)具有可感染非分裂细胞、转移基因片段容量较大、目的基因表达时间长、不易诱发宿主免疫反应等优点，成为目前较有前途的病毒载体。本以人类免疫缺陷病毒I型(HIV—I)为代表，综述对LV的改进及其在血液病基因治疗转移基因中的应用。     

An Inducible Caspase-9 Suicide Gene to Improve the Safety of Therapy Using Human Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (hiPSC) hold promise for regenerative therapies, though there are several safety concerns including the risk of oncogenic transformation or unwanted adverse effects associated with hiPSC or their differentiated progeny. Introduction of the inducible caspase-9 (iC9) suicide gene, which is activated by a specific chemical inducer of dimerization (CID), is one of the most appealing safety strategies for cell therapies and is currently being tested in multicenter clinical trials. Here, we show that the iC9 suicide gene with a human EF1α promoter can be introduced into hiPSC by lentiviral transduction. The transduced hiPSC maintain their pluripotency, including their capacity for unlimited self-renewal and the potential to differentiate into three germ layer tissues. Transduced hiPSC are eliminated within 24 hours of exposure to pharmacological levels of CID in vitro, with induction of apoptosis in 94–99% of the cells. Importantly, the iC9 suicide gene can eradicate tumors derived from hiPSC in vivo. In conclusion, we have developed a direct and efficient hiPSC killing system that provides a necessary safety mechanism for therapies using hiPSC. We believe that our iC9 suicide gene will be of value in clinical applications of hiPSC-based therapy.