RESUMO
BACKGROUND: X-linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the common cytokine-receptor gamma chain (gamma(c)), resulting in disruption of development of T lymphocytes and natural-killer cells. B-lymphocyte function is also intrinsically compromised. Allogeneic bone-marrow transplantation is successful if HLA-matched family donors are available, but HLA-mismatched procedures are associated with substantial morbidity and mortality. We investigated the application of somatic gene therapy by use of a gibbon-ape-leukaemia-virus pseudotyped gammaretroviral vector. METHODS: Four children with SCID-X1 were enrolled. Autologous CD34-positive haemopoietic bone-marrow stem cells were transduced ex vivo and returned to the patients without preceding cytoreductive chemotherapy. The patients were monitored for integration and expression of the gamma(c) vector and for functional immunological recovery. FINDINGS: All patients have shown substantial improvements in clinical and immunological features, and prophylactic medication could be withdrawn in two. No serious adverse events have been recorded. T cells responded normally to mitogenic and antigenic stimuli, and the T-cell-receptor (TCR) repertoire was highly diverse. Where assessable, humoral immunity, in terms of antibody production, was also restored and associated with increasing rates of somatic mutation in immunoglobulin genes. INTERPRETATION: Gene therapy for SCID-X1 is a highly effective strategy for restoration of functional cellular and humoral immunity.
Assuntos
Doenças Genéticas Ligadas ao Cromossomo X/terapia , Terapia Genética , Imunodeficiência Combinada Severa/terapia , Antígenos CD34/análise , Células da Medula Óssea/imunologia , Transplante de Medula Óssea , Pré-Escolar , Gammaretrovirus , Técnicas de Transferência de Genes , Doenças Genéticas Ligadas ao Cromossomo X/genética , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Vetores Genéticos , Humanos , Imunidade , Imunoglobulinas/sangue , Lactente , Subunidade gama Comum de Receptores de Interleucina , Ativação Linfocitária , Teste de Cultura Mista de Linfócitos , Mutação , Receptores de Interleucina-7/genética , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/imunologia , Linfócitos T/imunologia , Transdução GenéticaRESUMO
BACKGROUND: Retroviral vectors possess many advantages for use in gene therapy protocols, especially within the haematopoietic system. A number of attendant problems, however, still limit their safety in clinical application. The effects of the enhancer present in the retroviral long terminal repeat (LTR) are a major concern for the clinical usage of such vectors, as they can exert a powerful regulatory influence on the genes that surround them. METHODS: To improve the safety and widen the applicability of retroviral vectors for use in gene therapy we have developed an enhancer-deleted (Delta-LTR) retroviral vector that retained high titre and demonstrated transcriptional activity in myeloid cells. RESULTS: When used to correct a mouse model of autosomal recessive chronic granulomatous disease, the Delta-LTR vectors gave acceptable levels of gene transfer to mouse bone marrow cells. Evidence for a slight preferential expression in myeloid cells was obtained with all the vectors studied. Nitroblue tetrazolium assay of superoxide generation in mouse bone marrow derived haematopoietic colonies revealed that transduction with Delta-LTR vectors could restore functional NADPH oxidase to cells from these animals. Superoxide assay of peripheral blood confirmed that, although relatively low numbers of cells were transduced, the Delta-LTR vector was capable of reconstituting very high levels of oxidase activity, comparable to that obtained from normal cells. CONCLUSIONS: The Delta-LTR vector described here could provide the basis for a new generation of retroviral vectors with improved safety.
Assuntos
Elementos Facilitadores Genéticos , Terapia Genética/métodos , Vetores Genéticos/genética , Doença Granulomatosa Crônica/terapia , Superóxidos/metabolismo , Animais , Medula Óssea/virologia , Modelos Animais de Doenças , Citometria de Fluxo/métodos , Expressão Gênica , Doença Granulomatosa Crônica/genética , Doença Granulomatosa Crônica/metabolismo , Humanos , Camundongos , Camundongos Knockout , NADPH Oxidases , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Retroviridae/genética , Deleção de Sequência , Superóxidos/sangue , Sequências Repetidas TerminaisRESUMO
Prolonged exposure of human hematopoietic stem cells (HSC) to growth factors for efficient transduction by murine oncoretroviral vectors has major detrimental effects on repopulating activity. In this study, we have used a vesicular stomatitis virus G envelope protein (VSV-G)-pseudotyped human immunodeficiency virus type 1 (HIV-1) lentiviral-based vector system to transduce cord blood (CB) CD34+ cells over a limited time period (< or =24 hours). Under these conditions, significant gene marking was observed in engrafted human lymphoid, myeloid, and progenitor cells in all transplanted Severe Combined Immunodeficient (SCID) mice. To enhance the level of gene expression in hematopoietic cells, we also generated a series of lentiviral vectors incorporating the spleen focus forming virus (SFFV) long terminal repeat (LTR) sequences, and the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). By including the central polypurine tract (cPPT) sequence of HIV-1 we were then able to achieve high levels of transduction (over 80%) and gene expression in vivo after a single exposure to viral supernatant. These results demonstrate that lentiviral vectors are highly effective for gene transfer to human HSC, and that SFFV regulatory sequences can be successfully incorporated to enhance the long-term expression of a transgene in primary human hematopoietic cells in vivo.