RESUMO
Although gene transfer to hematopoietic stem cells (HSCs) has shown therapeutic efficacy in recent trials for several individuals with inherited disorders, transduction incompleteness of the HSC population remains a hurdle to yield a cure for all patients with reasonably low integrated vector numbers. In previous attempts at HSC selection, massive loss of transduced HSCs, contamination with non-transduced cells, or lack of applicability to large cell populations has rendered the procedures out of reach for human applications. Here, we fused codon-optimized puromycin N-acetyltransferase to herpes simplex virus thymidine kinase. When expressed from a ubiquitous promoter within a complex lentiviral vector comprising the ßAT87Q-globin gene, viral titers and therapeutic gene expression were maintained at effective levels. Complete selection and preservation of transduced HSCs were achieved after brief exposure to puromycin in the presence of MDR1 blocking agents, suggesting the procedure's suitability for human clinical applications while affording the additional safety of conditional suicide.
Assuntos
Terapia Genética , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Hemoglobinopatias/genética , Hemoglobinopatias/terapia , Transdução Genética , Globinas beta/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Modelos Animais de Doenças , Expressão Gênica , Ordem dos Genes , Genes Transgênicos Suicidas , Terapia Genética/métodos , Vetores Genéticos/genética , Humanos , Lentivirus/genética , Camundongos , Camundongos Transgênicos , TransgenesRESUMO
Recent marketing approval for genetically engineered hematopoietic stem and T cells bears witness to the substantial improvements in lentiviral vectors over the last two decades, but evaluations of the long-term efficacy and toxicity of gene and cell therapy products will, nevertheless, require further studies in nonhuman primate models. Macaca fascicularis monkeys from Mauritius have a low genetic diversity and are particularly useful for reproducible drug testing. In particular, they have a genetically homogeneous class I major histocompatibility complex system that probably mitigates the variability of the response to simian immunodeficiency virus infection. However, the transduction of simian cells with human immunodeficiency virus type 1 (HIV-1)-derived vectors is inefficient due to capsid-specific restriction factors, such as the tripartite motif-containing protein tripartite motif 5α, which prevent infection with non-host-adapted retroviruses. This study introduced the modified capsid of the macaque-trophic HIV-1 clone MN4/LSQD into the packaging system and compared transduction efficiencies between hematopoietic cells transduced with this construct and cells transduced with HIV-1 NL4-3-derived packaging constructs. Capsid modification increased transduction efficiency in all hematopoietic cells tested (by factors of up to 10), including hematopoietic progenitor cells, repopulating cells, and T cells from Mauritian Macaca fascicularis, regardless of vector structure or purification method. The study also established culture conditions similar to those used in clinical practice for the efficient transduction of hematopoietic stem and progenitor CD34+ cells. These results suggest that the procedure is suitable for use in Mauritian Macaca fascicularis, which can therefore be used as a model in preclinical studies for hematopoietic gene and cell therapy.
Assuntos
Capsídeo/imunologia , Vetores Genéticos/metabolismo , HIV-1/imunologia , Células-Tronco Hematopoéticas/imunologia , Macaca fascicularis/imunologia , Transdução Genética/métodos , Animais , Antígenos CD34/genética , Antígenos CD34/imunologia , Biomarcadores/metabolismo , Capsídeo/química , Feminino , Expressão Gênica , Vetores Genéticos/imunologia , HIV-1/genética , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/virologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Linfócitos T/imunologia , Linfócitos T/transplante , Linfócitos T/virologia , Transplante Heterólogo , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/imunologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/imunologiaRESUMO
A previously published clinical trial demonstrated the benefit of autologous CD34(+) cells transduced with a selfinactivating lentiviral vector (HPV569) containing an engineered ß-globin gene (ß(A-T87Q)-globin) in a subject with ß thalassemia major. This vector has been modified to increase transduction efficacy without compromising safety. In vitro analyses indicated that the changes resulted in both increased vector titers (3 to 4 fold) and increased transduction efficacy (2 to 3 fold). An in vivo study in which 58 ß-thalassemic mice were transplanted with vector- or mock-transduced syngenic bone marrow cells indicated sustained therapeutic efficacy. Secondary transplantations involving 108 recipients were performed to evaluate long-term safety. The six month study showed no hematological or biochemical toxicity. Integration site (IS) profile revealed an oligo/polyclonal hematopoietic reconstitution in the primary transplants and reduced clonality in secondary transplants. Tumor cells were detected in the secondary transplant mice in all treatment groups (including the control group), without statistical differences in the tumor incidence. Immunohistochemistry and quantitative PCR demonstrated that tumor cells were not derived from transduced donor cells. This comprehensive efficacy and safety data provided the basis for initiating two clinical trials with this second generation vector (BB305) in Europe and in the USA in patients with ß-thalassemia major and sickle cell disease.