Moreau T, Barlogis V, Bardin F, Nunes J A, Calmels B, Chabannon C, Tonnelle C
Institut Paoli-Calmettes, Centre de Thérapie Cellulaire et Génique, Centre Régional de Lutte Contre le Cancer, Marseille, France.
Gene Ther. 2008 Jun;15(12):942-52. doi: 10.1038/gt.2008.17. Epub 2008 Mar 6.
Further development of haematopoietic stem cell (HSC) gene therapy will depend on enhancement of gene transfer safety: ad hoc improvement of vector design relating to each particular disease is thus a crucial issue for HSC gene therapy. We modified a previously described lentiviral vector by adding the Emumar B-specific enhancer to a human CD19 promoter-derived sequence (Mol Ther 2004;10:45-56). We thus significantly improved the level of expression of the green fluorescent protein (GFP) reporter gene while retaining the specificity of expression in B-cell progeny of transduced human CD34+ progenitor cells obtained from cord blood or adult bone marrow. Indeed, GFP was strongly expressed from early medullary pro-B cells to splenic mature B cells whereas transgene expression remained low in transduced immature progenitors as in myeloid and T-lymphoid progeny retrieved from xenografted NOD/SCID/gammac(null) mice. Using this lentiviral vector, we further demonstrated the possibility to express a functional human BTK protein in long-term human CD34+ cell B-lymphoid progeny. This newly designed lentiviral vector fulfils one of the pre-requisites for the development of efficient and safe gene therapy for X-linked agammaglobulinaemia, the most common primary humoral immunodeficiency disorder.
造血干细胞（HSC）基因治疗的进一步发展将取决于基因转移安全性的提高：因此，针对每种特定疾病对载体设计进行特别改进是HSC基因治疗的关键问题。我们通过将Emumar B特异性增强子添加到源自人CD19启动子的序列中，对先前描述的慢病毒载体进行了修饰（《分子治疗》2004年；10：45 - 56）。我们因此显著提高了绿色荧光蛋白（GFP）报告基因的表达水平，同时保留了在从脐带血或成人骨髓获得的转导人CD34 +祖细胞的B细胞后代中的表达特异性。实际上，从早期骨髓前B细胞到脾成熟B细胞，GFP都有强烈表达，而在转导的未成熟祖细胞中，转基因表达仍然很低，就像从异种移植的NOD/SCID/γc（null）小鼠中回收的髓系和T淋巴细胞后代一样。使用这种慢病毒载体，我们进一步证明了在长期人CD34 +细胞B淋巴细胞后代中表达功能性人BTK蛋白的可能性。这种新设计的慢病毒载体满足了针对最常见的原发性体液免疫缺陷疾病——X连锁无丙种球蛋白血症开发高效安全基因治疗的先决条件之一。
