Nolta J A, Yu X J, Bahner I, Kohn D B
Division of Research Immunology/Bone Marrow Transplantation, Children's Hospital of Los Angeles, California 90027.
J Clin Invest. 1992 Aug;90(2):342-8. doi: 10.1172/JCI115868.
Gaucher disease, a lysosomal glycolipid storage disorder, results from the genetic deficiency of an acidic glucosidase, glucocerebrosidase (GC). The beneficial effects of allogeneic bone marrow transplantation (BMT) for Gaucher disease suggest that GC gene transduction and the transplantation of autologous hematopoietic stem cells (gene therapy) may similarly alleviate symptoms. We have constructed a retroviral vector, L-GC, produced by a clone of the amphotropic packaging cell line PA317, which transduces the normal human GC cDNA with high efficiency. Whole-marrow mononuclear cells and CD34-enriched cells from a 4-yr-old female with type 3 Gaucher disease were transduced by the L-GC vector and studied in long-term bone marrow culture (LTBMC). Prestimulation of marrow with IL-3 and IL-6, followed by co-cultivation with vector-producing fibroblasts, produced gene transfer into 40-45% of the hematopoietic progenitor cells. The levels of GC expression in progeny cells (primarily mature myelomonocytic) produced by the LTBMC were quantitatively analyzed by Northern blot, Western blot, and glucocerebrosidase enzyme assay. Normal levels of GC RNA, immunoreactive protein, and enzymatic activity were detected throughout the duration of culture. These studies demonstrate that retroviral vectors can efficiently transfer the GC gene into long-lived hematopoietic progenitor cells from the bone marrow of patients with Gaucher disease and express physiologically relevant levels of GC enzyme activity.
戈谢病是一种溶酶体糖脂贮积症，由酸性葡糖苷酶——葡萄糖脑苷脂酶（GC）的基因缺陷引起。同种异体骨髓移植（BMT）对戈谢病具有有益效果，这表明GC基因转导和自体造血干细胞移植（基因治疗）可能同样能缓解症状。我们构建了一种逆转录病毒载体L-GC，它由嗜性包装细胞系PA317的一个克隆产生，能高效转导正常人GC cDNA。用L-GC载体转导一名4岁3型戈谢病女性患者的全骨髓单个核细胞和富集CD34的细胞，并在长期骨髓培养（LTBMC）中进行研究。先用白细胞介素-3和白细胞介素-6对骨髓进行预刺激，然后与产生载体的成纤维细胞共培养，使基因转移到40% - 45%的造血祖细胞中。通过Northern印迹、Western印迹和葡萄糖脑苷脂酶活性测定对LTBMC产生的子代细胞（主要是成熟骨髓单核细胞）中GC的表达水平进行定量分析。在整个培养期间均检测到GC RNA、免疫反应性蛋白和酶活性的正常水平。这些研究表明，逆转录病毒载体能有效地将GC基因转移到戈谢病患者骨髓中长寿的造血祖细胞中，并表达具有生理相关性的GC酶活性水平。
