Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA.
Proc Natl Acad Sci U S A. 2009 Nov 24;106(47):19958-63. doi: 10.1073/pnas.0908528106. Epub 2009 Nov 10.
Restricting transgene expression to maturing erythroid cells can reduce the risk for activating oncogenes in hematopoietic stem cells (HSCs) and their progeny, yet take advantage of their robust protein synthesis machinery for high-level protein production. This study sought to evaluate the feasibility and efficacy of reprogramming erythroid cells for production of a lysosomal enzyme, alpha-L-iduronidase (IDUA). An erythroid-specific hybrid promoter provided inducible IDUA expression and release during in vitro erythroid differentiation in murine erythroleukemia cells, resulting in phenotypical cross-correction in an enzyme-deficient lymphoblastoid cell line derived from patients with mucopolysaccharidosis type I (MPS I). Stable and higher than normal plasma IDUA levels were achieved in vivo in primary and secondary MPS I chimeras for at least 9 months after transplantation of HSCs transduced with the erythroid-specific IDUA-containing lentiviral vector (LV). Moreover, long-term metabolic correction was demonstrated by normalized urinary glycosaminoglycan accumulation in all treated MPS I mice. Complete normalization of tissue pathology was observed in heart, liver, and spleen. Notably, neurological function and brain pathology were significantly improved in MPS I mice by erythroid-derived, higher than normal peripheral IDUA protein. These data demonstrate that late-stage erythroid cells, transduced with a tissue-specific LV, can deliver a lysosomal enzyme continuously at supraphysiological levels to the bloodstream and can correct the disease phenotype in both viscera and CNS of MPS I mice. This approach provides a paradigm for the utilization of RBC precursors as a depot for efficient and potentially safer systemic delivery of nonsecreted proteins by ex vivo HSC gene transfer.
将转基因表达限制在成熟的红细胞中可以降低激活造血干细胞（HSCs）及其后代中癌基因的风险，同时利用其强大的蛋白质合成机制进行高水平的蛋白质生产。本研究旨在评估对红细胞进行重编程以生产溶酶体酶α-L-艾杜糖苷酸酶（IDUA）的可行性和效果。在鼠红白血病细胞的体外红细胞分化过程中，一个红细胞特异性杂交启动子提供了诱导型 IDUA 表达和释放，导致在来自黏多糖贮积症 I 型（MPS I）患者的酶缺陷淋巴母细胞系中出现表型交叉校正。在 HSCs 转导了含有红细胞特异性 IDUA 的慢病毒载体（LV）后，在原发性和继发性 MPS I 嵌合体中，体内稳定且高于正常的血浆 IDUA 水平至少持续 9 个月。此外，通过所有接受治疗的 MPS I 小鼠的尿液糖胺聚糖积累正常化，证明了长期的代谢纠正。在心脏、肝脏和脾脏中观察到组织病理学的完全正常化。值得注意的是，通过红细胞衍生的、高于正常的外周 IDUA 蛋白，MPS I 小鼠的神经功能和脑病理学得到了显著改善。这些数据表明，经组织特异性 LV 转导的晚期红细胞可以持续以超生理水平将溶酶体酶递送至血液中，并可以纠正 MPS I 小鼠内脏和中枢神经系统的疾病表型。这种方法为利用 RBC 前体作为储存库，通过体外 HSC 基因转移，高效且潜在更安全地递送至全身的非分泌性蛋白提供了范例。
