Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229.
Proc Natl Acad Sci U S A. 2014 Feb 18;111(7):2680-5. doi: 10.1073/pnas.1323155111. Epub 2014 Feb 3.
Use of megakaryocytes/platelets for transgene expression may take advantage of their rapid turnover and protective storage in platelets and reduce the risk of activating oncogenes in hematopoietic stem and progenitor cells (HSCs). Here, we show that human megakaryocytic cells could overexpress the lysosomal enzyme, α-l-iduronidase (IDUA), which is deficient in patients with mucopolysaccharidosis type I (MPS I). Upon megakaryocytic differentiation, the amount of released enzyme increased rapidly and steadily by 30-fold. Using a murine MPS I model, we demonstrated that megakaryocyte/platelets were capable of producing, packaging, and storing large amounts of IDUA with proper catalytic activity, lysosomal trafficking, and receptor-mediated uptake. IDUA can be released directly into extracellular space or within microparticles during megakaryocyte maturation or platelet activation, while retaining the capacity for cross-correction in patient's cells. Gene transfer into 1.7% of HSCs led to long-term normalization of plasma IDUA and preferential distribution of enzyme in liver and spleen with complete metabolic correction in MPS I mice. Detection of GFP (coexpressed with IDUA) in Kupffer cells and hepatocytes suggested liver delivery of platelet-derived IDUA possibly via the clearance pathway for senile platelets. These findings provide proof of concept that cells from megakaryocytic lineage and platelets are capable of generating and storing fully functional lysosomal enzymes and can also lead to efficient delivery of both the enzymes released into the circulation and those protected within platelets/microparticles. This study opens a door for use of the megakaryocytes/platelets as a depot for efficient production, delivery, and effective tissue distribution of lysosomal enzymes.
利用巨核细胞/血小板进行转基因表达可能利用了它们在血小板中的快速周转和保护性储存，并降低了激活造血干细胞和祖细胞（HSCs）中癌基因的风险。在这里，我们表明，人类巨核细胞可以过度表达溶酶体酶α-L-艾杜糖苷酸酶（IDUA），这种酶在粘多糖贮积症 I 型（MPS I）患者中缺乏。在巨核细胞分化过程中，释放的酶的数量迅速且稳定地增加了 30 倍。使用 MPS I 小鼠模型，我们证明了巨核细胞/血小板能够产生、包装和储存大量具有适当催化活性、溶酶体运输和受体介导摄取的 IDUA。IDUA 可以在巨核细胞成熟或血小板激活过程中直接释放到细胞外空间或微颗粒中，同时保留在患者细胞中进行交叉校正的能力。将基因转移到 1.7%的 HSCs 中，可导致 MPS I 小鼠的血浆 IDUA 长期正常化，并优先分布在肝脏和脾脏中，实现完全的代谢校正。在库普弗细胞和肝细胞中检测到 GFP（与 IDUA 共表达），提示血小板衍生的 IDUA 可能通过衰老血小板的清除途径递送至肝脏。这些发现为巨核细胞谱系和血小板能够产生和储存完全功能性的溶酶体酶的概念提供了证据，并且还可以导致释放到循环中的酶和受血小板/微颗粒保护的酶的有效递送至组织。这项研究为利用巨核细胞/血小板作为高效生产、递送至组织以及有效分配溶酶体酶的储存库开辟了道路。
