使用生物反应器提高I型粘多糖贮积症患者原始造血祖细胞中的基因转移并使酶水平正常化。

Improved gene transfer and normalized enzyme levels in primitive hematopoietic progenitors from patients with mucopolysaccharidosis type I using a bioreactor.

作者信息

Pan Dao, Stroncek David F, Whitley Chester B

机构信息

Gene Therapy Program, Department of Pediatrics, and Institute of Human Genetics, University of Minnesota, Minneapolis, MN, USA.

出版信息

J Gene Med. 2004 Dec;6(12):1293-303. doi: 10.1002/jgm.621.

DOI:10.1002/jgm.621

PMID:15538732

Abstract

BACKGROUND

One of the major barriers to the clinical application of hematopoietic stem cell (HSC) gene therapy has been relatively low gene transfer efficiency. Other inadequacies of current transduction protocols are related to their multi-step procedures, e.g., using tissue-culture flasks, roller bottles or gas-permeable bags for clinical application.

METHODS

In comparison with a conventional bag transduction protocol, a 'closed' hollow-fiber bioreactor system (HBS) was exploited to culture and transduce human peripheral blood CD34(+) progenitor cells (PBPC(MPS)) from patients with mucopolysaccharidosis type I (MPS I) using an amphotropic retroviral vector based on a murine Moloney leukemia virus LN prototype. Both short-term colony-forming cell (CFC) and long-term culture initiating cell (LTCIC) assays were employed to determine transduction frequency and transgene expression in committed progenitor cells and primitive progenitors with multi-lineage potentials.

RESULTS

A novel ultrafiltration-transduction method was established to culture and transduce enzyme-deficient PBPC(MPS) over a 5-day period without loss in viability and CD34 identity (n = 5). Significantly higher transduction efficiencies were achieved in primary CFC that derived from the HBS (5.8-14.2%) in comparison with those from gas-permeable bags (undetectable to 1.7%; p < 0.01). Up to 15-fold higher-than-normal enzyme activity was found in selected PBPC(MPS)-LP1CD transductants. Moreover, higher gene transfer (4.4-fold) and expression in very primitive progenitors were observed in products from the HBS compared with bag experiments as indicated by CFC derived from primitive LTCIC. Remarkably, with relatively modest gene transfer levels in LTCIC from HBS experiments, the expression of the IDUA transgene corrected the enzyme-deficiency in 5-week long-term cultures (LTC).

CONCLUSIONS

MPS I progenitor cells achieved normalized enzyme levels in LTC after transduction in a HBS system. These studies demonstrate the advantages of a bioreactor-transduction system for viral-mediated stem cell gene transfer.

摘要

背景

造血干细胞（HSC）基因治疗临床应用的主要障碍之一是基因转移效率相对较低。当前转导方案的其他不足之处与其多步骤程序有关，例如，在临床应用中使用组织培养瓶、滚瓶或透气袋。

方法

与传统的袋装转导方案相比，利用一种“封闭”的中空纤维生物反应器系统（HBS），使用基于鼠莫洛尼白血病病毒LN原型的双嗜性逆转录病毒载体，培养并转导来自I型黏多糖贮积症（MPS I）患者的人外周血CD34(+)祖细胞（PBPC(MPS)）。采用短期集落形成细胞（CFC）和长期培养起始细胞（LTCIC）测定法，以确定定向祖细胞和具有多谱系潜能的原始祖细胞中的转导频率和转基因表达。

结果

建立了一种新型超滤转导方法，在5天时间内培养并转导酶缺陷型PBPC(MPS)，且活力和CD34身份均未丧失（n = 5）。与透气袋来源的原代CFC（未检测到至1.7%）相比，HBS来源的原代CFC实现了显著更高的转导效率（5.8 - 14.2%；p < 0.01）。在选定的PBPC(MPS)-LP1CD转导子中发现酶活性比正常水平高15倍。此外，如来自原始LTCIC的CFC所示，与袋装实验相比，HBS产物中在非常原始的祖细胞中观察到更高的基因转移（4.4倍）和表达。值得注意的是，在HBS实验的LTCIC中基因转移水平相对适度的情况下，IDUA转基因的表达在5周的长期培养（LTC）中纠正了酶缺陷。