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Evaluation of protective and adverse effects of gene transferred IPE cell transplantation on degenerative retinal diseases

基因转移IPE细胞移植对退行性视网膜疾病的保护作用和不良作用评价

基本信息

批准号：
15390524
负责人：
TAMAI Makoto
金额：
$ 8.19万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

We have been trying a clinical approach to the patients with AMD by transplantation of autologous cultured IPE cells. More than 80% of the patients showed an improvement of visual acuity after the transplantation. However the best visual acuity in these patients was still far from sufficient effect. These reports suggested that there is a limitation in the use of autologous IPE cells for transplantation. We investigated the stable and safety gene transduction to IPE cells. The purpose of this study is to demonstrate the effect of the neurotrophic factors-transduced cells and safety of vector to use gene transfer. The stability of transgene expression in the retina was needed for gene therapy. One of methods to transduce a gene into relevant cells is to use virus vectors. Now we can use various type of virus vectors. The recombinant adeno-associated virus (rAAV) has proven to be an efficient and effective vector. We studied the transduction efficiency to IPE cells by using 5 types of AAV serotypes. These results revealed that serotype 2 (rAAV2) is the most effective vector for gene therapy in IPE cells. But the trasduction efficiency was less than 20%. To improve this problem, we studied the manner of virus infection and revealed that the pre-treatment with tyrphostin-1 (epidermal growth factor receptor tyrosine kinase inhibitor) led to increase of AAV2-transduction. The neuroprotective effect of AAV BDNF transduced IPE cells (BDNF-IPE) on the retinal ganglion cells (RGCs) were assessed. In vitro, BDNF deprivation RGC death was significantly suppressed by HUSB-Tyr treated-BDNF-IPE. In vivo model of photoreceptor degeneration caused by continuous light exposure, transplantation of HUSB-Tyr-treated-BDNF-IPE inhibited photoreceptor degeneration.

我们一直在尝试通过移植自体培养的IPE细胞来治疗AMD患者的临床方法。超过80%的患者术后视力有改善。然而，这些患者的最佳视力仍远未达到足够的效果。这些报道表明，自体IPE细胞用于移植是有局限性的。我们研究了对IPE细胞的稳定和安全的基因转导。本研究的目的是验证神经营养因子转导细胞的作用和载体使用基因转移的安全性。转基因在视网膜中的稳定表达是基因治疗所必需的。将基因转导到相关细胞的方法之一是使用病毒载体。现在我们可以使用各种类型的病毒载体。重组腺相关病毒(RAAV)是一种高效的载体。我们用5种AAV血清型研究了其对IPE细胞的转导效率。这些结果表明，血清型2(RAAV2)是IPE细胞基因治疗的最有效载体。但转化率不到20%。为了改善这一问题，我们研究了病毒感染的方式，发现用表皮生长因子受体酪氨酸激酶抑制剂(tyrphostin-1)预处理会导致AAV2转导增加。观察AAV BDNF转导的IPE细胞对视网膜神经节细胞(RGCs)的神经保护作用。在体外，经HUSB-Tyr处理的BDNF-IPE可显著抑制剥夺BDNF的RGC死亡。在体光感受器变性模型中，移植HUSB-Tyr处理的BDNF-IPE抑制了光感受器退化。