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

我们一直在尝试用自体IPE细胞移植治疗黄斑变性的临床方法。超过80%的患者在移植后视力得到改善。然而，这些患者的最佳视力仍远未达到足够的效果。这些报告表明，自体IPE细胞用于移植有一定的局限性。我们研究了基因在IPE细胞中的稳定和安全转导。本研究的目的是证明神经营养因子转导细胞用于基因转移的效果和载体的安全性。基因治疗需要视网膜中转基因表达的稳定性。将基因导入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）是一种高效的病毒载体。我们用5种AAV血清型研究了其对IPE细胞的转导效率。结果表明血清型2 （rAAV2）是IPE细胞中最有效的基因治疗载体。但感应效率低于20%。为了改善这一问题，我们研究了病毒感染的方式，发现表皮生长因子受体酪氨酸激酶抑制剂tyrphotin -1（表皮生长因子受体酪氨酸激酶抑制剂）预处理导致aav2转导增加。观察AAV BDNF转导IPE细胞（BDNF-IPE）对视网膜神经节细胞（RGCs）的神经保护作用。体外，HUSB-Tyr处理的BDNF- ipe显著抑制BDNF剥夺RGC死亡。在连续光暴露引起的光感受器变性的体内模型中，移植husb - tyr处理的bdnf - ipe可抑制光感受器变性。

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.