Mechanistic Studies on Regenerative Medicine Approaches to Childhood Blindness

 描述（由申请人提供）：干细胞治疗在眼睛中找到了一个理想的试验场，这是一个具有相对免疫特权的器官，可以接近但又与世隔绝。对眼睛的治疗通常既不是侵入性的，也不是全身性的;由于眼睛是光学透明的，治疗可以在活体动物中容易地和非侵入性地监测-这是优于其他系统的主要优点。在视网膜疾病的临床前研究中，健康的视网膜色素上皮（RPE）移植物可以修复受损的视网膜。展望RPE移植的临床应用，我们认为从自体干细胞（患者自己的干细胞）生长的RPE移植物将是最佳的开发方法。尽管如此，这种方法的临床应用仍有待解决几个知识空白：成人干细胞的多能性足以使功能性RPE移植物发挥作用吗？即使病人的移植物是有功能的，体外培养会使它们具有抗原性吗？此外，即使自体RPE移植物起作用，基因治疗在RP的晚期阶段是否更有效？目标1、2和3分别涉及这些问题。这里探讨的疗法旨在修复遗传性视网膜变性。虽然我们的研究结果一般只涉及视网膜色素变性（RP），但这些研究主要集中在膜卷曲相关基因（MFRP）的罕见突变引起的RP。MFRP视网膜病变是我们研究的理想选择，原因有三。首先，已知的MFRP点突变导致RP表型。此外，MFRP视网膜病变有一个长达数十年的治疗机会窗口：在整个生命周期中，视网膜电图（ERG）的变化落后于感光细胞的损失，这表明尽管视网膜损伤，MFRP视网膜病变患者仍能保持视力，并且可能可以治疗到疾病的晚期。选择MFRP视网膜病变的第三个原因是我们可以利用充分表征的MFRP突变小鼠品系rd 6，其也患有致盲性RP。对于我们的研究，将分离、培养MFRP缺陷患者干细胞，并用腺相关病毒（AAV）转导以表达野生型MFRP。这些“MFRP基因修复”的患者干细胞将分化为RPE并移植到免疫缺陷（Scid）、MFRP缺陷（rd 6）小鼠的右眼中。将比较左眼和右眼的RP表型逆转。我们的长期目标是为遗传性视网膜疾病创造细胞和基因治疗方法，并开发扩展到其他疾病的策略。我们的目标是在这个建议是找到方法来使用病人的干细胞作为视网膜移植的来源。我们将测试我们的中心假设，即体外由AAV修复的患者源性RPE移植物已经是一种治疗选择。

 DESCRIPTION (provided by applicant): Stem cell therapy finds an ideal proving ground in the eye, an organ with relative immune privilege that is accessible yet isolated. Therapies for the eye are generally neither invasive nor systemic; and since the eye is optically transparent, treatment can be monitored easily and non-invasively in living animals-a major advantage over other systems. In preclinical studies of retinal disease, grafts of healthy retinal pigment epithela (RPE) can restore damaged retina. Looking ahead to clinical applications of RPE grafting, we posit that RPE grafts grown from autologous stem cells (a patient's own stem cells) would be the optimal approach to develop. Nevertheless, clinical use of this method awaits resolution of several knowledge gaps: Are an adult's stem cells pluripotent enough to render functional RPE grafts? Even if patient grafts are functional, will in vitro culture make them antigenic? Also, eve if autologous RPE grafts work, will gene therapy be more effective at late stages of RP? These questions are addressed by Aims 1, 2, and 3, respectively. The therapies explored here aim to repair hereditary retinal degeneration. Although, in general our results will pertain to retinitis pigmentosa (RP), these studies focus on RP caused by rare mutations in membrane frizzled- related gene (MFRP). MFRP retinopathy is ideal for our studies for three reasons. First, known MFRP point mutations cause an RP phenotype. In addition, MFRP retinopathy has a decades-long window of opportunity for treatment: Over the life span, electroretinogram (ERG) changes lag behind photoreceptor loss, suggesting that despite retinal damage, MFRP retinopathy patients retain sight and are likely treatable until late stages of the disease. The third reason fr choosing MFRP retinopathy is that we can take advantage of the well characterized, MFRP mutant mouse line, rd6 that also suffers blinding RP. For our study, MFRP-deficient patient stem cells will be isolated, cultured, and transduced with an Adeno-Associated-virus (AAV) to express wild-type MFRP. These "MFRP gene repaired" patient stem cells will be differentiated into RPE and grafted into right eyes of immunodeficient (Scid), MFRP-deficient (rd6) mice. Left and right eyes will be compared for reversal of the RP phenotype. Our long-term goals are to create cell- and gene-therapy cures for hereditary retinal diseases, and develop strategies that extend to other diseases. Our objective in this proposal is to find ways to use patient stem cells as a source of retinal grafts. We will test our central hypothesis that patient- derived RPE grafts, repaired by AAV in vitro, are already a therapeutic option.