Mechanistic Studies on Regenerative Medicine Approaches to Childhood Blindness

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 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.