CRB1 gene therapy coming of age: mechanistic insight and rAAV assays on mouse & human retinal organoid models

Biallelic CRB1 gene variations can cause retinitis pigmentosa (RP), Leber congenital amaurosis, or in some cases macular degeneration. This thesis describes the generation and analysis of RP-CRB1 mouse and human retinas (mouse: Crb1KOCrb2LowMGCs; chapter 2. Human RP-CRB1-patient-derived organoids (chapter 4 and 5). The data indicates that the human RP-CRB1 disease can be studied in mice and human organoids. Then, we show that recombinant adeno-associated viral (rAAV)-CRB gene supplementation therapy to Müller glial cells (MGCs) of the Crb1KOCrb2LowMGCs mouse retina can protect it from stress-induced vision loss, and that human CRB2 cDNA was superior to human CRB1 cDNA (chapter 2). We then developed an improved rAAV tropism assay on human donor eyes (chapter 3). This assay shows that rAAV5 can efficiently infect Müller glial cells and photoreceptors, the target cells of a RP-CRB1 gene therapy. Also, rAAV5 infection studies outperformed rAAV9 on human retinal organoids and human donor retinas (chapter 4). Finally, we find much more early endosomes and an increase of the degradative cellular vesicles which is linked to decrease of RAB11A-postive recycling endosomes in RP-CRB1 patient organoids (chapter 5). Thus, this thesis on both human and mouse models provides new insight into retinal degeneration and rAAV gene supplementation therapies.