Drack, Arlene V.1; Lobeck, Brianna1; Rankin, Tyler1; Stanley, Sarah; Kalmanek, Emily1; Anastasios, Georgiadis2; Holthaus, Sophia2; Thompson, Jacob1; Hassan, Salma1; Sheffield, Val3; Hsu, Ying1
1Department of Ophthalmology and Visual Science, University of Iowa; 2MeiraGTx; 3Department of Pediatrics, University of Iowa
Purpose: Biallelic mutations in BBS10 are the second most common cause of Bardet Biedl Syndrome (BBS), a ciliopathy associated with blindness in early life as well as obesity, polydactyly, and renal failure. Subretinal gene therapy using an AAV2/Anc80 capsid carrying the mouse Bbs10 gene showed efficacy in a BBS10 knock out mouse model. AAV8-RK-hBBS10 and AAV8-CAG-hBBS10, developed for clinical translation, were used to treat the same mouse model to assess safety and efficacy
Methods: The human BBS10 gene driven by either the rhodopsin kinase (RK) promoter or the CMV enhancer/chicken β-actin promoter (CAG) was cloned into a shuttle plasmid, packaged into AAV2/8, and titered by quantitative PCR for in vivo gene delivery. For testing the safety of these candidate vectors in mice, 2.48×1011 vg of either vector was subretinally injected into unaffected wild-type (WT) or heterozygous (HET) mice in a 2 µL volume, and optical coherence tomography (OCT) was performed at 1-, 3-, and 5-months post injection (MPI). A lead candidate vector was selected. For evaluating efficacy, Bbs10-/- mice were subretinally injected with 2.48×109, 2.48×1010, or 2.48×1011 vg/eye of the lead candidate vector, and outcome measures including electroretinography (ERG) and OCT were conducted at 1-, 2-, 3-, 5-, and 7-MPI. A visually-guided swim assay for evaluating rodent functional vision was conducted at 5- to 7-MPI.
Results: Unaffected HET or WT mice receiving 2.48×1011 vg/eye of AAV8-CAG-hBBS10 demonstrated retinal atrophy on OCT. Those receiving AAV8-RK-hBBS10 did not, and AAV8-RK-hBBS10 was designated the lead candidate vector. Subretinal injections of AAV8-RK-hBBS10 were performed in Bbs10-/- mice at 2.48×109, 2.48×1010, or 2.48×1011 vg/eye. Eyes receiving subretinal gene therapy developed a light adapted (LA) 5 Hz flicker ERG cone response, while no untreated eyes had a recordable LA 5 Hz flicker cone ERG. Eyes treated with high dose had higher average cone ERG amplitudes (17.8 ± 5.3 µV) compared to eyes that received the medium dose (9.6 ± 1.1 µV, p = 0.086), low dose (5.6 ± 1.0 µV, p < 0.001), or no treatment (5.0 ± 0.7 µV, p < 0.0001) at 5-MPI (averages ± SEM,two-way ANOVA, post-hoc Tukey’s multiple comparisons test). Treated Bbs10-/- mice averaged less time to find the platform in a visually-guided swim assay compared to untreated with no significant difference in times between different doses.
Conclusions: The choice of promoter to drive gene expression impacts safety profiles of the vector for in vivo gene delivery. AAV8-RK-hBBS10 had a superior safety profile and showed remarkable efficacy in vivo, causing eyes with no recordable cone ERG to develop one, and maintain it for at least 5 months after treatment. Mice with BBS10 retinal degeneration treated with high dose AAV8-RK-hBBS10 had higher ERG cone amplitudes and better functional vision compared to untreated BBS10 mice.
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