Abstract
Purpose:
Our previous work showed that BBS1 overexpression results in severe retinal cytotoxicity in vivo (Seo et al., IOVS 2013,11;54(9):6118-32) . This study sought to determine whether regulating expression levels of BBS1 using cell-type specific promoters and reduced multiplicity of infection could mitigate cytotoxicity and slow disease progression.
Methods:
Human wild type (WT) BBS1 was packaged in an AAV2 vector under control of the rho kinase promoter, and in a lentiviral (LV) vector under control of the CMV promoter. Subretinal injections of 1x107 to 1x109 viral genomes in 1 microliter total volume were delivered to right eyes and AAV-GFP to control left eyes of P30-P60 mice (either WT or Bbs1M390R/M390R knock-in). Retinal function was analyzed by electroretinography (ERG) and retinal structure by optical coherence tomography (OCT). For LV vectors, right eyes received subretinal injection at P2-4 with uninjected left eyes as controls. LV vector was also used to treat induced pluripotent stem cell (iPSC) derived photoreceptor precursor cells generated from Bbs1M390R/M390R mouse fibroblasts in vitro.
Results:
Transduction of iPSC derived photoreceptor precursor cells with LV particles containing human BBS1 resulted in widespread death, a result not detected in cultures transduced with an equal MOI of control LV-GFP. In vivo, the ERG of WT mice (n=3) eyes treated with LV-BBS1 was reduced by 50% at P191 compared to untreated eyes and OCT revealed a nodular retinal reaction. Subretinal AAV2-BBS1 with rho kinase promoter did not cause toxicity in WT mice eyes (n=2). In affected animals (n=6) a recordable difference in ERG amplitudes was detected, with eyes receiving therapeutic vector having slightly higher ERG amplitudes than sham injected eyes at both 1 and 8 weeks after injection.
Conclusions:
Modulating the expression level of WT BBS1 by using the weaker, cell-type specific promoter rho kinase, delivering smaller volumes of viral particles subretinally, and using lower viral titers per injection mitigates overexpression toxicity and demonstrates a detectable improvement in the ERG of Bbs1M390R/M390R mice. Disease-specific iPSCs may be used to test vectors for toxicity. These results are encouraging for future gene therapy in people with this most common type of BBS.
Keywords: 538 gene transfer/gene therapy •
696 retinal degenerations: hereditary •
721 stem cells