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Arlene V Drack, Sajag Bhattarai, Seongjin Seo, Edwin M Stone, Val Sheffield, Robert Mullins, Budd A Tucker; Overcoming the overexpression toxicity of gene replacement therapy for Bardet Biedl Syndrome type 1. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4378.
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© ARVO (1962-2015); The Authors (2016-present)
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.
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.
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.
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.
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