Abstract
Retinal gene transfer with adeno-associated viral (AAV) vectors holds great promise for the treatment of inherited retinal degenerations (IRDs). One limit of AAV is its transfer capacity of about 5 kb, which can be expanded to about 9 kb, using dual AAV vectors. This strategy would still not suffice for treatment of IRDs such as Usher syndrome type 1D or Alström syndrome type I (ALMS) due to mutations in CDH23 or ALMS1, respectively. To overcome this limitation, we generated triple AAV vectors, with a maximal transfer capacity of about 14 kb. Transcriptomic analysis following triple AAV transduction showed the expected full-length products along a number of aberrant transcripts. However, only the full-length transcripts are efficiently translated in vivo. We additionally showed that approximately 4% of mouse photoreceptors are transduced by triple AAV vectors and showed correct localization of recombinant ALMS1. The low-photoreceptor transduction levels might justify the modest and transient improvement we observe in the retina of a mouse model of ALMS. However, the levels of transduction mediated by triple AAV vectors in pig retina reached 40% of those observed with single vectors, and this bodes well for further improving the efficiency of triple AAV vectors in the retina.
Highlights
Inherited retinal degenerations (IRDs), with an overall global prevalence of 1/2,000,1 are a major cause of blindness worldwide
CDH23-3xflag and ALMS1-3xflag were split in three parts each cloned in a separate associated viral (AAV) vector (Figures 1C and 1D and Materials and Methods) from here called CDH-AAV 1, CDH-AAV 2, CDH-AAV 3, and Alström syndrome (ALMS)-AAV 1, ALMS-AAV 2, ALMS-AAV 3
While AAV-mediated gene therapy is effective in animal models and in patients with inherited blinding conditions,[3,4,5,6,7,52] its application to diseases affecting the retina and requiring transfer of sequences larger than 5 kb is limited by the small cargo capacity of AAV
Summary
Inherited retinal degenerations (IRDs), with an overall global prevalence of 1/2,000,1 are a major cause of blindness worldwide. Robust MYO7A reconstitution by dual AAV vectors was confirmed in the mouse retina by Dyka et al.,[27] while several examples of dual AAV systems for minidystrophin (CDS, 6.2 kb) expression in mouse muscle are available.[23,28] The efficacy of dual AAV vectors, with a maximum transfer capacity of around 9 kb, opens the venue for further expansion to triple AAV vectors with a theoretical transfer capacity of around 14 kb
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