Abstract
Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. However, AAV's limited cargo capacity prevents its application to therapies of inherited retinal diseases due to mutations of genes over 5 kb, like Stargardt's disease (STGD) and Usher syndrome type IB (USH1B). Previous methods based on ‘forced’ packaging of large genes into AAV capsids may not be easily translated to the clinic due to the generation of genomes of heterogeneous size which raise safety concerns. Taking advantage of AAV's ability to concatemerize, we generated dual AAV vectors which reconstitute a large gene by either splicing (trans-splicing), homologous recombination (overlapping), or a combination of the two (hybrid). We found that dual trans-splicing and hybrid vectors transduce efficiently mouse and pig photoreceptors to levels that, albeit lower than those achieved with a single AAV, resulted in significant improvement of the retinal phenotype of mouse models of STGD and USH1B. Thus, dual AAV trans-splicing or hybrid vectors are an attractive strategy for gene therapy of retinal diseases that require delivery of large genes.
Highlights
Inherited retinal degenerations (IRDs), with an overall global prevalence of 1/2.000 (Sohocki et al, 2001), are a major cause of blindness worldwide
This may be explained by: i. the homogeneous size of the dual associated viral (AAV) genome population when compared to AAV OZ genomes, which may favor the generation of transcriptionally active large transgene expression cassettes; ii. the small volume of the subretinal space, which we show favors infection and transduction of the same cell by two independent AAV vectors
We conclude that subretinal administration of dual AAV OV vectors should not be used for large gene transfer to PR, we can not exclude that sequences that are more recombinogenic than those included in our dual AAV OV ABCA4 and MYO7A vectors may allow efficient homologous recombination in PR
Summary
Inherited retinal degenerations (IRDs), with an overall global prevalence of 1/2.000 (Sohocki et al, 2001), are a major cause of blindness worldwide. A major limitation of AAV is its cargo capacity, which is thought to be limited to around 5 kb, the size of the parental viral genome (Dong et al, 2010a; Hermonat et al, 1997; Lai et al, 2010; Wang et al, 2012; Wu et al, 2010b) This limits the application of AAV gene therapy approaches for common IRDs that are caused by mutations in genes whose coding sequence (CDS) is larger than 5 kb ( referred to as large genes). The added region is placed downstream of the SD signal in the 5'-half vector and upstream of the SA signal in the 3'-half vector (Fig. 1) in order to increase recombination between the dual AAVs
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
