Utilizing circular, self-amplifying RNAs for prolonged expression

ABSTRACT Gene therapy to treat inherited retinal dystrophies (IRDs) is finally a clinical reality. Adeno-associated virus (AAV) has led the way as a medium to transport a corrective gene to various affected retinal cell types, as well as for CRISPR/Cas9-mediated genome editing. As a long-term solution, however, AAV is less than ideal. Clinically, the vector must be delivered by sub-retinal administration, which is relatively destructive because it results in retinal detachment, can only target a small portion of the retina, and is only approved to be administered once per eye. The plasmid containing the transgene of interest is capable of integrating into the host genome. While this can be problematic for the retina, the nearly ubiquitous bioavailability of AAV after sub-retinal injection suggests that the plasmid can integrate into other tissues and organs of the body. Finally, AAV is an expensive treatment. While expense may be worthwhile to save vision in the near-term, in the long-term, less destructive and more cost-effective treatments need to be developed. To this end, this proposal aims to combine self- amplifying and self-splicing RNA technologies to determine the longevity, immunogenicity, and potential in vivo side effects to retinal function. Self-amplifying messenger RNA (saRNA) is an area of intense investigation, as it relates to vaccine development. Studies have shown that saRNA can result in translation of the mRNA of interest for up to 7 weeks, but at a cost of increased immunogenicity. The associated immunogenicity can be suppressed, however, with co-translation of the interferon inactivating protein B18R. Self-splicing mRNA is an up-and-coming technology to develop circular mRNAs that increases both mRNA longevity and translation while reducing immunogenicity. These technologies on their own are exciting developments in mRNA therapeutic development, but have room for improvement. For example, B18R co-transfection is often utilized as a standalone mRNA without the ability to self-amplify, which reduces its longevity and efficacy, whereas the mRNA of interest is part of the self-amplifying construct. A more optimal approach would be to replace the mRNA of interest with B18R in the self-amplifying construct, and adding self-amplifying signal sequences to the mRNA of interest in a separate construct, where it can be trans-amplified. Circularizing these two constructs using type I self-splicing introns has the potential to increase the observed 7-week period of translation, while further decreasing immunogenicity. These proposed changes have never been combined, and never been tested in vivo, making this application innovative, yet feasible. If successful, this proposal will lay the foundation for mRNA- mediated gene augmentation therapy for all IRD genes, regardless of size, and without the issues that plague AAV-mediated gene therapies.