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Principal Investigator: Michael Farkas
Organization: STATE UNIVERSITY OF NEW YORK AT BUFFALO
Fiscal Year: 2024
Award: $346,500
Funding agency: National Eye Institute
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.
Terms: <AAV vector><AAV-based vector><Address><Adeno-Associated Viruses><Affect><Anti-sense RNA><Area><Benchmarking><Best Practice Analysis><Bioavailability><Biological Availability><Body Tissues><CRISPR approach><CRISPR based approach><CRISPR based therapeutics><CRISPR based treatment><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR therapeutics><CRISPR tools><CRISPR treatment><CRISPR-CAS-9><CRISPR-Cas based therapeutics><CRISPR-based disease therapeutics><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based therapy><CRISPR-based tool><CRISPR/CAS approach><CRISPR/Cas method><CRISPR/Cas technology><CRISPR/Cas therapeutics><CRISPR/Cas9><CRISPR/Cas9 technology><CRISPR/Cas9 therapeutics><CRISPR/Cas9 therapy><CRISPR/Cas9 treatment><CRISPR/Cas9-based therapy><Cas nuclease technology><Cas9 based therapeutics><Cell Body><Cells><Clinical><Clinical Trials><Clustered Regularly Interspaced Short Palindromic Repeats approach><Clustered Regularly Interspaced Short Palindromic Repeats based therapeutics><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><Clustered Regularly Interspaced Short Palindromic Repeats therapeutics><Codon><Codon Nucleotides><Common Rat Strains><DNA Therapy><Data><Dependoparvovirus><Dependovirus><Development><Engineering><Exhibits><Exonuclease><Eye><Eyeball><Foundations><Future><Gene Transfer Clinical><Genes><Genetic Intervention><Genome><Hereditary><IFN><Immune response><Immunological response><In Vitro><In vivo analysis><Inherited><Interferons><Intervening Sequences><Introns><Investigation><Length><Length of Life><Literature><Longevity><Mediating><Messenger RNA><Methods><Modality><Modeling><Non-Polyadenylated RNA><Nucleotides><Organ><Peptide Signal Sequences><Physiologic Availability><Plasmids><Proteins><RNA><RNA Gene Products><RNA Splicing><RNA Stability><RNA amplification><RNA based therapeutics><RNA based therapy><RNA therapy><RNA, Messenger, Splicing><RPE65><RPE65 protein><Rat><Rats Mammals><Rattus><Recombinant adeno-associated virus><Recombinant adeno-associated virus (rAAV)><Retina><Retinal Detachment><Retinal Dystrophy><Retinal Ganglion Cells><Ribonucleic Acid><Risk><Route><Sight><Signal Peptide><Signal Sequences><Specificity><Splicing><Sprague-Dawley Rats><Standardization><Techniques><Technology><Testing><Therapeutic><Tissues><Transgenes><Translating><Translations><Vision><adeno associated virus group><adeno-associated viral vector><adeno-associated virus vector><benchmark><cell type><circular RNA><closed circular RNA><cost><cost effective therapy><cost effective treatment><deliver mRNA><deliver messenger RNA><delivery system for mRNA><develop a vaccine><develop vaccines><development of a vaccine><developmental><gene augmentation intervention><gene augmentation therapy><gene repair therapy><gene therapy><gene-based therapy><genetic therapy><genome editing><genomic editing><genomic therapy><host response><immune system response><immunogenicity><immunoresponse><improved><in vitro Model><in vitro testing><in vivo><in vivo evaluation><in vivo testing><innovate><innovation><innovative><interest><intravitreal injection><lipid based nanoparticle><lipid nanoparticle><mRNA><mRNA delivery><messenger RNA delivery><nano particle delivery><nanoparticle delivered><nanoparticle delivery><new technology><novel><novel technologies><protein signal sequence><rAAV><recombinant AAV><retina detachment><retinal ganglion><side effect><stability testing><subretinal injection><therapeutic RNA><therapeutic agent development><therapeutic development><transgene><translation><vaccine development><vector><visual function>