Document text
Principal Investigator: Krzysztof Palczewski
Organization: UNIVERSITY OF CALIFORNIA-IRVINE
Fiscal Year: 2023
Award: $602,331
Funding agency: National Eye Institute
SUMMARY
Inherited retinal disorders are a genetically heterogeneous group of blinding diseases that have significant impact
on quality of life. Therapeutic approaches have lagged significantly behind initial identification of the genetic
bases for these diseases. However, there are some striking successes; e.g., RPE65 gene augmentation therapy
was the first FDA-approved gene therapy for any genetically inherited disease. Clinical translation of current
CRISPR-Cas9 technology has been impeded by its low editing efficiency, error-prone homology-directed repair
(HDR), and substantial indel formation. Precision genome editing is an advanced, innovative CRISPR-Cas9-
associated genome-editing tool that addresses the limitations of typical CRISPR-Cas9 implementation. Adenine
base editors (ABEs) enable conversion of a point mutation independently of Cas9-induced double-stranded DNA
breaks and HDR. When base editing is not applicable (e.g., due to transversion mutations, large deletions, or
insertions), prime editing technology offers feasible alternatives. Genome editing is highly specific; however,
prolonged expression of base editors could lead to undesired off-target alterations throughout the genome and
transcriptome. We hypothesize that transient delivery of genome editors via RNPs and synthetic RNAs can
achieve the same high editing rates as those for genome editors delivered via viral transduction with reduced
off-target and bystander editing. Accordingly, we propose two thematically linked aims.
Aim 1. Correct inherited retinal disease-causing mutations in the rhodopsin gene (RhoE150K/E150K)
associated with autosomal recessive retinitis pigmentosa (RP) via adenine base editing. Delivery of ABEs
will be optimized in the thoroughly characterized RhoE150K/E150K mouse model of RP. Proposed approaches will
provide a platform for ABEs to be quickly adapted to any suitable RPE or retinal mutation.
Aim 2. Repair the ABCA4 protein in Abca4PV/PV mice by prime editing. Using the PE3b prime editor and two
concurrent stabilized engineered prime-editing guide RNAs (epegRNA), we will restore functional ABCA4 protein
in Abca4PV/PV mice that carry double allelic mutations in photoreceptors and the RPE. Using immunoblotting and
next-generation sequencing for detecting rescued Abca4, and two-photon imaging techniques to detect A2E, we
will optimize genome editing efficiency in this animal model to improve prime-editing technology and its
application to treat inherited retinal diseases.
For both aims, we will test various means to deliver the editors transiently: (i) cell-penetrating peptides fused to
editors in purified ribonucleoprotein (RNP)-editing complexes; (ii) Coomassie-lipid tags on purified RNP-editing
complexes; (iii) viral-like particles containing RNP-editing complexes; or (iv) lipid nanoparticles containing
stabilized mRNAs of genome-editing materials for intracellular expression. These delivery systems will be
optimized first in engineered chromogenic cell lines. The efficacy of base and prime editing in mice will be
benchmarked against the level of expression of RPE65 in the rd12 animal model of Leber congenital amaurosis.
Terms: <1H-Purin-6-amine><2-photon><2-photon microscopy><220kDa rod outer segment rim protein><ABCA4 protein><ABCR protein><Address><Adenine><Alleles><Allelomorphs><Animal Model><Animal Models and Related Studies><Assay><Benchmarking><Best Practice Analysis><Bioassay><Biologic Assays><Biological Assay><Blindness><COVID-19><COVID19><CRISPR approach><CRISPR based approach><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR tools><CRISPR-CAS-9><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/CAS approach><CRISPR/Cas method><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 technology><CV-19><CV19><Cas nuclease technology><Cell Body><Cell Function><Cell Line><Cell Process><Cell physiology><CellLine><Cells><Cellular Function><Cellular Physiology><Cellular Process><Charge><Clinical Evaluation><Clinical Testing><Clustered Regularly Interspaced Short Palindromic Repeats approach><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><Color><Complex><Coomassie blue><DNA><DNA Double Strand Break><DNA Integration><DNA Therapy><Data><Deletion Mutation><Deoxyribonucleic Acid><Derivation><Derivation procedure><Development><Diminished Vision><Disease><Disorder><Engineering><Enzyme Gene><Enzymes><Eye><Eyeball><FDA approved><Familial juvenile macular degeneration syndrome><Gene Transfer Clinical><Genes><Genetic><Genetic Alteration><Genetic Change><Genetic Intervention><Genetic defect><Genome><Genomic DNA><Genomics><Guide RNA><Hereditary><Hereditary Disease><Histology><Human><Imaging Procedures><Imaging Technics><Imaging Techniques><Immunoblotting><Immunomodulation><Inborn Genetic Diseases><Inherited><Inherited disorder><Insertion Mutation><Juvenile onset macular degeneration><Label><Laboratories><Leber congenital amaurosis><Leber's amaurosis><Leber's congenital amaurosis><Light Signal Transduction><Link><Lipids><Low Vision><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Membrane><Messenger RNA><Methods><Mice><Mice Mammals><Modeling><Modern Man><Multienzyme Complexes><Murine><Mus><Mutate><Mutation><NGS Method><NGS system><Non-Polyadenylated RNA><Nucleosides><Outer pigmented layer of retina><Partial Sight><Penetration><Peptides><Photoreceptor Cell><Photoreceptors><Photosensitive Cell><Phototransduction><Pigment cell layer of retina><Pigmentary Retinopathy><Pigmented layer of retina><Point Mutation><Preclinical Testing><Protein Analysis><Protein Binding Domain><Protein Binding Motif><Protein-Protein Interaction Domain><Proteins><Publications><Publishing><QOL><Quality of life><RNA><RNA Gene Products><RNA Stability><RNA delivery><RNA vaccine><RNA-based vaccine><ROSRP><RPE65><RPE65 protein><Reagent><Reduced Vision><Reporter><Retina><Retinal Degeneration><Retinal Diseases><Retinal Disorder><Retinal Pigment Epithelium><Retinal pigment epithelial cells><Retinitis Pigmentosa><Rhodopsin><Ribonucleic Acid><Ribonucleoproteins><Route><STGD3><STGD3 disease><Safety><Scientific Publication><Stargardt disease><Stargardt macular dystrophy><Stargardt syndrome><Stargardt's disease><Stargardt-3><Stargardt-3 macular dystrophy><Stargardt-like macular dystrophy><Strains Cell Lines><Structure of retinal pigment epithelium><Subcellular Process><Subnormal Vision><System><Tapetoretinal Degeneration><Technology><Testing><Therapeutic><Therapeutic Intervention><Therapy Evaluation><Toxic effect><Toxicities><Viral><Visual Cortex><Visual Purple><Visual Receptor><Visual Transduction><Visual impairment><Vitamin B4><Western Blotting><Western Immunoblotting><amaurosis congenita of Leber><amino group><autosome><base><base editing><base editor><bases><benchmark><cell type><clinical test><clinical translation><clinically translatable><congenital amaurosis of retinal origin><corona virus disease 2019><coronavirus disease 2019><coronavirus disease-19><coronavirus infectious disease-19><cultured cell line><degenerative retina diseases><delivery vector><delivery vehicle><developmental><disease-causing mutation><effective therapy><effective treatment><enzyme complex><experiment><experimental research><experimental study><experiments><functional restoration><fundus flavimaculatus><gDNA><gRNA><gene augmentation intervention><gene augmentation therapy><gene function><gene repair therapy><gene therapy><gene-based therapy><gene-editing therapy><genetic therapy><genome editing><genome editing based therapy><genome editing therapy><genome editing treatment><genome editing-based therapeutics><genome mutation><genomic editing><genomic therapy><global gene expression><global transcription profile><hereditary disorder><heritable disorder><human disease><human model><immune modulation><immune regulation><immunologic reactivity control><immunomodulatory><immunoregulation><immunoregulatory><improved><in vivo><inborn error><indel><inherited diseases><inherited genetic disease><inherited genetic disorder><innovate><innovation><innovative><insertion-deletion><insertion-deletion mutation><insertion/deletion><insertion/deletion mutation><interest><intervention therapy><lipid based nanoparticle><lipid nanoparticle><mRNA><mRNA vaccine><mRNA-based vaccine><membrane structure><model of animal><model of human><mouse model><murine model><mutant><mutation correction><next gen sequencing><next generation sequencing><nextgen sequencing><novel><particle><photoreceptor degeneration><pre-clinical study><pre-clinical testing><preclinical study><prime editing><prime editor><protein blotting><protein expression><repair><repaired><research clinical testing><response><restore function><restore functionality><restore lost function><retina degeneration><retina disease><retina disorder><retinal degenerative><retinal degenerative diseases><retinoid isomerase><retinoid isomerohydrolase><retinol isomerase><retinopathy><risk minimization><rod and cone dystrophy><rod-cone dystrophy><screening><screenings><success><therapeutic editing><therapeutic genome editing><tool><transcriptome><transversion mutation><two photon excitation microscopy><two photon microscopy><two-photon><vision impairment><vision loss><visual cortical><visual cycle><visual loss><visually impaired>