Document text
Principal Investigator: Adam Tuttle
Organization: ENTERX BIOSCIENCES, INC.
Fiscal Year: 2024
Award: $294,052
Funding agency: National Eye Institute
Project Summary
Inherited retinal diseases (IRDs) are a heterogeneous group of genetic diseases that lead to loss of vision and
often progress to blindness. As a group, IRDs are linked to over 280 genes and affect ~4.5 million people
worldwide. In all IRDs, the ultimate cause of vision loss is degeneration of light-sensing photoreceptor (PR) cells
and, in most cases, genetic correction is needed specifically in PRs. Genome editing using the CRISPR-Cas9
toolkit has the potential to correct mutations directly in the patient’s DNA. Unfortunately, lack of a safe, efficient
delivery vehicle to PRs is a major barrier to developing gene therapies for IRDs. Viral gene therapies for IRDs
are in clinical testing but they have limited cargo size and induce a strong immune response. Lipid nanoparticles
(LNPs) are the most clinically advanced, non-viral nucleic acid delivery vehicle. LNPs form a “bubble” composed
of different lipids (and other compounds) that envelop nucleic acids and provide robust and safe delivery in vivo.
To address this PR-specific delivery barrier, EnterX Biosciences is developing novel PR-specific LNPs to
deliver RNA-based gene editing components based on our best-in-class, proprietary lipid technology.
Recently, we demonstrated the first and only use of lipid-based delivery to transfect PRs with mRNA in vivo,
which was accomplished by attaching a small proprietary peptide targeting moiety to the LNP. Our novel,
proprietary ionizable lipid, NTRX-7, can also generate LNPs that deliver mRNA to PRs. Combining this novel
lipid chemistry and targeting peptide could enable gene editing in PRs. To improve PR-specific delivery of gene
editors, we have two major Aims: 1) investigate efficiency of gene editor delivery via LNPs using NTRX lipids
and a targeting peptide in PRs; and 2) deploy top LNP candidates to quantify gene editing efficiency and retinal
toxicity. These experiments will identify the top LNP candidate(s) capable of safe, efficient delivery of RNA gene
editing components for immediate follow up studies of gene editing in specific IRDs. In Aim 1, we will screen LNP
delivery of gene editors to PRs. First, in Aim 1A, we will use DNA barcoding to rapidly identify the most potent
derivatives of NTRX-7 LNPs and evaluate which LNPs have suitable delivery efficiency to PRs via subretinal
injection in mice. The most potent LNP formulations identified by barcoding will then be formulated with Cas9
mRNA and gRNA and injected subretinally. By sectioning and immunostaining Ai9 reporter mouse retinas, we
will assay the number of tdTomato+ PRs to determine the spatial extent of gene editing (successful editing will
“allow” tdTomato expression in Ai9 mice) and determine the most potent lipids. In Aim 1B, we will similarly test if
adding the peptide targeting moiety to the LNP surface can further improve gene editing efficacy and fine-tune
PR specificity. Finally, in Aim 2, the top LNP candidates from Aim 1 will be tested longitudinally for retinal toxicity
and to quantify PR genome editing in reporter Nrl-GFP mice (here, gene editing will knock out GFP expression).
Successfully completing these Aims sets the table for further testing in animal models for retinal diseases and
unlocks therapeutic development for a major, unmet health need as well as the clinical market for IRD therapies.
Terms: <Active Follow-up><Address><Affect><Animal Disease Models><Animal Model><Animal Models and Related Studies><Area><Assay><Bar Codes><Bioassay><Biologic Characteristic><Biologic Sciences><Biological Assay><Biological Characteristics><Biological Sciences><Bioscience><Blindness><Body Tissues><COVID-19><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><Cas nuclease technology><Cell Body><Cell Communication and Signaling><Cell Signaling><Cells><Chemicals><Cholesterol><Clinical><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><Coronavirus Infectious Disease 2019><DNA><DNA Therapy><DNA cassette><DNA editor><Data><Deoxyribonucleic Acid><Deterioration><Development><Diminished Vision><Encapsulated><Ensure><Evaluation><FDA approved><Flow Cytofluorometries><Flow Cytofluorometry><Flow Cytometry><Flow Microfluorimetry><Flow Microfluorometry><Fluorescence><Follow-Up Studies><Followup Studies><Formulation><Future><Gene Delivery><Gene Transfer Clinical><Genes><Genetic><Genetic Diseases><Genetic Intervention><Genetic Medicine><Goals><Guide RNA><Head><Health><Hereditary><Hereditary Disease><Immune response><Immunological response><Inborn Genetic Diseases><Individual><Inherited><Inherited disorder><Intracellular Communication and Signaling><Knock-out><Knockout><Lead><Legal patent><Life Sciences><Light><Link><Lipid Chemistry><Lipids><Low Vision><Marketing><Measures><Messenger RNA><Methods><Mice><Mice Mammals><Mission><Modification><Murine><Mus><Nerve Cells><Nerve Unit><Neural Cell><Neurocyte><Neurons><Non-Polyadenylated RNA><Nucleic Acids><Partial Sight><Patents><Patients><Pb element><Peptides><Persons><Phase><Photoradiation><Photoreceptor Cell><Photoreceptors><Photosensitive Cell><Primates><Primates Mammals><Publishing><Qualifying><RNA><RNA Gene Products><RNA delivery><RNA vaccine><RNA-based vaccine><Reduced Vision><Reporter><Retina><Retinal Diseases><Retinal Disorder><Ribonucleic Acid><Rodent><Rodentia><Rodents Mammals><SBIR><Safety><Signal Transduction><Signal Transduction Systems><Signaling><Small Business Innovation Research><Small Business Innovation Research Grant><Specificity><Subnormal Vision><Surface><System><Technology><Testing><Therapeutic><Tissues><Transfection><Viral><Viral Genes><Visual Receptor><Visual impairment><Work><active followup><barcode><biological signal transduction><clinical test><commercialization><compare to control><comparison control><coronavirus disease 2019><coronavirus disease-19><coronavirus infectious disease-19><deliver mRNA><deliver messenger RNA><delivery system for mRNA><delivery vector><delivery vehicle><develop therapy><developmental><enhancer cassette><experiment><experimental research><experimental study><experiments><expression cassette><eye toxicity><flow cytophotometry><follow up><follow-up><followed up><followup><gRNA><gene cassette><gene editor><gene repair therapy><gene therapy><gene-based therapy><gene-based treatment><gene-directed therapy><gene-targeted therapy><gene-targeted treatment><genetic cassette><genetic condition><genetic disorder><genetic therapy><genome editing><genome editor><genomic editing><genomic therapy><heavy metal Pb><heavy metal lead><hereditary disorder><heritable disorder><host response><immune system response><immunogenicity><immunoresponse><improved><in vivo><inborn error><inherited diseases><inherited genetic disease><inherited genetic disorder><integration cassette><intervention development><knock-down><knockdown><knockout gene><lipid based nanoparticle><lipid nanoparticle><mRNA><mRNA delivery><mRNA vaccine><mRNA-based vaccine><messenger RNA delivery><model of animal><multidisciplinary><mutation correction><nano particle delivery><nanoparticle delivered><nanoparticle delivery><neuronal><novel><nucleic acid delivery><ocular toxicity><patient population><peptide aminoacid sequence><peptide sequence><prevent><preventing><promoter cassette><protein aminoacid sequence><reporter cassette><research clinical testing><resistance cassette><retina disease><retina disorder><retinal toxicity><retinopathy><screening><screenings><selectable cassette><selection cassette><stop cassette><subretinal injection><therapeutic agent development><therapeutic development><therapy development><transcription cassette><transcriptional cassette><transgene cassette><treatment development><vision impairment><vision loss><visual loss><visually impaired>