Slide 1: Systemic Delivery of a Mitochondria-Targeting ARCUS Gene Editing Nuclease by AAV
Systemic Delivery of a Mitochondria-Targeting ARCUS Gene Editing Nuclease by AAV Eliminates Mutant Mitochondrial DNA, Demonstrating Therapeutically Meaningful Heteroplasmy Shifts In Vivo Wendy K. Shoop, PhD ASGCT 2025 1
Slide 2: Forward-looking statements
Forward-looking statements This presentation contains forward-looking statements, as may any related presentations, within the meaning of the Private Securities Litigation Reform Act of 1995. The Company intends such forward- looking statements to be covered by the safe harbor provisions for forward
Slide 3: Disclosures
Disclosures Wendy Shoop is an employee of Precision BioSciences, Inc. (Nasdaq: DTIL) 3
Slide 4: ARCUS: Engineering nature's gene editing system
ARCUS: Engineering nature’s gene editing system 〉 ARCUS is derived from I-CreI, a homing endonuclease from algae 〉 Recognizes 22bp target sequence through extensive protein-DNA interactions 〉 Does not require guide RNA 〉 DNA recognition and cleavage activity are fully integrated into one protein for
Slide 5: Applications for ARCUS
Applications for ARCUS 〉Nuclear gene editing ― Targeted gene insertion ― OTC-HOPE clinical trial ongoing – oral presentation 5/14 8AM ― Elimination of viral DNA ― PBGENE-HBV/ELIMINATE-B clinical trial ongoing – oral presentation 5/16 4PM ― Excision of precise regions of genome ― PBGENE-DMD – poster
Slide 6: Multi-copy mitochondrial DNA (mtDNA) is critical for mitochondrial function
Multi-copy mitochondrial DNA (mtDNA) is critical for mitochondrial function Essential for energy production Mitochondrial DNA (mtDNA) Human cell Mitochondrion 6
Slide 7: Heteroplasmic mtDNA mutations exhibit a threshold at which symptoms manifest
Heteroplasmic mtDNA mutations exhibit a threshold at which symptoms manifest Clinically asymptomatic Clinically symptomatic 40% mutant 60% mutant 70% mutant 80% mutant Biochemical threshold Mutant mtDNA Wild-type mtDNA 7
Slide 8: Heteroplasmic mtDNA mutations exhibit a threshold at which symptoms manifest
Heteroplasmic mtDNA mutations exhibit a threshold at which symptoms manifest Clinically asymptomatic Clinically symptomatic Clinically asymptomatic Clinically symptomatic Less80% thanmutant 80% mutant Biochemical threshold mitoARCUS gene editing Mutant mtDNA Wild-type mtDNA 8
Slide 9: mitoARCUS therapeutic approach to shift heteroplasmy
mitoARCUS therapeutic approach to shift heteroplasmy Cytoplasm mitoARCUS MTS ARCUS NES mito-targeting nuclear export sequence signal WT MTS ARCUS NES mtDNA Mitochondrial matrix mutant ARCUS mtDNA
Slide 10: mitoARCUS gene editing seeks to eliminate mutant heteroplasmic mtDNA
mitoARCUS gene editing seeks to eliminate mutant heteroplasmic mtDNA 〉 Heteroplasmic point mutations Applications 〉 Heteroplasmic deletions Lead mitochondrial editing Research therapeutic program investigation Discovery stage Common Mouse deletion m.3243A>G m.5024C>T (del_mtDNA4977) 10
Slide 11: Developing PBGENE-3243 for patients with m.3243-associated mitochondrial disease
Developing PBGENE-3243 for patients with m.3243-associated mitochondrial disease m.3243A>G leads to defects in energy production affecting m.3243-associated mitochondrial high energy-demand tissues (e.g. skeletal muscle) disease estimated at ~20k patients in the US alone1 Chronic progressive externa
Slide 12: Objective and AAV delivery of PBGENE-3243
Systemically administer PBGENE-3243 to patients with m.3243-associated mitochondrial Objective disease to elicit a shift in heteroplasmy in skeletal muscle AAV Muscle-specific MTS and NES Nuclease Improved delivery promoter domains specificity activity Broad tissue tropism Drives nuclease expression
Slide 13: m.5024C>T heteroplasmic mouse demonstrates POC for therapeutic approach
m.5024C>T heteroplasmic mouse, in conjunction with surrogate nuclease, demonstrate POC for therapeutic approach m.5024C>T 〉 Mouse-specific point mutation 〉 No overt clinical phenotype 〉 Surrogate nuclease developed that is specific for mutant m.5024T mtDNA = mitoARCUS_5024 https://pubmed.ncbi.nlm.ni
Slide 14: Treatment of m.5024C>T mice with mitoARCUS_5024 to evaluate heteroplasmy shift
Treatment of m.5024C>T mice with mitoARCUS_5024 to evaluate Adult mice heteroplasmy shift Day 0 Day Week Week Week Week Week 3 1 2 4 12 24 Systemic AAV administration (5.0x1013 vg/kg) Necropsy and PURPOSE: 〉 Quadricep tissue collection Evaluate DNA isolated To evaluate the ability of 〉 Gastrocnemius
Slide 15: mitoARCUS_5024 effectively transduces target tissues
mitoARCUS_5024 effectively transduces target tissues AAV copy number 10 AAV/diploid genome 1 0.1 0.01 Quad Gastroc TA Heart Diaphragm Brain PBS mitoARCUS_5024 >1 AAV/diploid genome detected across all target tissues at day 3 Unpublished data 15
Slide 16: No sustained depletions in mtDNA copy number are observed in treated mice
No sustained depletions in mtDNA copy number are observed in treated mice Day 3 Week 1 Week 2 25 25 25 mtDNA/18S rDNA mtDNA/18S rDNA mtDNA/18S rDNA 20 20 20 15 15 15 10 10 10 5 5 5 0 0 0 Week 4 Week 12 Week 24 25 60 60 mtDNA/18S rDNA mtDNA/18S rDNA mtDNA/18S rDNA 20 50 50 40 40 15 30 30 10 20 20 5 1
Slide 17: Reductions in mutant mtDNA within target tissues sustained at week 24
Reductions in mutant mtDNA within target tissues are observed out to week 12 and sustained at week 24 Percent mutant mtDNA Percent reduction in mutant mtDNA from baseline 90 20 80 0 70 % mutant shift 60 -20 % mutant 50 -40 40 30 -60 20 -80 10 0 -100 Quad Gastroc TA Heart Diaphragm Brain Quad Gastroc
Slide 18: Concomitant increases in the percentage of wild-type mtDNA in treated mice
Concomitant increases in the percentage of wild-type mtDNA are observed in treated mice Percent WT mtDNA 100 90 80 70 60 50 % WT 40 30 20 10 0 Quad Gastroc TA Heart Diaphragm Brain PBS Week 24 WT mtDNA increases to 50-90% across all target tissues at week 24 Unpublished data 18
Slide 19: Clinical data indicates an absolute threshold of 50% mutant for phenotype
Clinical data from patients with m.3243-mitochondrial disease indicates an absolute threshold of 50% mutant to exhibit skeletal muscle phenotype Percent reduction in mutant mtDNA from baseline 20 0 % mutant shift -20 Target -40 Reduction Range -60 〉 Starting levels of heteroplasmy can vary between p
Slide 20: Conclusions
Conclusions ARCUS platform generates highly specific nucleases capable of discriminating a single nucleotide ARCUS can be trafficked to mitochondria to selectively eliminate mutant mtDNA, leading to shifts in heteroplasmy Systemic delivery of ARCUS using AAV transduces the skeletal muscle, heart, an
Slide 21: Thank You
Dedicated to Improving Life THANK YOU! 21