Precision BioSciences, Inc. · NASDAQ:DTIL
Precision BioSciences — UMDF 2024
37 slides from Precision BioSciences's conference presentation, Jun 27, 2024
Slide 1: Shifting Heteroplasmy with PBGENE-PMM: Gene Editing Therapy for m.3243A>G Associated Mitochondrial Myopathy
Shifting Heteroplasmy with PBGENE-PMM: Gene Editing Therapy for m.3243A>G Associated Mitochondrial Myopathy Wendy Shoop, PhD June 27, 2024
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 • I am an employee of Precision BioSciences. 3
Slide 4: Outline
Outline 〉Precision BioSciences and ARCUS gene editing 〉Applications for ARCUS gene editing of mitochondrial DNA ― Common deletion (del_mtDNA4977) ― m.3243A>G ― Mouse m.5024C>T 〉PBGENE-PMM 4
Slide 5: 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 6: ARCUS generates targeted DNA double-strand breaks
ARCUS generates targeted DNA double-strand breaks I-Crel ARCUS Protein engineering 5’-CAAAACGTCCTGA GACAGTTTG-3’ 5’-CAGGGCCCGGTAA TCGCATAAA-3’ 3’-GTTTTGCAG CACTCTGTCAAAC-5’ 3’-GTCCCGGGC CATTAGCGTATTT-5’ “Left” half of “Right” half of “Left” half of “Right” half of natural target natural target new t
Slide 7: ARCUS protein engineering is an iterative process
ARCUS protein engineering is an iterative process ARCUS I-Crel Protein Engineering Desired Therapeutic YES Clinical Candidate In silico Index Achieved? NO Design Repeat 1st Generation Off-Target Specificity Site Selection Optimization ARCUS Analysis Optimization Cycle 2-3X Directed Evolution Next Ge
Slide 8: Applications for ARCUS
Applications for ARCUS 〉Nuclear gene editing ― Targeted gene insertion ― ECUR-506 IND and CTA approved ― Elimination of viral DNA ― PBGENE-HBV IND/CTA expected 2024 ― Gene knockout ― Excision of precise regions of genome In vivo gene editing 〉Mitochondrial gene editing (mitoARCUS) 8
Slide 9: 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 9
Slide 10: 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 Less than 80% mutant Biochemical threshold mitoARCUS gene editing Mutant mtDNA Wild-type mtDNA 10
Slide 11: 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 12: mitoARCUS gene editing seeks to eliminate mutant heteroplasmic mtDNA
mitoARCUS gene editing seeks to eliminate mutant heteroplasmic mtDNA 〉 Heteroplasmic deletions Applications 〉 Heteroplasmic point mutations Discovery Lead mitochondrial editing Research stage therapeutic program investigation Common deletion Mouse (del_mtDNA4977) m.3243A>G m.5024C>T 12
Slide 13: Common deletion (del_mtDNA4977)
Common deletion (del_mtDNA4977) 13
Slide 14: 4977bp of mtDNA are missing in genomes with the common deletion
4977bp of mtDNA are missing in genomes with the common deletion Implicated in: 〉 Myopathies Wild-type Deleted 〉 Kearns-Sayre syndrome mtDNA mtDNA 〉 Alzheimer’s disease 16569-bp 11592-bp 4977-bp 〉 Chronic progressive deletion external ophthalmoplegia 〉 Pearson syndrome 14
Slide 15: 4977bp of mtDNA are missing in genomes with the common deletion
4977bp of mtDNA are missing in genomes with the common deletion Wild-type Deleted mtDNA mtDNA 16569-bp 11592-bp 4977-bp deletion mitoARCUS nuclease that recognizes deletion breakpoint = mitoARCUS_del4977 15
Slide 16: mitoARCUS_del4977 effectively eliminates mtDNA molecules containing common deletion in vitro
mitoARCUS_del4977 effectively eliminates mtDNA molecules containing common deletion in vitro 150% Ratio of mtDNA/18S, normalized 100% Transient Collect mRNA transfection cellular DNA 50% Day 0 Day 14 0% GFP control mitoARCUS del4977 Percentage mutant mtDNA Percentage WT mtDNA Unpublished data 16
Slide 17: mitoARCUS_del4977-edited cells demonstrates improved mitochondrial function
mitoARCUS_del4977-edited cells demonstrates improved mitochondrial function Cell Mito Stress Test Basal respiration Maximal respiration 200 3 4 OCR (pmol/min) 150 3 2 OCR, normalized OCR, normalized 100 2 1 50 1 0 0 0 0 20 40 60 80 GFP control mitoARCUS GFP control mitoARCUS Time (minutes) del4977 d
Slide 18: Nuclease optimization for mitoARCUS_del4977 is ongoing
Nuclease optimization for mitoARCUS_del4977 is ongoing ARCUS mitoARCUS_3243 I-Crel Protein Engineering Desired Therapeutic YES Clinical Candidate In silico Index Achieved? NO Design mitoARCUS_del4977 Repeat 1st Generation Off-Target Specificity Site Selection Optimization ARCUS Analysis Optimization
Slide 19: m.3243A>G
U _ Cc Gm1 m.3243A>G _ S AC Gm2 G UCGC GpnaA A A-U A-U A-U A Cc re A U (3) tm5U a A
Slide 20: m.3243A>G is the most common pathogenic mtDNA point mutation
m.3243A>G is the most common pathogenic mtDNA point mutation mt-tRNAleu(UUR) m.3243A>G m.3243A>G 〉 Estimated population prevalence of ~1 in 500 individuals1 A 〉 Impacts tRNA stability/modification and disrupts mitochondrial protein production 〉 Implicated in many disorders Sources: 1. Manwaring et a
Slide 21: Nuclease specificity is crucial to discriminate single nucleotide change
Nuclease specificity is crucial to discriminate single nucleotide change mitoARCUS nuclease specific to mutant m.3243G = mitoARCUS_3243 Mutant mtDNA sequence 5’-C A G G G C C C G G T A A T C G C A T A A A-3’ Sequence we want to cut Sequence we 5’-C A G A G C C C G G T A A T C G C A T A A A-3’ don’t
Slide 22: mitoARCUS_3243 efficiently eliminates mutant m.3243G, but does not cut wild-type m.3243A
mitoARCUS_3243 efficiently eliminates mutant m.3243G, but does not cut wild-type m.3243A 100% mutant 100% wild-type 8 m.3243G m.3243A 15 6 Ratio of mtDNA/18S Ratio of mtDNA/18S 10 4 5 2 0 0 0 6 24 48 72 0 6 24 48 72 Hour Hour GFP control mitoARCUS_3243 Shoop et al 2023 – Nature Metabolism 22
Slide 23: mitoARCUS_3243 shifts heteroplasmy in 95% mutant cybrid cells
mitoARCUS_3243 shifts heteroplasmy in 95% mutant cybrid cells Day 1 Day 3 Day 7 150% 150% 150% Ratio of mtDNA/18S, normalized Ratio of mtDNA/18S, normalized Ratio of mtDNA/18S, normalized 100% 100% 100% 50% 50% 50% 0% 0% 0% GFP control 1x103 1x104 1x105 GFP control 1x103 1x104 1x105 GFP control 1x10
Slide 24: Specific elimination of mutant m.3243G leads to improvements in mitochondrial function
Specific elimination of mutant m.3243G leads to improvements in mitochondrial function Cell Mito Stress Test Basal respiration Maximal respiration 15 2.5 2.5 OCR (pmol O2/min/1000 cells) 2 2 10 OCR, normalized OCR, normalized 1.5 1.5 5 1 1 0.5 0.5 0 0 0 0 25 50 75 100 GFP control 1x103 1x104 1x105 G
Slide 25: Nuclear off-target editing is eliminated with MTS and NES
Nuclear off-target editing is eliminated with MTS and NES On-target editing Nuclear off-target editing 150% 2.0% Ratio of mtDNA/18S, normalized 1.5% 100% Percent editing 1.0% 50% 0.5% LOD 0% 0.0% GFP control 1x104 1x105 1x106 2x106 1 2 3 4 5 6 7 8 9 10 11 12 13 14 RNA copies/cell Off site Percentage
Slide 26: Developing mitoARCUS_3243 for patients with m.3243 mitochondrial disease
Developing mitoARCUS_3243 for patients with m.3243 mitochondrial disease m.3243-associated mitochondrial diseases often lead to defects in energy production affecting high energy-demand tissues (e.g. skeletal muscle) m.3243A>G m.3243-associated mitochondrial Chronic progressive external disease esti
Slide 27: PBGENE-PMM is the first therapeutic designed to eliminate mutant m.3243G mtDNA
PBGENE-PMM is the first therapeutic designed to eliminate mutant m.3243G mtDNA AAV vector Muscle-specific MTS mitoARCUS_3243 NES promoter Systemically administer PBGENE-PMM to patients with m.3243-associated Objective mitochondrial disease to elicit a shift in heteroplasmy in skeletal muscle Use m.5
Slide 28: Mouse m.5024C>T
Mouse m.5024C>T m U A U G- A-U G-C G-C U-U © U au cAuuCcUCA AA I A uUuCcG™ GUAG Ga, G 1rti U U UAAAGC, ,A A-u © U-A U-A G-C A-U U OU U A UgC 28
Slide 29: Leveraging the m.5024C>T heteroplasmic mouse model to determine clinical dosing
Leveraging the m.5024C>T heteroplasmic mouse model to determine clinical dosing mt-tRNAAla m.5024C>T m.5024C>T A 〉 No overt clinical phenotype 〉 Model provides POC for therapeutic approach 〉 Surrogate mitoARCUS nuclease developed that is specific for mutant m.5024T mtDNA = mitoARCUS_5024 29
Slide 30: mitoARCUS_5024 effectively eliminates mutant m.5024T mtDNA in vitro
mitoARCUS_5024 effectively eliminates mutant m.5024T mtDNA in vitro Day 1 Day 4 150% 150% Ratio of mtDNA/18S, normalized Ratio of mtDNA/18S, normalized 100% 100% 50% 50% 0% 0% GFP control mitoARCUS GFP control mitoARCUS 5024 5024 Percentage mutant mtDNA Percentage WT mtDNA Unpublished data 30
Slide 31: mitoARCUS_5024 in vivo study design
mitoARCUS_5024 in vivo study design Group Treatment Dose N 1 PBS N/A 5 2 mitoARCUS_5024 2.5x1013 vg/kg 7 Necropsy and 3 mitoARCUS_5024 1x1014 vg/kg 7 Systemic AAV tissue administration collection PURPOSE: To evaluate ability of mitoARCUS_5024 to shift heteroplasmy in target tissues following Day 0 D
Slide 32: mitoARCUS_5024 effectively transduces target tissues and eliminates mutant mtDNA without inducing mtDNA depletion
mitoARCUS_5024 effectively transduces target tissues and eliminates mutant mtDNA without inducing mtDNA depletion AAV copy number mtDNA copy number Heteroplasmy 100 30 100% 10 80% AAV/diploid genome mtDNA/m18S rDNA % mutant mtDNA 1 20 60% 0.1 40% 0.01 10 20% 0.001 0.0001 0 0% c c c TA TA TA m m m rt
Slide 33: AAV biodistribution drives mutant mtDNA elimination in target tissues
AAV biodistribution drives mutant mtDNA elimination in target tissues 50% 25% 0% Minimal amounts % mutant change 0 1 2 3 4 AAV/diploid genome of AAV are needed -25% to drive heteroplasmy shift -50% in target tissues -75% -100% Unpublished data 33
Slide 34: PBGENE-PMM
PBGENE-PMM 34
Slide 35: PBGENE-PMM program overview
PBGENE-PMM program overview Final Clinical Candidate PoC and Dose Finding Studies underway on-track Initiate GLP Tox In-process Identify First-in-Human Study Sites on-track Expected CTA/IND in 2025 35
Slide 36: 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, both deletions and point mutations ARCUS-mediated rescue of mtDNA heteroplasmy improves mitochondrial respiratio
Slide 37: Acknowledgements
Acknowledgements • Emma Sevigny • Emily Manzon • Carlos Moraes • Adam Mischler • Joanna Dobson • Sandra Bacman • Spencer McKinstry • Dan Nazarenko • Ugne Zekonyte • Janel Lape • Traci Reddick • Jose Domingo Barrera-Paez • Jamie Juett • Dominique Burgess • Flavia Fontanesi • Jeff Smith • Crystal Gord