Generation Bio — OTS Webinar October 2023
36 slides from Generation Bio's conference presentation, Oct 12, 2023
Slide 1: Non-viral DNA delivery: Opportunities and challenges
Non-viral DNA delivery: Opportunities and challenges MATTHEW STANTON OTS WEBINAR OCTOBER 2023 CONFIDENTIAL
Slide 2: Forward-looking statements
Forward-looking statements Any statements in this presentation about future expectations, plans and prospects for the company, including statements about our strategic plans or objectives, our technology platform, our research and clinical development plans, and our preclinical data and other statem
Slide 3: GBIO is bringing DNA to non-viral genetic medicines
GBIO is bringing DNA to non-viral genetic medicines siRNA- mRNA- DNA- GalNAc LNP LNP Redosable Redosable Redosable Durable Transient Durable Loss of function Gain of function Gain of function 3
Slide 4: Three fundamental scientific problems must be addressed for non-viral DNA platform
To enable a non-viral DNA genetics medicine platform, solutions to three fundamental scientific problems must be addressed Innate Immunity Delivery Nuclear access • Strong innate immune • LNPs optimized for DNA • Non-viral delivery systems stimulation in immune cells cargo deliver cargo (DNA) to lim
Slide 5: Overcoming innate immunity: Optimizing cell-targeted LNPs
Overcoming innate immunity: Optimizing cell-targeted LNPs CONFIDENTIAL
Slide 6: Unlike RNA platforms, no known chemistry solutions to DNA innate immunity
Unlike RNA platforms, we started with no known chemistry solutions to DNA innate immunity DNA INNATE IMMUNITY siRNA IFN-alpha 1500 empty LNP 2’ ribose chemical modifications Mean Concentration DNA LNP 1000 (pg/ml) 500 mRNA 0 uridine chemical modifications/purity 6
Slide 7: Solving RNA innate immunity unlocked a global modality
Solving RNA innate immunity unlocked a global modality 7
Slide 8: Moderna – influence of manufacturing process and purity on innate immune signaling
Moderna – beyond just chemical modifications: influence of manufacturing process and purity on innate immune signaling 8
Slide 9: Standard LNPs are sufficient for RNA delivery but what about DNA?
Standard LNPs are sufficient for RNA delivery but what about DNA? ApoE-mediated trafficking Multiple mechanisms Myeloid cells in spleen, circulating Hepatocytes Approx. 1:1 distribution monocytes, Kupffer cells in liver 9
Slide 10: Consequences of biodistribution of traditional LNPs
Consequences of biodistribution of traditional LNPs: meh for RNA, existential for DNA 2.0 mg/kg i.v. injection (LNP) 6hrs Blood for multi-plex cytokine analysis CD-1 mice IFNα IL-6 2000 2500 PBS PBS mRNA 2000 mRNA 1500 IFNα (pg/mL) IL-6 (pg/mL) DNA DNA 1500 1000 1000 500 500 0 0 Consequences of cyto
Slide 11: Hepatocytes lack DNA pattern recognition receptors
Hepatitis B literature as inspiration: Hepatocytes lack DNA pattern recognition receptors Implications • Whole liver tissue contains STING, but isolated hepatocytes do not (STING is present in Kupffer cells) • Transfection of DNA in healthy hepatocytes does not result in IFNb stimulation. Control P
Slide 12: First generation solution: novel cell-targeted LNP
First generation solution: novel cell-targeted LNP minimizing off-target clearance ApoE-mediated trafficking; Reduce off-target clearance additional GalNAc targeting X Myeloid cells (Spleen, circulating monocytes, Kupffer cells in liver) 12
Slide 13: ceDNA physical properties create a barrier to reducing particle size
ceDNA physical properties create a barrier to reducing particle size and polydispersity ceDNA LNPs – more difficult to reduce particle Linear, B-form double stranded DNA size • Prefers extended conformations? • Increasing PEG% for flexible PolyA cargo dramatically decreases particle size: • For ceDN
Slide 14: Altering ceDNA structure to favor condensed LNPs: P-Form DNA
What can we do to alter ceDNA structure and favor condensed, tightly packaged LNPs: taking a lesson from literature on DNA structure • Studies aimed at understanding DNA structure upon denaturation in alcohol solvents identified a novel structure • This new structure was coined “P- Form” DNA – a ref
Slide 15: Adapting P-form to process: dehydrating ceDNA into ethanol
Adapting this to process – dehydrating and solubilizing ceDNA into ethanol – pDNA conformation • Q – can we dehydrate ceDNA in ethanolic LNP input stream (with lipids), favor P-form DNA in densely packed spheroids and use to create smaller, more homogeneous LNPs? 15
Slide 16: P-form process led to smaller, more homogeneous particle sizes
Adaptation of P-form DNA formation to LNP manufacturing process led to smaller, more homogeneous particle sizes Analytics of LNPs Blue = standard process Green/red = P-form process CryoEM: standard P-form 16
Slide 17: Smaller particles lead to better expression and tolerability
Adaptation of P-form DNA formation to LNP manufacturing process led to smaller, more homogeneous particle sizes, which leads to better expression and tolerability Analytics of LNPs Profile in mice Blue = standard process Green/red = P-form process • Mice dosed IV at 0.25 mg/kg • LNP compositions ide
Slide 18: Dose responsive, durable, redosable FVIII expression in mice
Dose responsive, durable, & redosable FVIII expression in immunocompetent FVIII KO mice (mFVIII KO/huFVIII-R593C) 1000 0.25 mg/kg 100 Plasma FVIII (% Normal) Plasma FVIII (% Normal) 0.25 mg/kg 0.5 mg/kg 0.5 mg/kg 100 1 mg/kg 10 0.25 mg/kg or 0.5 mg/kg + 1 mg/kg 2 mg/kg 10 1 1 0.1 0.1 0 50 100 150 0
Slide 19: 2nd generation ctLNP strategy: stealth LNPs with targeting ligand
Our 2nd generation ctLNP strategy focuses on reducing overall opsonin driven distribution to create stealth LNPs capable of directing with a targeting ligand ApoE-mediated trafficking Reduce off-target clearance X X Myeloid cells (Spleen, circulating monocytes, Kupffer cells in liver) Active, ligand
Slide 20: Stealth LNPs lack cell uptake across a number of cell types
Stealth LNPs lack cell uptake across a number of cell types after serum incubation Macrophages 1 ARPE19 2 cells LSECS 3 Hepatocytes 4 THP1 human monocytes Serum starved ARPE19 cells DiD labeled LNPs incubated Primary mouse hepatocytes were exposed to DiD LNPs differentiated into phagocytic upregulat
Slide 21: Lipid components screened and optimized for stealth profile
Lipid components rationally screened and optimized for ‘stealth’ profile with long blood t1/2 and minimal non-specific uptake without targeting Lipid SAR identified key determinants Reduced opsonin driven uptake to hepatocytes of enhanced half-life Stealth LNP Blood PK Stealth LNP Uptake in Primary
Slide 22: Lead GalNAc-targeted ctLNP attributes
Our lead GalNAc-targeted ctLNP embodies attributes informed by systematic approach to optimization Low non-specific clearance Ligand-directed biodistribution Cell-selective delivery leads to allows for GalNAc-directed drives on-target delivery and reduced immune cell activation hepatic extraction ex
Slide 23: scFv targeting to ASGPr provides improved alternative to GalNAc
Working towards more selective ligands: scFv targeting to ASGPr provides improved alternative to GalNAc Selectivity vs. GalNAc Conjugation to stealth LNP In vivo POC (ceDNA cargo) scFv-asgpr hASGPR 10 10 10 9 Flux [ph/s] 10 8 10 7 10 6 10 5 Ligand hASGPR CD301 KD (nM) KD (nM) Primary Hepatocyte mRNA
Slide 24: Cell-targeted LNPs avoid clearance by liver and spleen
Cell-targeted LNPs avoid clearance by liver and spleen to reach new targets Lipid Nanoparticles Clearance Organs Systemic Circulation Traditional LNPs Clearance by liver and spleen No systemic circulation Stealth Avoid liver and spleen Available for selective targeting 24
Slide 25: We're not the only ones interested in extrahepatic delivery
We’re not the only ones interested in extrahepatic delivery James Dahlman – barcoded libraries Dan Siegwart – 5th lipid component to Weissman/Parhiz – anti-CD5 targeted for empirical screening control distribution (SORT) LNPs for T cell delivery 25
Slide 26: Targeting platform is a modular system built on stealth LNP technology
Targeting platform is a modular system built on our foundational stealth LNP technology, capable of targeting a wide variety of extra-hepatic tissues ctLNP Targeting Platform Select Immune Cell Targeting Small format ligand ONCOLOGY Ligand discovery, panning, IMMUNOLOGY cell biology for INFECTIOUS D
Slide 27: Overcoming the nuclear barrier: ceDNA overview
Overcoming the nuclear barrier: ceDNA overview CONFIDENTIAL
Slide 28: Closed-end DNA (ceDNA) identified as capsid-free DNA structure for gene transfer
Closed-end DNA (ceDNA, originally termed CELiD) identified as a novel, recombinant AAV derived, capsid-free DNA structure for gene transfer ceDNA AFM image GFP expression in HEK293 cells 28
Slide 29: ceDNA accumulates in the nucleus in vitro and in vivo
ceDNA accumulates in the nucleus in vitro and in vivo In vitro – myocyte microinjection experiment In vivo – Quantification of nuclear copies by Halo • All constructs express well when injected directly into • Sectioned liver stained with ISH probes to nucleus ceDNA • ceDNA demonstrates much higher
Slide 30: ceDNA exhibits enhanced potency and tolerability over pDNA
ceDNA exhibits enhanced potency and tolerability over pDNA after ctLNP delivery in vivo In vivo Expression In vivo Tolerability Luciferase Activity Acute Body Weight Change (CD-1 Mice; Gen1 ctLNP; IV administration; Day 7) (CD-1 Mice; Gen1 ctLNP; IV administration; 24hr) 10 10 5 2mpk 0.5mpk 2mpk 0.5
Slide 31: What does the literature teach us about nuclear targeting of DNA?
What does the literature teach us about nuclear targeting of DNA? Behr et al. – ligating NLS peptide Dean et al. – DTS inclusion Cautionary counterpoints 31
Slide 32: Advantages of increased capacity of ceDNA for large constructs
Advantages of increased capacity of ceDNA for large constructs 1 2 3 4 Improved Expression Large transgenes Multiple transgenes Regulatable expression • Larger promoters/ • ABCA4 for Stargardt’s • Single constructs that • Large enhancer and enhancers and/or Disease encode for both heavy promoter reg
Slide 33: Unique RES process builds scale and drives structural DNA innovation
Unique RES process builds scale and is a driver of structural DNA innovation GLOBAL SCALE RAPID ENZYMATIC SYNTHESIS MATCHES SCALE OF PLATFORM POTENTIAL RES LARGE SCALE RES cGMP Efficiency Quality Speed MANUFACTURING Flexible, modular cGMP Consistent purity Robust, short cycle times manufacturing at
Slide 34: FVIII ceDNA optimization using semi-combinatorial construct screening
FVIII ceDNA optimization using a semi-combinatorial construct screening strategy • Rational design utilizes design rules and assumptions of module independence to efficiently screen construct variants • We generated and screened a semi-combinatorial library of > 300 constructs with following objecti
Slide 35: Semi-combinatorial optimization yields >30-fold increase in FVIII expression
A semi-combinatorial optimization strategy leverages the larger size capacity of ceDNA to engineer construct with > 30-fold increase in FVIII expression in vivo A) B) + TAKE HOME + • GB1 is the culmination of a semi- PBS combinatorial construct optimization 100 100 GB1 process using ceDNA Normalized
Slide 36: Thank you
Thank you QUESTIONS & ANSWERS