Non-viral Delivery of ZFN mrna Enables Highly Efficient In Vivo Genome Editing of Multiple Therapeutic Gene Targets

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1 Non-viral Delivery of ZFN mrna Enables Highly Efficient In Vivo Genome Editing of Multiple Therapeutic Gene Targets Anthony Conway, Ph.D. May 19, 2018

2 Outline Engineered zinc finger nuclease (ZFN) technology Lipid nanoparticles (LNP) for non-viral delivery of ZFN mrna Targeting the liver for potential therapeutic applications Single gene knockout In Vivo Protein Replacement Targeting lung epithelial cells via LNP 2

3 ZFNs enable genome editing Double Strand Break Insertion Non-Homologous End Joining (NHEJ) Homologous Recombination DNA Donor with Homology Deletion Gene Knockout Targeted Integration 3

4 Lipid nanoparticle (LNP) technology for efficient in vivo non-viral delivery of ZFN mrna to hepatocytes ~80 nm Alternative to AAV Delivery No pre-existing immunity Transient mrna delivery/expression Enables repeat dosing ApoE facilitates receptor binding, endocytosis in hepatocytes Endosomal escape of mrna by amino lipid protonation ApoE binds after loss of PEG-lipid 4

5 ZFN mrna delivery via LNP allows for accumulation of genome modification within the murine liver following repeat administration P < Linear regression analysis P < Progressive increase in indels out to 6 repeat doses 5

6 mrna-lnp primarily traffics to liver cells LNP PBS Genome editing highly targeted to liver Residual nucleated blood cells lower overall genome editing fraction in unperfused bulk liver 6

7 Systemic delivery of liver-targeted ZFP therapeutics via mrna-lnp for single gene knockout applications Protein Expression Indels (%) LNP Packaging ZFN mrna Repeat Administration Progressive Gene Knockout Lipids Therapeutic Level Doses Targets Include TTR, PCSK9 7

8 ZFNs targeting murine TTR delivered via mrna-lnp in mice achieves >90% gene disruption from the liver 60% gene knockout in bulk liver tissue 90% TTR protein knockdown in plasma 60% 90% 0.8 mg/kg dose 8

9 Optimized ZFNs targeting murine TTR results in highly efficient gene disruption at very low mrna-lnp doses with no signs of toxicity >60% gene knockout in bulk liver tissue No elevated liver toxicity ALT AST 4-Fold Lower Dose 9

10 ZFNs targeting murine PCSK9 delivered via mrna-lnp achieved >90% protein knockdown from the liver ZFNs targeting murine PCSK9 delivered as mrna-lnp also achieved >90% protein knockdown in plasma 10

11 ZFNs enable genome editing Double Strand Break Insertion Non-Homologous End Joining (NHEJ) Homologous Recombination DNA Donor with Homology Deletion Gene Knockout Targeted Integration 11

12 Systemic delivery of ZFP Therapeutics via AAV vectors may allow for in vivo correction of monogenic disease Currently Recruiting Patients in U.S. Hemophilia B (SB-FIX) MPS I (SB-318) MPS II (SB-913) 12

13 Lysosomal storage diseases MPS I and MPS II Glycoaminoglycans (GAGs) - Cognitive decline Iduronate 2-Sulfatase (IDS) in MPS II a-l-iduronidase (IDUA) in MPS I - Organomegaly - Cardiomyopathy - Respiratory complications - Carpal tunnel - Joint stiffness - Skeletal and orthopedic issues - Short stature Accumulation of GAGs (e.g., dermatan and heparan sulfates) in the lysosome of all cells leads to dysfunction in several tissues 13

14 GAG Levels (µg GAG/mg protein) IDS activity (nmol/hr/mg) IDS activity (nmol/hr/ml) ZFNs and hids transgene donor both delivered via AAV results in high levels of IDS protein secretion from the liver and significant GAG reduction in MPS II mice Mouse Plasma # Mouse Tissues (4 months) # Days post-injection # # ZFN+Donor vs. MPS II Untreated P-values: P < 0.01; # P <

15 Systemic delivery of ZFN mrna and transgene donor AAV vectors may allow for in vivo correction of monogenic disease Protein Expression Targeted Insertion (%) Packaging Delivery Progressive Targeted Gene Insertion LNP: ZFN mrna ZFN 1 One-time peripheral IV administration of donor vector ZFN 2 AAV Vector Repeat administration of ZFN mrna Therapeutic Level Homology hids Homology Doses Targets Include IDS, IDUA, FIX 15

16 Repeat dosing of ZFN mrna-lnp with a single dose of hids donor AAV leads to increasing levels of IDS enzymatic activity in mice Plasma IDS Enzymatic Activity (nmol/hr/ml) ~2 to 3-Fold Improvement Per Dose 0 16

17 Gene Regulation Genome Editing Gene Therapy Sangamo Technology Platform Versatile Delivery Gene Addition Tissue-Specific Viral & Non-Viral Gene Knockout ZFNs In Vivo Targeted Integration Gene Correction H TG Oligo H Liver CNS Lung Epithelium Hem. B, MPS I & II Tau, C9ORF72 CF Repression Ex Vivo Activation T / NK Cells HSCs Airway SCs Oncology, HIV β-thal/sca, HIV CF 17

18 Experimental Design: Evaluating lung-targeted LNP in mice mrna-lnp Delivery Collect Organs Lung Single Cell Dissociation and Sorting Analyze Genome Editing Epithelial Cells (CD326) mrna ZFNs targeting CFTR intron 1 14 Days Route Tail vein i.v. (200 ul, 1 injection) Delivery reagent Invivofectamine Lung (ThermoFisher Scientific) Liver Bulk Tissue 18

19 >10% genome editing of lung epithelial cells following i.v. delivery of ZFN mrna-lnp in wildtype mice Lung epithelial cells 19

20 Editing occurs preferentially in lung compared to liver Lung epithelial cells Bulk liver tissue 20

21 LNP delivery enables efficient ZFN-mediated genome editing of multiple therapeutic gene targets mrna-lnp delivery of ZFNs targeting TTR and PCSK9 results in >90% circulating protein knockdown in wildtype mice Repeat dosing of ZFN mrna-lnp with a single human IDS AAV donor dose enables progressively increasing enzymatic activity levels in mouse plasma mrna-lnp delivery of ZFNs targeting CFTR achieves >10% genome modification in wildtype mice lung epithelial cells following a single dose Ongoing work in non-viral in vivo genome editing field: Achieve therapeutically relevant levels of genome modification with an acceptable toxicity profile in non-human primate models 21

22 Research Matt Mendel Ken Kim John Jascur Gary Lee Mike Holmes Russ DeKelver Tech Ops AAV Team Clinical Ed Conner Development Acuitas Therapeutics Thermo Fisher Scientific Kathy Meyer Barb Mui Shikha Mishra Carolyn Gaspar Ying Tam Xavier de Mollerat du Jeu Lisa King Paulo Lin Technology Chris Barbosa Intellectual Property Ed Rebar Tom Redelmeier Susan Abrahamson Lei Zhang Tom Madden Dave Paschon Executive Project Management Jeff Miller Sandy Macrae Rainier Amora Miseq Team Production Team Acknowledgements