Therapeutic RNA delivery

Transcription

1 Therapeutic RNA delivery 05/02/2017 TIDES Dr. Adrien Weingärtner Head of Technology Development

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3 LNP delivery of RNA Lipid nanoparticle approach sirna mrna Gene editing payloads (CRISPR/Cas9) Vasculature Liver Macrophages 3

4 Time course and biodistribution of GN2205 functional luciferase mrna delivery % o f to ta l lu c ife ra s e a c tiv ity -19 days 0h 2h 4h 6h 8h 12h 24h 48h scid beige Hep3B (5x10 6 cells s.c.) IV administration of 1mg/kg mrna (FLuc; ᴪ; 5MeC; TriLink) (tumor size ~350mm³, n=6) ex vivo quantification of luciferase activity in tissue lysates lu c ife ra s e a c tiv ity r e la tiv e lu c ife r a s e a c tiv ity lu c ife ra s e a c tiv ity [p g /g tis s u e ] liv e r lu n g s p le e n k id n e y h e a r t b r a in a o r ta b o n e m a r r o w tu m o r 2 h 4 h 6 h 8 h h 4 8 h liv e r lu n g s p le e n k id n e y h e a r t b r a in a o r ta b o n e tu m o r m a r r o w This formulation essentially targets the liver 4

5 ITG B 6 /actin P D G F R b /a c tin G F A P /a c tin C D 6 8 /a c tin T T R /a c tin T ie 2 /a c tin GN2205 targets multiple cell types in the liver 0h 48h h e p a to c y te (T T R - liv e r) e n d o th e lia l c e lls (T ie 2 - liv e r) IV administration 0.5 mg/kg sirna (Silence) C57BL/6 (n=4-6) RT-PCR quantification of transcripts b u ffe r s it T R s il u c m g /K g b u ffe r s it ie 2 s ig F A P m g /K g s te la te c e lls (G FA P - Liver) m a c ro p h a g e (C D L iv e r) b u ffe r s it ie 2 s ig F A P b u ffe r s ic D 6 8 s ic D 4 5 m g /K g m g /K g c h o la n g io c y te (IT G B 6 - L iv e r) p e ric y te s (P D G F receptor beta - Liver) b u ffe r s ic o l1 a 1 s iit G B 6 m g /K g Physiol Rev 88: , 2008; doi: /physrev b u ffe r s ip D G F R a s ip D G F R b m g /K g 5 This formulation targets many cell types in the liver

6 T ie 2 /a c tin T ie 2 /a c tin T ie 2 /a c tin T ie 2 /a c tin T ie 2 /a c tin GN2205 targets vascular cells of liver only 0h 48h IV administration 0.5 mg/kg sirna (Silence) C57BL/6 (n=4-6) RT-PCR quantification of transcripts L iv e r T ie 2 (e n d o th e lia l c e lls ) 2.0 S p le e n T ie 2 (e n d o th e lia l c e lls ) b u ffe r s it ie 2 s ig F A P b u ffe r s it ie 2 s ig F A P m g /K g m g /K g L u n g T ie 2 (e n d o th e lia l c e lls ) K id n e y T ie 2 (e n d o th e lia l c e lls ) H e a r t T ie 2 (e n d o th e lia l c e lls ) b u ffe r s it ie 2 s ig F A P b u ffe r s it ie 2 s ig F A P b u ffe r s it ie 2 s ig F A P m g /K g m g /K g m g /K g This formulation targets endothelial cells in liver only 6

7 GN2205 RNA delivery platform Payloads: mrna sirna Liver target cells: x Hepatocytes Endothelial cells Stellate cells Macrophages Pericytes Cholangiocytes The LNP is suitable for RNA payloads and targets multiple liver cell types. 7

8 GN2205 co-delivery of guide RNA & spcas9- mrna m T T R [p g /m l] m P C S K 9 [p g /m l] day 1 day 35 day 63 day 91 day 119 day 147 day 175 day 203 day 238 day 266 day 294 IV administration of 1mg/kg and 0.5 mg/kg mrna & guide RNA (Cas9-mRNA: ᴪ & 5MeC, TriLink; guide RNA: Silence) c57bl/6 (n=6) quantification of serum protein at indicated time points by standardized ELISA & final genome analysis m T T R m P C S K d p i d p i d p i 6 3 d p i 9 1 d p i d p i d p i 6 3 d p i 9 1 d p i d p i d p i d p i d p i d p i d p i d p i c o n tr o l T T R P C S K 9 d p i d p i c o n tr o l T T R P C S K 9 d p i d p i C a s 9 m R N A C a s 9 m R N A Long lasting partial knock out of liver expressed genes achieved with an entirely RNA based approach, using our LNP delivery system 8

9 Summary LNP delivery approach We have developed a liver targeting LNP-formulation that targets multiple cell types Our LNP is suitable for efficient delivery of both sirna and mrna payloads Co-delivery of Cas9 mrna and guide RNA results in long lasting gene disruption in vivo 9

10 New Focus: sirna conjugates LNP approach Conjugation approach sirna mrna Gene editing payloads (CRISPR/Cas9) sirna w/ GalNAc cluster Vasculature Liver Macrophages Hepatocytes 10

11 Hepatocyte delivery by GalNAc Scheme based on information provided by Essentials of Glycobiology. 2nd edition. Varki A, Cummings RD, Esko JD, et al., editors. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; GalNAc linked to sirna GalNAc binds to a highly expressed ASGP receptor in hepatocytes, which is endocytosed and can shuttle cargo into the cytoplasm of the cell ASGPR is conserved across species GalNAc-siRNA conjugates can be administered by subcutaneous administration 11

12 Key considerations for GalNAc-siRNA sirna payload extensively chemically modified with 2 -O-Me, 2 -F, PS & other to stabilized against nucleases in biofluids and cytoplasm to minimize immune stimulation to improve overall performance (therapeutic window) Linker with optimized length and structure stabilized against degradation GalNAc GalNAc moiety polyvalent optimized for efficient transfer to the liver and hepatocytes 12

13 Knockdown of Target X using GalNAc-siRNA conjugates Target2/Actin X / Target2/PTEN X / Actin In vitro In vivo 0h 24h 0h 48h Murine 1 Hepatocytes GalNAc-siRNA conjugate RT-PCR quantification of transcripts s.c. administration GalNAc-siRNA conjugate c57bl/6 (n=4) RT-PCR quantification of transcripts 1,4 Target X gene expression 24h post treatment (mean ± SD) 1,4 Target X gene expression 48h post treatment (mean ± SD) 1,1 1,1 0,7 0,7 0,4 0,4 0,0 100nM 10nM 1nM 10nM ut GalNAc Target2X GalNAc control1 0,0 10mg/kg 3mg/kg 1mg/kg 10mg/kg Buffer GalNAc Target2 GalNAc GalNAc Target X control3 13

14 Duration of knockdown in mice (2nd vs 3rd gen. GalNAc) Target Y / PTEN day 0 day 7 day 14 day 21 day 28 2 nd & 3 rd gen. GalNAcsiRNA, s.c., 2 mg/kg c57bl/6 (n=4) RT-PCR quantification of transcripts 1,5 1,0 0,5 0,0 dpi 7 dpi 7 dpi 7 dpi 14 dpi 21 dpi 28 dpi 7 dpi 14 dpi 21 dpi 28 untreated control- GalNAc 2nd Duration of Knockdown: 2nd vs 3rd generation GalNAc (mean ± SD, knockdown in %) -59% -40% -15% -13% TargetX-GalNAc Y-GalNAc 2nd -94% -87% -62% TargetX-GalNAc Y-GalNAc 3rd -40% Improved knockdown achieved with 3 rd generation GalNAc-linker 14

15 Dose Response to GalNAc-siRNA (3 rd gen) in mice Target Y / PTEN 0h 48h 3 rd gen. GalNAc-siRNA, s.c., 0.1 to 3 mg/kg c57bl/6 (n=4) RT-PCR quantification of transcripts 1,5 1,0 Target gene expression in mouse liver 48h post s.c. application of indicated dose (mean ± SD, knock down in %) -32% -5% 0,5-94% -78% 0,0 buffer 3.0mg/kg 1.0mg/kg 0.3mg/kg 0.1mg/kg Target Y-GalNAc X 3rd 3rd Decent dose response with 3 rd generation GalNAc-linker 15

16 Long-lasting knockdown with 3 rd gen. GalNAc sirna conjugates in NHP..weekly.. day -7 day 0 day 4 day 7 day 14 day 84 day 91 s.c. administration of 3 rd gen. GalNAc-siRNA,1 and 0.3 mg/kg (n=4) quantification of serum protein Target Y at indicated time points Duration and dose response of knockdown translates from mouse to NHP 16

17 Safety parameters of 3 rd generation GalNAc conjugate in NHP Changes in cytokine levels expressed as fold ( ) of the predose group mean values GalNAc-Target Y GalNAc-control Cytokine 0.3 mg/kg b.w. 1.0 mg/kg b.w. 1.0 mg/kg b.w. males females males females males females IL-7 3 none none 2 none none 2 h after s.c. IL administratio Day 1 MIP-1b 3 3 none none none none 6 h after s.c. IL none none none 2 administratio MIP-1b none 2 none none 0.6 none Day 2 IL none h after IL none none none administratio MIP-1b none none Day 4 IL-7 none none h after IL-8 none none none none administratio MIP-1b 0.7 none 0.3 none 0.4 none no altered pro-inflammatory cytokines (IL-7, IL-8, MIP-1) no skin reaction no behavioral conspicuousness Well tolerated in higher species 17

18 Our pipeline Our programmes Out-licensed programmes (AtuRNAi TM ) 18

19 Summary GalNAc conjugates Progress made in our new focus area: Improved GalNAc linker results in increased potency and duration of effect GalNAc-siRNA conjugates successfully translated from mouse to NHP Well tolerated in all species tested Established a GalNAc-siRNA based pipeline across several therapeutic areas (included rare and metabolic) 19

20 Acknowledgements Silence Berlin & London Team 20

21 Thank you!