Gene Medicines for Exon Skipping

Transcription

1 Gene Medicines for Exon Skipping Steve Wilton, Penny Harding and Sue Fletcher Experimental Molecular Medicine Group & the NDC Genotyping Facility Centre for Neuromuscular and Neurological Disorders University of Western Australia

2 DystrophiN

3 DysX

4 DysX

5 Dys-rophiN

6 Normal Dystrophin: a molecular shock absorber DystrophiN

7 Duchenne MD: a broken shock absorber DysX

8

9

10

11 AOs by-pass defective instructions and allow semi-functional protein to be made

12 Induced dystrophin is still defective but should be better than nothing

13 Induced dystrophin could be of near normal function in some cases.

14 Exon skipping and DMD Exon skipping can not cure DMD May reduce severity some mutations should respond better than others more efficient exon skipping more functional protein being induced Not applicable to all dystrophin mutations large genomic deletions Dy N loss of crucial binding domains Dystrophi

15 Mild BMD cases. Exonic Deletion 3-9 * 9-22 * * * Special comments Playing competitive badminton at age 62 years. High CK, myalgia but well developed musculature and no evidence of muscle weakness Myalgia and cramps after normal activity Very mild BMD Source of the dystrophin mini-gene used in gene replacement studies Mild BMD with congenital cataracts Cramping after soccer or mountain climbing Elevated CK, otherwise asymptomatic Diagnosed age 60 Elevated CK, otherwise asymptomatic Reference Heald et al, 1994 Gospe et al, 1989 Ishigaki et al, 1996 Morandi et al, 1995 England et al, 2001 Mirabella et al, 1998 Koenig et al, 1989 Comi et al, 1994 Bosone et al, 2001 Comi et al, 1994

16 (only 3% deletions found from exon 55) Mild BMD cases. Exonic Deletion 3-9 * 9-22 * * * Special comments Playing competitive badminton at age 62 years. High CK, myalgia but well developed musculature and no evidence of muscle weakness Myalgia and cramps after normal activity Very mild BMD Source of the dystrophin mini-gene used in gene replacement studies Mild BMD with congenital cataracts Cramping after soccer or mountain climbing Elevated CK, otherwise asymptomatic Diagnosed age 60 Elevated CK, otherwise asymptomatic Reference Heald et al, 1994 Gospe et al, 1989 Ishigaki et al, 1996 Morandi et al, 1995 England et al, 2001 Mirabella et al, 1998 Koenig et al, 1989 Comi et al, 1994 Bosone et al, 2001 Comi et al, 1994

17 Antisense jargon Antisense oligonucleotides chemically synthesized genetic fragments complementary to specific RNA very specific genetic bandaid for dystrophin Binds to mrna (sense strand) Different chemistries available 2OMeAO Morpholino MOE

18 B a s e AO Chemistries B a s e Different chemistries confer different properties B a s e B a s e Different quality bandaids A U - increased stability and hence - persistence of action - rates of uptake - side effects G C T C G A Long term effects of any chemistry are not known

19 pre-mrna Splicing

20 pre-mrna Splicing

21 pre-mrna Splicing

22 pre-mrna Splicing

23 pre-mrna Splicing Cartegni L., Wang J., Zhu Z., Zhang M. Q., Krainer A. R.; ESEfinder: a web resource to identify exonic splicing enhancers, Nucleic Acid Research, 2003, Vol. 31, No

24 AO masking of splicing motifs Cartegni L., Wang J., Zhu Z., Zhang M. Q., Krainer A. R.; ESEfinder: a web resource to identify exonic splicing enhancers, Nucleic Acid Research, 2003, Vol. 31, No

25 AO masking of splicing motifs Cartegni L., Wang J., Zhu Z., Zhang M. Q., Krainer A. R.; ESEfinder: a web resource to identify exonic splicing enhancers, Nucleic Acid Research, 2003, Vol. 31, No

26 AO masking of splicing motifs Cartegni L., Wang J., Zhu Z., Zhang M. Q., Krainer A. R.; ESEfinder: a web resource to identify exonic splicing enhancers, Nucleic Acid Research, 2003, Vol. 31, No

27 AO masking of splicing motifs Cartegni L., Wang J., Zhu Z., Zhang M. Q., Krainer A. R.; ESEfinder: a web resource to identify exonic splicing enhancers, Nucleic Acid Research, 2003, Vol. 31, No

28 AO masking of splicing motifs Cartegni L., Wang J., Zhu Z., Zhang M. Q., Krainer A. R.; ESEfinder: a web resource to identify exonic splicing enhancers, Nucleic Acid Research, 2003, Vol. 31, No

29 AO induced exon skipping Cartegni L., Wang J., Zhu Z., Zhang M. Q., Krainer A. R.; ESEfinder: a web resource to identify exonic splicing enhancers, Nucleic Acid Research, 2003, Vol. 31, No

30 Dystrophin deletions of exons 3-7 (minor deletion hotspot)

31 AO Induced exon 8 (& 9*) skipping for exon 3-7 deletion *

32 Restored reading frame for 3-7 dystrophin deletions Exon 3-9 deletion associated with a very mild BMD phenotype 2 10

33 Genomic Duplications (Exons 6 and 7)

34 Addressing Exon 6 and 7 Duplications Targeting exons 6 and

35 Addressing Exon 6 and 7 Duplications Targeting exons 6 and 7... reading frame still disrupted

36 Targeting one more exon (8) *

37 Tailored treatment to address duplications (Exons 6 and 7)

38 Exon skipping to address other DMD mutations Deletion of exons 49 &50 disrupts the reading frame

39 Exon 51 skipping restores the reading frame

40 Exon 51 skipping restores the reading frame

41 Exon 51 skipping will not address nonsense mutations in that exon

42 Exon 51 skipping disrupts the reading frame

43 Target adjacent exon to restore the reading frame

44 Exon 51 & 52 skipping restores reading frame

45 Challenges for Exon Skipping Proof of Principle: Safety Studies and Clinical Trials Delivery AO Design for all targets Long term safety studies and application to all responsive DMD mutations Establishing dosage regimen in response to different induced dystrophins

46 Where we were July last year... Morpholinos seemed better than other chemistries Intramuscular injections work but are not practical Systemic delivery is needed for whole body treatment

47 RNA like AOs Morpholino AOs O O B a s e O O B a s e O H O - R -CH 3 (2OMe) -CH 2 -CH 2 -O-CH 3 (MOE) N O P S P N O O B a s e O O B a s e H O O - R N P N

48 Antisense chemistries Morpholino AOs are more stable than 2OMeAO Morpholino delivery limitations in vitro Once in better biological action 300 nm Morph lipoplex 300 nm 2OMeAO lipoplex

49 Western blotting of mdx muscle after im injection naked 2OMeAO, morpholino and PNA

50 Dystrophin expression : 6 weeks after injection of morpholino AO into TA of 11-day old mdx mouse 4x 10x inset 8% central nuclei 22% central nuclei

51 Enhanced morpholino injected into mdx mice 14 days after single ip injection, 10mg/kg

52 Enhanced morpholino* single ip injection into neonatal mdx mice: 2 weeks post single injection Dose 1mg/kg 2mg/kg 5mg/kg Diaphragm 10 mg/kg 25mg/kg mdx u/t Unfixed cryosections stained for dystrophin -Dys2 and Zenon AlexaFluor 488

53 Enhanced morpholino*: four ip injections into neonatal mdx mice: 5 mg/kg/week for 4 weeks Diaphragm Tibialis anterior Triceps brachialis Gluteus maximus Colon Heart Quadriceps femoris mdx mouse CK of 200!! Unfixed cryosections stained for dystrophin -Dys2 and Zenon AlexaFluor 488 * cytoporter, from AVI Biopharma

54 Morpholinos Can be made to clinical grade Uncharged backbone VERY stable Not metabolised Already in clinical trials as antiviral compounds (AVI Biopharma) Impressive safety record 50, 100, 300 mg doses of anti Hepatitis PMO administered over 2 weeks (subcutaneous, daily) with no adverse events (10 Jan, 06)

55 Issue of other dystrophin exons Full patent lodged 6th Jan, 2006 Over 200 antisense oligonucleotide sequences listed Includes selection of cocktails and weasels

56 AO design: there are no targeting rules! Predicted ESE sites Human exon p 200M 100M 40nM 25nM 10nM u/t neg 100p 200M 100M 40nM 25nM 10nM u/t neg 100p 200M 100M 40nM 25nM 10nM u/t neg 716 bp 536 bp H16A bp 536 bp H16A bp H16A-20mer

57 AO design: there are no targeting rules! Predicted ESE sites Human exon p 200M 100M 40nM 25nM 10nM u/t neg 100p 200M 100M 40nM 25nM 10nM u/t neg 100p 200M 100M 40nM 25nM 10nM u/t neg 716 bp 536 bp H16A bp 536 bp H16A bp H16A-20mer

58 AO design: there are no targeting rules! Predicted ESE sites Human exon p 200M 100M 40nM 25nM 10nM u/t neg 100p 200M 100M 40nM 25nM 10nM u/t neg 100p 200M 100M 40nM 25nM 10nM u/t neg 716 bp 536 bp H16A bp 536 bp H16A bp H16A-20mer

59 Type 1 Exon Skipping +++ greater than 30% at 100nM

60 Type 2 Exons Skipping ++ (moderate: 10-30% at 100 nm) Exon 21 Exon 36

61 Type 3 Exon Skipping + (poor: less than 10% at 100 nm) Exon 26 More work needed or Plan B?? Exon 76

62 Type 4 Exon Skipping: Special cases UT Two Exon 20 AOs used individually Consistently inconsistent exon skipping * *

63 Type 4 Exon Skipping: AO Cocktails L2K UT neg Exon 20 AOs individually Cocktail of 2 AOs targeting exon 20

64 Multi-exon skipping with AO cocktails (human cells) ut neg Exon 20 AOs individually L2K ut neg Exon 19 and 20 excision with 3AOs (19 and 20:20)

65 Multi-exon skipping in GRMD Have removed exon 6 and 8(+9) in GRMD model (McClorey et al, 2006) UT M UT

66 Multi-exon skipping Have removed exon 6 and 8(+9) in GRMD model (McClorey et al, 2006) UT M UT Could obtain some dystrophin protein in cultured cells only after morpholino treatment!! 5 10

67 Multi-exon skipping in mdx mice Have induced exon skipping in mdx mice morpholino resulted in protein produced in vivo. (Fall et al, 2006)

68 Multi-exon skipping Have induced exon skipping in mdx mice (morpholino only). (Fall et al, 2006) Found occasionally in untreated mice (revertant fibres)

69 Exon skipping described 2005 Exon / * u/t 18 u/t Exon u/t 24 u/t 25 u/t Exon u/t u/t u/t 49 u/t

70 Since then... Every exon can now be skipped (2-78) over 460 AOs tested for human dystrophin pre-mrna Some exons removed more efficiently than others Types 1, 2, 3 and 4. All exons will be removed at a high efficiency How efficiently is yet to be established

71 Exon Skipping June 2006 Exon / / Exon Exon Exon Exon

72 Exon skipping Summary Proof of Principle / Safety Studies and Clinical Trials ongoing Exon skipping confirmed in animal models, human cells and human muscle explants (McClorey et al, 2006) safety studies for morpholinos (unrelated sequences) show these compounds are well tolerated

73 Exon skipping Summary Proof of Principle Safety Studies & Clinical Trials Delivery intramuscular injections work well systemic delivery is possible and promising in mice Unrelated morpholinos have been administered to humans adults short term treatment

74 Exon skipping Summary Proof of Principle/Safety Studies & Clinical Trials Delivery AO Design for all targets all dystrophin exons can be now be skipped variable efficiency of removal expect all will be optimized

75 Exon skipping Summary Long term safety studies and application to all responsive DMD mutations Establishing dosage regimen in response to different induced dystrophins can only be addressed after trials started

76 Acknowledgements EMMG Sue Fletcher Kaite Honeyman Russell Johnsen Abbie Fall Penny Harding Marisa Gibbs Graham McClorey Josh Steinhaus Heidi Madden Cath Coleman Chalermchai Mitrpant Jodie King Australian Andrew Hoey Kathy North Andrew Kornberg Monique Ryan Frank Mastaglia John Rasko Phillipa Lamont Alan Emery*

77 Acknowledgements International Anthony Akkari (USA) Mike Sheehan (UK) Allen Roses (USA) Tony Morris (UK) Eric Lai (USA) Lefkos Middleton (UK) Francesco Muntoni (UK) Kate Bushby (UK) Ryszard Kole (USA) Dominic & Kim Wells (UK) Pat Iversen & Denis Burger (AVI USA) Kevin Flanigan (USA) Jerry Mendell (USA) Jeff Chamberlain (USA) Joe Kornegay (USA) Vincenzo Nigro (Italy) Woon Chee Yee (Singapore) Rakesh Patel (NZ) Pat Furlong (USA) Support from : GSK, NHMRC, MDA USA, MDA WA, PPMD, AVI Biopharma, NIH, Neil Brandom,Trahar Foundation, Jett Foundation, Aktion benni & co, MHRIF.