Little Steps Aartsma-Rus Rare Disease Israel 2012

Exon skipping for DMDAnnemieke Aartsma‐Rus

February 29 2012

Annemieke Aartsma-RusDepartment of Human Genetics 2

Duchenne Muscular Dystrophy (DMD)

Annemieke Aartsma-RusDepartment of Human Genetics 3

Becker Muscular Dystrophy (BMD)

Age

Clin

ical

sym

ptom

s B

MD

Genes and proteins

Annemieke Aartsma-Rus

•• ProteinsProteins building building blocksblocks of of ourour bodybody

•• GenesGenes containcontain blueprintblueprint forfor proteinsproteins

•• MistakeMistake in gene in gene mistakemistake in in proteinprotein

•• GenesGenes have a volume switch have a volume switch

OnlyOnly onon in in necessarynecessary tissuetissue

•• Dystrophin Dystrophin proteinprotein has has functionfunction in in musclemuscle

•• MistakeMistake in dystrophin in dystrophin musclemuscle problemsproblems

Muscles


•• 3030‐‐40% of 40% of ourour body body consistsconsists of of musclemuscle

•• MusclesMuscles cancan growgrow biggerbigger and smallerand smaller

•• MusclesMuscles useuse lot of lot of energyenergy

•• OnlyOnly maintainedmaintained whenwhen neededneeded

•• MusclesMuscles damageddamaged afterafter excessiveexcessive exerciseexercise

•• MusclesMuscles veryvery efficientefficient at at repairingrepairing damagedamage

BiggerBigger whenwhen neededneeded

Muscles


Muscle fibers


SkeletonSkeleton

Connective tissueConnective tissue

Dystrophin


DystrophinDystrophin

Genes and proteins


•• ProteinsProteins building building blocksblocks of of ourour bodybody

•• GenesGenes containcontain blueprintblueprint forfor proteinsproteins

•• GenesGenes consistconsist of DNAof DNA

•• CellCell cancan translate DNA code translate DNA code intointo proteinprotein

Gene Protein

Annemieke Aartsma‐Rus

DNA

Protein factory

Temporary gene copy (RNA)

Cell nucleus

Cell body(cytoplasm)

Dystrophin gene


Splicing


Exons Introns1

35

6

7 Gene (DNA)

RNA copy (pre mRNA)

messenger RNA

1 - - - - - - - - - 79

dystrophin protein

Splicing

2

4

3 4 5 6 71 21 2 3 4 5 6 7 8

Duchenne: reading frame disrupted


Duchenne: reading frame disrupted


Protein translation stops prematurelyProtein translation stops prematurely

Dystrophin not functionalDystrophin not functional

Exon 46 Exon 47 Exon 51 Exon 52?

Becker: reading frame maintained


Becker: reading frame maintained


Protein translation continuesProtein translation continues

Dystrophin partly functionalDystrophin partly functionalLess damageLess damage

Exon 46 Exon 47 Exon 52 Exon 53

Exon skipping: restore reading frame


Exon 51 skipping (del exon 48Exon 51 skipping (del exon 48‐‐50)50)

AnnemiekeAnnemieke AartsmaAartsma‐‐RusRus

52524747

525251514747

MM NTNT 5151 ‐‐RTRT HCHC0h0h 4h4h 8h8h 16h16h 24h24h 48h48h

NT 48 post transfection

MANDYS1 DYS2MANDYS1

Aartsma‐Rus HumMolGen 2003 12: 907‐14

Deletion exon 45‐55


‐AON

+AON +AON

Exon 44 skipping

Annemieke Aartsma‐Rus

‐AON

+AON +AON

MM NTNT 4444 ‐‐RTRT

44444343 5555

55554343

NTNT HC(1:10)HC(1:10)2d2d 4d4d 7d7d

NT

MANDYS1 DYS2

48 hours post transfection

MANDYS1

Double exon skipping


Double Exon Skipping


MM NTNT 45 &

45 &

5151 ‐‐ RTRT

4444 5252

4444 4545 5151 52524444 4545 5252

NTNT 2d2d 3d3d HCHC

Not Treated 48 uur post transfection

Aartsma‐Rus AmJHumGen 2004, 74: 83‐91

Mdxmouse


•• NaturalNatural mousemouse modelmodel

•• DisruptingDisrupting mutationmutation exon 23exon 23

•• Dystrophin Dystrophin deficiencydeficiency

•• MuscleMuscle functionfunction impairedimpaired

•• LessLess severesevere diseasedisease thanthan humanshumans

•• Exon 23 Exon 23 skippingskipping cancan restorerestore geneticgenetic code, code, dystrophin and dystrophin and leadsleads to to functionalfunctional improvementimprovementafterafter intramuscularintramuscular injectioninjection of AONsof AONs

Exon skipping

Annemieke Aartsma-Rus Alter et al. Nat Med 2006, 12: 175‐7

•• Exon Exon skippingskipping and dystrophin and dystrophin restorationrestorationconfirmedconfirmed in in patientpatient‐‐derivedderived cellscells ((deletionsdeletions, , duplicationsduplications, , smallsmall mutationsmutations))

•• Exon Exon skippingskipping, dystrophin , dystrophin restorationrestoration and and musclemusclefunctionfunction improvementimprovement confirmedconfirmed in in mdxmdx mousemousemodel and GRMD model and GRMD dogsdogs ((locallocal and and systemicsystemic!)!)

Applicability

Annemieke Aartsma-Rus Aartsma‐Rus Hum Mutat 2009, 30:293‐9

hotspot

AON chemistry


•• TwoTwo different different chemistrieschemistries developeddeveloped furtherfurther

•• ProsensaProsensa: 2: 2’’‐‐OO‐‐methylmethyl phosphorothioatephosphorothioate

•• AVIAVI‐‐BiopharmaBiopharma: : phosphorodiamidatephosphorodiamidate morpholinomorpholinooligomersoligomers

•• TargetingTargeting exon 51exon 51

First clinical trial – set up


Set upSet up

•• Single Single dosedose, single center , single center studystudy

•• 4 4 prepre‐‐screenedscreened DMD DMD patientspatients agedaged 88‐‐1616

•• Single IM Single IM injectioninjection PRO051 in PRO051 in shinshin musclemuscle

•• BiopsyBiopsy 4 weeks 4 weeks afterafter injectioninjection

•• Exon Exon skipskip? Dystrophin? Safe?? Dystrophin? Safe?

Pre‐screening Baseline Assessment Treatment Follow‐up Safety & Efficacy Assessment

Day ‐60 to ‐7 Day 0 Day 0 Day 1 to 28 Day 28 to 35

First clinical trial – pre‐screening


First clinical trial – analysis RNA



Department of Human Genetics 30


Department of Human Genetics 31Pt.3120 fibers

Pt.2725 fibers

Dystrophin quantification


Second intramuscular trial (AVI)

Annemieke Aartsma-Rus Kinali Lancet Neurol 2009, 8: 918‐28

•• TwoTwo doses doses testedtested (0.09 and 0.9 mg)(0.09 and 0.9 mg)

•• EDB EDB musclemuscle

•• Exon Exon skipskip in all dosesin all doses

•• Dystrophin Dystrophin restorationrestoration onlyonly forfor high high dosedose

Intramuscular trials


•• Exon Exon skippingskipping observedobserved in all in all patientspatients

•• No No toxictoxic effectseffects observedobserved!!

•• Dystrophin Dystrophin levelslevels veryvery comparablecomparable

1717‐‐35% 35% vsvs 2222‐‐32%32%

•• NumberNumber of dystrophin of dystrophin positivepositive cellscells comparablecomparable

6464‐‐97% 97% vsvs 4444‐‐79%79%

•• SystemicSystemic treatmenttreatment neededneeded

Systemic treatment


WT Mice

Mdx Mice

AON levels in muscle and Liver Exon 23 Skipping

Gastrocn.

Systemic treatment


AON Tissue Levels(ug/g)

Systemic treatment


TibialisTibialis AnteriorAnterior

02468

10121416

25 mg/kg 50 mg/kg 100 mg/kg

Dose

% e

xon

23 s

kipp

ing

Tissue Tissue levelslevels RNARNA

0

5

10

15

20

25


Dose

Tiss

ue le

vels

(ug/

g)

02

468

1012

1416


Dose

Dys

trop

hin

%

8 weeks4 weeks

ProteinProtein

SimilarSimilar resultsresults forfor all all skeletalskeletal musclesmusclesHeartHeart lowerlower exon exon skipskip and and proteinprotein levelslevels

Systemic trial 2OMePS (GSK/Prosensa)


GSK2402968 GSK2402968 (0.5, 2, 4 & 6 mg/kg)(0.5, 2, 4 & 6 mg/kg)

•• SubcutaneousSubcutaneous injectionsinjections (abdomen)(abdomen)•• WeeklyWeekly forfor 5 weeks5 weeks•• 3 3 patientspatients per per groupgroup•• BiopsyBiopsy taken 1 taken 1 monthmonth afterafter last last injectioninjection•• No No severesevere sideside effectseffects reportedreported

Open label Open label extensionextension studystudy initiatedinitiated•• All All patientspatients receivereceive 6 mg/kg/week 6 mg/kg/week subcutaneoussubcutaneous



DosePre‐Treatment

*Revertant Fiber

*

Dystrophin (ManDys 106)

Goemans et al, NEJM 2011, 364: 1513‐22


Annemieke Aartsma-Rus Goemans et al, NEJM 2011, 364: 1513‐22



•• Dystrophin Dystrophin restorationrestoration observedobserved in 10/12 in 10/12 patientspatients

•• 6060‐‐100% 100% musclemuscle fibers dystrophin fibers dystrophin positivepositive

•• Dystrophin Dystrophin levelslevels up to 15.6% of up to 15.6% of controlcontrol

•• DoseDose dependentdependent increaseincrease of dystrophin of dystrophin levelslevels

•• Open label Open label extensionextension studystudy initiatedinitiated

•• All All patientspatients enrolledenrolled

•• WeeklyWeekly treatmenttreatment forfor 90+ weeks 90+ weeks

Goemans et al, NEJM 2011, 364: 1513‐22

Systemic trial MDEX/AVI


AVIAVI‐‐4658 4658 (0.5, 1, 2, 4, 10 & 20 mg/kg)(0.5, 1, 2, 4, 10 & 20 mg/kg)

•• IntravenousIntravenous infusionsinfusions

•• WeeklyWeekly forfor 12 weeks12 weeks

•• 22‐‐4 4 patientspatients per per groupgroup ((totaltotal 19)19)

•• BiopsiesBiopsies

•• BeforeBefore treatmenttreatment

•• 2 weeks 2 weeks afterafter last last injectioninjection

•• No No severesevere sideside effectseffects reportedreported

Cirak et al, Lancet 2011, 378: 595‐605

Systemic trials MDEX/AVI


•• ClearClear dystrophin in 7/19 dystrophin in 7/19 patientspatients

•• 77‐‐10% of 10% of controlcontrol prepre‐‐treatmenttreatment

•• 1111‐‐22% of 22% of controlcontrol postpost‐‐treatmenttreatment

•• 3 3 goodgood respondersresponders (2, 10 & 20 mg/kg (2, 10 & 20 mg/kg groupgroup))

•• Up to 55% dystrophin Up to 55% dystrophin positivepositive fibersfibers

•• Up to 18% dystrophin (Western Up to 18% dystrophin (Western blotblot))

•• InflammatroyInflammatroy infiltrateinfiltrate musclemuscle decreaseddecreased

•• No effect No effect onon CK CK oror functionfunction

•• DosingDosing needsneeds to to bebe optimizedoptimized furtherfurtherCirak et al, Lancet 2011, 378: 595‐605

Systemic trials MDEX/AVI

Annemieke Aartsma-Rus Cirak et al, Lancet 2011, 378: 595‐605

Planned/ongoing trials


•• Exon 51 Exon 51 skippingskipping•• Non ambulant trial Non ambulant trial ongoingongoing USA (GSK/USA (GSK/ProsensaProsensa))•• DoseDose frequencyfrequency trial trial ongoingongoing (GSK/(GSK/ProsensaProsensa))•• ComparativeComparative dosedose trial trial plannedplanned (GSK/(GSK/ProsensaProsensa))•• PhasePhase 3 trial 3 trial ongoingongoing (GSK/(GSK/ProsensaProsensa))•• AVIAVI‐‐4658 4658 higherhigher dosedose startedstarted (AVI/NWH USA)(AVI/NWH USA)

•• Exon 44 Exon 44 skippingskipping PhasePhase I/II I/II ongoingongoing ((ProsensaProsensa))•• Plans Plans forfor exon 45 and 53 trials (exon 45 and 53 trials (ProsensaProsensa))•• OptimizationOptimization otherother exonsexons ongoingongoing (52 & 55) (52 & 55)

Documents

Little Steps Aartsma-Rus Rare Disease Israel 2012