Calcium Regulators as Enhancers of Exon Skipping. M. Carrie Miceli, PhD Stanley F Nelson, MD Melissa Spencer, PhD

Transcription

1 Calcium Regulators as Enhancers of Exon Skipping M. Carrie Miceli, PhD Stanley F Nelson, MD Melissa Spencer, PhD

2 An#sense- mediated exon skipping for DMD Goyenvalle A et al. Hum. Mol. Genet Becker like protein

3 Can we screen for drugs that increase the efficiency of exon skipping? We know that most boys have revertant fibers, which often result from natural skipping. Therefore, cells have the machinery to skip. Perhaps we can find a drug that targets and promotes that machinery. Increasing dystrophin levels even two fold is reasonably likely to result in clinical gain.

4 Exon-50 GFP Reporter in C2C12 Screened in the Absence and Presence of Suboptimal AON by High Content Imaging β- ac#n pr EGFPa β- globin I Intron 49 Exon 50 Intron 50 β- globin I EGFPb No AON6 AON6 Plus AON6 (Construct developed by Q. Lu) EGFPa Exon 50 EGFPb EGFPa EGFPb (3 GFP sequence out of frame) (GFP in frame) Prestwick Biomol Z = triplicate plates # of nuclei, cell number % GFP posi;ve

5 10-20% of op;mal

6 Strategy for tes;ng dantrolene synergy with systemic e23 PMO in mul;ple skeletal muscles in the mdx mouse 10mg/kg or 100mg/kg AO Day 1 Day 2-6 am Day 7 am pm am pm = Systemic AO (morpholino) in saline (tail vein injec;on) = Dantrolene in 20% DMSO in sterile saline injected intraperitoneally Harvest Skeletal Muscles RT- PCR Western Blot Immunofluorescence

7 Dantrolene synergizes with systemic delivery of PMO to increase skipped Dmd RNA and dystrophin protein expression in mdx Dmd RNA Quan;ta;on: Dystrophin Protein Quan;ta;on:

8 Saline + Dantrolene 10mg/kg PMO + DMSO 10mg/kg PMO + Dantrolene 100mg/kg PMO + DMSO Quadriceps Quadriceps Quadriceps Quadriceps Gastrocnemius Gastrocnemius Gastrocnemius Gastrocnemius Diaphragm Diaphragm Diaphragm Diaphragm

9 Saline + Dantrolene 10mg/kg PMO + DMSO 10mg/kg PMO + Dantrolene 100mg/kg PMO + DMSO Tibialis Anterior Tibialis Anterior Tibialis Anterior Tibialis Anterior Triceps Triceps Triceps Triceps Heart Heart Heart Heart

10 Dantrolene synergizes with systemic delivery of PMO to rescue skipped DMD mrna and dyrstrophin protein

11 Rescued dystrophin func;ons to rescue expression of other DGC components at the sarcolemma

12 Dantrolene synergizes with intravascularly delivered AO to increase Dmd exon 23 skipping and improve muscle strength in mdx mice (1 weekly injec;ons over 3 weeks)

13 DMD Muta#on Specific Fibroblasts Directly Reprogrammable to Muscle Lineage Cells (idrm) + LV- htert + LV- MyoD- ER(T) (J. Chamberlain) + Tamoxifen DMD Pa#ent Fibroblasts Immortalized DMD fibroblasts + differen;a;on med DMD Pa#ent Derived Myotubes MHC/DNA Custom 8x15k CGH Array

14 Dantrolene Synergizes with AO to Promote DMD Exon 51 Skipping in Reprogrammed Human Myotubes (Δ45-50 dele;on)

15 Model of Small Molecule Antagonism of RyR, a Shared Molecular Target

16 Ryanodine receptor antagonists enhance AO directed DMD exon 51 in Reprogrammed Human Myotubes (Δ45-50 dele;on)

17 Dantrolene is an attractive developmental candidate for use with AON as a combined therapeutic In literature by 1967: FDA approved with established safety profile. Used in children both acutely and chronically for muscle spasticity and Malignant Hyperthermia Effect of dantrolene in Duchenne muscular dystrophy Bertorini TE, Palmieri GM, Groffin J, Igarashi, M Hinton A, Karas, JG, Muscle Nerve Jun;14(6): patients Dose of 8mg/kg/day for two years: One child Reduced to 6mg/kg/day due to mild muscle weakness. 2 years no meds; 2 years dantrolene 3X decrease in CK relative to natural history controls (3X)] and relative to yr 1 Trend in decrease in slope of MMT loss No change in function No serious side effects.

18 Oral dosing using human compa;ble Dantrium enhances PMO e23 skipping in mdx Dantrium by oral gavage 2X daily ; e23pmo 1X/week over 3 weeks treatment

19 Dantrolene in chow can enhance low and higher dose PMO over 3 week assay ( calculated daily dose) Viable for long term dosing

20 Screening on the Prestwick library Cluster 222: Lead Hits from the +AO/C Ra;o Analysis

21 A Model for Calcium Regula;on of Exon Skipping Ac;vity

22 Next steps moving toward testing of Dantrolene/AON combination therapy Dantrolene is already FDA approved- available single agent: toxicity and dosing data available in the public record Demonstrated efficacy with exon 51 skip in reprogrammed myotubes Needs: to optimize dosing/delivery for combined therapeutic Assess ability to enhance suboptimal and optimal AON dose Long-term functional efficacy-6 months (chow dosing) to assess applicability of CDMD51Plus to multiple DMD mutations amenable to exon 51 skipping Can dantrolene promote exon skipping in the context of all deletions amenable to exon 51 skipping? Other exons; other drugs

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