A new model of Duchenne muscular dystrophy in rat Séverine REMY Plate-forme TRIP Thibaut LARCHER Plate-forme APEX 30/11/15 24 / 11 / 2015
Duchenne muscular dystrophy Genetic disease X-linked recessive disorder of skeletal and cardiac muscles 1/3,500 male at birth Non-sense mutation in the dystrophin gene Physipathogeny Lack of dystrophin Healthy muscle DMD muscle Clinical presentation - Progressive muscular impairment - Premature death (cardiac insufficiency) Muscle fiber necrosis Replacement by connective and adipose tissues Healthy muscle DMD muscle Gower s sign Treatment - No therapy.02 30/11/15
Animal models of Duchenne muscular dystrophy mdx mouse and variants Dmd -/- pig HFMD cat GRMD dog Genec basis dystrophin gene (stop codon in exon 23) dystrophin gene (stop codon in exon 52) dystrophin gene (promotor deletion) dystrophin gene (stop codon in exon 8) Breeding Easy Difficult Moderatly difficult Difficult Life span Muscle lesions Muscle funcon Highly shorten Shorten Slightly shorten respiratory insufficiency renal insufficiency Primary (no or late fibrosis) Strength reduction Normal locomotion Primary and secundary Strength reduction Impaired locomotion Primary (hypertrophy predominant) Strength reduction Impaired locomotion Shorten (# 1.5 yrs) Primary and secundary Strength reduction Impaired locomotion Cardiac funcon Normal Normal (young) Cardiomyopathy Mild cardiomyopathy Adult weight 25 g 20 kg 4 kg 15 kg Main use Physiopathology, preclinical testing Recent description Short term studies No use Preclinical testing.03
_01 Development of the strain.04
TALENs targeted Dmd mutation in Rat Transcripon Acvator Like Effector (TALE) ~34aa repeat moduls Variable diresidues - Tandem repeat domains - Individual TALE repeat contains ~34 aa that recognize a single base pair via two Variable residues in posions 12 and 13,responsible for DNA-binding specificity.05
TALENs targeted Dmd mutation in Rat TALEN Targeted DNA sequence DNA double strand break (DSB) Targeted DNA sequence (Exogenous template) Non-homologous end joining (NHEJ) Imperfect repair (small Inserons or Deleons = Indels) Targeted gene mutaon or complete KO Homology-directed repair (HDR) Precise repair (user defined) Targeted Gene Integraon 5.06
TALENs targeted Dmd mutation in Rat TALEN pair Exon 23 Rat Dmd gene one-cell embryo 387 micro-injected zygotes 294 eggs tranferred into oviduct of pseudopregnant females 11 mutants = 3.74% of the tranferred eggs = 12.5% of the offspring DNA sequencing 88 offspring From Tesson et al., Nat Biotechnol, 2011.07
TALENs targeted Dmd mutation in Rat Mutation identification mainly deletions of 1 to 24 bp and rare insertions of few nucleotides 9 mutants with disruption of the open reading frame with premature stop codon Irregular transmission of the mutation to the off-spring Line 61 selected upon its clinical and histological presentation WT sequence 2886-CTG CAA AGC TCT TTG AAA GAG CAA CAA AAT GGC TTC AAC TAT CTG AAT - - - - * at 10965 L Q S S L K E Q Q N G F N Y L N *codon stop Male # 61 (del 11 pb) 2886-CTG CAA AGC TCT TTG AAA GAG xxx xxx xxx xxctt CAA CTA TCT GAA - - - - - * 81bp aer the mutaon L Q S S L K E L Q L S E + 22 aa Observations on founders Dmd mdx Female carriers = no clinical signs potential off target mutations were analyzed in founder 61and its offspring (2 candidate sites located on the X-chromosome) no mutations detected.08
Growth and development in Dmd mdx rats WT (n=10) Dmd mdx (n=11) - 29 % - 34 % - 40 % Dmd mdx - 21 % WT - 15% Altered growth visible from 4 weeks Body weight: Dmd mdx rats < WT rats (n=11) So, moist food delivered from 34 weeks of age (#8 months).09
_02 Analysis of dystrophin expression.011
Dystrophin expression in F2 Western blot and PCR protein undetected Ab NCL-DYS2 & MANEX10-11C mrna detected.012
Dystrophin expression in F2 Immunohistochemistry MANDYS110 Anbody Tissue expression tested with Ab DYSB, MANDYS110 and DYS2 Rare revertant fibers in skeletal muscles 4.9 +/- 0.8 % (n=5) p < 0.05.013
_03 Histopathological evaluation.016
Skeletal muscle lesions Lower weight of fast muscles (tibialis anterior and extensor digitorum longus) at 7 months Serum CK, ASAT and ALAT higher in Dmd mdx rats creatine kinase (m ± sd) 3 months 7 months WT (n=5 each) 185 ± 17 U/L 588 ± 397 U/L Dmd -/- (n=5 each) 1945 ± 6620 U/L 3965 ± 817 U/L Similar appearance of all observed skeletal muscles (Tibialis cranialis, extensor digitorum longus, biceps femoris, biceps brachii, soleus and diaphragm) Dmd mdx rat, 7 months, Soleus Dmd mdx rat, 7 months, Extensor digitorum longus Dmd mdx rat, 7 months, Biceps bracchialis.017
Skeletal muscle lesions Biceps femoris muscle, anisocytosis 30% 25% 20% Dmd mdx rat, 3 months 30% 25% 20% Dmd mdx rat, 7 months KO WT 15% 15% 10% 10% 5% 5% 0% 0% diameter (m ± sd) 3 months 7 months WT 54.7 ± 16.7 µm Dmd mdx 41.1 ± 20.9 µm 54.7 ± 18.5 µm 45.0 ± 22.5 µm p < 0.05 p < 0.05.019
Skeletal muscle lesions Biceps femoris muscle, regeneration MyoHC dev + (m ± sd) 3 months 7 months WT 0 ± 0 % 0 ± 0 % Dmd mdx 13.1 ± 6.7 % 10.2 ± 4.4 % Myosin heavy chain, developmental isoform (MyoHCd) Anbody WT rat, 7 months Dmd mdx rat, 3 months Dmd mdx rat, 7 months.020
Skeletal muscle lesions Biceps femoris muscle, fibrosis WT rat, 7 months Total area ratio (m ± sd) 3 months 7 months WT 6.7 ± 3.2 % 6.4 ± 2.3 % Dmd mdx 16.9 ± 4.7 % 24.3 ± 8.7 % p < 0.05 p < 0.05 Dmd mdx rat, 3 months Dmd mdx rat, 7 months Dmd mdx rat, 18 months.022
_04 Clinical and functional evaluation.024
Muscle function evaluation Grip test, 3 months of age - 20% DMD mdx vs WT - 50% DMD mdx vs WT Dmd mdx rats versus WT control rats Weaker global and relative force exerted by the forelimbs Significant decrease over five successive pulls.025
Spontaneous activity Actimetry at 3 months of age.026
_05 Cardiac exploration.027
Cardiac muscle lesion Gross appearance heart weight (m ± sd) 3 months 7 months WT 1380 ± 85 mg 2050 ± 73 mg Dmd mdx 1732 ± 271 mg 1766 ± 84 mg WT rat, 3 months Dmd mdx rat, 3 months.028
Cardiac muscle lesion Progressive cardiomyopathy p/vg (m ± sd) 7 months WT 43.1 ± 1.8 % Dmd mdx 35.8 ± 3.9 % Ventricular wall hypertrophy at 3 months Ventricular wall atrophy and ventricular lumen dilatation at 7 months Scattered foci of fibrosis Foci of necrosis Dmd mdx rat, 7 months p < 0.0001 WT rat, 7 months Dmd mdx rat, 3 months Dmd mdx rat, 7 months Dmd mdx rat, 12 months.029
Cardiac function Echocardiography and doppler imaging Ventricular wall hypertrophy at 3 months Diastolic function alteration.030
_06 Life span.031
Life span and cause of death Shorter life span of Dmd mdx rats Death of 50% of Dmd mdx rats versus no death at 12 months of age (median survival : 382 days).032
Life span and cause of death Main causes of death during life span study (n=11) Cardiac insufficiency (n=4; 8, 9, 12 and 13 months) Cachexia and dilated cardiomyopathy (n=1; 14 months) Cachexia without cardiac alteration (n=1; 15 months) Dental malocclusion and cachexia (n=3; 11, 12 and 13 months) associated with cardiomyopathy Rhabdomyosarcoma (n=2, 11 and 17 months) Rem. Spontaneous deaths from acute cardiac insufficiency (2 and 7 months).033
_07 Conclusion.035
Take-home messages Dmd mdx rats using TALENs Shorter life span No dystrophin in muscles Early muscle tissue alteration (on-going characterisation of early lesions at 1.5 months) Benefits compared to other mouse and dog models Clinically: Growth retardation, impaired locomotion, shorter life span Histology: early and extended muscle fibrosis and adipose tissue deposit Cardiac function and tissue alteration Promising alternative model preclinical evaluation of new treatments physiopathology (heart).037
MYORAT study INRA UMR703 PAnTher Oniris Plate forme APEX - Nantes Yan CHEREL Hélicia GOUBIN Maeva DUTILLEUL Lydie GUIGAND Thibaut LARCHER INSERM UMR 1064 Centre pour la recherche en Transplantaon & Immunolo-intervenon Plate forme TRIP - Nantes Séverine REMY Laurent TESSON Virginie THEPENIER Ignacio ANEGON INSERM UMR 1089 Atlanc Gene Therapies - Nantes INSERM U1154 - CNRS UMR 7196 Muséum Naonal d Histoire Naturelle Plate forme TACGENE - Paris Anne DE CIAN Charloe BOIX Jean-Bapste RENAUD Carine GIOVANNANGELI Jean Paul CONCORDET Virginie FRANÇOIS Caroline LE GUINER INSERM UMR 1087 - CNRS UMR 6291 Instut du Thorax Plate forme Therassay Nantes Aude LAFOUX Gilles TOUMANIANTZ Corinne HUCHET.038