Exon skipping in a DCM mouse model mimicking a human mutation in titin

Transcription

1 Exon skipping in a DCM mouse model mimicking a human mutation in titin Dr. Michael Gramlich Department of Cardiology, University of Tuebingen, Germany

2 I do not have a financial interest/arrangement or affiliation with one or more organizations that could be perceived as a real or apparent conflict of interest in the context of the subject of this presentation.

3 The Giant Muscle Protein Titin - a major player for sarcomeric assembly Biggest known gene in humans,spans half the sarcomere from the Z-disc to the M-line Expression in heart and skeletal muscle tissue

4 The Giant Muscle Protein Titin - a major player for sarcomeric assembly Biggest known gene in humans,spans half the sarcomere from the Z-disc to the M-line Expression in heart and skeletal muscle tissue Acts as a molecular blueprint and directs sarcomere assembly

5 The Giant Muscle Protein Titin - a major player for sarcomeric assembly Biggest known gene in humans,spans half the sarcomere from the Z-disc to the M-line Expression in heart and skeletal muscle tissue Acts as a molecular blueprint and directs sarcomere assembly Contains repetitive domains that behave as a system of serially linked springs, defining the elasticity of myofibrils

6 The Giant Muscle Protein Titin - a major player for sarcomeric assembly Biggest known gene in humans,spans half the sarcomere from the Z-disc to the M-line Expression in heart and skeletal muscle tissue Acts as a molecular blueprint and directs sarcomere assembly Contains repetitive domains that behave as a system of serially linked springs, defining the elasticity of myofibrils Role in myofibrillar signal transduction (hypertrophy)

7 The Australian DCM Family A1 (from Brisbane) 2 bp insertion (AT) in sequence coding for A-band titin - segregates with the disease - not observed in 340 control chromosomes -causes a frame shift with a premature stop Gramlich, Gerull et al., Nat Gen 2002

8 2 Mutations in TTN are a common cause for DCM MAO family I II American DCM family MAO: Missense mutation in exon 18 III Gramlich et al., Nat Gen 2002 IV Australian DCM family A3: 1 bp deletion in exon 336 Gerull et al., JMM 2006 Israeli DCM family A4: 2 bp insertion in exon 326 Gramlich et al., Am J Cardiol 2012

9 NEJM, 2012

10 Generation of a TTN knock-in mouse model that imitates the human TTN mutation

11 Cardiac Stress Model: Angiotensin II day 0 day 1 echocardiography implantation of angiotensin II osmotic pumps (1,4 mg/kg/d) day 7 echocardiography day 14 echocardiography histology

12 Stressed heterozygous Ttn knock-in mice develop features of DCM

13 Skipping of mutant exon 326 with specific antisense oligonucleotids (AON)

14 Exon skipping in Duchenne Muscular Dystrophy (DMD)

15 Exon 326 can be skipped in cell culture (HL1) EXON 325 EXON 327 skipped EXON 325 EXON 326 unskipped

16 Loss of exon 326 does not negatively affect cell function (HL1) HL-1 + AO HL1 AO

17 Homozygous Ttn embryos show severe defects in sarcomeric assembly

18 AON treatment rescues sarcomeric assembly in cultured embryos

19 AON treated Ttn embryos develop a beating heart WT E KO E AON KO E UNTREATED

20 Exon skipping in adult heterozygous DCM mice day 0 day 1 echocardiography implantation of angiotensin II osmotic pumps (1,4 mg/kg/d) +/- i.v. injection of AON day 7 Echocardiography +/- i.v. injection of AON day 14 echocardiography histology

21 AON treatment prevents from DCM het + AngII skipped unskipped het + AngII + AO

22 CONCLUSION 1. Titin exon 326 can be specifically skipped in cell culture, embryos, and adult mice 2. Restoring the reading frame (partially) rescues the phenotype of Titin knock-in mice 3. Exon skipping should also be a feasible strategy for the treatment of DCM patients with a mutation in TTN

23 Acknowledgment University of Tübingen, Germany Qifeng Zhou Sonja Schötterl Lisha Zha Dr. Tanja Schönberger Tobias Jürgens Prof. Meinrad Gawaz Deutsches Herzzentrum Munich, Germany Dr. Luna Simona Pane Prof. Karl-Ludwig Laugwitz University of Leiden, the Netherlands Annemieke Aartsma-Rus

24 Outlook: Exon-skipping in human ips cells from a DCM patient KLF4 SOX2 ips Cardiogenic differentiation OCT4 C-myc Fibroblast culture of affected family member