Overview of my dissertation research

1. Towards Gene Therapy for Duchenne Muscular Dystrophy Heart Disease Brian Bostick MD/PhD Student Duan Lab June 25, 2008 Department Seminar University of Missouri School of Medicine Department of Molecular Microbiology & Immunology

12. Experimental Design 2. We then compared the dystrophin expression in the hearts of the female BL10, mdx, and carrier mice . 3. Next, we performed a comprehensive battery of anatomical, histopathological and functional studies of cardiovascular function. 4. Finally, we investigated potential mechanisms for protection. 1. We crossed C57BL/10 mice with mdx mice BL10 mdx Dystrophin +/+ or Dystrophin +/y Dystrophin -/y or Dystrophin -/- X Heterozygous Females (Carrier Mice) Dystrophin +/-

13. Carrier mice expressed 50% dystrophin in the heart in a mosaic pattern. Bostick et al 2008 Circ Res BL10 Female (Dystrophin +/+) Mdx Female (Dystrophin -/-) Carrier Female (Dystrophin +/-)

14. Carrier heart expression mimics gene or cell therapy. Bostick et al 2008 Circ Res Low Expressing Region of Carrier Heart High Expressing Region of Carrier Heart

15. 50% mosaic expression normalized structural and histopathological defects. Bostick et al 2008 Circ Res

16. ECG changes were completely prevented by 50% mosaic expression. Bostick et al 2008 Circ Res

17. Left ventricular catheterization revealed normal heart function with 50% mosaic expression. Bostick et al 2008 Circ Res

18. A small population of carrier mice exhibited focal inflammation which did not impact heart function. Bostick et al 2008 Circ Res

19. Selective expansion of dystrophin positive cells was not responsible for protection. Bostick et al 2008 Circ Res

20. Utrophin was up-regulated in dystrophin negative regions of carrier mouse heart. Bostick et al 2008 Circ Res

25. Experimental Design 1. Generate a heart-specific ∆ H2-R19 mini-dystrophin expression cassette 3. Characterize ∆H2-R19 mini-dystrophin expression 4. Perform comprehensive structure-function analysis of heart restricted ∆H2-R19 mini-dystrophin transgenic mdx mice 2. Create founder transgenic lines and breed to congenic mdx background (5 ~ 7 generations)

26. Transgenic expression of ∆H2-R19 mini-dystrophin utilizing the α -MHC promoter yielded cardiac specific expression. Bostick et al 2009 Molecular Therapy Southern Blot Western Blot Immunofluorescence

27. The ∆H2-R19 mini-dystrophin restored sarcolemmal integrity in the mdx heart. Bostick et al 2009 Molecular Therapy Evan’s Blue Dye Uptake Assay

28. Heart specific ∆H2-R19 mini-dystrophin prevented fibrosis in the mdx heart. Bostick et al 2009 Molecular Therapy Masson Trichrome

29. Uphill treadmill endurance was enhanced in mdx mice with heart specific ∆ H2-R19 mini-dystrophin expression. Bostick et al 2008 HMG under review

30. The ECG of ∆H2-R19 transgenic mdx mice was improved but not completely corrected. Bostick et al 2008 HMG under review

31. LV catheterization revealed a rescue of systolic function but not diastolic function. Bostick et al 2008 HMG under review

32. Overall left ventricular function was improved, but not completely rescued. Bostick et al 2009 Molecular Therapy

33. Heart specific ∆ H2-R19 mini-dystrophin restored dobutamine stress response and improved survival under stress. Bostick et al 2009 Molecular Therapy

34. Expressing ∆ H2-R19 mini-dystrophin on normal BL10 background profoundly displaces wild-type dystrophin.

35. Cardiovascular function is completely normalized with the heart specific ∆ H2-R19 mini-dystrophin expressed on wild-type BL10 background. N = 10 for 8-m-old BL10, N = 21 for 22-m-old BL10, N = 15 for 8-m-old transgenic BL10, and N = 7 for 22-m-old transgenic BL10 mice ECG LV Catheterization D

40. AAV-9 microdystrophin efficiently transduces the heart at both the latent and symptomatic stage . Bostick et al 2008 Human Gene Therapy Latent Stage Symptomatic Stage Low Power View High Expressing Region High Expressing Region Low Expressing Region

41. Treatment with AAV-9 microdystrophin during the latent stage improves the electrocardiographic function . Bostick et al 2008 Human Gene Therapy

42. Treatment with AAV-9 microdystrophin during the symptomatic stage reduces fibrosis and improves electrocardiographic function. Symptomatic Mdx AAV Treated Mdx BL10 PR (ms) * † QT (ms) Untreated Mdx (n=5) BL10 (n=6) Treated Mdx (n=5) HR (bpm) *

43. Treatment with AAV-9 microdystrophin during the symptomatic stage improves left ventricular performance. Volume (  l) Pressure (mmHg) Untreated Mdx BL10 Treated Mdx * dP/dt Max (KmmHg/sec) * Ejection Fraction (Percent) † Stroke Volume (  l ) Untreated Mdx (n=6) BL10 (n=8) Treated Mdx (n=6) BL10 (n=8) Treated Mdx (n=6) Untreated Mdx (n=6) *