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. 2014 Sep;148(3):1065-72, 1073e1-2; discussion1072-3.
doi: 10.1016/j.jtcvs.2014.05.070. Epub 2014 Jun 7.

Safety and efficacy of high-dose adeno-associated virus 9 encoding sarcoplasmic reticulum Ca(2+) adenosine triphosphatase delivered by molecular cardiac surgery with recirculating delivery in ovine ischemic cardiomyopathy

Michael G Katz  1 , Anthony S Fargnoli  1 , Richard D Williams  1 , Nury M Steuerwald  2 , Alice Isidro  1 , Anna V Ivanina  3 , Inna M Sokolova  3 , Charles R Bridges  4
Affiliations

Safety and efficacy of high-dose adeno-associated virus 9 encoding sarcoplasmic reticulum Ca(2+) adenosine triphosphatase delivered by molecular cardiac surgery with recirculating delivery in ovine ischemic cardiomyopathy

Michael G Katz et al. J Thorac Cardiovasc Surg. 2014 Sep.

Abstract

Objective: Therapeutic safety and efficacy are the basic prerequisites for clinical gene therapy. We investigated the effect of high-dose molecular cardiac surgery with recirculating delivery (MCARD)-mediated adeno-associated virus 9 (AAV9)/sarcoplasmic reticulum Ca(2+) adenosine triphosphatase (SERCA2a) gene delivery on clinical parameters, oxidative stress, humoral and cellular immune responses, and cardiac remodeling.

Methods: Ischemic cardiomyopathy was generated in a sheep model. The sheep were assigned to 1 of 2 groups: control (n = 10) and study (MCARD, n = 6). The control group underwent no intervention and the study group received 10(14) genome copies of AAV9/SERCA2a 4 weeks after infarction.

Results: Our ischemic model produced reliable infarcts leading to heart failure. The baseline ejection fraction in the MCARD group was 57.6% ± 1.6% versus 61.2% ± 1.9% in the control group (P > .05). At 12 weeks after infarction, the MCARD group had superior left ventricular function compared with the control group: stroke volume index, 46.6 ± 1.8 versus 35.8 ± 2.5 mL/m(2) (P < .05); ejection fraction, 46.2% ± 1.9% versus 38.7% ± 2.5% (P < .05); and left ventricular end-systolic and end-diastolic dimensions, 41.3 ± 1.7 versus 48.2 ± 1.4 mm and 51.2 ± 1.5 versus 57.6 ± 1.7 mm, respectively (P < .05). The markers of oxidative stress were significantly reduced in the infarct zone in the MCARD group. No positive T-cell-mediated immune response was seen in the MCARD group at any point. Myocyte hypertrophy was also significantly attenuated in the MCARD group compared with the control group.

Conclusions: Cardiac overexpression of the SERCA2a gene by way of MCARD is a safe therapeutic intervention. It significantly improves left ventricular function, decreases markers of oxidative stress, abrogates myocyte hypertrophy, arrests remodeling, and does not induce a T-cell-mediated immune response.

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Conflict of interest statement

No related disclosures or conflicts of interest to report

Figures

Figure 1
Figure 1
A. QPCR of SERCA2a biodistribution in heart and liver tissue (genome copies per 100ng DNA). B. Relative expression by RT-qPCR of SERCA2a/GAPDH in heart and liver tissue. MCARD-AAV9/SERCA2a group: Grey columns. Control group: Black columns. *p0.05 difference between groups. ╪p<0.05 difference within groups.
Figure 2
Figure 2
Baseline, 3 weeks post-infarction, and 12 weeks post-infarction values for A. Left ventricular ejection fraction (%), B. stroke volume index (mL/m2), C. end systolic diameter (mm), D. end diastolic diameter (mm), E. left ventricular wall thickness (mm), F. left ventricular fractional wall thickening (%). MCARD-AAV9/SERCA2a group: Grey. Control group: Black. *p<0.05 difference between groups.
Figure 3
Figure 3
Morphometric analysis of myocytes and mitochondria. Hematoxylin and eosin stained infarct border zone tissue cross section (scale bar = 40μm) from A. Control, B. MCARD-AAV9/SERCA2a. C. Average cross sectional cardiac myocyte area in infarct border zone (μm2). D. Transmission electron microscopy of infarct border zone tissue (scale bar = 1μm) (control, arrows indicate mitochondrial swelling). E, MCARD-AAV9/SERCA2a (arrows indicate normal-appearing mitochondria). F. Average stereological estimates of: F. mitochondrial fractional volume (%) and, G. mitochondrial density (per μm2). MCARD-AAV9/SERCA2a group: Grey columns. Control group: Black columns. *p<0.05 difference between groups.
Figure 4
Figure 4
Oxidative markers. In infarct and remote tissue zones: A. 4-hydroxynonenal (HNE)-protein conjugates, in HNE-bovine serum albumin equivalents (μg/μg protein), B. Malondialdehyde (MDA) concentration (pmole/mg protein. MCARD-AAV9/SERCA2a group: Grey columns. Control group: Black columns. *p<0.05 difference within groups.
Figure 5
Figure 5
Time course of transgene specific T cell responses to pools of AAV9 capsid peptides and pools of SERCA2a transgene in peripheral blood mononuclear cells (PBMCs) after administration ofAAV9/SERCA2a. SFU - Number of IFN-γ spot-forming units in ELISPOT assay of LV tissue samples. No stim: negative control. PMA+ION: positive control. Red line: positive result threshold. A, B, C and D each refer to a specific animal in the MCARD-AAV9/SERCA2a group.
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