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Cardiac-targeted RNA interference mediated by an AAV9 vector improves cardiac function in coxsackievirus B3 cardiomyopathy

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Abstract

RNA interference (RNAi) has potential to be a novel therapeutic strategy in diverse areas of medicine. In this paper, we report on targeted RNAi for the treatment of a viral cardiomyopathy, which is a major cause of sudden cardiac death or terminal heart failure in children and young adults. RNAi therapy employs small regulatory RNAs to achieve its effect, but in vivo use of synthetic small interfering RNAs is limited by instability in plasma and low transfer into target cells. We instead evaluated an RNAi strategy using short hairpin RNA (shRdRp) directed at the RNA polymerase (RdRP) of coxsackievirus B3 (CoxB3) in HeLa cells, primary rat cardiomyocytes (PNCMs) and CoxB3-infected mice in vivo. A conventional AAV2 vector expressing shRdRp protected HeLa against virus-induced death, but this vector type was unable to transduce PNCMs. In contrast, an analogous pseudotyped AAV2.6 vector was protective also in PNCMs and reduced virus replication by >3 log10 steps. Finally, we evaluated the intravenous treatment of mice with an AAV2.9-shRdRp vector because AAV9 carries the most cardiotropic AAV capsid currently known for in vivo use. Mice with CoxB3 cardiomyopathy had disturbed left ventricular (LV) function with impaired parameters of contractility (dP/dt max = 3,006 ± 287 vs. 7,482 ± 487 mmHg/s, p < 0.01) and diastolic relaxation (dP/dt min = −2,224 ± 195 vs. −6,456 ± 356 mmHg/s, p < 0.01 and Tau = 16.2 ± 1.1 vs. 10.7 ± 0.6 ms, p < 0.01) compared to control mice. AAV2.9-shRdRp treatment significantly attenuated the cardiac dysfunction compared to control vector-treated mice on day 10 after CoxB3 infection: dP/dt max = 3,865 ± 354 vs. 3,006 ± 287 mmHg/s (p < 0.05), dP/dt min = −3,245 ± 231 vs. −2,224 ± 195 mmHg/s (p < 0.05) and Tau = 11.9 ± 0.5 vs. 16.2 ± 1.1 ms (p < 0.01). The data show, for the first time, that intravenously injected AAV9 has the potential to target RNAi to the heart and suggest AAV9-shRNA vectors as a novel therapeutic approach for cardiac disorders.

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Abbreviations

AAV:

adeno-associated virus

AAV2.6:

pseudotyped AAV2.6 vector

CoxB3:

coxsackievirus B3

dP/dt max :

maximal rate of pressure increase over time

LV:

left ventricle

moi:

multiplicity of infection

pfu:

plaque-forming unit

PNCM:

primary neonatal cardiomyocyte

RdRp:

RNA-dependent RNA polymerase

RNAi:

RNA interference

shRNA:

short hairpin RNA

siRNA:

short interfering RNA

Tau:

isovolumetric relaxation time constant

vg/c:

vector genomes per cell

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Acknowledgements

We thank Dr. J. Kleinschmidt and Dr. J.M. Wilson for kindly providing the AAV packaging plasmids and Denise Werk und Steffen Schubert for the excellent assistance. This work has been supported by the Deutsche Forschungsgemeinschaft through SFB Transregio 19 by project grants C5 to WP and HF, C1 to JK and VAE and Z3 to CT.

Note added in proof

An important recent study of AAV 1–9 tropism after intravenous injecton comprehensively demonstrated that AAV9 is superior to the other serotypes by 1–3 orders of magnitude with respect to both cardiotropism and CMV promotor-driven GFP expression level [31]. Although the current paper employs U6 promoter-driven shRNA transcription instead this new study suggests that currently only AAV9 is suitable to generate therapeutic RNAi levels within the heart.

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Author notes

  1. Jens Kurreck

    Present address: Institut für Industrielle Genetik, Universität Stuttgart, Berlin, Germany

Authors and Affiliations

  1. Department of Cardiology and Pneumology, Campus Benjamin Franklin, Charité Universitätsmedizin Berlin, Hindenburgdamm 30, 12200, Berlin, Germany

    Henry Fechner, Isaac Sipo, Dirk Westermann, Sandra Pinkert, Xiaomin Wang, Lennart Suckau, Carsten Tschope & Wolfgang Poller

  2. Institute of Chemistry (Biochemistry), Freie Universität Berlin, Berlin, Germany

    Jens Kurreck & Volker Erdmann

  3. Department of Virology, Institute of Infectious Diseases, Campus Benjamin Franklin, Charité Universitätsmedizin Berlin, Berlin, Germany

    Heinz Zeichhardt

  4. Innere Medizin III, Universitätsklinikum, Heidelberg, Germany

    Oliver Müller

  5. Institute of Clinical Pharmacology and Toxicology, Campus Benjamin Franklin, Charité Universitätsmedizin Berlin, Berlin, Germany

    Roland Vetter

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  1. Henry Fechner
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  2. Isaac Sipo
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Corresponding author

Correspondence to Wolfgang Poller.

Additional information

Henry Fechner and Isaac Sipo contributed equally to this paper.

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Fechner, H., Sipo, I., Westermann, D. et al. Cardiac-targeted RNA interference mediated by an AAV9 vector improves cardiac function in coxsackievirus B3 cardiomyopathy. J Mol Med 86, 987–997 (2008). https://doi.org/10.1007/s00109-008-0363-x

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  • DOI: https://doi.org/10.1007/s00109-008-0363-x

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