ORIGINAL ARTICLE Efficiency of eight different AAV serotypes in transducing rat myocardium in vivo J Palomeque 1 , ER Chemaly 1 , P Colosi 2 , JA Wellman 2 , S Zhou 2 , F del Monte 1 and RJ Hajjar 1 1 Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA and 2 Avigen Inc., Alameda, CA, USA Recombinant adeno-associated (AAV) viruses have unique properties, which make them ideal vectors for gene transfer targeting the myocardium. Numerous serotypes of AAV have been identified with variable tropisms towards cardiac tissue. In the present study, we investigated the time course of expression of eight different AAV serotypes in rat myocar- dium and the nature of the immunity against these serotypes. We first assessed whether neutralizing antibodies (NAb) were present for any of the serotype in the rats. We injected 100 ml of each AAV 1–8 serotype (10 12 DNAse resistant particles/ml), encoding LacZ gene, into the apical wall of rat myocardium. At 1, 4, 12 and 24 weeks after gene delivery, the animals were killed and b-galactosidase (b-gal) activity was assessed by luminometry. Additionally, LacZ genomic copies and AAV capsids copies were measured through standard polymerase chain reaction analysis and cryo- sections from the area of viral injection were stained for X-gal detection at the same time points. No NAbs were detected against any of AAV serotypes. At all the time points studied, AAV1, 6 and 8 demonstrated the highest efficiency in transducing rat hearts in vivo. Parallel to the results with b-gal activity, the highest levels LacZ and AAV DNA genomic copies were with AAV1, 6 and 8. The positive X-gal staining depicted by these serotypes confirmed these results. These results indicate that among the various AAV serotypes, AAV1, 6 and 8 have differential tropism for the heart unaffected by pre-existing NAb in the rat. Although AAV 1 and 6 vectors induced rapid and robust expression and reach a plateau at 4 weeks, AAV 8 continued increasing until the end of the study. AAV 2, 5 and 7 vectors were slower to induce expression of the reporter gene, but did reach levels of expression comparable to AAV1 and AAV6 vectors after 3 months. Gene Therapy (2007) 14, 989–997. doi:10.1038/ sj.gt.3302895; published online 25 January 2007 Keywords: gene transfer; AAV vectors; cardiovascular gene therapy Introduction Cardiovascular diseases and specifically congestive heart failure (CHF) remain a leading cause of morbidity and mortality around the world. Over the last few years, a number of molecular abnormalities have been described in the pathogenesis of CHF. These cellular and molecular abnormalities are all potential targets for gene modifica- tions. For this reason, gene therapy has been considered as an alternative and novel experimental therapeutic approach to target the various underlying mechanisms responsible for the decreased contractility in the failing heart. The feasibility of in vivo cardiac gene transfer by viral vectors has been consistently demonstrated over the last few years. In addition, in short-term experiments, gene transfer studies targeting calcium-handling pro- teins such as SERCA2a 1,2 and phospholamban 3 have proven that this approach can improve cardiac function. For CHF applications, the requirements for a gene transfer vector include non-pathogenicity, sustained transgene expression throughout the myocardium and enhanced cardiac tropism. These desirable features for cardiac gene transfer seem to reside in the adeno- associated virus (AAV) vectors. For this reason, AAV derived technology has recently been identified as the most promising means to target myocardial contractile dysfunction. AAVs belong to the viral family Parvoviridae (parvo- viruses). AAV has a non-enveloped proteinaceous capsid with a diameter of approximately 20–25 nm 4 containing single-stranded DNA genome of approximately 4.7 kb encoding two genes. 5 One is the rep gene, which encodes for proteins involved in DNA replication. The other is the cap gene, which by differential splicing, encodes for three proteins that make up the protein coat of the virus. AAV is a dependovirus, so named because members of this group cannot replicate autonomously. AAVrequires co-infection with a helper virus, such as herpesvirus or adenovirus, to provide necessary helper functions in trans to allow for AAV replication. 6 AAV vectors have eliminated all the viral genes. This adds a safety feature that not only prevents the generation of replication- competent pseudo wild-type AAV via homologous recombination, 7 but also mitigates the possibility of immune reactions caused by undesired viral gene Received 19 June 2006; revised 20 September 2006; accepted 26 October 2006; published online 25 January 2007 Correspondence: Dr RJ Hajjar, Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, Building 149, 13th Street, 4 floor, CNY-4, Charlestown, MA 02129, USA. E-mail: rhajjar@partners.org Gene Therapy (2007) 14, 989–997 & 2007 Nature Publishing Group All rights reserved 0969-7128/07 $30.00 www.nature.com/gt