title: Cardiac gene transfer of short hairpin RNA directed against phospholamban effectively knocks down gene expression but causes cellular toxicity in canines.
abstract: Derangements in calcium cycling have been described in failing hearts, and preclinical studies have suggested that therapies aimed at correcting this defect can lead to improvements in cardiac function and survival. One strategy to improve calcium cycling would be to inhibit phospholamban (PLB), the negative regulator of SERCA2a that is upregulated in failing hearts. The goal of this study was to evaluate the safety and efficacy of using adeno-associated virus (AAV)-mediated cardiac gene transfer of short hairpin RNA (shRNA) to knock down expression of PLB. Six dogs were treated with self-complementary AAV serotype 6 (scAAV6) expressing shRNA against PLB. Three control dogs were treated with empty AAV6 capsid, and two control dogs were treated with scAAV6 expressing dominant negative PLB. Vector was delivered via a percutaneously inserted cardiac injection catheter. PLB mRNA and protein expression were analyzed in three of six shRNA dogs between days 16 and 26. The other three shRNA dogs and five control dogs were monitored long-term to assess cardiac safety. PLB mRNA was reduced 16-fold, and PLB protein was reduced 5-fold, with treatment. Serum troponin elevation and depressed cardiac function were observed in the shRNA group only at 4 weeks. An enzyme-linked immunospot assay failed to detect any T cells reactive to AAV6 capsid in peripheral blood mononuclear cells, heart, or spleen. Microarray analysis revealed alterations in cardiac expression of several microRNAs with shRNA treatment. AAV6-mediated cardiac gene transfer of shRNA effectively knocks down PLB expression but is associated with severe cardiac toxicity. Toxicity may result from dysregulation of endogenous microRNA pathways.
authors.affiliation: Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA. bish@mail.med.upenn.edu
authors.forename: Lawrence T
authors.initials: LT
authors.lastname: Bish
authors.forename: Meg M
authors.initials: MM
authors.lastname: Sleeper
authors.forename: Caryn
authors.initials: C
authors.lastname: Reynolds
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Gazzara
authors.forename: Elanor
authors.initials: E
authors.lastname: Withnall
authors.forename: Gretchen E
authors.initials: GE
authors.lastname: Singletary
authors.forename: George
authors.initials: G
authors.lastname: Buchlis
authors.forename: Daniel
authors.initials: D
authors.lastname: Hui
authors.forename: Katherine A
authors.initials: KA
authors.lastname: High
authors.forename: Guangping
authors.initials: G
authors.lastname: Gao
authors.forename: James M
authors.initials: JM
authors.lastname: Wilson
authors.forename: H Lee
authors.initials: HL
authors.lastname: Sweeney
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics; D004285:Dogs; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D018014:Gene Transfer Techniques / Q000009:adverse effects; D005822:Genetic Vectors; D009000:Monocytes / Q000166:cytology / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D014336:Troponin / Q000097:blood; D000099211:Phospholamban
chemical_list: D002118:Calcium; D002135:Calcium-Binding Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014336:Troponin; D000099211:Phospholamban