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.

Studies of Efficacy and Liver Toxicity Related to Adeno-Associated Virus–Mediated RNA Interference

“…They proposed a micro-RNA saturation model suggesting that the toxic side effect was caused by competition by the transduced shRNAs for the endogenous microRNA pathway, thereby interfering with endogenous microRNA biogenesis and functionality. Subsequently, several other groups reported that shRNA overexpression from AAV vectors, most using the strong U6 promoter, causes cytotoxicity in different cell types and organs (McBride et al, 2008;Boudreau et al, 2009;Bish et al, 2011;Martin et al, 2011). Lowering the AAV dose reduced the adverse effects (Grimm et al, 2006;Ulusoy et al, 2009;Ehlert et al, 2010), suggesting that the toxic effect is dependent upon shRNA levels, a result supported by our findings.…”

Adeno-Associated Virus Vectors as Therapeutic and Investigational Tools in the Cardiovascular System

“…Because of size constrains, scAAVs seem especially useful for the expression of small genes, including shRNAs and microRNAs. 52,53 …”

“…66 Albeit less efficient, these promoters offer the possibility to obtain tissue-specific or inducible expression and to avoid the risk of saturating the RNA interference pathway, with possible side effects. 53,67,68 In the cardiovascular field, particular efforts have been made in the development of AAV carrying shRNAs targeting genes that regulate the cardiac Ca 2+ cycle. In a rat model of heart failure, a scAAV9 encoding an shRNA against phospholamban was able to restore cardiac function and reduce pathological hypertrophy and dilation after systemic administration.…”

“…69,116,117 The enthusiasm for these promising results has been recently lessened by a study in which an shRNA against phospholamban delivered by an scAAV6 vector in canines resulted in serum troponin elevation and depressed cardiac function, possibly attributed to the dysregulation of endogenous microRNA pathways. 53 AAV-based experiments have proven the important role of additional proteins in controlling the levels of functional SERCA2a. Among these, SUMO1 is abundantly expressed βARKct indicates β-adrenergic receptor kinase carboxyl-terminus; AAV, adeno-associated virus; CMV, cytomegalovirus; GMRD, Golden Retriever muscular dystrophy; HDL, high-density lipoprotein; LDLR, low-density lipoprotein receptor; MCARD, molecular cardiac surgery with recirculating delivery; SERCA2a, sarcoplasmic reticulum Ca 2+ -ATPase; shApoB, shRNA for apolipoproteinB; snRNA, small nuclear RNA; and VEGF, vascular endothelial growth factor.…”

Cardiac gene therapy in large animals: bridge from bench to bedside

“…PLB expression was effectively reduced, however it was associated with cardiac dysfunction with serum cardiac enzyme elevation. 81 Together with development of severe dilated cardiomyopathy in PLB null human patients, 82 PLB modulation needs further assessment before clinical application. Gene transfer of S100A1, another calcium regulating protein, which impacts not only SERCA2a but also ryanodine receptors, was conducted in a pig myocardial infarction model.…”