A Novel Artificial MicroRNA Expressing AAV Vector for Phospholamban Silencing in Cardiomyocytes Improves Ca2+ Uptake into the Sarcoplasmic Reticulum

Abstract

In failing rat hearts, post-transcriptonal inhibition of phospholamban (PLB) expression by AAV9 vector-mediated cardiac delivery of short hairpin RNAs directed against PLB (shPLBr) improves both impaired SERCA2a controlled Ca2+ cycling and contractile dysfunction. Cardiac delivery of shPLB, however, was reported to cause cardiac toxicity in canines. Thus we developed a new AAV vector, scAAV6-amiR155-PLBr, expressing a novel engineered artificial microRNA (amiR155-PLBr) directed against PLB under control of a heart-specific hybrid promoter. Its PLB silencing efficiency and safety were compared with those of an AAV vector expressing shPLBr (scAAV6-shPLBr) from an ubiquitously active U6 promoter. Investigations were carried out in cultured neonatal rat cardiomyocytes (CM) over a period of 14 days. Compared to shPLBr, amiR155-PLBr was expressed at a significantly lower level, resulting in delayed and less pronounced PLB silencing. Despite decreased knockdown efficiency of scAAV6-amiR155-PLBr, a similar increase of the SERCA2a-catalyzed Ca2+ uptake into sarcoplasmic reticulum (SR) vesicles was observed for both the shPLBr and amiR155-PLBr vectors. Proteomic analysis confirmed PLB silencing of both therapeutic vectors and revealed that shPLBr, but not the amiR155-PLBr vector, increased the proinflammatory proteins STAT3, STAT1 and activated STAT1 phosphorylation at the key amino acid residue Tyr701. Quantitative RT-PCR analysis detected alterations in the expression of several cardiac microRNAs after treatment of CM with scAAV6-shPLBr and scAAV6-amiR155-PLBr, as well as after treatment with its related amiR155- and shRNAs-expressing control AAV vectors. The results demonstrate that scAAV6-amiR155-PLBr is capable of enhancing the Ca2+ transport function of the cardiac SR PLB/SERCA2a system as efficiently as scAAV6-shPLBr while offering a superior safety profile.

Anti-adenoviral Artificial MicroRNAs Expressed from AAV9 Vectors Inhibit Human Adenovirus Infection in Immunosuppressed Syrian Hamsters

“…Thus, here we developed scAAV vectors expressing anti-adenoviral amiRs and analyzed their efficiency in the inhibition of hAd5 infection, first in vitro and then in vivo in immunosuppressed Syrian hamsters. This approach was chosen for several reasons: (1) scAAV vectors have a broad tissue tropism and enable rapid and strong transgene expression after transduction of the target cells in vitro and in vivo, 40 , 43 , 45 (2) amiRs can efficiently silence target genes and can easily be delivered from viral vectors including AAV vectors in vitro and in vivo, 46 , 47 , 48 , 49 but were reported to have lower toxicity than shRNAs, which are more commonly used in the context of viral vector-based gene-silencing investigations, 46 , 48 , 50 , 51 , 52 and (3) immunosuppressed Syrian hamsters represent an animal model that is permissive for hAd5. 18 , 53 …”

Rheostatic Regulation of the SERCA/Phospholamban Membrane Protein Complex Using Non-Coding RNA and Single-Stranded DNA oligonucleotides

“…Irrespective of the possible role of the endogenous miRNAs toward the SERCA/PLN complex in cardiomyocytes, the nanomolar affinity of ssDNA for PLN and its ability to reverse its inhibitory effects on SERCA constitutes a unique opportunity to exploit oligonucleotides as scaffolds for the design of small molecules to target Ca 2+ regulation. While DNA aptamers or miRNA constructs have been successfully used to target and regulate the PLN gene 11 41 , silencing or ablation of the PLN gene has resulted in cardiomyopathies. In contrast, tunable regulation of PLN inhibitory function of SERCA by oligonucleotide-based drugs may represent a more promising therapeutic avenue in which the extent of SERCA activation can be fine-tuned to match the pathological insult.…”

Artificial miRNAs as therapeutic tools: Challenges and opportunities

“…Among cardiovascular diseases, hypertension and heart failure have been treated with amiRNAs (Fan et al, 2012; Größl et al, 2014). The aim of the first study was to determine whether mir‐155‐based AT1aR (angiotensin II type 1 receptor, overexpressed in spontaneously hypertensive rats) amiRNA could impair hypertension and improve cardiovascular remodeling in spontaneously hypertensive rats.…”

“…Despite decreased amiRNA expression, both RNAi triggers reduced PLB expression equally and enhanced Ca2+ transport. Proteomic analysis showed that the shRNA induced the expression of interferon‐regulated and proinflammatory genes whereas artificial miRNA did not induce any of them (Größl et al, 2014).…”