Gene expression and whole-transcriptome analysis of heart tissue isolated from mutant transgenic mice after 16 weeks of treatment with AAV9-M7.8L shRNA

Purpose: Assess the effects of the myocardial transcriptional program and off-target delivery or endogenous RNA interference pathways by AAV9-M7.8L shRNA Results: RNA sequencing including small RNA profiling was used to assess i) the presence of delivered therapeutic, ii) effects on the mutant and wild type alleles, iii) gene expression network effects of disease and silencing, and iv) disruption of the native small RNA processing apparatus and microRNA function. Transcriptome analysis showed that M7.8L shRNA and CFP was highly expressed in the hearts of humanized mutant transgenic mice treated at 3 days old of age and allele specific silencing 27% of the human MYL2-47K (RLC-47K) mutation without affecting the mouse endogenous myl2 gene. miRNA profiling showed a pathological signature in untreated and control RLC-47K mice, with the upregulation of mir208a, and mir208b and downregulation of mir-499, which is a common response to cardiac injury, however no significant changes were observed for mir208a and mir-499 in the M7.8L treated group compared to the untreated and control groups. On the other hand, the expression of mir208b was significantly downregulated in the treated group, which was consistent with a positive therapeutic benefit. This emphasizes the complexity of cardiac remodeling and the time-dependent nature of the changes in different signaling networks. To assess downstream broader transcriptional effects of the treatment, we adopted a network approach similar to those we have previously published.We obtained the top 30 differentially expressed genes between treated and control groups and extracted interacting genes and relationships from the STRING database. We then plotted the resulting interaction network with each gene's expression fold change . This visualization revealed reversal of the stress/remodeling myocardial gene program and related markers (including Myh7, Nppa, Nppb, Acta1) as well as up-regulation of gene networks that included acetyl-CoA transferases (Ech1, Ecl1, Acaa1), indicating a wide, systemic, transcriptional effect of the treatment in reverting the disease gene expression program. We also observed minimal impact of off-target delivery or endogenous RNA interference pathways. Thus, we assessed the expression level of the enzymes argonaute and dicer and found a slight increase of argonaute and no significant changes in the expression of dicer in the 3 days old treated mutant transgenic mice as compared to the untreated and control animals suggesting that shRNA/miRNA-shared pathways were modestly affected during four months of constitutive expression of M7.8L shRNA in the hearts of adult mice. These effects and the lack of in vivo cytotoxicity in RLC-4K treated mice are consistent with the effects of the H1 promoter that drives M7.8L shRNA expression in a more restrained manner than the U6 promoter. Similar findings have been observed in mouse liver after knockdown of hepatitis B virus with shRNA driven by weak promoters. Conclusions: in vivo allele-knockdown of MYL2-47K at 4 months was 27% by RNA sequencing. This sustained but modest reduction appeared sufficient to revert the disease phenotype during four months of observation without affecting the normal allele. In addition, we observed that shRNA expression declined after four months of treatment, however this course of treatment was sufficient to ameliorate the disease with minimal off-target effects, while efficacy when mice were treated at older age was reduced. In humans, there is often progression of disease seen during adolescence. Based on this observation it would seem that RNAi therapies should target early disease. Overall design: Methods: RLC transgenic mice were injected via the jugular vein at three days old of age with AAV9 H1-M7.8L RSVp-Cerulean. For the neonatal group, 25ul of virus were injected (3x10^11 viral particles). Echocardiography and treadmill studies were done at 2 and 3.5 months of age. At 4 months of age, animals were sacrificed and hearts were isolated for hyperttophic biomarker quatification, collagene deposited and microRNAseq and mRNAseq. Two illumina RNA libraries were prepared: one Small RNA library ran in MiSeq Rapid Mode 50 nt-single end and an Poly A library in HiSeq Rapid mode 100 nt -pair end Heart_wtTg= Heart tissue isolated from wild type transgenic mice (wtTg) carrying the normal human allele of the MYL2 gene. Heart_mutTg=Heart tissue isolated from mutant transgenic mice (mutTg) carrying the mutated human allele of the MYL2 gene. Control virus= AAV9 virus non-expressing M7.8L shRNA.