Biology subjects

Dainis, A.

Publications and source records attributed to Dainis, A..

3 recordsLinked to original sources

bioRxiv2019

Dissociation of disease phenotype and allele silencing in hypertrophic cardiomyopathy

Allele-specific RNA silencing has been shown to be an effective therapeutic treatment in a number of diseases, including neurodegenerative disorders. Studies of allele-specific silencing in hypertrophic cardiomyopathy to date have focused on mouse models of disease. Here, we investigate two methods of allele-specific silencing, short hairpin RNA (shRNA) and antisense oligonucleotide (ASO) silencing, using a human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model of disease. We used cellular micropatterning devices with traction force microscopy and automated video analysis to examine each strategys effects on contractile defects underlying disease. We find that shRNA silencing ameliorates contractile phenotypes of disease, reducing disease-associated increases in cardiomyocyte velocity, force, and power. We find that ASO silencing, while better able to target and knockdown a specific disease-associated allele, showed more modest improvements in contractile phenotypes. We find a dissociation between allelic-specificity and functional improvements between the two tested therapeutic strategies, suggesting a more complex method of allelic control underlying HCM-associated transcripts.\n\nAuthor summaryAllele-specific silencing, whereby a therapeutic molecule is used to lower the expression of just one of the two copies or alleles of a gene, may be a potential therapeutic strategy in diseases caused by a single mutation. In this paper, we examine two such strategies in hypertrophic cardiomyopathy, a disease characterized by an overgrowth of the left-ventricular heart muscle as well as contractile dysfunction. We used a human cell model of disease, creating induced pluripotent stem cell derived cardiomyocytes from a patient with HCM caused by a single base pair change in just one allele of the gene MYH7. We used two strategies to silence the disease-associated copy of MYH7, both focused on reducing RNA expression from the mutated allele, as well as state-of-the-art biophysical techniques for measuring contractility. We found that one silencing strategy, which reduced expression of both the disease-associated and the healthy alleles of MYH7, showed great improvements in contractility between treated and untreated cells. Our second strategy, which silenced only the disease-associated copy of MYH7, showed more modest improvements in contractility. This suggests that the disease mechanism underlying this type of hypertrophic cardiomyopathy may be more complex than just presence or absence of the mutated RNA.

genetics↗

bioRxiv2019

Allele-specific silencing ameliorates restrictive cardiomyopathy due to a human myosin regulatory light chain mutation

BackgroundRestrictive cardiomyopathy (RCM) is a rare heart disease associated with mutations in sarcomeric genes and with phenotypic overlap with hypertrophic cardiomyopathy. There is no approved therapy. Here, we explore the potential of an interfering RNA (RNAi) therapeutic for a human sarcomeric mutation in MYL2 causative of restrictive cardiomyopathy in a mouse model. MethodsAAV9-M7.8L shRNA was selected from a pool of RNAi oligonucleotides containing the SNV in different positions to specifically target the mutated allele causative of RCM by FACS screening. Two groups of RLC-N47K transgenic mice were injected with a single dose of AAV9-M7.8L shRNA at 3 days of age and at 60 days of age. Mice were subjected to treadmill exercise and echocardiography after treatment to determine VO2max and left ventricular mass. At the end of treatment, heart, lung, liver and kidney tissue was harvested to determine viral tropism and for transcriptome and proteomic analysis. Cardiomyocytes were isolated for single cell studies. ResultsOne time injection of AAV9-M7.8L RNAi in 3-day-old humanized RLC mutant transgenic mice silenced the mutated allele (RLC-47K) with minimal effects on the normal allele (RLC-47N) assayed 16 weeks post-injection. AAV9-M7.8L RNAi suppressed the expression of hypertrophic biomarkers, reduced heart weight and attenuated a pathological increase in left ventricular mass (LVM). Single adult cardiac myocytes from mice treated with AAV9-M7.8L showed partial restoration of the maximal contraction velocity with marked reduction in hypercontractility as well as relaxation kinetics and improved time to maximal calcium reuptake velocity. In addition, cardiac stress protein biomarkers, such as calmodulin-dependent protein kinase II (CAMKII) and the transcription activator Brg1 were reduced suggesting recovery towards a healthy myocardium. Transcriptome analyses further revealed no significant changes of argonaute (AGO1, AGO2) and endoribonuclease dicer (DICER1) transcripts while endogenous microRNAs were preserved suggesting the RNAi pathway was not saturated. ConclusionsOur results show the feasibility, efficacy, and safety of RNAi therapeutics directed at human restrictive cardiomyopathy. This is a promising step towards targeted therapy for a prevalent human disease. Clinical PerspectiveWhat is new? O_LIRestrictive cardiomyopathy due to a mutation of human MYL2 modeled in cells and mice can be treated with RNA interference C_LIO_LIReduction of disease-causing allele improves function at the cellular and organ level C_LIO_LIOff target effects evaluated and not found to be significant C_LI What are the clinical implications? O_LIAllele-specific RNA silencing of human alleles may be effective in treating inherited restrictive cardiomyopathy C_LIO_LIRNA therapies targeting individual mutations may need to be developed prior to consideration of clinical translation C_LI

molecular biology↗

bioRxiv2019

Targeted Long-Read RNA Sequencing Demonstrates Transcriptional Diversity Driven by Splice-Site Variation in MYBPC3

BackgroundClinical sequencing has traditionally focused on genomic DNA through the use of targeted panels and exome sequencing, rather than investigating the potential transcriptomic consequences of disease-associated variants. RNA sequencing has recently been shown to be an effective additional tool for identifying disease-causing variants. We here use targeted long-read genome and transcriptome sequencing to efficiently and economically identify molecular consequences of a rare, disease-associated variant in hypertrophic cardiomyopathy (HCM).\n\nMethods and ResultsOur study, which employed both Pacific Biosciences SMRT sequencing and Oxford Nanopore Technologies MinION sequencing, as well as two RNA targeting strategies, identified alternatively-spliced isoforms that resulted from a splice-site variant containing allele in HCM. These included a predicted in-frame exon-skipping event, as well as an abundance of additional isoforms with unexpected intron-inclusion, exon-extension, and pseudo-exon events. The use of long-read RNA sequencing allowed us to not only investigate full length alternatively-spliced transcripts but also to phase them back to the variant-containing allele.\n\nConclusionsWe suggest that targeted, long-read RNA sequencing in conjunction with genome sequencing may provide additional molecular evidence of disease for rare or de novo variants in cardiovascular disease, as well as providing new information about the consequence of these variants on downstream RNA and protein expression.

genetics↗