Allele-specific control of rodent and human lncRNA KMT2E-AS1 promotes hypoxic endothelial pathology in pulmonary hypertension.

Hypoxic reprogramming of vasculature relies on genetic, epigenetic, and metabolic circuitry, but the control points are unknown. In pulmonary arterial hypertension (PAH), a disease driven by hypoxia inducible factor (HIF)-dependent vascular dysfunction, HIF-2α promoted expression of neighboring genes, long noncoding RNA (lncRNA) histone lysine N-methyltransferase 2E-antisense 1 (KMT2E-AS1) and histone lysine N-methyltransferase 2E (KMT2E). KMT2E-AS1 stabilized KMT2E protein to increase epigenetic histone 3 lysine 4 trimethylation (H3K4me3), driving HIF-2α-dependent metabolic and pathogenic endothelial activity. This lncRNA axis also increased HIF-2α expression across epigenetic, transcriptional, and posttranscriptional contexts, thus promoting a positive feedback loop to further augment HIF-2α activity. We identified a genetic association between rs73184087, a single-nucleotide variant (SNV) within a KMT2E intron, and disease risk in PAH discovery and replication patient cohorts and in a global meta-analysis. This SNV displayed allele (G)-specific association with HIF-2α, engaged in long-range chromatin interactions, and induced the lncRNA-KMT2E tandem in hypoxic (G/G) cells. In vivo, KMT2E-AS1 deficiency protected against PAH in mice, as did pharmacologic inhibition of histone methylation in rats. Conversely, forced lncRNA expression promoted more severe PH. Thus, the KMT2E-AS1/KMT2E pair orchestrates across convergent multi-ome landscapes to mediate HIF-2α pathobiology and represents a key clinical target in pulmonary hypertension.

Novel AAV-mediated genome editing therapy improves health and survival in a mouse model of methylmalonic acidemia.

Methylmalonic acidemia (MMA) is an inborn error of metabolism mostly caused by mutations in the mitochondrial methylmalonyl-CoA mutase gene (MMUT). MMA patients suffer from frequent episodes of metabolic decompensation, which can be life threatening. To mimic both the dietary restrictions and metabolic decompensation seen in MMA patients, we developed a novel protein-controlled diet regimen in a Mmut deficient mouse model of MMA and demonstrated the therapeutic benefit of mLB-001, a nuclease-free, promoterless recombinant AAV GeneRideTM vector designed to insert the mouse Mmut into the endogenous albumin locus via homologous recombination. A single intravenous administration of mLB-001 to neonatal or adult MMA mice prevented body weight loss and mortality when challenged with a high protein diet. The edited hepatocytes expressed functional MMUT protein and expanded over time in the Mmut deficient mice, suggesting a selective growth advantage over the diseased cells. In mice with a humanized liver, treatment with a human homolog of mLB-001 resulted in site-specific genome editing and transgene expression in the transplanted human hepatocytes. Taken together, these findings support the development of hLB-001 that is currently in clinical trials in pediatric patients with severe forms of MMA.

Lipid Nanoparticle-Mediated Delivery of Anti-miR-17 Family Oligonucleotide Suppresses Hepatocellular Carcinoma Growth.

Hepatocellular carcinoma (HCC) is one of the most common human malignancies with poor prognosis and urgent unmet medical need. Aberrant expression of multiple members of the miR-17 family are frequently observed in HCC, and their overexpression promotes tumorigenic properties of HCC cells. However, whether pharmacologic inhibition of the miR-17 family inhibits HCC growth remains unknown. In this study, we validated that the miR-17 family was upregulated in a subset of HCC tumors and cell lines and its inhibition by a tough decoy inhibitor suppressed the growth of Hep3B and HepG2 cells, which overexpress the miR-17 family. Furthermore, inhibition of the miR-17 family led to a global derepression of direct targets of the family in all three HCC cell lines tested. Pathway analysis of the deregulated genes indicated that the genes associated with TGFß signaling pathway were highly enriched in Hep3B and HepG2 cells. A miR-17 family target gene signature was established and used to identify RL01-17(5), a lipid nanoparticle encapsulating a potent anti-miR-17 family oligonucleotide. To address whether pharmacologic modulation of the miR-17 family can inhibit HCC growth, RL01-17(5) was systemically administrated to orthotopic Hep3B xenografts. Suppression of Hep3B tumor growth in vivo was observed and tumor growth inhibition correlated with induction of miR-17 family target genes. Together, this study provides proof-of-concept for targeting the miR-17 family in HCC therapy. Mol Cancer Ther; 16(5); 905-13. ©2017 AACR.

Gene activation of SMN by selective disruption of lncRNA-mediated recruitment of PRC2 for the treatment of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by progressive motor neuron loss and caused by mutations in SMN1 (Survival Motor Neuron 1). The disease severity inversely correlates with the copy number of SMN2, a duplicated gene that is nearly identical to SMN1. We have delineated a mechanism of transcriptional regulation in the SMN2 locus. A previously uncharacterized long noncoding RNA (lncRNA), SMN-antisense 1 (SMN-AS1), represses SMN2 expression by recruiting the Polycomb Repressive Complex 2 (PRC2) to its locus. Chemically modified oligonucleotides that disrupt the interaction between SMN-AS1 and PRC2 inhibit the recruitment of PRC2 and increase SMN2 expression in primary neuronal cultures. Our approach comprises a gene-up-regulation technology that leverages interactions between lncRNA and PRC2. Our data provide proof-of-concept that this technology can be used to treat disease caused by epigenetic silencing of specific loci.

Polysome shift assay for direct measurement of miRNA inhibition by anti-miRNA drugs.

Anti-miRNA (anti-miR) oligonucleotide drugs are being developed to inhibit overactive miRNAs linked to disease. To help facilitate the transition from concept to clinic, new research tools are required. Here we report a novel method--miRNA Polysome Shift Assay (miPSA)--for direct measurement of miRNA engagement by anti-miR, which is more robust than conventional pharmacodynamics using downstream target gene derepression. The method takes advantage of size differences between active and inhibited miRNA complexes. Active miRNAs bind target mRNAs in high molecular weight polysome complexes, while inhibited miRNAs are sterically blocked by anti-miRs from forming this interaction. These two states can be assessed by fractionating tissue or cell lysates using differential ultracentrifugation through sucrose gradients. Accordingly, anti-miR treatment causes a specific shift of cognate miRNA from heavy to light density fractions. The magnitude of this shift is dose-responsive and maintains a linear relationship with downstream target gene derepression while providing a substantially higher dynamic window for aiding drug discovery. In contrast, we found that the commonly used 'RT-interference' approach, which assumes that inhibited miRNA is undetectable by RT-qPCR, can yield unreliable results that poorly reflect the binding stoichiometry of anti-miR to miRNA. We also demonstrate that the miPSA has additional utility in assessing anti-miR cross-reactivity with miRNAs sharing similar seed sequences.

Matrix Remodeling Promotes Pulmonary Hypertension through Feedback Mechanoactivation of the YAP/TAZ-miR-130/301 Circuit.

Pulmonary hypertension (PH) is a deadly vascular disease with enigmatic molecular origins. We found that vascular extracellular matrix (ECM) remodeling and stiffening are early and pervasive processes that promote PH. In multiple pulmonary vascular cell types, such ECM stiffening induced the microRNA-130/301 family via activation of the co-transcription factors YAP and TAZ. MicroRNA-130/301 controlled a PPAR?-APOE-LRP8 axis, promoting collagen deposition and LOX-dependent remodeling and further upregulating YAP/TAZ via a mechanoactive feedback loop. In turn, ECM remodeling controlled pulmonary vascular cell crosstalk via such mechanotransduction, modulation of secreted vasoactive effectors, and regulation of associated microRNA pathways. In vivo, pharmacologic inhibition of microRNA-130/301, APOE, or LOX activity ameliorated ECM remodeling and PH. Thus, ECM remodeling, as controlled by the YAP/TAZ-miR-130/301 feedback circuit, is an early PH trigger and offers combinatorial therapeutic targets for this devastating disease.

Genetic and hypoxic alterations of the microRNA-210-ISCU1/2 axis promote iron-sulfur deficiency and pulmonary hypertension.

Iron-sulfur (Fe-S) clusters are essential for mitochondrial metabolism, but their regulation in pulmonary hypertension (PH) remains enigmatic. We demonstrate that alterations of the miR-210-ISCU1/2 axis cause Fe-S deficiencies in vivo and promote PH. In pulmonary vascular cells and particularly endothelium, hypoxic induction of miR-210 and repression of the miR-210 targets ISCU1/2 down-regulated Fe-S levels. In mouse and human vascular and endothelial tissue affected by PH, miR-210 was elevated accompanied by decreased ISCU1/2 and Fe-S integrity. In mice, miR-210 repressed ISCU1/2 and promoted PH. Mice deficient in miR-210, via genetic/pharmacologic means or via an endothelial-specific manner, displayed increased ISCU1/2 and were resistant to Fe-S-dependent pathophenotypes and PH. Similar to hypoxia or miR-210 overexpression, ISCU1/2 knockdown also promoted PH. Finally, cardiopulmonary exercise testing of a woman with homozygous ISCU mutations revealed exercise-induced pulmonary vascular dysfunction. Thus, driven by acquired (hypoxia) or genetic causes, the miR-210-ISCU1/2 regulatory axis is a pathogenic lynchpin causing Fe-S deficiency and PH. These findings carry broad translational implications for defining the metabolic origins of PH and potentially other metabolic diseases sharing similar underpinnings.

Anti-microRNA-21 oligonucleotides prevent Alport nephropathy progression by stimulating metabolic pathways.

MicroRNA-21 (miR-21) contributes to the pathogenesis of fibrogenic diseases in multiple organs, including the kidneys, potentially by silencing metabolic pathways that are critical for cellular ATP generation, ROS production, and inflammatory signaling. Here, we developed highly specific oligonucleotides that distribute to the kidney and inhibit miR-21 function when administered subcutaneously and evaluated the therapeutic potential of these anti-miR-21 oligonucleotides in chronic kidney disease. In a murine model of Alport nephropathy, miR-21 silencing did not produce any adverse effects and resulted in substantially milder kidney disease, with minimal albuminuria and dysfunction, compared with vehicle-treated mice. miR-21 silencing dramatically improved survival of Alport mice and reduced histological end points, including glomerulosclerosis, interstitial fibrosis, tubular injury, and inflammation. Anti-miR-21 enhanced PPARα/retinoid X receptor (PPARα/RXR) activity and downstream signaling pathways in glomerular, tubular, and interstitial cells. Moreover, miR-21 silencing enhanced mitochondrial function, which reduced mitochondrial ROS production and thus preserved tubular functions. Inhibition of miR-21 was protective against TGF-ß-induced fibrogenesis and inflammation in glomerular and interstitial cells, likely as the result of enhanced PPARα/RXR activity and improved mitochondrial function. Together, these results demonstrate that inhibition of miR-21 represents a potential therapeutic strategy for chronic kidney diseases including Alport nephropathy.

The microRNA-130/301 family controls vasoconstriction in pulmonary hypertension.

Pulmonary hypertension (PH) is a complex disorder, spanning several known vascular cell types. Recently, we identified the microRNA-130/301 (miR-130/301) family as a regulator of multiple pro-proliferative pathways in PH, but the true breadth of influence of the miR-130/301 family across cell types in PH may be even more extensive. Here, we employed targeted network theory to identify additional pathogenic pathways regulated by miR-130/301, including those involving vasomotor tone. Guided by these predictions, we demonstrated, via gain- and loss-of-function experimentation in vitro and in vivo, that miR-130/301-specific control of the peroxisome proliferator-activated receptor γ regulates a panel of vasoactive factors communicating between diseased pulmonary vascular endothelial and smooth muscle cells. Of these, the vasoconstrictive factor endothelin-1 serves as an integral point of communication between the miR-130/301-peroxisome proliferator-activated receptor γ axis in endothelial cells and contractile function in smooth muscle cells. Thus, resulting from an in silico analysis of the architecture of the PH disease gene network coupled with molecular experimentation in vivo, these findings clarify the expanded role of the miR-130/301 family in the global regulation of PH. They further emphasize the importance of molecular cross-talk among the diverse cellular populations involved in PH.

Let-7 coordinately suppresses components of the amino acid sensing pathway to repress mTORC1 and induce autophagy.

Macroautophagy (hereafter autophagy) is the major pathway by which macromolecules and organelles are degraded. Autophagy is regulated by the mTOR signaling pathway-the focal point for integration of metabolic information, with mTORC1 playing a central role in balancing biosynthesis and catabolism. Of the various inputs to mTORC1, the amino acid sensing pathway is among the most potent. Based upon transcriptome analysis of neurons subjected to nutrient deprivation, we identified let-7 microRNA as capable of promoting neuronal autophagy. We found that let-7 activates autophagy by coordinately downregulating the amino acid sensing pathway to prevent mTORC1 activation. Let-7 induced autophagy in the brain to eliminate protein aggregates, establishing its physiological relevance for in vivo autophagy modulation. Moreover, peripheral delivery of let-7 anti-miR repressed autophagy in muscle and white fat, suggesting that let-7 autophagy regulation extends beyond CNS. Hence, let-7 plays a central role in nutrient homeostasis and proteostasis regulation in higher organisms.

Anti-miRs competitively inhibit microRNAs in Argonaute complexes.

MicroRNAs (miRNAs), small RNA molecules that post-transcriptionally regulate mRNA expression, are crucial in diverse developmental and physiological programs and their misregulation can lead to disease. Chemically modified oligonucleotides have been developed to modulate miRNA activity for therapeutic intervention in disease settings, but their mechanism of action has not been fully elucidated. Here we show that the miRNA inhibitors (anti-miRs) physically associate with Argonaute proteins in the context of the cognate target miRNA in vitro and in vivo. The association is mediated by the seed region of the miRNA and is sensitive to the placement of chemical modifications. Furthermore, the targeted miRNAs are stable and continue to be associated with Argonaute. Our results suggest that anti-miRs specifically associate with Argonaute-bound miRNAs, preventing association with target mRNAs, which leads to subsequent stabilization and thus increased expression of the targeted mRNAs.

Systems-level regulation of microRNA networks by miR-130/301 promotes pulmonary hypertension.

Development of the vascular disease pulmonary hypertension (PH) involves disparate molecular pathways that span multiple cell types. MicroRNAs (miRNAs) may coordinately regulate PH progression, but the integrative functions of miRNAs in this process have been challenging to define with conventional approaches. Here, analysis of the molecular network architecture specific to PH predicted that the miR-130/301 family is a master regulator of cellular proliferation in PH via regulation of subordinate miRNA pathways with unexpected connections to one another. In validation of this model, diseased pulmonary vessels and plasma from mammalian models and human PH subjects exhibited upregulation of miR-130/301 expression. Evaluation of pulmonary arterial endothelial cells and smooth muscle cells revealed that miR-130/301 targeted PPARγ with distinct consequences. In endothelial cells, miR-130/301 modulated apelin-miR-424/503-FGF2 signaling, while in smooth muscle cells, miR-130/301 modulated STAT3-miR-204 signaling to promote PH-associated phenotypes. In murine models, induction of miR-130/301 promoted pathogenic PH-associated effects, while miR-130/301 inhibition prevented PH pathogenesis. Together, these results provide insight into the systems-level regulation of miRNA-disease gene networks in PH with broad implications for miRNA-based therapeutics in this disease. Furthermore, these findings provide critical validation for the evolving application of network theory to the discovery of the miRNA-based origins of PH and other diseases.

Non-inhibited miRNAs shape the cellular response to anti-miR.

Identification of primary microRNA (miRNA) gene targets is critical for developing miRNA-based therapeutics and understanding their mechanisms of action. However, disentangling primary target derepression induced by miRNA inhibition from secondary effects on the transcriptome remains a technical challenge. Here, we utilized RNA immunoprecipitation (RIP) combined with competitive binding assays to identify novel primary targets of miR-122. These transcripts physically dissociate from AGO2-miRNA complexes when anti-miR is spiked into liver lysates. mRNA target displacement strongly correlated with expression changes in these genes following in vivo anti-miR dosing, suggesting that derepression of these targets directly reflects changes in AGO2 target occupancy. Importantly, using a metric based on weighted miRNA expression, we found that the most responsive mRNA target candidates in both RIP competition assays and expression profiling experiments were those with fewer alternative seed sites for highly expressed non-inhibited miRNAs. These data strongly suggest that miRNA co-regulation modulates the transcriptomic response to anti-miR. We demonstrate the practical utility of this 'miR-target impact' model, and encourage its incorporation, together with the RIP competition assay, into existing target prediction and validation pipelines.

Method for widespread microRNA-155 inhibition prolongs survival in ALS-model mice.

microRNAs (miRNAs) are dysregulated in a variety of disease states, suggesting that this newly discovered class of gene expression repressors may be viable therapeutic targets. A microarray of miRNA changes in ALS-model superoxide dismutase 1 (SOD1)(G93A) rodents identified 12 miRNAs as significantly changed. Six miRNAs tested in human ALS tissues were confirmed increased. Specifically, miR-155 was increased 5-fold in mice and 2-fold in human spinal cords. To test miRNA inhibition in the central nervous system (CNS) as a potential novel therapeutic, we developed oligonucleotide-based miRNA inhibitors (anti-miRs) that could inhibit miRNAs throughout the CNS and in the periphery. Anti-miR-155 caused global derepression of targets in peritoneal macrophages and, following intraventricular delivery, demonstrated widespread functional distribution in the brain and spinal cord. After treating SOD1(G93A) mice with anti-miR-155, we significantly extended survival by 10 days and disease duration by 15 days (38%) while a scrambled control anti-miR did not significantly improve survival or disease duration. Therefore, antisense oligonucleotides may be used to successfully inhibit miRNAs throughout the brain and spinal cord, and miR-155 is a promising new therapeutic target for human ALS.

Disease-linked microRNA-21 exhibits drastically reduced mRNA binding and silencing activity in healthy mouse liver.

MicroRNAs (miRNAs) bind to mRNAs and fine-tune protein output by affecting mRNA stability and/or translation. miR-21 is a ubiquitous, highly abundant, and stress-responsive miRNA linked to several diseases, including cancer, fibrosis, and inflammation. Although the RNA silencing activity of miR-21 in diseased cells has been well documented, the roles of miR-21 under healthy cellular conditions are not well understood. Here, we show that pharmacological inhibition or genetic deletion of miR-21 in healthy mouse liver has little impact on regulation of canonical seed-matched mRNAs and only a limited number of genes enriched in stress response pathways. These surprisingly weak and selective regulatory effects on known and predicted target mRNAs contrast with those of other abundant liver miRNAs such as miR-122 and let-7. Moreover, miR-21 shows greatly reduced binding to polysome-associated target mRNAs compared to miR-122 and let-7. Bioinformatic analysis suggests that reduced thermodynamic stability of seed pairing and target binding may contribute to this deficiency of miR-21. Significantly, these trends are reversed in human cervical carcinoma (HeLa) cells, where miRNAs including miR-21 show enhanced target binding within polysomes and where miR-21 triggers strong degradative activity toward target mRNAs. Taken together, our results suggest that, under normal cellular conditions in liver, miR-21 activity is maintained below a threshold required for binding and silencing most of its targets. Consequently, enhanced association with polysome-associated mRNA is likely to explain in part the gain of miR-21 function often found in diseased or stressed cells.

Functional genomics identifies therapeutic targets for MYC-driven cancer.

MYC oncogene family members are broadly implicated in human cancers, yet are considered "undruggable" as they encode transcription factors. MYC also carries out essential functions in proliferative tissues, suggesting that its inhibition could cause severe side effects. We elected to identify synthetic lethal interactions with c-MYC overexpression (MYC-SL) in a collection of ~3,300 druggable genes, using high-throughput siRNA screening. Of 49 genes selected for follow-up, 48 were confirmed by independent retesting and approximately one-third selectively induced accumulation of DNA damage, consistent with enrichment in DNA-repair genes by functional annotation. In addition, genes involved in histone acetylation and transcriptional elongation, such as TRRAP and BRD4, were identified, indicating that the screen revealed known MYC-associated pathways. For in vivo validation we selected CSNK1e, a kinase whose expression correlated with MYCN amplification in neuroblastoma (an established MYC-driven cancer). Using RNAi and available small-molecule inhibitors, we confirmed that inhibition of CSNK1e halted growth of MYCN-amplified neuroblastoma xenografts. CSNK1e had previously been implicated in the regulation of developmental pathways and circadian rhythms, whereas our data provide a previously unknown link with oncogenic MYC. Furthermore, expression of CSNK1e correlated with c-MYC and its transcriptional signature in other human cancers, indicating potential broad therapeutic implications of targeting CSNK1e function. In summary, through a functional genomics approach, pathways essential in the context of oncogenic MYC but not to normal cells were identified, thus revealing a rich therapeutic space linked to a previously "undruggable" oncogene.

MicroRNA-21 promotes fibrosis of the kidney by silencing metabolic pathways.

Scarring of the kidney is a major public health concern, directly promoting loss of kidney function. To understand the role of microRNA (miRNA) in the progression of kidney scarring in response to injury, we investigated changes in miRNA expression in two kidney fibrosis models and identified 24 commonly up-regulated miRNAs. Among them, miR-21 was highly elevated in both animal models and in human transplanted kidneys with nephropathy. Deletion of miR-21 in mice resulted in no overt abnormality. However, miR-21(-/-) mice suffered far less interstitial fibrosis in response to kidney injury, a phenotype duplicated in wild-type mice treated with anti-miR-21 oligonucleotides. Global derepression of miR-21 target mRNAs was readily detectable in miR-21(-/-) kidneys after injury. Analysis of gene expression profiles up-regulated in the absence of miR-21 identified groups of genes involved in metabolic pathways, including the lipid metabolism pathway regulated by peroxisome proliferator-activated receptor-α (Pparα), a direct miR-21 target. Overexpression of Pparα prevented ureteral obstruction-induced injury and fibrosis. Pparα deficiency abrogated the antifibrotic effect of anti-miR-21 oligonucleotides. miR-21 also regulated the redox metabolic pathway. The mitochondrial inhibitor of reactive oxygen species generation Mpv17l was repressed by miR-21, correlating closely with enhanced oxidative kidney damage. These studies demonstrate that miR-21 contributes to fibrogenesis and epithelial injury in the kidney in two mouse models and is a candidate target for antifibrotic therapies.