Biology subjects

Musunuru, K.

Publications and source records attributed to Musunuru, K..

13 recordsLinked to original sources

bioRxiv2026

Correction of a recurrent pathogenic variant in methylmalonic acidemia using adenine base editing

Methylmalonic acidemia (MMA) is a recessive genetic disease caused by variants in the MMUT (mitochondrial enzyme methylmalonyl-CoA mutase) gene or by defects in transport or metabolism of MMUT cofactor (5 deoxyadenosylcobalamin), including variants in the MMAB gene. For the most recurrent pathogenic MMAB variant, c.556C>T (R186W), we identified a corrective editing strategy using adenine base editing. Deploying an adenine base editor mRNA and optimized hybrid guide RNA with lipid nanoparticles, we observed efficient in vitro corrective editing of the variant to wild-type, with minimized bystander editing and off-target editing in hepatocytes. These observations lay the groundwork for a gene editing therapy for patients with MMA resulting from at least one copy of the MMAB c.556C>T (R186W) variant, as well as a platform of similar therapies for patients with MMA caused by other variants amenable to adenine base editing.

genetics↗

bioRxiv2025

OxLDL-targeted Chimeric Antigen Receptor T Regulatory Cells Reduce Atherosclerotic Plaque Development

Cardiovascular disease caused by atherosclerosis is responsible for 18 million deaths annually, highlighting a significant need for new medical therapies, especially for patients ineligible for surgical interventions. Atherosclerosis is driven by the accumulation of low-density lipoprotein (LDL) and the formation of foam cells, accompanied by oxidative stress and the deposition of oxidized LDL (OxLDL), a pro-inflammatory molecule. Lowering LDL is the mainstay of current medical treatment in addition to blood pressure control and lifestyle changes, but to date, specifically targeting the inflammatory pathways contributing to plaque development without significant systemic side effects has not been feasible. Over the past decade, chimeric antigen receptor (CAR) T cells have treated cancer and restored immune imbalance in autoimmune diseases in patients and resolved cardiac fibrosis in preclinical models. Using an inducible T regulatory cell (Treg) platform, we created an anti-OxLDL-specific CAR Treg therapy that exerts cell- and cytokine-mediated immunosuppression to reduce macrophage-foam cell formation in vitro. Murine anti-OxLDL CAR Tregs inhibited 80% of atherosclerotic plaque formation in immunocompetent mouse models of hyperlipidemia and atherosclerosis. These studies illustrate the potential of anti-OxLDL CAR Tregs to mitigate the inflammation and plaque deposition associated with OxLDL, potentially offering a new therapeutic option for atherosclerosis.

synthetic biology↗

bioRxiv2024

Improved specificity and efficacy of base-editing therapies with hybrid guide RNAs

Phenylketonuria (PKU), pseudoxanthoma elasticum (PXE), and hereditary tyrosinemia type 1 (HT1) are autosomal recessive disorders linked to the PAH, ABCC6, and FAH and HPD genes, respectively. Here we evaluated the off-target editing profiles of clinical lead guide RNAs (gRNAs) that, when combined with adenine base editors (ABEs), correct the recurrent PAH P281L variant, PAH R408W variant, or ABCC6 R1164X variant or disrupt either of two sites in the HPD gene (a modifier gene of HT1) in human hepatocytes. To mitigate off-target mutagenesis, we systematically screened hybrid gRNAs with DNA nucleotide substitutions. Comprehensive and variant-aware specificity profiling of these hybrid gRNAs revealed dramatically reduced off-target editing and reduced bystander editing in cells. In humanized PAH P281L and ABCC6 R1164X mouse models of PKU and PXE, we showed that when formulated in lipid nanoparticles (LNPs) with ABE mRNA, selected hybrid gRNAs reverted disease phenotypes, reduced off-target editing, increased on-target editing, and reduced bystander editing in vivo. These studies highlight the utility of hybrid gRNAs to improve the safety and efficiency of adenine base editing therapies.

molecular biology↗

bioRxiv2023

Epigenome Editing Durability Varies Widely Across Cardiovascular Disease Target Genes

BackgroundHepatic knockdown of the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene or the angiopoietin-like 3 (ANGPTL3) gene has been demonstrated to reduce blood low-density lipoprotein cholesterol (LDL-C) levels, and hepatic knockdown of the angiotensinogen (AGT) gene has been demonstrated to reduce blood pressure. Genome editing can productively target each of these three genes in hepatocytes in the liver, offering the possibility of durable "one-and-done" therapies for hypercholesterolemia and hypertension. However, concerns around making permanent gene sequence changes via DNA strand breaks might hinder acceptance of these therapies. Epigenome editing offers an alternative approach to gene inactivation, via silencing of gene expression by methylation of the promoter region, but the long-term durability of epigenome editing remains to be established. MethodsWe assessed the ability of epigenome editing to durably reduce the expression of the human PCSK9, ANGPTL3, and AGT genes in HuH-7 hepatoma cells. Using the CRISPRoff epigenome editor, we identified guide RNAs that produced efficient gene knockdown immediately after transfection. We assessed the durability of gene expression and methylation changes through serial cell passages. ResultsCells treated with CRISPRoff and PCSK9 guide RNAs were maintained for up to 124 cell doublings and demonstrated durable knockdown of gene expression and increased CpG dinucleotide methylation in the promoter, exon 1, and intron 1 regions. In contrast, cells treated with CRISPRoff and ANGPTL3 guide RNAs experienced only transient knockdown of gene expression. Cells treated with CRISPRoff and AGT guide RNAs also experienced transient knockdown of gene expression; although initially there was increased CpG methylation throughout the early part of the gene, this methylation was geographically heterogeneous--transient in the promoter, and stable in intron 1. ConclusionsThis work demonstrates precise and durable gene regulation via methylation, supporting a new therapeutic approach for protection against cardiovascular disease via knockdown of genes such as PCSK9. However, the durability of knockdown with methylation changes is not generalizable across target genes, likely limiting the therapeutic potential of epigenome editing compared to other modalities.

genomics↗

bioRxiv2023

A Resource of Gene Expression Data from a Multiethnic Population Cohort of Induced Pluripotent Cell-Derived Cardiomyocytes

Induced pluripotent stem cells (iPSCs), upon differentiation into somatic cell types, offer the ability to model the genetic states of tissues in the individuals from whom the iPSCs were derived.1 A prior study used a multiethnic cohort of healthy individuals to derive iPSCs and iPSC-hepatocytes and perform gene expression and expression quantitative trait locus (eQTL) analyses, identifying causal genes and variants linked to blood lipid levels.2 We sought to use this cohort of iPSC lines to generate a similar resource with iPSC-cardiomyocytes (iPSC-CMs) and make the results available to the community.

genomics↗

bioRxiv2022

CRISPR/Cas9 screens implicate RARA and SPNS1 in doxorubicin cardiotoxicity

Doxorubicin (DOX) is an efficacious chemotherapy compound used to treat various cancers which elicits severe side effects, including heart failure. Uptake of DOX by cardiomyocytes causes metabolic dysfunction and cell death but causal mechanisms remain largely undefined. We applied genome-wide CRISPR/Cas9 knockout screens to discover genetic modifiers of DOX-induced cardiomyocyte cell death, and independently, DOX uptake and clearance. Both screens discovered known and novel factors. In cell death screens and validation studies, loss of retinoic acid receptor- (RARA) predisposed cardiomyocytes to DOX-mediated cell death. Conversely, RARA activation reduced DOX cytotoxicity in wild type cardiomyocytes. RNA-Seq analysis revealed that whilst DOX caused large-scale suppression of metabolic and mitochondrial gene expression, RARA activation mitigated this effect. In DOX accumulation screens, an essential role for lysosomes in DOX clearance was observed. Loss of Sphingolipid Transporter 1 (SPNS1) led to DOX hyperaccumulation, suppression of autophagy, increased DNA damage, and increased cell death. Hence, SPNS1 plays a key role in buffering against DOX accumulation and toxicity. Collectively, our study nominated hundreds of drug-gene interactions, providing a springboard for exploration of causal mechanisms, and a technical framework for future screening campaigns.

cell biology↗

bioRxiv2021

Lipid nanoparticles incorporating a GalNAc ligand enable in vivo liver ANGPTL3 editing in wild-type and somatic LDLR knockout non-human primates

Standard lipid nanoparticles (LNPs) deliver gene editing cargoes to hepatocytes through receptor-mediated uptake via the low-density lipoprotein receptor (LDLR). Homozygous familial hypercholesterolemia (HoFH) is a morbid genetic disease characterized by complete or near-complete LDLR deficiency, markedly elevated blood low-density lipoprotein cholesterol (LDL-C) levels, and premature atherosclerotic cardiovascular disease. In order to enable in vivo liver gene editing in HoFH patients, we developed a novel LNP delivery technology that incorporates a targeting ligand--N-acetylgalactosamine (GalNAc)--which binds to the asialoglycoprotein receptor (ASGPR). In a cynomolgus monkey (Macaca fascicularis) non-human primate (NHP) model of HoFH created by somatic knockout of the LDLR gene via CRISPR-Cas9, treatment with GalNAc-LNPs formulated with an adenine base editor mRNA and a guide RNA (gRNA) targeting the ANGPTL3 gene yielded ~60% whole-liver editing and ~94% reduction of blood ANGPTL3 protein levels, whereas standard LNPs yielded minimal editing. Moreover, in wild-type NHPs, the editing achieved by GalNAc-LNPs compared favorably to that achieved by standard LNPs, suggesting that GalNAc-LNP delivery technology may prove useful across a range of in vivo therapeutic applications targeting the liver.

genetics↗

bioRxiv2021

Adipocyte-specific modulation of KLF14 expression in mice leads to sex-dependent impacts in adiposity and lipid metabolism

Genome-wide association studies identified single nucleotide polymorphisms on chromosome 7 upstream of KLF14 to be associated with metabolic syndrome traits and increased risk for Type 2 Diabetes (T2D). The associations were more significant in women than in men. The risk allele carriers expressed lower levels of the transcription factor KLF14 in adipose tissues than non-risk allele carriers. To investigate how adipocyte KLF14 regulates metabolic traits in a sex-dependent manner, we characterized high-fat diet fed male and female mice with adipocyte-specific Klf14 deletion or overexpression. Klf14 deletion resulted in increased fat mass in female mice and decreased fat mass in male mice. Female Klf14-deficient mice had overall smaller adipocytes in subcutaneous fat depots but larger adipocytes in parametrial depots, indicating a shift in lipid storage from subcutaneous to visceral fat depots. They had reduced metabolic rates and increased respiratory exchange ratios consistent with increased utilization of carbohydrates as an energy source. Fasting and isoproterenol-induced adipocyte lipolysis was defective in female Klf14-deficient mice and concomitantly adipocyte triglycerides lipase mRNA levels were downregulated. Female Klf14-deficient mice cleared blood triglyceride and NEFA less efficiently than wild type. Finally, adipocyte-specific overexpression of Klf14 resulted in lower total body fat in female but not male mice. Taken together, consistent with human studies, adipocyte KLF14 deficiency in female but not in male mice causes increased adiposity and redistribution of lipid storage from subcutaneous to visceral adipose tissues. Increasing KLF14 abundance in adipocytes of females with obesity and T2D may provide a novel treatment option to alleviate metabolic abnormalities.

physiology↗

bioRxiv2021

Global-scale CRISPR gene editor specificity profiling by ONE-seq identifies population-specific, variant off-target effects

Defining off-target profiles of gene-editing nucleases and CRISPR base editors remains an important challenge for use of these technologies, therapeutic or otherwise. Existing methods can identify off-target sites induced by these gene editors on an individual genome but are not designed to account for the broad diversity of genomic sequence variation that exists within populations of humans or other organisms. Here we describe OligoNucleotide Enrichment and sequencing (ONE-seq), a novel in vitro method that leverages customizable, high-throughput DNA synthesis technology instead of purified genomic DNA (gDNA) from individual genomes to profile gene editor off-target sites. We show that ONE-seq matches or exceeds the sensitivity of existing single-genome methods for identifying bona fide CRISPR-Cas9 off-target sites in cultured human cells and in vivo in a liver-humanized mouse model. In addition, ONE-seq outperforms existing best-in-class single-genome methods for defining off-target sites of CRISPR-Cas12a nucleases, cytosine base editors (CBEs), and adenine base editors (ABEs), unveiling previously undescribed bona fide off-target sites for all these editors in human cells. Most importantly, we leveraged ONE-seq to generate the first experimentally-derived population-scale off-target profiles for Cas9 nucleases that define the impacts of sequence variants from >2,500 individual human genome sequences in the 1000 Genomes Project database. Notably, some of the variants we identified that lead to increased mutation frequencies at off-target sites are enriched in specific human populations. We validated that novel population-specific, variant-sensitive off-target sites nominated by ONE-seq in vitro can show increased frequencies of mutations in human lymphoblastoid cells (LCLs) harboring these sequence variants. Collectively, our results demonstrate that ONE-seq is a highly sensitive off-target nomination method that can uniquely be used to identify population subgroup-linked differences in off-target profiles of gene editors. ONE-seq provides an important new pathway by which to assess the impacts of global human genetic sequence diversity on the specificities of gene editors, thereby enabling a broader and more all-inclusive approach for profiling off-target effects of these transformative therapeutic technologies.

molecular biology↗

bioRxiv2021

Randomized controlled studies comparing traditional lectures versus online modules

We assessed the efficacy of traditional lectures versus online modules with respect to student learning in an undergraduate introductory biochemistry course in two successive years. In the first year, students had the options of attending live lectures by the course instructor and viewing online modules pre-recorded by the instructor, with the lectures and modules covering identical content; in addition, all students had a mandatory weekly application session. Utilizing pre-course and post-course tests as an instrument with which to measure learning during the course, we observed significantly increased learning (0.7 standard deviations) with attendance of traditional lectures and decreased learning with use of online modules, even after adjustment for grade point average. In the second year, the course had the same curriculum, but students were randomized to either live lectures or online modules for the first half of the course, crossing over to the other modality during the second half. With randomization, no difference in learning was observed between the two groups. Furthermore, we found that students self-reported greater engagement when viewing online modules than when attending lectures in person. These findings suggest some aspects of the lecture experience can be shifted to online modules in STEM courses without impacts on student learning so as to use classroom time more fully for application-based active learning interventions.

scientific communication and education↗

bioRxiv2019

EDEM3 modulates plasma triglyceride level through its regulation of LRP1 expression

Human genetics studies have uncovered genetic variants that can be used to guide biological research and prioritize molecular targets for therapeutic intervention for complex diseases and metabolic conditions. We have identified a missense variant (P746S) in EDEM3 associated with lower blood triglyceride (TG) levels in >300,000 individuals. Functional analyses in cell and mouse models show that EDEM3 deficiency strongly increased the uptake of very low-density lipoprotein and thereby reduced the plasma TG level, as a result of up-regulated expression of LRP1 receptor. We demonstrate that EDEM3 deletion up-regulated the pathways for RNA and ER protein processing and transport, and consequently increased the cell surface mannose-containing glycoproteins, including LRP1. Metabolomics analyses reveal a cellular TG accumulation under EDEM3 deficiency, a profile consistent with individuals with carrying EDEM3 P746S. Our study identifies EDEM3 as a regulator of blood TG, and targeted inhibition of EDEM3 may provide a complementary approach for lowering elevated blood TG concentrations.

genetics↗

bioRxiv2019

Evaluating the contribution of cell-type specific alternative splicing to variation in lipid levels

BackgroundGenome-wide association studies have identified 150+ loci associated with lipid levels. However, the genetic mechanisms underlying most of these loci are not well-understood. Recent work indicates that changes in the abundance of alternatively spliced transcripts contributes to complex trait variation. Consequently, identifying genetic loci that associate with alternative splicing in disease-relevant cell types and determining the degree to which these loci are informative for lipid biology is of broad interest.\n\nMethods and ResultsWe analyze gene splicing in 83 sample-matched induced pluripotent stem cell (iPSC) and hepatocyte-like cell (HLC) lines (n=166), as well as in an independent collection of primary liver tissues (n=96). We observe that transcript splicing is highly cell-type specific, and the genes that are differentially spliced between iPSCs and HLCs are enriched for metabolism pathway annotations. We identify 1,381 HLC splicing quantitative trait loci (sQTLs) and 1,462 iPSC sQTLs and find that sQTLs are often shared across cell types. To evaluate the contribution of sQTLs to variation in lipid levels, we conduct colocalization analysis using lipid genome-wide association data. We identify 19 lipid-associated loci that colocalize either with an HLC expression quantitative trait locus (eQTL) or sQTL. Only one locus colocalizes with both an sQTL and eQTL, indicating that sQTLs contribute information about GWAS loci that cannot be obtained by analysis of steady-state gene expression alone.\n\nConclusionsThese results provide an important foundation for future efforts that use iPSC and iPSC-derived cells to evaluate genetic mechanisms influencing both cardiovascular disease risk and complex traits in general.

genetics↗

bioRxiv2018

RNA-binding protein A1CF modulates plasma triglyceride levels through posttranscriptional regulation of stress-induced VLDL secretion

BackgroundA recent human exome-chip study on plasma lipids identified a missense mutation in the A1CF (APOBEC1 complementation factor) gene that is associated with elevated triglyceride (TG) levels, but how A1CF, an RNA binding protein, influences plasma TG is unknown.\n\nMethodsWe generated A1cf knockout (A1cf -/-) mice and knock-in mice homozygous for the TG-associated Gly398Ser mutation (A1cfGS/GS), determined lipid phenotypes, and assessed TG physiology through measurements of clearance and secretion. We further identified A1CFs RNA binding targets using enhanced cross-linking and immunoprecipitation sequencing of cultured HepG2 cells and investigated pathways enriched for these targets. Transcriptomic effects of A1CF deficiency were evaluated through RNA sequencing and analyses for differential expression, alternative splicing, and RNA editing.\n\nResultsBoth A1cf -/-and A1cfGS/GS mice exhibited increased fasting plasma TG, establishing that the TG phenotype is due to A1CF loss of function. In vivo TG secretion and clearance studies revealed increased TG secretion without changes in clearance in A1cf -/-mice. Increased VLDL-apoB secretion was also seen in A1cf -/-rat hepatoma cells, but no increase in apoB synthesis was observed. This phenotype was seen without significant shifts in apoB-100/apoB-48 in A1CF deficiency. To discover novel pathways for A1CFs role in TG metabolism, we identified A1CFs RNA binding targets, which were enriched for pathways related to proteasomal catabolism and endoplasmic reticulum (ER) stress. Indeed, proteasomal inhibition led to increased cellular stress in A1cf -/-cells, and higher expression of ER-stress protein GRP78 was observed in resting A1cf -/-cells. RNA-seq of whole livers from wild-type and A1cf -/-mice revealed that pro-inflammatory, not lipogenesis, genes were upregulated as a secondary effect of A1CF deficiency. Differential alternative splicing (AS) analysis and RNA editing analysis revealed that genes involved in cellular stress and metabolism underwent differential changes in A1CF deficiency, and top A1CF binding target proteins with relevance to intracellular stress were differentially expressed on the protein but not mRNA level, implicating multiple mechanisms by which A1CF influences TG secretion.\n\nConclusionsThese data suggest an important role for A1CF in mediating VLDL-TG secretion through regulating intracellular stress.

genomics↗