Biology subjects

Ekker, S. C.

Publications and source records attributed to Ekker, S. C..

At least 19 recordsLinked to original sources

bioRxiv2026

Systematic Engineering of Loss-of-Function Alleles in the Zebrafish Mitochondrial Proteome

Pathogenic variants in the 13 protein-coding genes of the mitochondrial genome underlie clinically and biochemically heterogeneous disorders. Most mtDNA-encoded genes lack defined loss-of-function (LOF) models in vivo. To address this gap, we have generated Z-Terminator, a systematic in vivo atlas of loss-of-function alleles covering all the mtDNA-encoded OXPHOS subunits in zebrafish (Danio rerio). We used mitochondrial TALE base editors to introduce premature termination codon (PTC) alleles via C-to-T transitions across Complexes I, III, IV, and V. Larvae harboring PTC alleles displayed bioenergetic defects and elevated lactate. While mtDNA mutations are associated with sensorineural hearing loss, the cellular basis has remained unclear. We show that engineered mtDNA LOF alleles directly impair hair cell function in proportion to heteroplasmy in a living vertebrate. We investigated the germline transmission and tissue-specific heteroplasmy of these LOF alleles and observed that a subset of variants was transmitted to the F1 generation and displayed distinct mutant loads across organs. These findings establish Z-Terminator as a vertebrate platform for interrogating the role of mtDNA protein-coding genes in cellular dysfunction and for elucidating the pathophysiology of mitochondrial disorders.

genetics↗

bioRxiv2026

A Persistent Fleet of AI Scientists Exhibits Cooperative and Autopoietic Behavior

Scientific work depends on memory, provenance, and continuity across projects, yet most agentic scientist systems are evaluated in bounded workflows or short benchmark runs. We describe a persistent fleet of cooperative AI scientist agents that operated continuously for nearly six months using shared memory, tools, and cross-agent communication. Critically, failures identified during longitudinal scientific research in this fleet prompted an advanced and recursively improving persistent memory architecture (MoE) that, with agentic science workflows, induced the generation of a novel trust architecture for the enablement of full provenance across all agentic scientific operations. An identity-level fabrication constraint reduced delusion-reinforcement probe failures from 91.7% to 0%, and a verification pipeline reduced wrong-topic citation hallucination more than 14-fold in companion benchmarks. This high provenance enabled the use of project memory systems to improve a local open-weight model on internal benchmarks from 44% to [~]90% through the deployment of fleet-specific institutional knowledge. This high-fidelity data environment also supported to date 104 recurring multi-phase reasoning cycles and produced 43 manually curated hypotheses, including cross-domain convergence events and a self-correcting rare-disease pharmacological chaperone-design case. While by design the fleet did not achieve unconstrained autonomous self-improvement or full autopoiesis, we term this bounded pattern AI Autopoietic Behavior due to the recurring operational improvement mediated by internal feedback and retained through institutional records with high confidence. Together, persistent memory, trusted provenance, and recursive learning shifted these agents from episodic assistants toward accountable, long-term scientific collaborators.

bioinformatics↗

bioRxiv2026

Tracing developmental and adult hematopoiesis with an endogenous zebrafish runx1-2A-CreERT2 CRISPR knock-in

Zebrafish combines the power of genetics and unparalleled in vivo imaging to investigate the dynamics of vertebrate hematopoietic development. The conserved transcription factor Runx1 is essential for definitive hematopoiesis. We generated an endogenous zebrafish runx1-2A-creERT2 CRISPR knock-in for tamoxifen-regulated Runx1 lineage tracing and characterized its activity using recombinase-dependent fluorescence reporters, live imaging and flow cytometry. Tamoxifen induction of Cre in the early embryo labeled all expected Runx1 lineages, including neuroectodermal olfactory placode and Rohan-Beard neurons, primitive hematopoietic blood cells, and emerging definitive hematopoietic stem and progenitor cells (HSPCs) in the dorsal aorta. Short windows of tamoxifen treatment allowed separation of Runx1 primitive from definitive hematopoiesis and visualization of HSPC colonization of the larval caudal hematopoietic tissue, thymus and kidney. Runx1 HSPCs labeled during development persisted into adulthood and contributed to hematopoietic precursors, myeloid, lymphoid, and peripheral blood lineages. Labeling of all blood lineages was also effective by tamoxifen treatment of 5-month-old adults. The runx1-2A-creERT2 knock-in line provides a novel genetic tool for inducible spatial and temporal analysis of Runx1 progenitor mechanisms in developmental and adult hematopoiesis.

developmental biology↗

bioRxiv2026

Validation and analysis of 12,000 AI-driven CAR-T designs in the Bits to Binders competition

Artificial intelligence (AI) methods for proteins have advanced rapidly, improving structure prediction and design, particularly for de novo binders. However, most evaluations emphasize binding affinity rather than higher-order biological function. We present Bits to Binders, a global competition benchmarking de novo binder design in the context of chimeric antigen receptor (CAR) T cells. Teams from 42 countries submitted 12,000 designs of 80-amino acid binders targeting human CD20 as CAR binding domains. Designs were screened by pooled CAR-T proliferation, identifying 707 designs exhibiting significant CD20-specific enrichment, with team hit rates from 0.6% to 38.4%. Top-performing candidates were validated as individual constructs, measuring CD20-specific proliferation, expansion, cytokine production, and targeted cell lysis. We identified common design methodologies and factors correlated with DNA synthesis, expression, and target-specific T cell activation which nearly double the success rates when applied as a retrospective filter. We release this dataset as an open resource, with practical recommendations to support more effective AI-driven binder design.

bioinformatics↗

bioRxiv2025

Non-Destructive Larval Genotyping of Danio rerio for Mitochondrial DNA Genetics

The rapid advancement of nuclear and mitochondrial genomic editing tools has created an urgent need for efficient, non-lethal larval genotyping methods in zebrafish (Danio rerio) research. This study optimizes and validates a non-destructive proteinase K digestion method for mitochondrial and nuclear DNA genotyping while characterizing its impact on larval survival and gene expression. Using optimized protocol parameters, we demonstrate successful amplification of different mitochondrial and nuclear genetic loci with consistently high sensitivity. Molecular validation through PCR, restriction fragment length polymorphism analysis, and Sanger sequencing confirmed the specificity and reliability of the extracted DNA. The method successfully detected C-to-T base edits in the mt-tl1 gene introduced using the FusX TALE Base editor system, demonstrating its applicability to gene editing studies. Both 48-well and optimized 96-well formats were used, enabling this approach to be deployed at scale. This optimized method enables researchers to correlate genotypes with phenotypes in longitudinal studies while maintaining specimen viability particularly valuable for investigating early-onset mitochondrial diseases and utilizes standard laboratory equipment and reagents, facilitating widespread adoption in zebrafish research while adhering to ethical principles in reducing animal mortality.

genetics↗

bioRxiv2025

VEGF-B/NRP1 Signaling Modulates Mitochondrial Homeostasis and Cardiac Function After Myocardial Infarction

BackgroundMyocardial infarction (MI) remains a leading cause of mortality worldwide. Recent studies suggest a cardioprotective role for vascular endothelial growth factor-B (VEGF-B) in MI. However, the molecular mechanisms of VEGF-B-mediated signaling via its co-receptor Neuropilin-1 (NRP1) in MI are poorly understood. In this study, we investigated the intricate signaling mechanisms involving VEGF-B and NRP1 in cardiomyocytes (CMs) using ischemic injury as a model of MI. And secondly, we further validated the protective role of VEFG-B in ischemic heart disease, shedding light on their roles not only in cardiac function but also in its therapeutic potential. MethodsIn this study, we utilized both in vitro and in vivo approaches to elucidate the role of VEGF-B and NRP1 signaling in MI and how it manifests a protective role in mitochondrial functions and cardiac regeneration following ischemic injury. We used two different cardiomyocyte cell lines, H9c2 (rat ventricular cardiomyocytes) and HL-1 (mouse ventricular cardiomyocytes), and induced hypoxia conditions, using either 1% oxygen or 200{micro}M cobalt chloride (CoCl2) to mimic the myocardial infarction-induced ischemic injury in the heart. In addition, we developed a novel heat shock inducible zebrafish model of a cardiomyocyte-specific VEGF-B overexpression system to further examine the protective role of VEGF-B in vivo. ResultsOur findings indicate that both VEGF-B and NRP1 are predominantly expressed in heart tissue compared to other tissues, and their expression is altered in response to hypoxia/ischemic injury. Our results demonstrate that VEGF-B treatment prior to hypoxia enhances cardiomyocyte survival, while NRP1 knockdown abolishes this protective effect, highlighting a prominent role of NRP1 signaling in VEGF-B-mediated cardio protection. Furthermore, we found that VEGF-B promotes cardiomyocyte survival by improving mitochondrial function, as evidenced by reduced oxidative stress and ROS accumulation, decreased oxidative stress, preserved mitochondrial membrane potential, and increased ATP levels. Lastly, using our VEGF-B transgenic zebrafish model, we demonstrated that VEGF-B overexpression in zebrafish cardiomyocytes protects the heart from ischemic injury and enhances cardiac regeneration in an NRP1-dependent manner. ConclusionOur study has uncovered an important role of VEGF-B-NRP1 signaling axis in VEGF-B mediated cell survival and in beneficial mitochondrial functions in the CMs. Importantly, we also demonstrated that VEGF-B is protective against ischemic injury in vivo using a novel zebrafish model Novelty and SignificanceO_ST_ABSWhat Is Known?C_ST_ABSO_LIMyocardial infarction (MI) results in mitochondrial dysfunction, cardiomyocyte death, and adverse ventricular remodeling. C_LIO_LIVascular Endothelial Growth Factor B (VEGF-B) is traditionally associated with cardiac metabolism, survival, vascular biology, particularly in angiogenesis and lipid metabolism. C_LIO_LIThe neurophilin-1 (NRP1) receptor, a co-receptor for VEGF-B, is expressed in cardiomyocytes and implicated in mitochondrial signaling pathways. C_LIO_LIEmerging evidence suggests that VEGF-B may influence mitochondrial integrity and function under stress conditions. C_LI What New Information Does This Article Contribute?O_LIVEGF-B exerts a protective effect on cardiomyocyte mitocondria following ischemic injury by preserving mitochondrial function and reducing oxidative stress. C_LIO_LINRP1 receptor as a key mediator of VEGF-B modulates mitochondrial homeostasis in cardiomyocytes. C_LIO_LIVEGF-B signaling can attenuate apoptosis and enhance mitochondrial bioenergetics post-MI, suggesting a novel cardioprotective mechanism. C_LIO_LIClinical Implication: These findings suggest that therapeutic strategies aimed at enhancing VEGF-B/NRP1 signaling may improve mitochondrial function and cardiac recovery after MI, offering a novel avenue for limiting heart failure progression in ischemic heart disease. C_LI

molecular biology↗

bioRxiv2024

Lineage labeling with zebrafish hand2 Cre and CreERT2 recombinase CRISPR knock-ins

BackgroundThe ability to generate endogenous Cre recombinase drivers using CRISPR- Cas9 knock-in technology allows lineage tracing, cell type specific gene studies, and in vivo validation of inferred developmental trajectories from phenotypic and gene expression analyses. This report describes endogenous zebrafish hand2 Cre and CreERT2 drivers generated with GeneWeld CRISPR-Cas9 precision targeted integration. Resultshand2-2A-cre and hand2-2A-creERT2 knock-ins crossed with ubiquitous loxP-based Switch reporters led to broad labeling in expected mesodermal and neural crest-derived lineages in cardiac, pectoral fins, pharyngeal arch, liver, intestine, and mesothelial tissues, as well as enteric neurons. Novel patterns of hand2 lineage tracing appeared in venous blood vessels. CreERT2 induction at 24 hours reveals late emerging hand2 progenitors in the 24 - 48 hour embryo contribute to the venous and intestinal vasculature. Induction in 3 dpf larva restricts hand2 lineage labeling to mesoderm-derived components of the branchial arches, heart, liver and enteric neurons. Conclusionshand2 progenitors from the lateral plate mesoderm and ectoderm contribute to numerous lineages in the developing embryo. Later emerging hand2 progenitors become restricted to a subset of lineages in the larva. The hand2 Cre and CreERT2 drivers establish critical new tools to investigate hand2 lineages in zebrafish embryogenesis and larval organogenesis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/626907v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@12f58cborg.highwire.dtl.DTLVardef@16e95c8org.highwire.dtl.DTLVardef@12b8e9dorg.highwire.dtl.DTLVardef@a01848_HPS_FORMAT_FIGEXP M_FIG C_FIG Zebrafish hand2 Cre and CreERT2 drivers generated with GeneWeld CRISPR/Cas9 precision targeted integration label numerous lineages from the mesoderm and ectoderm. Temporal CreERT2 Tamoxifen regulated switching re{-} veals late emerging hand2 progenitors contribute to the embryonic venous vasculature and larval organs.

developmental biology↗

bioRxiv2024

Unconstrained Precision Mitochondrial Genome Editing with αDdCBEs

DddA-derived cytosine base editors (DdCBEs) enable the targeted introduction of C*G-to-T*A conversions in mitochondrial DNA (mtDNA). DdCBEs are often deployed as pairs, with each arm comprised of a transcription activator-like effector (TALE), a split double-stranded DNA deaminase half, and a uracil glycosylase inhibitor. This pioneering technology has helped improve our understanding of cellular processes involving mtDNA and has paved the way for the development of models and therapies for genetic disorders caused by pathogenic mtDNA variants. Nonetheless, given the intrinsic properties of TALE proteins, several target sites in human mtDNA remain out of reach to DdCBEs and other TALE-based technologies. Specifically, due to the conventional requirement for a thymine immediately upstream of the TALE target sequences (i.e., the 5-T constraint), over 150 loci in the human mitochondrial genome are presumed to be inaccessible to DdCBEs. Previous attempts at circumventing this constraint, either by developing monomeric DdCBEs or utilizing DNA-binding domains alternative to TALEs, have resulted in suboptimal specificity profiles with reduced therapeutic potential. Here, aiming to challenge and elucidate the relevance of the 5-T constraint in the context of DdCBE-mediated mtDNA editing, and to expand the range of motifs that are editable by this technology, we generated DdCBEs that contain modified TALE proteins engineered to recognize all 5 bases. Notably, 5-T-noncompliant, canonical DdCBEs efficiently edited mtDNA at diverse loci. However, DdCBEs were frequently outperformed by DdCBEs, which consistently displayed significant improvements in activity and specificity, regardless of the 5-most bases of their TALE binding sites. Furthermore, we showed that DdCBEs are compatible with DddAtox and its derivatives DddA6, and DddA11, and we validated TALE shifting with DdCBEs as an effective approach to optimize base editing outcomes at a single target site. Overall, DdCBEs enable efficient, specific, and unconstrained mitochondrial base editing.

bioengineering↗

bioRxiv2022

A phenotype-based forward genetic screen identifies Dnajb6 as a sick sinus syndrome gene

Sick sinus syndrome (SSS) is a group of heart rhythm disorders caused by malfunction of the sinus node, the hearts primary pacemaker. Partially owing to its aging-associated phenotypic manifestation and low expressivity, molecular mechanisms of SSS remain difficult to decipher. Here, we aim to develop a phenotype-based forward genetic approach in the zebrafish (Danio rerio) animal model for discovering essential genes which dysfunction could result in SSS-like phenotypes. Previously we showed the generation of protein trap library by using a revertible gene-breaking transposon (GBT)-based insertional mutagenesis system. Here, we reported the generation of a collection of 35 zebrafish insertional cardiac lines derived from this protein trap library, which was screened using electrocardiographic measurements. As a result, three mutants with SSS-like phenotypes were identified. We then focused on one of these 3 GBT mutants called GBT411 in which dnajb6b gene was disrupted, and conducted expressional, genetic, transcriptome, and electrophysiological studies using both zebrafish and mouse models. These studies confirmed the identity of Dnajb6 as a novel SSS causative gene with a unique expression pattern within the specialized population of sinus node pacemaker cardiomyocytes that lack the expression of HCN4 channels. Together, this study demonstrates the feasibility of a genetic screening approach in an adult vertebrate animal model for discovering new genetic factors for a heart rhythm disorder such as SSS.

genetics↗

bioRxiv2021

Zebrafish Cre/lox regulated UFlip alleles generated by CRISPR/Cas targeted integration provide cell-type specific conditional gene inactivation

The ability to regulate gene activity spatially and temporally is essential to investigate cell type specific gene function during development and in postembryonic processes and disease models. The Cre/lox system has been widely used for performing cell and tissue-specific conditional analysis of gene function in zebrafish, but simple and efficient methods for isolation of stable, Cre/lox regulated alleles are lacking. Here we applied our GeneWeld CRISPR/Cas9 short homology-directed targeted integration strategy to generate floxed conditional alleles that provide robust gene knockdown and strong loss of function phenotypes. A universal targeting vector, UFlip, with sites for cloning short 24-48 bp homology arms flanking a floxed mRFP gene trap plus secondary reporter cassette, was integrated into an intron in hdac1, rbbp4, and rb1. Active, gene off orientation hdac1-UFlip-Off and rb1-UFlip-Off integration alleles result in >99% reduction of gene expression in homozygotes and recapitulate known indel loss of function phenotypes. Passive, gene on orientation rbbp4-UFlip-On and rb1-UFlip-On integration alleles do not cause phenotypes in trans-heterozygous combination with an indel mutation. Cre recombinase injection leads to recombination at alternating pairs of loxP and lox2272 sites, inverting and locking the cassette into the active, gene off orientation, and the expected mutant phenotypes. In combination with our endogenous neural progenitor Cre drivers we demonstrate rbbp4-UFlip-On and rb1-UFlip-On gene inactivation phenotypes can be restricted to specific neural cell populations. Replacement of the UFlip mRFP primary reporter gene trap with a 2A-RFP in rbbp4-UFlip-Off, or 2A-KalTA4 in rb1-UFlip-Off, shows strong RFP expression in wild type or UAS:RFP injected embryos, respectively. Together these results validate a simplified approach for efficient isolation of highly mutagenic Cre/lox responsive conditional gene alleles to advance zebrafish Cre recombinase genetics.

genetics↗

bioRxiv2021

Chimeric RNA:DNA Donorguide Improves HDR in vitro and in vivo

Introducing small genetic changes to study specific mutations or reverting clinical mutations to wild type has been an area of interest in precision genomics for several years. In fact, it has been found that nearly 90% of all human pathogenic mutations are caused by small genetic variations, and the methods to efficiently and precisely correct these errors are critically important. One common way to make these small DNA changes is to provide a single stranded DNA (ssDNA) donor containing the desired alteration together with a targeted double-strand break (DSB) at the genomic target. The donor is typically flanked by regions of homology that are often ~30-100bp in length to leverage the homology directed repair (HDR) pathway. Coupling a ssDNA donor with a CRISPR-Cas9 to produce a targeted DSB is one of the most streamlined approaches to introduce small changes. However, in many cell types this approach results in a low rate of incorporation of the desired alteration and has undesired imprecise repair at the 5 or 3 junction due to artifacts of the DNA repair process. We herein report a technology that couples the spatial temporal localization of an ssDNA repair template and leverages the nucleic acid components of the CRISPR-Cas9 system. We show that by direct fusion of an ssDNA template to the trans activating RNA (tracrRNA) to generate an RNA-DNA chimera, termed Donorguide, we recover precise integration of genetic alterations, with both increased integration rates and decreased imprecision at the 5 or 3 junctions relative to an ssODN donor in vitro in HEK293T cells as well as in vivo in zebrafish. Further, we show that this technology can be used to enhance gene conversion with other gene editing tools such as TALENs.

genetics↗

bioRxiv2021

The FusX TALE Base Editor (FusXTBE) for rapid mitochondrial DNA programming of human cells in vitro and zebrafish disease models in vivo

Functional analyses of mitochondria have been hampered by few effective approaches to manipulate mtDNA and a lack of existing animal models. Recently a TALE-derived base editor was shown to induce C-to-T (or G-to-A) sequence changes in mtDNA. We report here the FusX TALE Base Editor (FusXTBE) to facilitate broad-based access to TALE mitochondrial base editing technology. TALE Writer is a de novo in silico design tool to map potential mtDNA base editing sites. FusXTBE was demonstrated to function with comparable activity to the initial base editor in human cells in vitro. Zebrafish embryos were used as a pioneering in vivo test system, with FusXTBE inducing 90+% editing efficiency in mtDNA loci, the first example of majority mtDNA heteroplasmy induction in any system. Gene editing specificity as precise as a single nucleotide was observed in vivo for a protein-coding gene. Non-destructive genotyping enables single animal mtDNA analyses for downstream biological functional genomics applications. FusXTBE is a new gene editing toolkit for exploring important questions in mitochondrial biology and genetics.

genetics↗

bioRxiv2021

Rapid adaptation and remote delivery of undergraduate research training during the COVID 19 Pandemic

COVID-19 continues to alter daily life around the globe. Education is particularly affected by shifts to distance learning. This change has poignant effects on all aspects of academic life, including the consequence of increased mental stress reported specifically for students. COVID-19 cancellations of many summer fellowships and internships for undergraduates across the country increased students uncertainty about their educational opportunities and careers. When the pandemic necessitated elimination of on-campus programming at Mayo Clinic, a new program was developed for remote delivery. Summer Foundations in Research (SFIR) was drafted around 4 aims: 1) support the academic trajectory gap in research science created by COVID-19; 2) build sustainable scientific relationships with mentors, peers, and the community; 3) create opportunities for participants to share and address concerns with their own experiences in the pandemic; and 4) provide support for individual wellbeing. SFIR included research training, but also training in communication through generative Dialogue and resilience through Amit Soods SMART program. 170 participants were followed for outcomes in these spaces. Knowledge of and interest in careers involving biomedical research rose significantly following SFIR. Participants mean confidence levels in 12 Key areas of research rose between 0.08 to 1.32 points on a 7-point scale. The strongest gains in mean confidence levels were seen in designing a study and collaborating with others. SFIR participants demonstrated gains in perceived happiness, and measured resilience and a reduction in stress. Participants qualitative responses indicated exceptionally positive mentor relationships and specific benefit of both the SMART program and Dialogue.

scientific communication and education↗

bioRxiv2021

Imaging cytoplasmic lipid droplets in vivo with fluorescent perilipin 2 and perilipin 3 knockin zebrafish

Cytoplasmic lipid droplets are highly dynamic storage organelles; their rapid synthesis, expansion, and degradation, as well as their varied interactions with other organelles allow cells to maintain lipid homeostasis. While the molecular details of lipid droplet dynamics are currently a very active area of investigation, this work has been primarily performed in cultured cells and in vitro systems. By taking advantage of the powerful transgenic and in vivo imaging opportunities afforded by the zebrafish model system, we have built a suite of tools to allow lipid droplets to be studied in real-time from the subcellular to the whole organism level. Fluorescently-tagging the lipid droplet associated proteins, perilipin 2 and perilipin 3, in the endogenous loci, permits visualization of lipid droplets in the intestine, liver, lateral line and adipose tissue. Using these transgenic lines we have found that perilipin 3 is rapidly loaded on intestinal lipid droplets following a high fat meal and then largely replaced by perilipin 2 a few hours later. These powerful new tools will facilitate studies on the role of lipid droplets in different tissues and under different genetic and physiological manipulations.

cell biology↗

bioRxiv2020

Endogenous zebrafish neural Cre drivers generated by CRISPR/Cas9 short homology directed targeted integration

The Cre/lox recombinase system has been widely used for spatiotemporal control of gene expression in animal model systems, however, efficient methods to isolate zebrafish Cre drivers that reliably recapitulate endogenous gene expression patterns are needed. Here, we apply CRISPR/Cas9 targeting to integrate a 2A-Cre recombinase transgene with 48bp homology arms into proneural genes ascl1b, olig2 and neurod1. We observed high rates of germline transmission ranging from 10%-100% (2/20 olig2; 1/5 neurod1; 3/3 ascl1b). The transgenic lines Tg(ascl1b-2A-Cre)is75, Tg(olig2-2A-Cre)is76, and Tg(neurod1-2A-Cre)is77 expressed functional Cre recombinase in the expected proneural cell populations. The results demonstrate Cre recombinase expression is driven by the native promoter and regulatory elements of the targeted genes. This approach provides a straightforward, efficient, and cost-effective method to generate cell type specific zebrafish Cre drivers whose spatial and temporal restricted expression mimics endogenous genes, surmounting the challenges associated with promoter BAC cloning and transposon mediated transgenesis.

genetics↗

bioRxiv2020

Vascular Endothelial Growth Factor as an Immediate-Early Activator of UV-induced Skin Injury

The negative health consequences of acute ultraviolet (UV) exposure are evident, with reports of 30,000 emergency room visits annually to treat the effects of sunburn in the United States alone. Acute effects of sunburn include erythema, edema, and severe pain, and chronic overexposure to UV radiation can lead to skin cancer. While the pain associated with the acute effects of sunburn may be relieved by current interventions, existing post-sunburn treatments are not capable of reversing the cumulative and long-term pathological effects of UV exposure, an unmet clinical need. Here we show that activation of the vascular endothelial growth factor (VEGF) pathway is a direct and immediate consequence of acute UV exposure, and activation of VEGF signaling is necessary for the initiating the acute pathological effects of sunburn. In UV-exposed human subjects, VEGF signaling is activated within hours. Topical delivery of VEGF pathway inhibitors, targeted against the ligand VEGF-A (gold nanoparticles conjugated with anti-VEGF antibodies) and small molecule antagonists of VEGF receptor signaling, prevent the development of erythema and edema in UV-exposed mice. Collectively, these findings suggest targeting VEGF signaling may reduce the subsequent inflammation and pathology associated with UV-induced skin damage, which reveals a new post-exposure therapeutic window to potentially inhibit the known detrimental effects of UV on human skin.

molecular biology↗

bioRxiv2020

A Genetic Model Therapy Proposes a Critical Role for Liver Dysfunction in Mitochondrial Biology and Disease

The clinical and largely unpredictable heterogeneity of phenotypes in patients with mitochondrial disorders demonstrates the ongoing challenges in the understanding of this semi-autonomous organelle in biology and disease. Here we present a new animal model that recapitulates key components of Leigh Syndrome, French Canadian Type (LSFC), a mitochondrial disorder that includes diagnostic liver dysfunction. LSFC is caused by allelic variations in the Leucine Rich Pentatricopeptide repeat-containing motif (LRPPRC) gene. LRPPRC has native functions related to mitochondrial mRNA polyadenylation and translation as well as a role in gluconeogenesis. We used the Gene-Breaking Transposon (GBT) cassette to create a revertible, insertional mutant zebrafish line in the LRPPRC gene. lrpprc zebrafish homozygous mutants displayed impaired muscle development, liver function and lowered levels of mtDNA transcripts and are lethal by 12dpf, all outcomes similar to clinical phenotypes observed in patients. Investigations using an in vivo lipidomics approach demonstrated accumulation of non-polar lipids in these animals. Transcript profiling of the mutants revealed dysregulation of clinically important nuclearly encoded and mitochondrial transcripts. Using engineered liver-specific rescue as a genetic model therapy, we demonstrate survival past the initial larval lethality, as well as restored normal gut development, mitochondrial morphology and triglyceride levels functionally demonstrating a critical role for the liver in the pathophysiology of this model of mitochondrial disease. Understanding the molecular mechanism of the liver-mediated genetic rescue underscores the potential to improve the clinical diagnostic and therapeutic developments for patients suffering from these devastating disorders.

genetics↗

bioRxiv2020

MMEJ-based Precision Gene Editing for applications in Gene Therapy and Functional Genomics

Experiments in gene editing commonly elicit error-prone non-homologous end joining for DNA double-strand break (DSB) repair. Microhomology-mediated end joining (MMEJ) can generate more predictable outcomes for functional genomic and somatic therapeutic applications. MENTHU is a computational tool that predicts nuclease-targetable sites likely to result in MMEJ-repaired, homogeneous genotypes (PreMAs) in zebrafish. We deployed MENTHU on 5,885 distinct Cas9-mediated DSBs in mouse embryonic stem cells, and compared the predictions to those by inDelphi, another DSB repair predictive algorithm. MENTHU correctly identified 46% of all PreMAs available, doubling the sensitivity of inDelphi. We also introduce MENTHU@4, an MENTHU update trained on this large dataset. We trained two MENTHU-based algorithms on this larger dataset and validated them against each other, MENTHU, and inDelphi. Finally, we estimated the frequency and distribution of SpCas9-targetable PreMAs in vertebrate coding regions to evaluate MMEJ-based targeting for gene discovery. 44 out of 54 genes (81%) contained at least one early out-of-frame PreMA and 48 out of 54 (89%) did so when also considering Cas12a. We suggest that MMEJ can be deployed at scale for reverse genetics screenings and with sufficient intra-gene density rates to be viable for nearly all loss-of-function based gene editing therapeutic applications.

genomics↗