Biology subjects

Hartman, A.

Publications and source records attributed to Hartman, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Ultra-large targeted DNA integrations in primary human cells

Genetic engineering experiments and therapies are constrained by the size of DNA integrations into human cells genomes. Existing AAV, lentiviral, and non-viral methods rapidly decrease in integration efficiency beyond [~]5kb of sequence. Through systematic evaluation of non-viral DNA template formats, we identified circular ssDNA and dsDNA as capable of mediating >5kb integrations. Large circular DNA delivery efficiency and its impacts on cell viability and payload expression could be significantly improved with small DNA "helper" plasmids, mRNA-encoded nucleases, and sequence design optimizations. Collectively, these modifications enabled ultra-large--up to 10 kb DNA--integrations at >20% efficiency in primary human T cells at the TRAC locus and at >60% efficiency in human iPSCs at the AAVS1 locus. Finally, we demonstrate that GMP clinical-manufactured T cells with ultra-large integrations are functional in vitro and in vivo. Overall, we identified optimal template architectures, delivery modes, and sequence design rules for ultra-large DNA integrations in both research and clinical settings to accelerate basic genetic research and next-generation cellular therapies.

genetics↗

bioRxiv2026

Systematic identification of seed-driven off-target effects in Perturb-seq experiments

Genome-wide Perturb-seq (GWPS) has emerged as a powerful approach for unbiased mapping of gene regulatory networks. A key assumption underlying many Perturb-seq analyses is that each guide RNA exclusively perturbs a single target locus. Without methods to identify and filter off-target events, erroneous gene-pathway associations driven by off-target activity can propagate into downstream analyses. Here, we present a workflow for the systematic identification of candidate off-target events in CRISPRi Perturb-seq experiments. Our approach exploits the observation that cells harboring a guide which represses an off-target gene display transcriptional similarity to cells in which that gene is directly targeted by an on-target guide. We apply our workflow to multiple GWPS datasets and nominate off-target events in which a guide nominally targeting one gene also represses a distinct gene producing a phenotype likely attributable to the off-target perturbation. We use both off-target gene repression and guide seed sequence alignments at the off-target promoter locus as evidence for off-target effects and find independent evidence of putative off-target events in separate GWPS datasets. Together, these results establish a principled framework for the identification and filtering of off-target guide effects in Perturb-seq experiments.

genomics↗

bioRxiv2025

A unified genetic perturbation language for human cellular programming

Evolution simultaneously and combinatorially explores complex genetic changes across perturbation classes, including gene knockouts, knockdowns, overexpression, and the creation of new genes from existing domains. Separate technologies are capable of genetic perturbations at scale in human cells, but these methods are largely mutually incompatible. Here we present CRISPR-All, a unified genetic perturbation language for programming of any major type of genetic perturbation simultaneously, in any combination, at genome scale, in primary human cells. This is enabled by a standardized molecular architecture for each major perturbation class, development of a functional syntax for combining arbitrary numbers of elements across classes, and linkage to unique single cell compatible barcodes. To facilitate use, CRISPR-All converts high level descriptions of desired complex genetic changes into a single DNA sequence that can rewire genomic programs within a cell. Using the CRISPR-All language allowed for head-to-head functional comparisons across perturbation types in a comprehensive analysis of all previously identified genetic enhancements of human CAR-T cells. Combining CRISPR-All programs with single cell RNA sequencing revealed a greater diversity of phenotypic states, including improved functional performance, only accessible through distinct perturbation classes. Finally, CRISPR-All combinatorial genome scale screening of up to four distinct perturbations simultaneously revealed additive functional improvements in human T cells accessible only through iterative multiplexing of modifications across perturbation classes. CRISPR-All enables exploration of a combinatorial genetic perturbation space, which may be impactful for biological and clinical applications.

genetics↗

bioRxiv2025

Modulating Cardiac-Gut Microbiome Interaction Post-Myocardial Infarction with Engineered Bacteria

The gut microbiome plays a critical role in the pathophysiology of acute myocardial infarction (MI). MI events significantly impact intestinal integrity which results in leakage of bacterial products into the systemic circulation. We demonstrate that MI not only compromises intestinal integrity, leading to systemic leakage of bacterial products like LPS, but also results in the translocation and colonization of live, intact gut bacteria in the MI heart - a novel aspect of the heart-gut axis. Our initial findings with natural murine gut microbiome were substantiated using orally administered E. coli Nissle 1917 (EcN), as a tracer bacterium. Furthermore, we engineered EcN to express the microbial anti-inflammatory molecule (MAM) derived from the probiotic Faecalibacterium prausnitzii. Treatment with this engineered strain, EcN-MAM, led to significantly improved survival and cardiac function in MI mice. This was attributed to enhanced gut barrier integrity, resulting in reduced systemic bacterial permeation and subsequent inflammation. These findings shed light on a previously unrecognized dimension of the heart-gut axis and highlight the potential of microbiome-based interventions in MI management.

synthetic biology↗

bioRxiv2025

Prenatal Zika Virus Exposure Disrupts Social-Emotional Development and Cortical Visual Function in Infant Macaques

Prenatal exposure to Zika virus (ZIKV) results in a spectrum of outcomes, ranging from severe birth defects and early childhood developmental delays to no apparent deficits. However, the mechanisms underlying these outcomes, particularly in the context of different maternal infection conditions, remain poorly defined. In this study, we employed a translational rhesus macaque model of prenatal ZIKV infection to evaluate longitudinal visual and auditory development from 1 to 12 months of age and characterize neurodevelopmental outcomes at 12 months. Pregnant macaques, either flavivirus-naive or with prior dengue virus (DENV) exposure, were inoculated in the first trimester ([~]30 or 45 gestational days) with an Asian or African lineage of ZIKV or received a saline injection as controls. Maternal plasma viremia duration, viral RNA burden at the maternal-fetal interface, and neutralizing antibody titers did not differ between inoculation cohorts or controls. At 12 months, ZIKV-exposed infants demonstrated altered maternal attachment behaviors and reduced inhibition when approaching sensory stimuli compared to controls. Visual pathway function, assessed by electrophysiology, was significantly impaired at 3 months but normalized by 12 months. Hearing loss was more common among ZIKV-exposed infants, although not statistically significant. Developmental outcomes were associated with prenatal ZIKV exposure itself, independent of viremia duration, neutralizing antibody titer, viral lineage, or maternal DENV immunity, and were not mediated by visual impairment or hearing loss. These findings highlight that any prenatal ZIKV exposure can disrupt early neurobehavioral development and visual function, underscoring the need for prevention strategies focused on maternal infection and early intervention.

microbiology↗

bioRxiv2024

Evolutionarily guided transcription factor design programs novel T cell states

Protein-coding genes in the human genome evolved via modular rearrangement of domains from ancestral genes1. Here, we develop a scalable, evolutionarily guided method to assemble novel protein-coding genes from constituent domains within a protein family, termed DESynR (Domain Engineered via Synthesis and Recombination) genes. Using primary human chimeric antigen receptor T cells as a model system, we find that the expression of DESynR Activator Protein-1 (AP-1) transcription factors (TFs) significantly outperforms the overexpression of natural AP-1 TFs in multiple functional assays in vitro and in vivo. Top DESynR AP-1 TFs exhibit non-intuitive architectures of constituent domains, including from TFs that are not canonically expressed in T cells. DESynR AP-1 TFs induce broad transcriptional and epigenetic reprogramming of T cells and, in some cases, lead to the development of non-natural T cell states, engaging gene expression modules from disparate human cell types. Taken together, we demonstrate that novel configurations of existing protein domains may uncover non-evolved genes that program cell states with therapeutically relevant functions.

genetics↗

bioRxiv2024

A Reproducibility Focused Meta-Analysis Method for Single-Cell Transcriptomic Case-Control Studies Uncovers Robust Differentially Expressed Genes

We assessed the reproducibility of differentially expressed genes (DEGs) in previously published Alzheimers (AD), Parkinsons (PD), Huntingtons (HD), Schizophrenia (SCZ), and COVID-19 scRNA-seq studies. While transcriptional scores from DEGs of individual PD, HD, and COVID-19 datasets had moderate predictive power for case-control status of other datasets, genes from individual AD and SCZ datasets had poor predictive power. We developed a non-parametric meta-analysis method, SumRank, based on reproducibility of relative differential expression ranks across datasets, and found DEGs with improved predictive power. By multiple metrics, specificity and sensitivity of these genes were substantially higher than those discovered by dataset merging and inverse variance weighted p-value aggregation methods and had significant enrichment in snATAC-seq peaks and human disease gene associations. The DEGs revealed known and novel biological pathways, such as up-regulation of chaperone-mediated protein processing in PD glia and lipid transport in AD and PD microglia, and down-regulation of glutamatergic processes in AD astrocytes and glutamatergic neurons and synaptic processing and neuron projection genes in HD FOXP2 neurons. We find 56 DEGs shared amongst AD, PD, and HD, and validate BCAT1 as down-regulated in AD mouse oligodendrocytes. Lastly, we evaluate factors influencing reproducibility of individual studies as a prospective guide for experimental design.

genomics↗

bioRxiv2024

Comparative analysis of multiplexed in situ gene expression profiling technologies

The burgeoning interest in in situ multiplexed gene expression profiling technologies has opened new avenues for understanding cellular behavior and interactions. In this study, we present a comparative benchmark analysis of six in situ gene expression profiling methods, including both commercially available and academically developed methods, using publicly accessible mouse brain datasets. We find that standard sensitivity metrics, such as the number of unique molecules detected per cell, are not directly comparable across datasets due to substantial differences in the incidence of off-target molecular artifacts impacting specificity. To address these challenges, we explored various potential sources of molecular artifacts, developed novel metrics to control for them, and utilized these metrics to evaluate and compare different in situ technologies. Finally, we demonstrate how molecular false positives can seriously confound spatially-aware differential expression analysis, requiring caution in the interpretation of downstream results. Our analysis provides guidance for the selection, processing, and interpretation of in situ spatial technologies.

genomics↗

bioRxiv2023

No evidence for enhanced disease with polyclonal SARS-CoV-2 antibody in the ferret model.

Since SARS-CoV-2 emerged in late 2019, it spread from China to the rest of the world. An initial concern was the potential for vaccine- or antibody-enhanced disease (AED) as had been reported with other coronaviruses. To evaluate this, we first developed a ferret model by exposing ferrets to SARS-CoV-2 by either mucosal inoculation (intranasal/oral) or inhalation using a small particle aerosol. Mucosal inoculation caused a mild fever and weight loss that resolved quickly; inoculation via either route resulted in virus shedding detected in the nares, throat, and rectum for 7-10 days post-infection. To evaluate the potential for AED, we then inoculated groups of ferrets intravenously with 0.1, 0.5, or 1 mg/kg doses of a human polyclonal anti-SARS-CoV-2 IgG from hyper-immunized transchromosomic bovines (SAB-185). Twelve hours later, ferrets were challenged by mucosal inoculation with SARS-CoV-2. We found no significant differences in fever, weight loss, or viral shedding after infection between the three antibody groups or the controls. Signs of pathology in the lungs were noted in infected ferrets but no differences were found between control and antibody groups. The results of this study indicate that healthy, young adult ferrets of both sexes are a suitable model of mild COVID-19 and that low doses of specific IgG in SAB-185 are unlikely to enhance the disease caused by SARS-CoV-2.

microbiology↗

bioRxiv2023

Phospho-seq: Integrated, multi-modal profiling of intracellular protein dynamics in single cells

Cell signaling plays a critical role in regulating cellular behavior and fate. While multimodal single-cell sequencing technologies are rapidly advancing, scalable and flexible profiling of cell signaling states alongside other molecular modalities remains challenging. Here we present Phospho-seq, an integrated approach that aims to quantify phosphorylated intracellular and intranuclear proteins, and to connect their activity with cis-regulatory elements and transcriptional targets. We utilize a simplified benchtop antibody conjugation method to create large custom antibody panels for simultaneous protein and scATAC-seq profiling on whole cells, and integrate this information with scRNA-seq datasets via bridge integration. We apply our workflow to cell lines, induced pluripotent stem cells, and 3-month-old brain organoids to demonstrate its broad applicability. We demonstrate that Phospho-seq can define cellular states and trajectories, reconstruct gene regulatory relationships, and characterize the causes and consequences of heterogeneous cell signaling in neurodevelopment.

genomics↗

bioRxiv2023

CPA-Perturb-seq: Multiplexed single-cell characterization of alternative polyadenylation regulators

Most mammalian genes have multiple polyA sites, representing a substantial source of transcript diversity that is governed by the cleavage and polyadenylation (CPA) regulatory machinery. To better understand how these proteins govern polyA site choice we introduce CPA-Perturb-seq, a multiplexed perturbation screen dataset of 42 known CPA regulators with a 3 scRNA-seq readout that enables transcriptome-wide inference of polyA site usage. We develop a statistical framework to specifically identify perturbation-dependent changes in intronic and tandem polyadenylation, and discover modules of co-regulated polyA sites exhibiting distinct functional properties. By training a multi-task deep neural network (APARENT-Perturb) on our dataset, we delineate a cis-regulatory code that predicts responsiveness to perturbation and reveals interactions between distinct regulatory complexes. Finally, we leverage our framework to re-analyze published scRNA-seq datasets, identifying new regulators that affect the relative abundance of alternatively polyadenylated transcripts, and characterizing extensive cellular heterogeneity in 3 UTR length amongst antibody-producing cells. Our work highlights the potential for multiplexed single-cell perturbation screens to further our understanding of post-transcriptional regulation in vitro and in vivo.

genomics↗

bioRxiv2022

Frequent first-trimester pregnancy loss in rhesus macaques infected with African-lineage Zika virus

In the 2016 Zika virus (ZIKV) pandemic, a previously unrecognized risk of birth defects surfaced in babies whose mothers were infected with Asian-lineage ZIKV during pregnancy. Less is known about the impacts of gestational African-lineage ZIKV infections. Given high human immunodeficiency virus (HIV) burdens in regions where African-lineage ZIKV circulates, we evaluated whether pregnant rhesus macaques infected with simian immunodeficiency virus (SIV) have a higher risk of African-lineage ZIKV-associated birth defects. Remarkably, in both SIV+ and SIV-animals, ZIKV infection early in the first trimester caused a high incidence (78%) of spontaneous pregnancy loss within 20 days. These findings suggest a significant risk for early pregnancy loss associated with African-lineage ZIKV infection and provide the first consistent ZIKV-associated phenotype in macaques for testing medical countermeasures.

microbiology↗

bioRxiv2022

Dictionary learning for integrative, multimodal, and scalable single-cell analysis

Mapping single-cell sequencing profiles to comprehensive reference datasets represents a powerful alternative to unsupervised analysis. Reference datasets, however, are predominantly constructed from single-cell RNA-seq data, and cannot be used to annotate datasets that do not measure gene expression. Here we introduce bridge integration, a method to harmonize singlecell datasets across modalities by leveraging a multi-omic dataset as a molecular bridge. Each cell in the multi-omic dataset comprises an element in a dictionary, which can be used to reconstruct unimodal datasets and transform them into a shared space. We demonstrate that our procedure can accurately harmonize transcriptomic data with independent single cell measurements of chromatin accessibility, histone modifications, DNA methylation, and protein levels. Moreover, we demonstrate how dictionary learning can be combined with sketching techniques to substantially improve computational scalability, and harmonize 8.6 million human immune cell profiles from sequencing and mass cytometry experiments. Our approach aims to broaden the utility of single-cell reference datasets and facilitate comparisons across diverse molecular modalities. AvailabilityInstallation instructions, documentations, and vignettes are available at http://www.satijalab.org/seurat

genomics↗

bioRxiv2021

Long-term persistence of viral RNA and inflammation in the CNS of macaques exposed to aerosolized Venezuelan equine encephalitis virus.

Venezuelan equine encephalitis virus (VEEV) is a positively-stranded RNA arbovirus of the genus Alphavirus that causes encephalitis in humans. Cynomolgus macaques are a relevant model of the human disease caused by VEEV and are useful in exploring pathogenic mechanisms and the host response to VEEV infection. Macaques were exposed to small-particle aerosols containing virus derived from an infectious clone of VEEV strain INH-9813, a subtype IC strain isolated from a human infection. VEEV-exposed macaques developed a biphasic fever after infection similar to that seen in humans. Maximum temperature deviation correlated with the inhaled dose, but fever duration did not. Neurological signs, suggestive of virus penetration into the CNS, were predominantly seen in the second febrile period. Electroencephalography data indicated a statistically significant decrease in all power bands and circadian index during the second febrile period that returned to normal after fever resolved. Intracranial pressure increased late in the second febrile period. On day 6 post-infection macaques had high levels of MCP-1 and IP-10 chemokines in the CNS, as well as a marked increase of T lymphocytes and activated microglia. More than four weeks after infection, viral genomic RNA was found in the brain, cerebrospinal fluid and cervical lymph nodes. Pro-inflammatory cytokines & chemokines, infiltrating leukocytes and pathological changes were seen in the CNS tissues of macaques euthanized at these times. These data are consistent with persistence of virus replication and/or genomic RNA and potentially, inflammatory sequelae in the central nervous system after resolution of acute VEEV disease.

immunology↗

bioRxiv2020

Reprogramming progressive cells display low CAG promoter activity

There is wide variability in the propensity of somatic cells to reprogram into pluripotency in response to the Yamanaka factors. How to segregate these variability to enrich for cells of specific traits that reprogram efficiently remains challenging. Here we report that the variability in reprogramming propensity is associated with the activity of the MKL1/SRF transcription factor and concurs with small cell size as well as rapid cell cycle. Reprogramming progressive cells can be prospectively identified by their low activity of a widely used synthetic promoter, CAG. CAGlow cells arise and expand during cell cycle acceleration in the early reprogramming culture of both mouse and human fibroblasts. Our work illustrate a molecular scenario underlying the distinct reprogramming propensities and demonstrate a convenient practical approach for their enrichment.

cell biology↗

bioRxiv2019

Pharyngeal motor cortex grey matter abnormalities and retinal photoreceptor layer dysfunction in macaques exposed to Zika virus in utero

Congenital Zika virus (ZIKV) exposure results in a spectrum of disease ranging from severe birth defects to delayed onset neurodevelopmental deficits. ZIKV-related neuropathogenesis, predictors of birth defects, and neurodevelopmental deficits are not well defined in people. Here we assess the methodological and statistical feasibility of a congenital ZIKV exposure macaque model for identifying infant neurobehavior and brain abnormalities that may underlie neurodevelopmental deficits. We inoculated five pregnant macaques with ZIKV and mock-inoculated one macaque in the first trimester. Following birth, growth, ocular structure/function, brain structure, hearing, histopathology, and neurobehavior were quantitatively assessed during the first week of life. We identified the typical pregnancy outcomes of congenital ZIKV infection, with fetal demise and placental abnormalities. We estimated sample sizes needed to define differences between groups and demonstrated that future studies quantifying brain region volumes, retinal structure, hearing, and visual pathway function require a sample size of 14 animals per group (14 ZIKV, 14 control) to detect statistically significant differences in at least half of the infant exam parameters. Establishing the parameters for future studies of neurodevelopmental outcomes following congenital ZIKV exposure in macaques is essential for robust and rigorous experimental design.

microbiology↗

bioRxiv2018

MLL-AF9 Initiates Transformation From Fast-Proliferating Myeloid Progenitors

Cancer is a hyper-proliferative clonal disease. Whether the proliferative state originates from the cell-of-origin or emerges later remains elusive. By tracking de novo transformation from normal hematopoietic progenitors expressing an acute myeloid leukemia (AML) oncogene MLL-AF9, we reveal that the cell cycle rate heterogeneity among granulocyte-macrophage progenitors (GMPs) determines their probability of transformation. An intrinsic fast cell cycle kinetics at the time of oncogene expression provide permissiveness for transformation, with the fastest cycling 3% of GMPs ([~]0.006% of bone marrow nucleated cells) acquiring malignancy with nearly 100% efficiency. Molecularly, we propose that MLL-AF9 preserves the gene expression of the cellular states in which it is expressed. As such, when expressed in the naturally-existing, rapidly-cycling myeloid progenitors, this cell state is perpetuated, yielding malignancy. Our work elucidates one of the earliest steps toward malignancy and suggests that modifying the cycling state of the cell-of-origin could be an effective approach to prevent malignancy.

cancer biology↗

bioRxiv2018

Non-cell-autonomous promotion of pluripotency induction mediated by YAP

While Yes-associated protein (YAP) antagonizes pluripotency during early embryogenesis, it has also been shown to promote stemness of multiple stem cell types, including pluripotent stem cells. Whether cellular context underlies these distinct functions of YAP in pluripotency remains unclear. Here, we establish that depending on the specific cells in which it is expressed, YAP exhibits opposing effects on pluripotency induction from somatic cells. Specifically, YAP inhibits pluripotency induction cell-autonomously but promotes it non-cell-autonomously. For its non-cell-autonomous role, YAP alters the expression of many secreted and matricellular proteins including CYR61, which recapitulates the promotional effect when added as a recombinant protein. Thus, we define a unique YAP-driven non-cell-autonomous process that enhances pluripotency induction. Our work highlights the importance of considering the distinct contributions from heterologous cell types in deciphering the mechanism of cell fate control and calls for careful re-examination of the co-existing bystander cells in complex cultures or tissues.

cell biology↗