Defining the activity of pro-reparative extracellular vesicles in wound healing based on miRNA payloads and cell type-specific lineage mapping.

Small extracellular vesicles (EVs) are released by cells and deliver biologically active payloads to coordinate the response of multiple cell types in cutaneous wound healing. Here we used a cutaneous injury model as a donor of pro-reparative EVs to treat recipient diabetic obese mice, a model of impaired wound healing. We established a functional screen for microRNAs (miRNAs) that increased the pro-reparative activity of EVs and identified a down-regulation of miR-425-5p in EVs in vivo and in vitro associated with the regulation of adiponectin. We tested a cell type-specific reporter of a tetraspanin CD9 fusion with GFP to lineage map the release of EVs from macrophages in the wound bed, based on the expression of miR-425-5p in macrophage-derived EVs and the abundance of macrophages in EV donor sites. Analysis of different promoters demonstrated that EV release under the control of a macrophage-specific promoter was most abundant and that these EVs were internalized by dermal fibroblasts. These findings suggested that pro-reparative EVs deliver miRNAs, such as miR-425-5p, that stimulate the expression of adiponectin that has insulin-sensitizing properties. We propose that EVs promote intercellular signaling between cell layers in the skin to resolve inflammation, induce proliferation of basal keratinocytes, and accelerate wound closure.

Grand Challenges at the Interface of Engineering and Medicine.

Over the past two decades Biomedical Engineering has emerged as a major discipline that bridges societal needs of human health care with the development of novel technologies. Every medical institution is now equipped at varying degrees of sophistication with the ability to monitor human health in both non-invasive and invasive modes. The multiple scales at which human physiology can be interrogated provide a profound perspective on health and disease. We are at the nexus of creating "avatars" (herein defined as an extension of "digital twins") of human patho/physiology to serve as paradigms for interrogation and potential intervention. Motivated by the emergence of these new capabilities, the IEEE Engineering in Medicine and Biology Society, the Departments of Biomedical Engineering at Johns Hopkins University and Bioengineering at University of California at San Diego sponsored an interdisciplinary workshop to define the grand challenges that face biomedical engineering and the mechanisms to address these challenges. The Workshop identified five grand challenges with cross-cutting themes and provided a roadmap for new technologies, identified new training needs, and defined the types of interdisciplinary teams needed for addressing these challenges. The themes presented in this paper include: 1) accumedicine through creation of avatars of cells, tissues, organs and whole human; 2) development of smart and responsive devices for human function augmentation; 3) exocortical technologies to understand brain function and treat neuropathologies; 4) the development of approaches to harness the human immune system for health and wellness; and 5) new strategies to engineer genomes and cells.

Transcriptional control of leukemogenesis by the chromatin reader SGF29.

ABSTRACT: Aberrant expression of stem cell-associated genes is a common feature in acute myeloid leukemia (AML) and is linked to leukemic self-renewal and therapy resistance. Using AF10-rearranged leukemia as a prototypical example of the recurrently activated "stemness" network in AML, we screened for chromatin regulators that sustain its expression. We deployed a CRISPR-Cas9 screen with a bespoke domain-focused library and identified several novel chromatin-modifying complexes as regulators of the TALE domain transcription factor MEIS1, a key leukemia stem cell (LSC)-associated gene. CRISPR droplet sequencing revealed that many of these MEIS1 regulators coordinately controlled the transcription of several AML oncogenes. In particular, we identified a novel role for the Tudor-domain-containing chromatin reader protein SGF29 in the transcription of AML oncogenes. Furthermore, SGF29 deletion impaired leukemogenesis in models representative of multiple AML subtypes in multiple AML subtype models. Our studies reveal a novel role for SGF29 as a nononcogenic dependency in AML and identify the SGF29 Tudor domain as an attractive target for drug discovery.

Interface-guided phenotyping of coding variants in the transcription factor RUNX1 with SEUSS.

Understanding the consequences of single amino acid substitutions in cancer driver genes remains an unmet need. Perturb-seq provides a tool to investigate the effects of individual mutations on cellular programs. Here we deploy SEUSS, a Perturb-seq like approach, to generate and assay mutations at physical interfaces of the RUNX1 Runt domain. We measured the impact of 115 mutations on RNA profiles in single myelogenous leukemia cells and used the profiles to categorize mutations into three functionally distinct groups: wild-type (WT)-like, loss-of-function (LOF)-like and hypomorphic. Notably, the largest concentration of functional mutations (non-WT-like) clustered at the DNA binding site and contained many of the more frequently observed mutations in human cancers. Hypomorphic variants shared characteristics with loss of function variants but had gene expression profiles indicative of response to neural growth factor and cytokine recruitment of neutrophils. Additionally, DNA accessibility changes upon perturbations were enriched for RUNX1 binding motifs, particularly near differentially expressed genes. Overall, our work demonstrates the potential of targeting protein interaction interfaces to better define the landscape of prospective phenotypes reachable by amino acid substitutions.

Multimodal perturbation analyses of cyclin-dependent kinases reveal a network of synthetic lethalities associated with cell-cycle regulation and transcriptional regulation.

Cell-cycle control is accomplished by cyclin-dependent kinases (CDKs), motivating extensive research into CDK targeting small-molecule drugs as cancer therapeutics. Here we use combinatorial CRISPR/Cas9 perturbations to uncover an extensive network of functional interdependencies among CDKs and related factors, identifying 43 synthetic-lethal and 12 synergistic interactions. We dissect CDK perturbations using single-cell RNAseq, for which we develop a novel computational framework to precisely quantify cell-cycle effects and diverse cell states orchestrated by specific CDKs. While pairwise disruption of CDK4/6 is synthetic-lethal, only CDK6 is required for normal cell-cycle progression and transcriptional activation. Multiple CDKs (CDK1/7/9/12) are synthetic-lethal in combination with PRMT5, independent of cell-cycle control. In-depth analysis of mRNA expression and splicing patterns provides multiple lines of evidence that the CDK-PRMT5 dependency is due to aberrant transcriptional regulation resulting in premature termination. These inter-dependencies translate to drug-drug synergies, with therapeutic implications in cancer and other diseases.

Systematic discovery of transcription factors that improve hPSC-derived cardiomyocyte maturation via temporal analysis of bioengineered cardiac tissues.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have the potential to become powerful tools for disease modeling, drug testing, and transplantation; however, their immaturity limits their applications. Transcription factor (TF) overexpression can improve hPSC-CM maturity, but identifying these TFs has been elusive. Toward this, we establish here an experimental framework for systematic identification of maturation enhancing factors. Specifically, we performed temporal transcriptome RNAseq analyses of progressively matured hPSC-derived cardiomyocytes across 2D and 3D differentiation systems and further compared these bioengineered tissues to native fetal and adult-derived tissues. These analyses revealed 22 TFs whose expression did not increase in 2D differentiation systems but progressively increased in 3D culture systems and adult mature cell types. Individually overexpressing each of these TFs in immature hPSC-CMs identified five TFs (KLF15, ZBTB20, ESRRA, HOPX, and CAMTA2) as regulators of calcium handling, metabolic function, and hypertrophy. Notably, the combinatorial overexpression of KLF15, ESRRA, and HOPX improved all three maturation parameters simultaneously. Taken together, we introduce a new TF cocktail that can be used in solo or in conjunction with other strategies to improve hPSC-CM maturation and anticipate that our generalizable methodology can also be implemented to identify maturation-associated TFs for other stem cell progenies.

RNA editing: Expanding the potential of RNA therapeutics.

RNA therapeutics have had a tremendous impact on medicine, recently exemplified by the rapid development and deployment of mRNA vaccines to combat the COVID-19 pandemic. In addition, RNA-targeting drugs have been developed for diseases with significant unmet medical needs through selective mRNA knockdown or modulation of pre-mRNA splicing. Recently, RNA editing, particularly antisense RNA-guided adenosine deaminase acting on RNA (ADAR)-based programmable A-to-I editing, has emerged as a powerful tool to manipulate RNA to enable correction of disease-causing mutations and modulate gene expression and protein function. Beyond correcting pathogenic mutations, the technology is particularly well suited for therapeutic applications that require a transient pharmacodynamic effect, such as the treatment of acute pain, obesity, viral infection, and inflammation, where it would be undesirable to introduce permanent alterations to the genome. Furthermore, transient modulation of protein function, such as altering the active sites of enzymes or the interface of protein-protein interactions, opens the door to therapeutic avenues ranging from regenerative medicine to oncology. These emerging RNA-editing-based toolsets are poised to broadly impact biotechnology and therapeutic applications. Here, we review the emerging field of therapeutic RNA editing, highlight recent laboratory advancements, and discuss the key challenges on the path to clinical development.

Whole-genome CRISPR screening identifies PI3K/AKT as a downstream component of the oncogenic GNAQ-focal adhesion kinase signaling circuitry.

G proteins and G protein-coupled receptors activate a diverse array of signal transduction pathways that promote cell growth and survival. Indeed, hot spot-activating mutations in GNAQ/GNA11, encoding Gαq proteins, are known to be driver oncogenes in uveal melanoma (UM), for which there are limited effective therapies currently available. Focal adhesion kinase (FAK) has been recently shown to be a central mediator of Gαq-driven signaling in UM, and as a result, is being explored clinically as a therapeutic target for UM, both alone and in combination therapies. Despite this, the repertoire of Gαq/FAK-regulated signaling mechanisms have not been fully elucidated. Here, we used a whole-genome CRISPR screen in GNAQ-mutant UM cells to identify mechanisms that, when overactivated, lead to reduced sensitivity to FAK inhibition. In this way, we found that the PI3K/AKT signaling pathway represented a major resistance driver. Our dissection of the underlying mechanisms revealed that Gαq promotes PI3K/AKT activation via a conserved signaling circuitry mediated by FAK. Further analysis demonstrated that FAK activates PI3K through the association and tyrosine phosphorylation of the p85 regulatory subunit of PI3K and that UM cells require PI3K/AKT signaling for survival. These findings establish a novel link between Gαq-driven signaling and the stimulation of PI3K as well as demonstrate aberrant activation of signaling networks underlying the growth and survival of UM and other Gαq-driven malignancies.

Insulin-regulated serine and lipid metabolism drive peripheral neuropathy.

Diabetes represents a spectrum of disease in which metabolic dysfunction damages multiple organ systems including liver, kidneys and peripheral nerves1,2. Although the onset and progression of these co-morbidities are linked with insulin resistance, hyperglycaemia and dyslipidaemia3-7, aberrant non-essential amino acid (NEAA) metabolism also contributes to the pathogenesis of diabetes8-10. Serine and glycine are closely related NEAAs whose levels are consistently reduced in patients with metabolic syndrome10-14, but the mechanistic drivers and downstream consequences of this metabotype remain unclear. Low systemic serine and glycine are also emerging as a hallmark of macular and peripheral nerve disorders, correlating with impaired visual acuity and peripheral neuropathy15,16. Here we demonstrate that aberrant serine homeostasis drives serine and glycine deficiencies in diabetic mice, which can be diagnosed with a serine tolerance test that quantifies serine uptake and disposal. Mimicking these metabolic alterations in young mice by dietary serine or glycine restriction together with high fat intake markedly accelerates the onset of small fibre neuropathy while reducing adiposity. Normalization of serine by dietary supplementation and mitigation of dyslipidaemia with myriocin both alleviate neuropathy in diabetic mice, linking serine-associated peripheral neuropathy to sphingolipid metabolism. These findings identify systemic serine deficiency and dyslipidaemia as novel risk factors for peripheral neuropathy that may be exploited therapeutically.

Scalable Functional Assays for the Interpretation of Human Genetic Variation.

Scalable sequence-function studies have enabled the systematic analysis and cataloging of hundreds of thousands of coding and noncoding genetic variants in the human genome. This has improved clinical variant interpretation and provided insights into the molecular, biophysical, and cellular effects of genetic variants at an astonishing scale and resolution across the spectrum of allele frequencies. In this review, we explore current applications and prospects for the field and outline the principles underlying scalable functional assay design, with a focus on the study of single-nucleotide coding and noncoding variants.

Methods for recruiting endogenous and exogenous ADAR enzymes for site-specific RNA editing.

Adenosine deaminases acting on RNA (ADARs) can be repurposed to achieve site-specific A-to-I RNA editing by recruiting them to a target of interest via an ADAR-recruiting guide RNA (adRNA). In this chapter, we present details towards experimental methods to enable this via two orthogonal strategies: one, via recruitment of endogenous ADARs (i.e. ADARs already natively expressed in cells); and two, via recruitment of exogenous ADARs (i.e. ADARs delivered into cells). Towards the former, we describe the use of circular adRNAs to recruit endogenous ADARs to a desired mRNA target. This results in robust, persistent and highly transcript specific editing both in vitro and in vivo. Towards the latter, we describe the use of a split-ADAR2 system, which allows for overexpression of ADAR2 variants that can be utilized to edit adenosines with high specificity, including at challenging to edit adenosines in non-preferred motifs such as those flanked by a 5' guanosine. We anticipate the described methods should facilitate RNA editing applications across research and biotechnology settings.

Efficient in vitro and in vivo RNA editing via recruitment of endogenous ADARs using circular guide RNAs.

Recruiting endogenous adenosine deaminases using exogenous guide RNAs to edit cellular RNAs is a promising therapeutic strategy, but editing efficiency and durability remain low using current guide RNA designs. In this study, we engineered circular ADAR-recruiting guide RNAs (cadRNAs) to enable more efficient programmable adenosine-to-inosine RNA editing without requiring co-delivery of any exogenous proteins. Using these cadRNAs, we observed robust and durable RNA editing across multiple sites and cell lines, in both untranslated and coding regions of RNAs, and high transcriptome-wide specificity. Additionally, we increased transcript-level specificity for the target adenosine by incorporating interspersed loops in the antisense domains, reducing bystander editing. In vivo delivery of cadRNAs via adeno-associated viruses enabled 53% RNA editing of the mPCSK9 transcript in C57BL/6J mice livers and 12% UAG-to-UGG RNA correction of the amber nonsense mutation in the IDUA-W392X mouse model of mucopolysaccharidosis type I-Hurler syndrome. cadRNAs enable efficient programmable RNA editing in vivo with diverse protein modulation and gene therapeutic applications.

Comprehensive interrogation of the ADAR2 deaminase domain for engineering enhanced RNA editing activity and specificity.

Adenosine deaminases acting on RNA (ADARs) can be repurposed to enable programmable RNA editing, however their exogenous delivery leads to transcriptome-wide off-targeting, and additionally, enzymatic activity on certain RNA motifs, especially those flanked by a 5' guanosine is very low thus limiting their utility as a transcriptome engineering toolset. Towards addressing these issues, we first performed a novel deep mutational scan of the ADAR2 deaminase domain, directly measuring the impact of every amino acid substitution across 261 residues, on RNA editing. This enabled us to create a domain-wide mutagenesis map while also revealing a novel hyperactive variant with improved enzymatic activity at 5'-GAN-3' motifs. As overexpression of ADAR enzymes, especially hyperactive variants, can lead to significant transcriptome-wide off-targeting, we next engineered a split-ADAR2 deaminase which resulted in >100-fold more specific RNA editing as compared to full-length deaminase overexpression. Taken together, we anticipate this systematic engineering of the ADAR2 deaminase domain will enable broader utility of the ADAR toolset for RNA biotechnology applications.

Integrated genome and tissue engineering enables screening of cancer vulnerabilities in physiologically relevant perfusable ex vivo cultures.

Genetic screens are powerful tools for both resolving biological function and identifying potential therapeutic targets, but require physiologically accurate systems to glean biologically useful information. Here, we enable genetic screens in physiologically relevant ex vivo cancer tissue models by integrating CRISPR-Cas-based genome engineering and biofabrication technologies. We first present a novel method for generating perfusable tissue constructs, and validate its functionality by using it to generate three-dimensional perfusable dense cultures of cancer cell lines and sustain otherwise ex vivo unculturable patient-derived xenografts. Using this system we enable large-scale CRISPR screens in perfused tissue cultures, as well as emulate a novel point-of-care diagnostics scenario of a clinically actionable CRISPR knockout (CRISPRko) screen of genes with FDA-approved drug treatments in ex vivo PDX cell cultures. Our results reveal differences across in vitro and in vivo cancer model systems, and highlight the utility of programmable tissue engineered models for screening therapeutically relevant cancer vulnerabilities.

Charting oncogenicity of genes and variants across lineages via multiplexed screens in teratomas.

Deconstructing tissue-specific effects of genes and variants on proliferation is critical to understanding cellular transformation and systematically selecting cancer therapeutics. This requires scalable methods for multiplexed genetic screens tracking fitness across time, across lineages, and in a suitable niche, since physiological cues influence functional differences. Towards this, we present an approach, coupling single-cell cancer driver screens in teratomas with hit enrichment by serial teratoma reinjection, to simultaneously screen drivers across multiple lineages in vivo. Using this system, we analyzed population shifts and lineage-specific enrichment for 51 cancer associated genes and variants, profiling over 100,000 cells spanning over 20 lineages, across two rounds of serial reinjection. We confirmed that c-MYC alone or combined with myristoylated AKT1 potently drives proliferation in progenitor neural lineages, demonstrating signatures of malignancy. Additionally, mutant MEK1 S218D/S222D provides a proliferative advantage in mesenchymal lineages like fibroblasts. Our method provides a powerful platform for multi-lineage longitudinal study of oncogenesis.

Programmatic introduction of parenchymal cell types into blood vessel organoids.

Pluripotent stem cell-derived organoids have transformed our ability to recreate complex three-dimensional models of human tissue. However, the directed differentiation methods used to create them do not afford the ability to introduce cross-germ-layer cell types. Here, we present a bottom-up engineering approach to building vascularized human tissue by combining genetic reprogramming with chemically directed organoid differentiation. As a proof of concept, we created neuro-vascular and myo-vascular organoids via transcription factor overexpression in vascular organoids. We comprehensively characterized neuro-vascular organoids in terms of marker gene expression and composition, and demonstrated that the organoids maintain neural and vascular function for at least 45 days in culture. Finally, we demonstrated chronic electrical stimulation of myo-vascular organoid aggregates as a potential path toward engineering mature and large-scale vascularized skeletal muscle tissue from organoids. Our approach offers a roadmap to build diverse vascularized tissues of any type derived entirely from pluripotent stem cells.

Peptide-tiling screens of cancer drivers reveal oncogenic protein domains and associated peptide inhibitors.

Gene fragments derived from structural domains mediating physical interactions can modulate biological functions. Utilizing this, we developed lentiviral overexpression libraries of peptides comprehensively tiling high-confidence cancer driver genes. Toward inhibiting cancer growth, we assayed ~66,000 peptides, tiling 65 cancer drivers and 579 mutant alleles. Pooled fitness screens in two breast cancer cell lines revealed peptides, which selectively reduced cellular proliferation, implicating oncogenic protein domains important for cell fitness. Coupling of cell-penetrating motifs to these peptides enabled drug-like function, with peptides derived from EGFR and RAF1 inhibiting cell growth at IC50s of 27-63 µM. We anticipate that this peptide-tiling (PepTile) approach will enable rapid de novo mapping of bioactive protein domains and associated interfering peptides.