Embryo and fetal gene editing: Technical challenges and progress toward clinical applications.

Gene modification therapies (GMTs) are slowly but steadily making progress toward clinical application. As the majority of rare diseases have an identified genetic cause, and as rare diseases collectively affect 5% of the global population, it is increasingly important to devise gene correction strategies to address the root causes of the most devastating of these diseases and to provide access to these novel therapies to the most affected populations. The main barriers to providing greater access to GMTs continue to be the prohibitive cost of developing these novel drugs at clinically relevant doses, subtherapeutic effects, and toxicity related to the specific agents or high doses required. In vivo strategy and treating younger patients at an earlier course of their disease could lower these barriers. Although currently regarded as niche specialties, prenatal and preconception GMTs offer a robust solution to some of these barriers. Indeed, treating either the fetus or embryo benefits from economy of scale, targeting pre-pathological tissues in the fetus prior to full pathogenesis, or increasing the likelihood of complete tissue targeting by correcting pluripotent embryonic cells. Here, we review advances in embryo and fetal GMTs and discuss requirements for clinical application.

Extracorporeal Membrane Oxygenation-Dependent Fulminant Melioidosis From Caspase 4 Mutation Reversed by Interferon Gamma Therapy.

We describe bedside-to-bench immunological and genetic elucidation of defective pyroptosis attributable to novel caspase 4 defect mediating pathogen-triggered inflammatory programmed cell death, in the setting of severe pneumonia and abscess-forming melioidosis in an overtly healthy host failing to clear Burkholderia pseudomallei infection, and how targeted adjunctive biological therapy led to a successful outcome.

Redox-Responsive Phase-Separating Peptide as a Universal Delivery Vehicle for CRISPR/Cas9 Genome Editing Machinery.

CRISPR/Cas9-based genome editing tools have enormous potential for the development of various therapeutic treatments due to their reliability and broad applicability. A central requirement of CRISPR/Cas9 is the efficient intracellular delivery of the editing machinery, which remains a well-recognized challenge, notably to deliver Cas9 in its native protein form. Herein, a phase-separating peptide with intracellular redox-triggered release properties is employed to encapsulate and deliver all three forms of CRISRP-Cas9 editing machinery, namely, pDNA, mRNA/sgRNA, and the ribonucleoprotein complex. These modalities are readily recruited within peptide coacervates during liquid-liquid phase separation by simple mixing and exhibit higher transfection and editing efficiency compared to highly optimized commercially available transfection reagents currently used for genome editing.

AAV-CRISPR-Cas13 eliminates human enterovirus and prevents death of infected mice.

BACKGROUND: RNA viruses account for many human diseases and pandemic events but are often not targetable by traditional therapeutics modalities. Here, we demonstrate that adeno-associated virus (AAV) -delivered CRISPR-Cas13 directly targets and eliminates the positive-strand EV-A71 RNA virus in cells and infected mice. METHODS: We developed a Cas13gRNAtor bioinformatics pipeline to design CRISPR guide RNAs (gRNAs) that cleave conserved viral sequences across the virus phylogeny and developed an AAV-CRISPR-Cas13 therapeutics using in vitro viral plaque assay and in vivo EV-A71 lethally-infected mouse model. FINDINGS: We show that treatment with a pool of AAV-CRISPR-Cas13-gRNAs designed using the bioinformatics pipeline effectively blocks viral replication and reduces viral titers in cells by >99.99%. We further demonstrate that AAV-CRISPR-Cas13-gRNAs prophylactically and therapeutically inhibited viral replication in infected mouse tissues and prevented death in a lethally challenged EV-A71-infected mouse model. INTERPRETATION: Our results show that the bioinformatics pipeline designs efficient CRISPR-Cas13 gRNAs for direct viral RNA targeting to reduce viral loads. Additionally, this new antiviral AAV-CRISPR-Cas13 modality represents an effective direct-acting prophylactic and therapeutic agent against lethal RNA viral infections. FUNDING: Agency for Science, Technology and Research (A∗STAR) Assured Research Budget, A∗STAR Central Research Fund UIBR SC18/21-1089UI, A∗STAR Industrial Alignment Fund Pre-Positioning (IAF-PP) grant H17/01/a0/012, MOE Tier 2 2017 (MOE2017-T2-1-078; MOE-T2EP30221-0005), and NUHSRO/2020/050/RO5+5/NUHS-COVID/4.

Multiplex viral tropism assay in complex cell populations with single-cell resolution.

Gene therapy constitutes one of the most promising mode of disease treatments. Two key properties for therapeutic delivery vectors are its transduction efficiency (how well the vector delivers therapeutic cargo to desired target cells) and specificity (how well it avoids off-target delivery into unintended cells within the body). Here we developed an integrated bioinformatics and experimental pipeline that enables multiplex measurement of transduction efficiency and specificity, particularly by measuring how libraries of delivery vectors transduce libraries of diverse cell types. We demonstrated that pairing high-throughput measurement of AAV identity with high-resolution single-cell RNA transcriptomic sequencing maps how natural and engineered AAV variants transduce individual cells within human cerebral and ocular organoids. We further demonstrate that efficient AAV transduction observed in organoids is recapitulated in vivo in non-human primates. This library-on-library technology will be important for determining the safety and efficacy of therapeutic delivery vectors.

Disrupting the LINC complex by AAV mediated gene transduction prevents progression of Lamin induced cardiomyopathy.

Mutations in the LaminA gene are a common cause of monogenic dilated cardiomyopathy. Here we show that mice with a cardiomyocyte-specific Lmna deletion develop cardiac failure and die within 3-4 weeks after inducing the mutation. When the same Lmna mutations are induced in mice genetically deficient in the LINC complex protein SUN1, life is extended to more than one year. Disruption of SUN1's function is also accomplished by transducing and expressing a dominant-negative SUN1 miniprotein in Lmna deficient cardiomyocytes, using the cardiotrophic Adeno Associated Viral Vector 9. The SUN1 miniprotein disrupts binding between the endogenous LINC complex SUN and KASH domains, displacing the cardiomyocyte KASH complexes from the nuclear periphery, resulting in at least a fivefold extension in lifespan. Cardiomyocyte-specific expression of the SUN1 miniprotein prevents cardiomyopathy progression, potentially avoiding the necessity of developing a specific therapeutic tailored to treating each different LMNA cardiomyopathy-inducing mutation of which there are more than 450.

Ethics and regulatory considerations for the clinical translation of somatic cell human epigenetic editing.

Altering the human epigenome with gene-editing technology in attempt to treat a variety of diseases and conditions seems scientifically feasible. We explore some of the ethical and regulatory issues related to the clinical translation of human epigenetic editing arguing that such approaches should be considered akin to somatic therapies.

Coding and non-coding roles of MOCCI (C15ORF48) coordinate to regulate host inflammation and immunity.

Mito-SEPs are small open reading frame-encoded peptides that localize to the mitochondria to regulate metabolism. Motivated by an intriguing negative association between mito-SEPs and inflammation, here we screen for mito-SEPs that modify inflammatory outcomes and report a mito-SEP named "Modulator of cytochrome C oxidase during Inflammation" (MOCCI) that is upregulated during inflammation and infection to promote host-protective resolution. MOCCI, a paralog of the NDUFA4 subunit of cytochrome C oxidase (Complex IV), replaces NDUFA4 in Complex IV during inflammation to lower mitochondrial membrane potential and reduce ROS production, leading to cyto-protection and dampened immune response. The MOCCI transcript also generates miR-147b, which targets the NDUFA4 mRNA with similar immune dampening effects as MOCCI, but simultaneously enhances RIG-I/MDA-5-mediated viral immunity. Our work uncovers a dual-component pleiotropic regulation of host inflammation and immunity by MOCCI (C15ORF48) for safeguarding the host during infection and inflammation.

Programmable C:G to G:C genome editing with CRISPR-Cas9-directed base excision repair proteins.

Many genetic diseases are caused by single-nucleotide polymorphisms. Base editors can correct these mutations at single-nucleotide resolution, but until recently, only allowed for transition edits, addressing four out of twelve possible DNA base substitutions. Here, we develop a class of C:G to G:C Base Editors to create single-base genomic transversions in human cells. Our C:G to G:C Base Editors consist of a nickase-Cas9 fused to a cytidine deaminase and base excision repair proteins. Characterization of >30 base editor candidates reveal that they predominantly perform C:G to G:C editing (up to 90% purity), with rAPOBEC-nCas9-rXRCC1 being the most efficient (mean 15.4% and up to 37% without selection). C:G to G:C Base Editors target cytidine in WCW, ACC or GCT sequence contexts and within a precise three-nucleotide window of the target protospacer. We further target genes linked to dyslipidemia, hypertrophic cardiomyopathy, and deafness, showing the therapeutic potential of these base editors in interrogating and correcting human genetic diseases.

Translating CRISPR-Cas Therapeutics: Approaches and Challenges.

CRISPR-Cas clinical trials have begun, offering a first glimpse at how DNA and RNA targeting could enable therapies for many genetic and epigenetic human diseases. The speedy progress of CRISPR-Cas from discovery and adoption to clinical use is built on decades of traditional gene therapy research and belies the multiple challenges that could derail the successful translation of these new modalities. Here, we review how CRISPR-Cas therapeutics are translated from technological systems to therapeutic modalities, paying particular attention to the therapeutic cascade from cargo to delivery vector, manufacturing, administration, pipelines, safety, and therapeutic target profiles. We also explore potential solutions to some of the obstacles facing successful CRISPR-Cas translation. We hope to illuminate how CRISPR-Cas is brought from the academic bench toward use in the clinic.

Author Correction: Immune-orthogonal orthologues of AAV capsids and of Cas9 circumvent the immune response to the administration of gene therapy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Immunity to CRISPR Cas9 and Cas12a therapeutics.

Genome-editing therapeutics are poised to treat human diseases. As we enter clinical trials with the most promising CRISPR-Cas9 and CRISPR-Cas12a (Cpf1) modalities, the risks associated with administering these foreign biomolecules into human patients become increasingly salient. Preclinical discovery with CRISPR-Cas9 and CRISPR-Cas12a systems and foundational gene therapy studies indicate that the host immune system can mount undesired responses against the administered proteins and nucleic acids, the gene-edited cells, and the host itself. These host defenses include inflammation via activation of innate immunity, antibody induction in humoral immunity, and cell death by T-cell-mediated cytotoxicity. If left unchecked, these immunological reactions can curtail therapeutic benefits and potentially lead to mortality. Ways to assay and reduce the immunogenicity of Cas9 and Cas12a proteins are therefore critical for ensuring patient safety and treatment efficacy, and for bringing us closer to realizing the vision of permanent genetic cures. WIREs Syst Biol Med 2018, 10:e1408. doi: 10.1002/wsbm.1408 This article is categorized under: Laboratory Methods and Technologies > Genetic/Genomic Methods Translational, Genomic, and Systems Medicine > Translational Medicine Translational, Genomic, and Systems Medicine > Therapeutic Methods.

Corrigendum: Engineering and optimising deaminase fusions for genome editing.

This corrects the article DOI: 10.1038/ncomms13330.

Insulin-Like Growth Factor 1 Receptor-Dependent Pathway Drives Epicardial Adipose Tissue Formation After Myocardial Injury.

BACKGROUND: Epicardial adipose tissue volume and coronary artery disease are strongly associated, even after accounting for overall body mass. Despite its pathophysiological significance, the origin and paracrine signaling pathways that regulate epicardial adipose tissue's formation and expansion are unclear. METHODS: We used a novel modified mRNA-based screening approach to probe the effect of individual paracrine factors on epicardial progenitors in the adult heart. RESULTS: Using 2 independent lineage-tracing strategies in murine models, we show that cells originating from the Wt1+ mesothelial lineage, which includes epicardial cells, differentiate into epicardial adipose tissue after myocardial infarction. This differentiation process required Wt1 expression in this lineage and was stimulated by insulin-like growth factor 1 receptor (IGF1R) activation. IGF1R inhibition within this lineage significantly reduced its adipogenic differentiation in the context of exogenous, IGF1-modified mRNA stimulation. Moreover, IGF1R inhibition significantly reduced Wt1 lineage cell differentiation into adipocytes after myocardial infarction. CONCLUSIONS: Our results establish IGF1R signaling as a key pathway that governs epicardial adipose tissue formation in the context of myocardial injury by redirecting the fate of Wt1+ lineage cells. Our study also demonstrates the power of modified mRNA -based paracrine factor library screening to dissect signaling pathways that govern progenitor cell activity in homeostasis and disease.

Engineering and optimising deaminase fusions for genome editing.

Precise editing is essential for biomedical research and gene therapy. Yet, homology-directed genome modification is limited by the requirements for genomic lesions, homology donors and the endogenous DNA repair machinery. Here we engineered programmable cytidine deaminases and test if we could introduce site-specific cytidine to thymidine transitions in the absence of targeted genomic lesions. Our programmable deaminases effectively convert specific cytidines to thymidines with 13% efficiency in Escherichia coli and 2.5% in human cells. However, off-target deaminations were detected more than 150 bp away from the target site. Moreover, whole genome sequencing revealed that edited bacterial cells did not harbour chromosomal abnormalities but demonstrated elevated global cytidine deamination at deaminase intrinsic binding sites. Therefore programmable deaminases represent a promising genome editing tool in prokaryotes and eukaryotes. Future engineering is required to overcome the processivity and the intrinsic DNA binding affinity of deaminases for safer therapeutic applications.

A multifunctional AAV-CRISPR-Cas9 and its host response.

CRISPR-Cas9 delivery by adeno-associated virus (AAV) holds promise for gene therapy but faces critical barriers on account of its potential immunogenicity and limited payload capacity. Here, we demonstrate genome engineering in postnatal mice using AAV-split-Cas9, a multifunctional platform customizable for genome editing, transcriptional regulation, and other previously impracticable applications of AAV-CRISPR-Cas9. We identify crucial parameters that impact efficacy and clinical translation of our platform, including viral biodistribution, editing efficiencies in various organs, antigenicity, immunological reactions, and physiological outcomes. These results reveal that AAV-CRISPR-Cas9 evokes host responses with distinct cellular and molecular signatures, but unlike alternative delivery methods, does not induce extensive cellular damage in vivo. Our study provides a foundation for developing effective genome therapeutics.

In vivo gene editing in dystrophic mouse muscle and muscle stem cells.

Frame-disrupting mutations in the DMD gene, encoding dystrophin, compromise myofiber integrity and drive muscle deterioration in Duchenne muscular dystrophy (DMD). Removing one or more exons from the mutated transcript can produce an in-frame mRNA and a truncated, but still functional, protein. In this study, we developed and tested a direct gene-editing approach to induce exon deletion and recover dystrophin expression in the mdx mouse model of DMD. Delivery by adeno-associated virus (AAV) of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonucleases coupled with paired guide RNAs flanking the mutated Dmd exon23 resulted in excision of intervening DNA and restored the Dmd reading frame in myofibers, cardiomyocytes, and muscle stem cells after local or systemic delivery. AAV-Dmd CRISPR treatment partially recovered muscle functional deficiencies and generated a pool of endogenously corrected myogenic precursors in mdx mouse muscle.

TRPV4-mediated calcium influx into human bronchial epithelia upon exposure to diesel exhaust particles.

BACKGROUND: Human respiratory epithelia function in airway mucociliary clearance and barrier function and have recently been implicated in sensory functions. OBJECTIVE: We investigated a link between chronic obstructive pulmonary disease (COPD) pathogenesis and molecular mechanisms underlying Ca2+ influx into human airway epithelia elicited by diesel exhaust particles (DEP). METHODS AND RESULTS: Using primary cultures of human respiratory epithelial (HRE) cells, we determined that these cells possess proteolytic signaling machinery, whereby proteinase-activated receptor-2 (PAR-2) activates Ca2+-permeable TRPV4, which leads to activation of human respiratory disease-enhancing matrix metalloproteinase-1 (MMP-1), a signaling cascade initiated by diesel exhaust particles (DEP), a globally relevant air pollutant. Moreover, we observed ciliary expression of PAR-2, TRPV4, and phospholipase-Cß3 in human airway epithelia and their DEP-enhanced protein-protein complex formation. We also found that the chronic obstructive pulmonary disease (COPD)-predisposing TRPV4P19S variant enhances Ca2+ influx and MMP 1 activation, providing mechanistic linkage between man-made air pollution and human airway disease. CONCLUSION: DEP evoked protracted Ca2+ influx via TRPV4, enhanced by the COPD-predisposing human genetic polymorphism TRPV4P19S. This mechanism reprograms maladaptive inflammatory and extracellular-matrix-remodeling responses in human airways. The novel concept of air pollution-responsive ciliary signal transduction from PAR-2 to TRPV4 in human respiratory epithelia will accelerate rationally targeted therapies, possibly via the inhalatory route.