Early host defense against virus infections.

Early host defense eliminates many viruses before infections are established while clearing others so they remain subclinical or cause only mild disease. The field of immunology has been shaped by broad concepts, including the pattern recognition theory that currently dominates innate immunology. Focusing on early host responses to virus infections, we analyze the literature to build a working hypothesis for the principles that govern the early line of cellular antiviral defense.

Cell-targeted gene modification by delivery of CRISPR-Cas9 ribonucleoprotein complexes in pseudotyped lentivirus-derived nanoparticles.

To fully utilize the potential of CRISPR-Cas9-mediated genome editing, time-restricted and targeted delivery is crucial. By modulating the pseudotype of engineered lentivirus-derived nanoparticles (LVNPs), we demonstrate efficient cell-targeted delivery of Cas9/single guide RNA (sgRNA) ribonucleoprotein (RNP) complexes, supporting gene modification in a defined subset of cells in mixed cell populations. LVNPs pseudotyped with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein resulted in angiotensin-converting enzyme 2 (ACE2)-dependent insertion or deletion (indel) formation in an ACE2/ACE2 population of cells, whereas Nipah virus glycoprotein pseudotyping resulted in Ephrin-B2/B3-specific gene knockout.

Gene Editing by Ferrying of CRISPR/Cas Ribonucleoprotein Complexes in Enveloped Virus-Derived Particles.

The invention of next-generation CRISPR/Cas gene editing tools, like base and prime editing, for correction of gene variants causing disease, has created hope for use in patients leading to wider clinical translation. To realize this potential, delivery vehicles that can ferry gene editing tool kits safely and effectively into specific cell populations or tissues are in great demand. In this review, we describe the development of enveloped retrovirus-derived particles as carriers of "ready-to-work" ribonucleoprotein complexes consisting of Cas9-derived editor proteins and single guide RNAs.

SAM68 directs STING signaling to apoptosis in macrophages.

DNA is a danger signal sensed by cGAS to engage signaling through STING to activate innate immune functions. The best-studied downstream responses to STING activation include expression of type I interferon and inflammatory genes, but STING also activates other pathways, including apoptosis. Here, we report that STING-dependent induction of apoptosis in macrophages occurs through the intrinsic mitochondrial pathway and is mediated via IRF3 but acts independently of gene transcription.

STING activation counters glioblastoma by vascular alteration and immune surveillance.

Glioblastoma (GBM) is an aggressive brain tumor with a median survival of 15 months and has limited treatment options. Immunotherapy with checkpoint inhibitors has shown minimal efficacy in combating GBM, and large clinical trials have failed. New immunotherapy approaches and a deeper understanding of immune surveillance of GBM are needed to advance treatment options for this devastating disease.

Engineered lentivirus-derived nanoparticles (LVNPs) for delivery of CRISPR/Cas ribonucleoprotein complexes supporting base editing, prime editing and in vivo gene modification.

Implementation of therapeutic in vivo gene editing using CRISPR/Cas relies on potent delivery of gene editing tools. Administration of ribonucleoprotein (RNP) complexes consisting of Cas protein and single guide RNA (sgRNA) offers short-lived editing activity and safety advantages over conventional viral and non-viral gene and RNA delivery approaches. By engineering lentivirus-derived nanoparticles (LVNPs) to facilitate RNP delivery, we demonstrate effective administration of SpCas9 as well as SpCas9-derived base and prime editors (BE/PE) leading to gene editing in recipient cells.

Restriction of C1-inhibitor activity in hereditary angioedema by dominant-negative effects of disease-associated SERPING1 gene variants.

Background: Patients with hereditary angioedema experience recurrent, sometimes life-threatening, attacks of edema. It is a rare genetic disorder characterized by genetic and clinical heterogenicity. Most cases are caused by genetic variants in the SERPING1 gene leading to plasma deficiency of the encoded protein C1 inhibitor (C1INH).

Resistance to vincristine in DLBCL by disruption of p53-induced cell cycle arrest and apoptosis mediated by KIF18B and USP28.

The frontline therapy R-CHOP for patients with diffuse large B-cell lymphoma (DLBCL) has remained unchanged for two decades despite numerous Phase III clinical trials investigating new alternatives. Multiple large studies have uncovered genetic subtypes of DLBCL enabling a targeted approach. To further pave the way for precision oncology, we perform genome-wide CRISPR screening to uncover the cellular response to one of the components of R-CHOP, vincristine, in the DLBCL cell line SU-DHL-5.

Prime editing in hematopoietic stem cells-From to CRISPR-based treatment of blood disorders.

Prime editing of human hematopoietic stem cells has the potential to become a safe and efficient way of treating diseases of the blood directly in patients. By allowing site-targeted gene intervention without homology-directed repair donor templates and DNA double-stranded breaks, the invention of prime editing fuels the exploration of alternatives to conventional recombination-based genome editing of hematopoietic stem cells. Prime editing is as close as we get today to a true genome editing drug that does not require a separate DNA donor.

CRISPR-screen identifies ZIP9 and dysregulated Zn2+ homeostasis as a cause of cancer-associated changes in glycosylation.

Introduction: In epithelial cancers, truncated O-glycans, such as the Thomson-nouveau antigen (Tn) and its sialylated form (STn), are upregulated on the cell surface and associated with poor prognosis and immunological escape. Recent studies have shown that these carbohydrate epitopes facilitate cancer development and can be targeted therapeutically; however, the mechanism underpinning their expression remains unclear.

Methods: To identify genes directly influencing the expression of cancer-associated O-glycans, we conducted an unbiased, positive-selection, whole-genome CRISPR knockout-screen using monoclonal antibodies against Tn and STn.

Delivering genes with human immunodeficiency virus-derived vehicles: still state-of-the-art after 25 years.

Viruses are naturally endowed with the capacity to transfer genetic material between cells. Following early skepticism, engineered viruses have been used to transfer genetic information into thousands of patients, and genetic therapies are currently attracting large investments. Despite challenges and severe adverse effects along the way, optimized technologies and improved manufacturing processes are driving gene therapy toward clinical translation.

CRISPR-Cas9-directed gene tagging using a single integrase-defective lentiviral vector carrying a transposase-based Cas9 off switch.

Locus-directed DNA cleavage induced by the CRISPR-Cas9 system triggers DNA repair mechanisms allowing gene repair or targeted insertion of foreign DNA. For gene insertion to be successful, availability of a homologous donor template needs to be timed with cleavage of the DNA by the Cas9 endonuclease guided by a target-specific single guide RNA (sgRNA). We present a novel approach for targeted gene insertion based on a single integrase-defective lentiviral vector (IDLV) carrying a Cas9 off switch.

A genome-wide CRISPR-Cas9 knockout screen identifies novel PARP inhibitor resistance genes in prostate cancer.

DNA repair gene mutations are frequent in castration-resistant prostate cancer (CRPC), suggesting eligibility for poly(ADP-ribose) polymerase inhibitor (PARPi) treatment. However, therapy resistance is a major clinical challenge and genes contributing to PARPi resistance are poorly understood. Using a genome-wide CRISPR-Cas9 knockout screen, this study aimed at identifying genes involved in PARPi resistance in CRPC.

A truncated reverse transcriptase enhances prime editing by split AAV vectors.

Prime editing is a new CRISPR-based, genome-editing technology that relies on the prime editor (PE), a fusion protein of Cas9-nickase and M-MLV reverse transcriptase (RT), and a prime editing guide RNA (pegRNA) that serves both to target PE to the desired genomic locus and to carry the edit to be introduced. Here, we make advancements to the RT moiety to improve prime editing efficiencies and truncations to mitigate issues with adeno-associated virus (AAV) viral vector size limitations, which currently do not support efficient delivery of the large prime editing components. These efforts include RT variant screening, codon optimization, and PE truncation by removal of the RNase H domain and further trimming.

Life-threatening viral disease in a novel form of autosomal recessive IFNAR2 deficiency in the Arctic.

Type I interferons (IFN-I) play a critical role in human antiviral immunity, as demonstrated by the exceptionally rare deleterious variants of IFNAR1 or IFNAR2. We investigated five children from Greenland, Canada, and Alaska presenting with viral diseases, including life-threatening COVID-19 or influenza, in addition to meningoencephalitis and/or hemophagocytic lymphohistiocytosis following live-attenuated viral vaccination. The affected individuals bore the same homozygous IFNAR2 c.

Single-Cell Monitoring of Activated Innate Immune Signaling by a d2eGFP-Based Reporter Mimicking Time-Restricted Activation of Expression.

The innate immune system represents a balanced first line of defense against infection. Type I interferons (IFNs) are key regulators of the response to viral infections with an essential early wave of IFN-β expression, which is conditional, time-restricted, and stochastic in its nature. The possibility to precisely monitor individual cells with active transcription during innate signaling requires a robust reporter system that mimics the endogenous IFN-β signal.

piggyPrime: High-Efficacy Prime Editing in Human Cells Using piggyBac-Based DNA Transposition.

Prime editing is a novel genome editing technology that allows a wide range of tailored genomic alterations. Prime editing does not involve homologous recombination, but suffers from low efficacy. Here, we demonstrate piggyPrime, a transfected single-vector system based on piggyBac DNA transposition for genomic integration of all prime editing components in human cells allowing easy and effective transgenesis with prime editing efficacies up to 100% in cell lines.

pegIT - a web-based design tool for prime editing.

Prime editing (PE) is a novel CRISPR-derived genome editing technique facilitating precision editing without double-stranded DNA breaks. PE, mediated by a Cas9-reverse transcriptase fusion protein, is based on dual-functioning prime editing guide RNAs (pegRNAs), serving both as guide molecules and as templates carrying the desired edits. Due to such diverse functions, manual pegRNA design is a subject to error and not suited for large-scale setups.

Defects in and underlie HSV2 meningitis and reveal a critical role for autophagy in antiviral defense in humans.

Recurrent herpesvirus infections can manifest in different forms of disease, including cold sores, genital herpes, and encephalitis. There is an incomplete understanding of the genetic and immunological factors conferring susceptibility to recurrent herpes simplex virus 2 (HSV2) infection in the central nervous system (CNS). Here, we describe two adult patients with recurrent HSV2 lymphocytic Mollaret's meningitis that each carry a rare monoallelic variant in the autophagy proteins ATG4A or LC3B2.

Characterization of distinct molecular interactions responsible for IRF3 and IRF7 phosphorylation and subsequent dimerization.

IRF3 and IRF7 are critical transcription factors in the innate immune response. Their activation is controlled by phosphorylation events, leading to the formation of homodimers that are transcriptionally active. Phosphorylation occurs when IRF3 is recruited to adaptor proteins via a positively charged surface within the regulatory domain of IRF3.

Targeted Knockout of the Vegfa Gene in the Retina by Subretinal Injection of RNP Complexes Containing Cas9 Protein and Modified sgRNAs.

The therapeutic effect of retinal gene therapy using CRISPR/Cas9-mediated genome editing and knockout applications is dependent on efficient and safe delivery of gene-modifying tool kits. Recently, transient administration of single guide RNAs (sgRNAs) and SpCas9 proteins delivered as ribonucleoproteins (RNPs) has provided potent gene knockout in vitro. To improve efficacy of CRISPR-based gene therapy, we delivered RNPs containing SpCas9 protein complexed to chemically modified sgRNAs (msgRNAs).

Identification of BLNK and BTK as mediators of rituximab-induced programmed cell death by CRISPR screens in GCB-subtype diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is characterized by extensive genetic heterogeneity, and this results in unpredictable responses to the current treatment, R-CHOP, which consists of a cancer drug combination supplemented with the humanized CD20-targeting monoclonal antibody rituximab. Despite improvements in the patient response rate through rituximab addition to the treatment plan, up to 40% of DLBCL patients end in a relapsed or refractory state due to inherent or acquired resistance to the regimen. Here, we employ a lentiviral genome-wide clustered regularly interspaced short palindromic repeats library screening approach to identify genes involved in facilitating the rituximab response in cancerous B cells.

MicroRNA-106b Regulates Expression of the Tumour Suppressors p21 and TXNIP and Promotes Tumour Cell Proliferation in Mycosis Fungoides.

A prognostic 3-miRNA classifier for early-stage mycosis fungoides has been developed recently, with miR-106b providing the strongest prognostic power. The aim of this study was to investigate the molecular function of miR-106b in mycosis fungoides disease progression. The cellular localization of miR-106b in mycosis fungoides skin biopsies was determined by in situ hybridization.