A simultaneous knockout knockin genome editing strategy in HSPCs potently inhibits CCR5- and CXCR4-tropic HIV-1 infection.

Allogeneic hematopoietic stem and progenitor cell transplant (HSCT) of CCR5 null (CCR5Δ32) cells can be curative for HIV-1-infected patients. However, because allogeneic HSCT poses significant risk, CCR5Δ32 matched bone marrow donors are rare, and CCR5Δ32 transplant does not confer resistance to the CXCR4-tropic virus, it is not a viable option for most patients. We describe a targeted Cas9/AAV6-based genome editing strategy for autologous HSCT resulting in both CCR5- and CXCR4-tropic HIV-1 resistance. Edited human hematopoietic stem and progenitor cells (HSPCs) maintain multi-lineage repopulation capacity in vivo, and edited primary human T cells potently inhibit infection by both CCR5-tropic and CXCR4-tropic HIV-1. Modification rates facilitated complete loss of CCR5-tropic replication and up to a 2,000-fold decrease in CXCR4-tropic replication without CXCR4 locus disruption. This multi-factor editing strategy in HSPCs could provide a broad approach for autologous HSCT as a functional cure for both CCR5-tropic and CXCR4-tropic HIV-1 infections.

CRISPR/Cas9 model of prostate cancer identifies Kmt2c deficiency as a metastatic driver by Odam/Cabs1 gene cluster expression.

Metastatic prostate cancer (PCa) poses a significant therapeutic challenge with high mortality rates. Utilizing CRISPR-Cas9 in vivo, we target five potential tumor suppressor genes (Pten, Trp53, Rb1, Stk11, and RnaseL) in the mouse prostate, reaching humane endpoint after eight weeks without metastasis. By further depleting three epigenetic factors (Kmt2c, Kmt2d, and Zbtb16), lung metastases are present in all mice. While whole genome sequencing reveals few mutations in coding sequence, RNA sequencing shows significant dysregulation, especially in a conserved genomic region at chr5qE1 regulated by KMT2C. Depleting Odam and Cabs1 in this region prevents metastasis. Notably, the gene expression signatures, resulting from our study, predict progression-free and overall survival and distinguish primary and metastatic human prostate cancer. This study emphasizes positive genetic interactions between classical tumor suppressor genes and epigenetic modulators in metastatic PCa progression, offering insights into potential treatments.

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. By intersecting four mass spectrometry datasets, we identify SAM68 as crucial for the induction of apoptosis downstream of STING activation. SAM68 is essential for the full activation of apoptosis. Still, it is not required for STING-mediated activation of IFN expression or activation of NF-κB. Mechanistic studies reveal that protein trafficking is required and involves SAM68 recruitment to STING upon activation, with the two proteins associating at the Golgi or a post-Golgi compartment. Collectively, our work identifies SAM68 as a STING-interacting protein enabling induction of apoptosis through this DNA-activated innate immune pathway.

CRISPR activation to characterize splice-altering variants in easily accessible cells.

Genetic variants that affect mRNA splicing are a major cause of hereditary disorders, but the spliceogenicity of variants is challenging to predict. RNA diagnostics of clinically accessible tissues enable rapid functional characterization of splice-altering variants within their natural genetic context. However, this analysis cannot be offered to all individuals as one in five human disease genes are not expressed in easily accessible cell types. To overcome this problem, we have used CRISPR activation (CRISPRa) based on a dCas9-VPR mRNA-based delivery platform to induce expression of the gene of interest in skin fibroblasts from individuals with suspected monogenic disorders. Using this ex vivo splicing assay, we characterized the splicing patterns associated with germline variants in the myelin protein zero gene (MPZ), which is exclusively expressed in Schwann cells of the peripheral nerves, and the spastin gene (SPAST), which is predominantly expressed in the central nervous system. After overnight incubation, CRISPRa strongly upregulated MPZ and SPAST transcription in skin fibroblasts, which enabled splice variant profiling using reverse transcription polymerase chain reaction, next-generation sequencing, and long-read sequencing. Our investigations show proof of principle of a promising genetic diagnostic tool that involves CRISPRa to activate gene expression in easily accessible cells to study the functional impact of genetic variants. The procedure is easy to perform in a diagnostic laboratory with equipment and reagents all readily available.

Single-cell transcriptome analysis of epithelial, immune, and stromal signatures and interactions in human ovarian cancer.

A comprehensive investigation of ovarian cancer (OC) progression at the single-cell level is crucial for enhancing our understanding of the disease, as well as for the development of better diagnoses and treatments. Here, over half a million single-cell transcriptome data were collected from 84 OC patients across all clinical stages. Through integrative analysis, we identified heterogeneous epithelial-immune-stromal cellular compartments and their interactions in the OC microenvironment. The epithelial cells displayed clinical subtype features with functional variance. A significant increase in distinct T cell subtypes was identified including Tregs and CD8+ exhausted T cells from stage IC2. Additionally, we discovered antigen-presenting cancer-associated fibroblasts (CAFs), with myofibroblastic CAFs (myCAFs) exhibiting enriched extracellular matrix (ECM) functionality linked to tumor progression at stage IC2. Furthermore, the NECTIN2-TIGIT ligand-receptor pair was identified to mediate T cells communicating with epithelial, fibroblast, endothelial, and other cell types. Knock-out of NECTIN2 using CRISPR/Cas9 inhibited ovarian cancer cell (SKOV3) proliferation, and increased T cell proliferation when co-cultured. These findings shed light on the cellular compartments and functional aspects of OC, providing insights into the molecular mechanisms underlying stage IC2 and potential therapeutic strategies for OC.

Progress and harmonization of gene editing to treat human diseases: Proceeding of COST Action CA21113 GenE-HumDi.

The European Cooperation in Science and Technology (COST) is an intergovernmental organization dedicated to funding and coordinating scientific and technological research in Europe, fostering collaboration among researchers and institutions across countries. Recently, COST Action funded the "Genome Editing to treat Human Diseases" (GenE-HumDi) network, uniting various stakeholders such as pharmaceutical companies, academic institutions, regulatory agencies, biotech firms, and patient advocacy groups. GenE-HumDi's primary objective is to expedite the application of genome editing for therapeutic purposes in treating human diseases. To achieve this goal, GenE-HumDi is organized in several working groups, each focusing on specific aspects. These groups aim to enhance genome editing technologies, assess delivery systems, address safety concerns, promote clinical translation, and develop regulatory guidelines. The network seeks to establish standard procedures and guidelines for these areas to standardize scientific practices and facilitate knowledge sharing. Furthermore, GenE-HumDi aims to communicate its findings to the public in accessible yet rigorous language, emphasizing genome editing's potential to revolutionize the treatment of many human diseases. The inaugural GenE-HumDi meeting, held in Granada, Spain, in March 2023, featured presentations from experts in the field, discussing recent breakthroughs in delivery methods, safety measures, clinical translation, and regulatory aspects related to gene editing.

ER stress induces caspase-2-tBID-GSDME-dependent cell death in neurons lytically infected with herpes simplex virus type 2.

Neurotropic viruses, including herpes simplex virus (HSV) types 1 and 2, have the capacity to infect neurons and can cause severe diseases. This is associated with neuronal cell death, which may contribute to morbidity or even mortality if the infection is not controlled. However, the mechanistic details of HSV-induced neuronal cell death remain enigmatic. Here, we report that lytic HSV-2 infection of human neuron-like SH-SY5Y cells and primary human and murine brain cells leads to cell death mediated by gasdermin E (GSDME). HSV-2-induced GSDME-mediated cell death occurs downstream of replication-induced endoplasmic reticulum stress driven by inositol-requiring kinase 1α (IRE1α), leading to activation of caspase-2, cleavage of the pro-apoptotic protein BH3-interacting domain death agonist (BID), and mitochondria-dependent activation of caspase-3. Finally, necrotic neurons released alarmins, which activated inflammatory responses in human iPSC-derived microglia. In conclusion, lytic HSV infection in neurons activates an ER stress-driven pathway to execute GSDME-mediated cell death and promote inflammation.

Enrichment strategies to enhance genome editing.

Genome editing technologies hold great promise for numerous applications including the understanding of cellular and disease mechanisms and the development of gene and cellular therapies. Achieving high editing frequencies is critical to these research areas and to achieve the overall goal of being able to manipulate any target with any desired genetic outcome. However, gene editing technologies sometimes suffer from low editing efficiencies due to several challenges. This is often the case for emerging gene editing technologies, which require assistance for translation into broader applications. Enrichment strategies can support this goal by selecting gene edited cells from non-edited cells. In this review, we elucidate the different enrichment strategies, their many applications in non-clinical and clinical settings, and the remaining need for novel strategies to further improve genome research and gene and cellular therapy studies.

The p53 challenge of hematopoietic stem cell gene editing.

Ex vivo gene editing in hematopoietic stem and progenitor cells (HSPCs) represents a promising curative treatment strategy for monogenic blood disorders. Gene editing using the homology-directed repair (HDR) pathway enables precise genetic modifications ranging from single base pair correction to replacement or insertion of large DNA segments. Hence, HDR-based gene editing could facilitate broad application of gene editing across monogenic disorders, but the technology still faces challenges for clinical translation. Among these, recent studies demonstrate induction of a DNA damage response (DDR) and p53 activation caused by DNA double-strand breaks and exposure to recombinant adeno-associated virus vector repair templates, resulting in reduced proliferation, engraftment, and clonogenic capacity of edited HSPCs. While different mitigation strategies can reduce this DDR, more research is needed on this phenomenon to ensure safe and efficient implementation of HDR-based gene editing in the clinic.

Dual intron-targeted CRISPR-Cas9-mediated disruption of the AML RUNX1-RUNX1T1 fusion gene effectively inhibits proliferation and decreases tumor volume in vitro and in vivo.

Oncogenic fusion drivers are common in hematological cancers and are thus relevant targets of future CRISPR-Cas9-based treatment strategies. However, breakpoint-location variation in patients pose a challenge to traditional breakpoint-targeting CRISPR-Cas9-mediated disruption strategies. Here we present a new dual intron-targeting CRISPR-Cas9 treatment strategy, for targeting t(8;21) found in 5-10% of de novo acute myeloid leukemia (AML), which efficiently disrupts fusion genes without prior identification of breakpoint location. We show in vitro growth rate and proliferation reduction by 69 and 94% in AML t(8;21) Kasumi-1 cells, following dual intron-targeted disruption of RUNX1-RUNX1T1 compared to a non t(8;21) AML control. Furthermore, mice injected with RUNX1-RUNX1T1-disrupted Kasumi-1 cells had in vivo tumor growth reduction by 69 and 91% compared to controls. Demonstrating the feasibility of RUNX1-RUNX1T1 disruption, these findings were substantiated in isolated primary cells from a patient diagnosed with AML t(8;21). In conclusion, we demonstrate proof-of-principle of a dual intron-targeting CRISPR-Cas9 treatment strategy in AML t(8;21) without need for precise knowledge of the breakpoint location.

Combined lineage tracing and scRNA-seq reveals unexpected first heart field predominance of human iPSC differentiation.

During mammalian development, the left and right ventricles arise from early populations of cardiac progenitors known as the first and second heart fields, respectively. While these populations have been extensively studied in non-human model systems, their identification and study in vivo human tissues have been limited due to the ethical and technical limitations of accessing gastrulation-stage human embryos. Human-induced pluripotent stem cells (hiPSCs) present an exciting alternative for modeling early human embryogenesis due to their well-established ability to differentiate into all embryonic germ layers. Here, we describe the development of a TBX5/MYL2 lineage tracing reporter system that allows for the identification of FHF- progenitors and their descendants including left ventricular cardiomyocytes. Furthermore, using single-cell RNA sequencing (scRNA-seq) with oligonucleotide-based sample multiplexing, we extensively profiled differentiating hiPSCs across 12 timepoints in two independent iPSC lines. Surprisingly, our reporter system and scRNA-seq analysis revealed a predominance of FHF differentiation using the small molecule Wnt-based 2D differentiation protocol. We compared this data with existing murine and 3D cardiac organoid scRNA-seq data and confirmed the dominance of left ventricular cardiomyocytes (>90%) in our hiPSC-derived progeny. Together, our work provides the scientific community with a powerful new genetic lineage tracing approach as well as a single-cell transcriptomic atlas of hiPSCs undergoing cardiac differentiation.

CRISPR-Cas-mediated transcriptional modulation: The therapeutic promises of CRISPRa and CRISPRi.

The CRISPR-Cas system is commonly known for its ability to cleave DNA in a programmable manner, which has democratized gene editing and facilitated recent breakthroughs in gene therapy. However, newer iterations of the technology using nuclease-disabled Cas enzymes have spurred a variety of different types of genetic engineering platforms such as transcriptional modulation using the CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) systems. This review introduces the creation of these programmable transcriptional modulators, various methods of delivery utilized for these systems, and recent technological developments. CRISPRa and CRISPRi have also been implemented in genetic screens for interrogating gene function and discovering genes involved in various biological pathways. We describe recent compelling examples of how these tools have become powerful means to unravel genetic networks and uncovering important information about devastating diseases. Finally, we provide an overview of preclinical studies in which transcriptional modulation has been used therapeutically, and we discuss potential future directions of these novel modalities.

Impact of U2AF1 mutations on circular RNA expression in myelodysplastic neoplasms.

Mutations in U2AF1 are relatively common in myelodysplastic neoplasms (MDS) and are associated with an inferior prognosis, but the molecular mechanisms underlying this are not fully elucidated. Circular RNAs (circRNAs) have been implicated in cancer, but it is unknown how mutations in splicing factors may impact on circRNA biogenesis. Here, we used RNA-sequencing to investigate the effects of U2AF1 mutations on circRNA expression in K562 cells with a doxycycline-inducible U2AF1S34 mutation, in a mouse model with a doxycycline-inducible U2AF1S34 mutation, and in FACS-sorted CD34+ bone marrow cells from MDS patients with either U2AF1S34 or U2AF1Q157 mutations. In all contexts, we found an increase in global circRNA levels in the U2AF1-mutated setting, which was independent of expression changes in the cognate linear host genes. In patients, the U2AF1S34 and U2AF1Q157 mutations were both associated with an overall increased expression of circRNAs. circRNAs generated by a non-Alu-mediated mechanism generally showed the largest increase in expression levels. Several well-described cancer-associated circRNAs, including circZNF609 and circCSNK1G3, were upregulated in MDS patients with U2AF1 mutations compared to U2AF1-wildtype MDS controls. In conclusion, high circRNA expression is observed in association with U2AF1 mutations in three biological systems, presenting an interesting possibility for biomarker and therapeutic investigation.

Enrichment of transgene integrations by transient CRISPR activation of a silent reporter gene.

CRISPR-Cas-mediated site-specific integration of transgenes by homology-directed repair (HDR) is challenging, especially in primary cells, where inferior editing efficiency may impede the development of gene- and cellular therapies. Various strategies for enrichment of cells with transgene integrations have been developed, but most strategies either generate unwanted genomic scars or rely on permanent integration and expression of a reporter gene used for selection. However, stable expression of a reporter gene may perturb cell homeostasis and function. Here we develop a broadly applicable and versatile enrichment strategy by harnessing the capability of CRISPR activation (CRISPRa) to transiently induce expression of a therapeutically relevant reporter gene used for immunomagnetic enrichment. This strategy is readily adaptable to primary human T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs), where enrichment of 1.8- to 3.3-fold and 3.2- to 3.6-fold was achieved, respectively. Furthermore, chimeric antigen receptor (CAR) T cells were enriched 2.5-fold and demonstrated improved cytotoxicity over non-enriched CAR T cells. Analysis of HDR integrations showed a proportion of cells harboring deletions of the transgene cassette arising either from impartial HDR or truncated adeno-associated virus (AAV) vector genomes. Nonetheless, this novel enrichment strategy expands the possibility to enrich for transgene integrations in research settings and in gene and cellular therapies.

Development of HIV-Resistant CAR T Cells by CRISPR/Cas-Mediated CAR Integration into the CCR5 Locus.

Adoptive immunotherapy using chimeric antigen receptor (CAR) T cells has been highly successful in treating B cell malignancies and holds great potential as a curative strategy for HIV infection. Recent advances in the use of anti-HIV broadly neutralizing antibodies (bNAbs) have provided vital information for optimal antigen targeting of CAR T cells. However, CD4+ CAR T cells are susceptible to HIV infection, limiting their therapeutic potential. In the current study, we engineered HIV-resistant CAR T cells using CRISPR/Cas9-mediated integration of a CAR cassette into the CCR5 locus. We used a single chain variable fragment (scFv) of the clinically potent bNAb 10-1074 as the antigen-targeting domain in our anti-HIV CAR T cells. Our anti-HIV CAR T cells showed specific lysis of HIV-infected cells in vitro. In a PBMC humanized mouse model of HIV infection, the anti-HIV CAR T cells expanded and transiently limited HIV infection. In conclusion, this study provides proof-of-concept for developing HIV-resistant CAR T cells using CRISPR/Cas9 targeted integration.

Plasmacytoid Dendritic Cells as a Novel Cell-Based Cancer Immunotherapy.

Plasmacytoid dendritic cells (pDCs) are multifaceted immune cells with a wide range of innate and adaptive immunological functions. They constitute the first line of defence against multiple viral infections and have also been reported to actively participate in antitumor immune responses. The clinical implication of the presence of pDCs in the tumor microenvironment (TME) is still ambiguous, but it is clear that pDCs possess the ability to modulate tumor-specific T cell responses and direct cytotoxic functions. Therapeutic strategies designed to exploit these qualities of pDCs to boost tumor-specific immune responses could represent an attractive alternative compared to conventional therapeutic approaches in the future, and promising antitumor effects have already been reported in phase I/II clinical trials. Here, we review the many roles of pDCs in cancer and present current advances in developing pDC-based immunotherapeutic approaches for treating cancer.

Recent Advances in the Development of Anti-FLT3 CAR T-Cell Therapies for Treatment of AML.

Following the success of the anti-CD19 chimeric antigen receptor (CAR) T-cell therapies against B-cell malignancies, the CAR T-cell approach is being developed towards other malignancies like acute myeloid leukemia (AML). Treatment options for relapsed AML patients are limited, and the upregulation of the FMS-like tyrosine kinase 3 (FLT3) in malignant T-cells is currently not only being investigated as a prognostic factor, but also as a target for new treatment options. In this review, we provide an overview and discuss different approaches of current anti-FLT3 CAR T-cells under development. In general, these therapies are effective both in vitro and in vivo, however the safety profile still needs to be further investigated. The first clinical trials have been initiated, and the community now awaits clinical evaluation of the approach of targeting FLT3 with CAR T-cells.

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. This led to a codon-optimized and size-minimized PE that has an expression advantage (1.4-fold) and size advantage (621 bp shorter). In addition, we optimize the split intein PE system and identify Rma-based Cas9 split sites (573-574 and 673-674) that combined with the truncated PE delivered by dual AAVs result in superior AAV titer and prime editing efficiency. We also show that this minimized PE gives rise to superior lentiviral vector titers (46-fold) over the regular PE in an all-in-one PE lentiviral vector. We finally deliver the minimized PE to mouse liver by dual AAV8 vectors and show up to 6% precise editing of the PCSK9 gene, thereby demonstrating the value of this truncated split PE system for in vivo applications.

CRISPR/Cas-Based Gene Editing Strategies for DOCK8 Immunodeficiency Syndrome.

Defects in the DOCK8 gene causes combined immunodeficiency termed DOCK8 immunodeficiency syndrome (DIDS). DIDS previously belonged to the disease category of autosomal recessive hyper IgE syndrome (AR-HIES) but is now classified as a combined immunodeficiency (CID). This genetic disorder induces early onset of susceptibility to severe recurrent viral and bacterial infections, atopic diseases and malignancy resulting in high morbidity and mortality. This pathological state arises from impairment of actin polymerization and cytoskeletal rearrangement, which induces improper immune cell migration-, survival-, and effector functions. Owing to the severity of the disease, early allogenic hematopoietic stem cell transplantation is recommended even though it is associated with risk of unintended adverse effects, the need for compatible donors, and high expenses. So far, no alternative therapies have been developed, but the monogenic recessive nature of the disease suggests that gene therapy may be applied. The advent of the CRISPR/Cas gene editing system heralds a new era of possibilities in precision gene therapy, and positive results from clinical trials have already suggested that the tool may provide definitive cures for several genetic disorders. Here, we discuss the potential application of different CRISPR/Cas-mediated genetic therapies to correct the DOCK8 gene. Our findings encourage the pursuit of CRISPR/Cas-based gene editing approaches, which may constitute more precise, affordable, and low-risk definitive treatment options for DOCK8 deficiency.