nCas9 Engineering for Improved Target Interaction Presents an Effective Strategy to Enhance Base Editing.

Base editors (BEs) are a recent generation of genome editing tools that couple a cytidine or adenosine deaminase activity to a catalytically impaired Cas9 moiety (nCas9) to enable specific base conversions at the targeted genomic loci. Given their strong application potential, BEs are under active developments toward greater levels of efficiency and safety. Here, a previously overlooked nCas9-centric strategy is explored for enhancement of BE. Based on a cytosine BE (CBE), 20 point mutations associated with nCas9-target interaction are tested. Subsequently, from the initial positive X-to-arginine hits, combinatorial modifications are applied to establish further enhanced CBE variants (1.1-1.3). Parallel nCas9 modifications in other versions of CBEs including A3A-Y130F-BE4max, YEE-BE4max, CGBE, and split-AncBE4max, as well as in the context of two adenine BEs (ABE), likewise enhance their respective activities. The same strategy also substantially improves the efficiencies of high-fidelity nCas9/BEs. Further evidence confirms that the stabilization of nCas9-substrate interactions underlies the enhanced BE activities. In support of their translational potential, the engineered CBE and ABE variants respectively enable 82% and 25% higher rates of editing than the controls in primary human T-cells. This study thus demonstrates a highly adaptable strategy for enhancing BE, and for optimizing other forms of Cas9-derived tools.

PFKFB3 controls acinar IP3R-mediated Ca2+ overload to regulate acute pancreatitis severity.

Acute pancreatitis (AP) is among the most common hospital gastrointestinal diagnoses; understanding the mechanisms underlying the severity of AP is critical for development of new treatment options for this disease. Here, we evaluate the biological function of phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) in AP pathogenesis in 2 independent genetically engineered mouse models of AP. PFKFB3 was elevated in AP and severe AP (SAP), and KO of Pfkfb3 abrogated the severity of alcoholic SAP (FAEE-SAP). Using a combination of genetic, pharmacological, and molecular studies, we defined the interaction of PFKFB3 with inositol 1,4,5-trisphosphate receptor (IP3R) as a key event mediating this phenomenon. Further analysis demonstrated that the interaction between PFKFB3 and IP3R promotes FAEE-SAP severity by altering intracellular calcium homeostasis in acinar cells. Together, our results support a PFKFB3-driven mechanism controlling AP pathobiology and define this enzyme as a therapeutic target to ameliorate the severity of this condition.

PE-STOP: A versatile tool for installing nonsense substitutions amenable for precise reversion.

The rapid advances in genome editing technologies have revolutionized the study of gene functions in cell or animal models. The recent generation of double-stranded DNA cleavage-independent base editors has been suitably adapted for interrogation of protein-coding genes on the basis of introducing premature stop codons or disabling the start codons. However, such versions of stop/start codon-oriented genetic tools still present limitations on their versatility, base-level precision, and target specificity. Here, we exploit a newly developed prime editor (PE) that differs from base editors by its adoption of a reverse transcriptase activity, which enables incorporation of various types of precise edits templated by a specialized prime editing guide RNA. Based on such a versatile platform, we established a prime editing-empowered method (PE-STOP) for installation of nonsense substitutions, providing a complementary approach to the present gene-targeting tools. PE-STOP is bioinformatically predicted to feature substantially expanded coverage in the genome space. In practice, PE-STOP introduces stop codons with good efficiencies in human embryonic kidney 293T and N2a cells (with medians of 29% [ten sites] and 25% [four sites] editing efficiencies, respectively), while exhibiting minimal off-target effects and high on-target precision. Furthermore, given the fact that PE installs prime editing guide RNA-templated mutations, we introduce a unique strategy for precise genetic rescue of PE-STOP-dependent nonsense mutation via the same PE platform. Altogether, the present work demonstrates a versatile and specific tool for gene inactivation and for functional interrogation of nonsense mutations.

IL-6/ERK signaling pathway participates in type I IFN-programmed, unconventional M2-like macrophage polarization.

Type I interferons (IFN-Is) have been harnessed for cancer therapies due to their immunostimulatory functions. However, certain tumor-tolerating activities by IFN-Is also exist, and may potentially thwart their therapeutic effects. In this respect, our previous studies have demonstrated a monocyte-orchestrated, IFN-I-to-IL-4 cytokine axis, which can subsequently drive M2-skewed pro-tumoral polarization of macrophages. Whether other IFN-dependent signals may also contribute to such an unconventional circumstance of M2-like macrophage skewing remain unexplored. Herein, we first unveil IL-6 as another ligand that participates in IFN-dependent induction of a typical M2 marker (ARG1) in transitional monocytes. Indeed, IL-6 significantly promotes IL-4-dependent induction of a major group of prominent M2 markers in mouse bone marrow-derived macrophages (BMDMs) and human peripheral blood-derived macrophages, while it alone does not engage marked increases of these markers. Such a pattern of regulation is confirmed globally by RNAseq analyses in BMDMs, which in turn suggests an association of IL-6-amplified subset of M2 genes with the ERK1/2 signaling pathway. Interestingly, pharmacological experiments establish the role of SHP2-ERK cascade in mediating IL-6's enhancement effect on these M2 targets. Similar approaches also validate the involvement of IL-6/ERK signaling in promoting the IFN-dependent, unconventional M2-skewing phenotype in transitional monocytes. Furthermore, an inhibitor of ERK signaling cooperates with an IFN-I inducer to enable a greater antitumor effect, which correlates with suppression of treatment-elicited ARG1. The present work establishes a role of IL-6/ERK signaling in promoting M2-like macrophage polarization, and suggests this axis as a potential therapeutic target for combination with IFN-I-based cancer treatments.

Development of a versatile nuclease prime editor with upgraded precision.

The applicability of nuclease-based form of prime editor (PEn) has been hindered by its complexed editing outcomes. A chemical inhibitor against DNA-PK, which mediates the nonhomologous end joining (NHEJ) pathway, was recently shown to promote precise insertions by PEn. Nevertheless, the intrinsic issues of specificity and toxicity for such a chemical approach necessitate development of alternative strategies. Here, we find that co-introduction of PEn and a NHEJ-restraining, 53BP1-inhibitory ubiquitin variant potently drives precise edits via mitigation of unintended edits, framing a high-activity editing platform (uPEn) apparently complementing the canonical PE. Further developments involve exploring the effective configuration of a homologous region-containing pegRNA (HR-pegRNA). Overall, uPEn can empower high-efficiency installation of insertions (38%), deletions (43%) and replacements (52%) in HEK293T cells. When compared with PE3/5max, uPEn demonstrates superior activities for typically refractory base substitutions, and for small-block edits. Collectively, this work establishes a highly efficient PE platform with broad application potential.

Precise somatic genome editing for treatment of inborn errors of immunity.

Rapid advances in high throughput sequencing have substantially expedited the identification and diagnosis of inborn errors of immunity (IEI). Correction of faulty genes in the hematopoietic stem cells can potentially provide cures for the majority of these monogenic immune disorders. Given the clinical efficacies of vector-based gene therapies already established for certain groups of IEI, the recently emerged genome editing technologies promise to bring safer and more versatile treatment options. Here, we review the latest development in genome editing technologies, focusing on the state-of-the-art tools with improved precision and safety profiles. We subsequently summarize the recent preclinical applications of genome editing tools in IEI models, and discuss the major challenges and future perspectives of such treatment modalities. Continued explorations of precise genome editing for IEI treatment shall move us closer toward curing these unfortunate rare diseases.

Enhancement of prime editing via xrRNA motif-joined pegRNA.

The prime editors (PEs) have shown great promise for precise genome modification. However, their suboptimal efficiencies present a significant technical challenge. Here, by appending a viral exoribonuclease-resistant RNA motif (xrRNA) to the 3'-extended portion of pegRNAs for their increased resistance against degradation, we develop an upgraded PE platform (xrPE) with substantially enhanced editing efficiencies in multiple cell lines. A pan-target average enhancement of up to 3.1-, 4.5- and 2.5-fold in given cell types is observed for base conversions, small deletions, and small insertions, respectively. Additionally, xrPE exhibits comparable edit:indel ratios and similarly minimal off-target editing as the canonical PE3. Of note, parallel comparison of xrPE to the most recently developed epegRNA-based PE system shows their largely equivalent editing performances. Our study establishes a highly adaptable platform of improved PE that shall have broad implications.

Precise tumor immune rewiring via synthetic CRISPRa circuits gated by concurrent gain/loss of transcription factors.

Reinvigoration of antitumor immunity has recently become the central theme for the development of cancer therapies. Nevertheless, the precise delivery of immunotherapeutic activities to the tumors remains challenging. Here, we explore a synthetic gene circuit-based strategy for specific tumor identification, and for subsequently engaging immune activation. By design, these circuits are assembled from two interactive modules, i.e., an oncogenic TF-driven CRISPRa effector, and a corresponding p53-inducible off-switch (NOT gate), which jointly execute an AND-NOT logic for accurate tumor targeting. In particular, two forms of the NOT gate are developed, via the use of an inhibitory sgRNA or an anti-CRISPR protein, with the second form showing a superior performance in gating CRISPRa by p53 loss. Functionally, the optimized AND-NOT logic circuit can empower a highly specific and effective tumor recognition/immune rewiring axis, leading to therapeutic effects in vivo. Taken together, our work presents an adaptable strategy for the development of precisely delivered immunotherapy.

A New Mechanism for Early-Time Plasmaspheric Refilling: The Role of Charge Exchange Reactions in the Transport of Energy and Mass Throughout the Ring Current-Plasmasphere System.

Cold H+ produced via charge exchange reactions between ring current ions and exospheric neutral hydrogen constitutes an additional source of cold plasma that further contributes to the plasmasphere and affects the plasma dynamics in the Earth's magnetosphere system; however, its production and associated effects on the plasmasphere dynamics have not been fully assessed and quantified. In this study, we perform numerical simulations mimicking an idealized three-phase geomagnetic storm to investigate the role of heavy ion composition in the ring current (O+ vs. N+) and exospheric neutral hydrogen density in the production of cold H+ via charge exchange reactions. It is found that ring current heavy ions produce more than 50% of the total cold H+ via charge exchange reactions, and energetic N+ is more efficient in producing cold H+ via charge exchange reactions than O+. Furthermore, the density structure of the cold H+ is highly dependent on the mass of the parent ion; that is, cold H+ deriving from charge exchange reactions involving energetic O+ with neutral hydrogen, populates the lower L-shells, while cold H+ deriving from charge exchange reactions involving energetic N+ with neutral hydrogen populates the higher L-shells. In addition, the density of cold H+ produced via charge exchange reactions involving N+ can be peak at values up to one order of magnitude larger than the local plasmaspheric density, suggesting that solely considering the supply of cold plasma from the ionosphere to the plasmasphere can lead to a significant underestimation of plasmasphere density.

A Monocyte-Orchestrated IFN-I-to-IL-4 Cytokine Axis Instigates Protumoral Macrophages and Thwarts Poly(I:C) Therapy.

Type I IFNs (IFN-I) are important for tumor immune surveillance and contribute to the therapeutic responses for numerous treatment regimens. Nevertheless, certain protumoral activities by IFN-I have been increasingly recognized. Indeed, our recent work showed that systemic poly(I:C)/IFN treatment can undesirably trigger high arginase (ARG1) expression within the tumor-associated monocyte/macrophage compartment. Using a line of CRISPR-generated Arg1-YFP reporter knock-in mice, we have determined that a subset of tumor-associated macrophages represent the major Arg1-expressing cell type following poly(I:C)/IFN stimulation. More detailed analyses from in vitro and in vivo models demonstrate a surprising IFN-to-IL-4 cytokine axis in transitional monocytes, which can subsequently stimulate IL-4 target genes, including Arg1, in macrophages. Intriguingly, IFN stimulation of transitional monocytes yielded concurrent M2 (YFP+)- and M1 (YFP-)-skewed macrophage subsets, correlated with an inhibitory crosstalk between IFN-I and IL-4. Genetic abrogation of IL-4 signaling in mice diminished poly(I:C)/IFN-induced ARG1 in tumors, leading to enhanced activation of CD8+ T cells and an improved therapeutic effect. The present work uncovered a monocyte-orchestrated macrophage phenotype conversion mechanism that may have broad implications.

Internalization Characterization of Si Nanorod with Camouflaged Cell Membrane Proteins Reveals ATXN2 as a Negative Regulator.

The fabrication of shape-controlled nanocarriers is critical for efficient delivery of biomolecules across the cell membrane. Surface coating of the nanocarrier can improve internalization efficiency. Here, we developed a facile method of silicon nanorod fabrication leading to a controlled size and shape. We then systematically evaluated five surface modifications with membrane proteins from different cancer cell lines including MCF7, MD231, Hela, Panc-PDX, and Panc-1. We demonstrated that silicon nanorods coated with either a homolytic or heterolytic membrane protein coating have significantly improved internalization efficiency as compared with uncoated Si nanorods. To elucidate the molecular mechanism of the improved efficiency associated with a modified coating, we analyzed the coating membrane proteins derived from five cell lines with proteomics and identified 601 proteins shared by different cell sources. These proteins may function as cell-substrate adhesion molecules that contribute to the enhanced internalization. We also tested the internalization efficiency of nanorods with different coatings in each of the five cell lines to determine the influencing factors from target cells. We found that the internalization efficiency varied among different target cells, and the ranking of the average efficiency was as follows: Hela > Panc-PDX > MD231 > MCF7 > Panc-1. The bioinformatics analysis suggested that the low internalization efficiency in Panc-1 cells might be associated with the upregulation of ATXN2, which is a negative regulator of endocytosis. We further demonstrated that ATXN2 knockdown with specific siRNA significantly improved nanorod internalization efficiency in Panc-1 cells suggesting that ATXN2 can be a reference for efficiency prediction of nanoparticle delivery to tumor cells. Thus, we studied the effect of different cancer cell membrane proteins on nanorod uptake efficiencies. These results can improve nanorod internalization to cancer cells, including a fundamental understanding of the internalization efficiency of cancer cells.

Golgi-resident TRIO regulates membrane trafficking during neurite outgrowth.

Neurite outgrowth requires coordinated cytoskeletal rearrangements in the growth cone and directional membrane delivery from the neuronal soma. As an essential Rho guanine nucleotide exchange factor (GEF), TRIO is necessary for cytoskeletal dynamics during neurite outgrowth, but its participation in the membrane delivery is unclear. Using co-localization studies, live-cell imaging, and fluorescence recovery after photobleaching analysis, along with neurite outgrowth assay and various biochemical approaches, we here report that in mouse cerebellar granule neurons, TRIO protein pools at the Golgi and regulates membrane trafficking by controlling the directional maintenance of both RAB8 (member RAS oncogene family 8)- and RAB10-positive membrane vesicles. We found that the spectrin repeats in Golgi-resident TRIO confer RAB8 and RAB10 activation by interacting with and activating the RAB GEF RABIN8. Constitutively active RAB8 or RAB10 could partially restore the neurite outgrowth of TRIO-deficient cerebellar granule neurons, suggesting that TRIO-regulated membrane trafficking has an important functional role in neurite outgrowth. Our results also suggest cross-talk between Rho GEF and Rab GEF in controlling both cytoskeletal dynamics and membrane trafficking during neuronal development. They further highlight how protein pools localized to specific organelles regulate crucial cellular activities and functions. In conclusion, our findings indicate that TRIO regulates membrane trafficking during neurite outgrowth in coordination with its GEF-dependent function in controlling cytoskeletal dynamics via Rho GTPases.

Concomitant type I IFN and M-CSF signaling reprograms monocyte differentiation and drives pro-tumoral arginase production.

BACKGROUND: Type I IFN-based therapies against solid malignancies have yielded only limited success. How IFN affects tumor-associated macrophage (TAM) compartment to impact the therapeutic outcomes are not well understood. METHODS: The effect of an IFN-inducer poly(I:C) on tumor-infiltrating monocytes and TAMs were analyzed using a transplantable mouse tumor model (LLC). In vitro culture systems were utilized to study the direct actions by poly(I:C)-IFN on differentiating monocytes. RESULTS: We found that poly(I:C)-induced IFN targets Ly6C+ monocytes and impedes their transition into TAMs. Such an effect involves miR-155-mediated suppression of M-CSF receptor expression, contributing to restricting tumor growth. Remarkably, further analyses of gene expression profile of IFN-treated differentiating monocytes reveal a strong induction of Arg1 (encoding arginase-1) in addition to other classical IFN targets. Mechanistically, the unexpected Arg1 arm of IFN action is mediated by a prolonged STAT3 signaling in monocytes, in conjunction with elevated macrophage colony-stimulating factor (M-CSF) signaling. Functionally, induction of ARG1 limited the therapeutic effect of IFN, as inhibition of arginase activity could strongly synergize with poly(I:C) to enhance CD8+ T cell responses to thwart tumor growth in mice. CONCLUSIONS: Taken together, we have uncovered two functionally opposing actions by IFN on the TAM compartment. Our work provides significant new insights on IFN-mediated immunoregulation that may have implications in cancer therapies.

PFKFB3-Driven Macrophage Glycolytic Metabolism Is a Crucial Component of Innate Antiviral Defense.

Signaling by viral nucleic acids and subsequently by type I IFN is central to antiviral innate immunity. These signaling events are also likely to engage metabolic changes in immune and nonimmune cells to support antiviral defense. In this study, we show that cytosolic viral recognition, by way of secondary IFN signaling, leads to upregulation of glycolysis preferentially in macrophages. This metabolic switch involves induction of glycolytic activator 6-phosphofructose-2-kinase and fructose-2,6-bisphosphatase (PFKFB3). Using a genetic inactivation approach together with pharmacological perturbations in mouse cells, we show that PFKFB3-driven glycolysis selectively promotes the extrinsic antiviral capacity of macrophages, via metabolically supporting the engulfment and removal of virus-infected cells. Furthermore, the antiviral function of PFKFB3, as well as some contribution of its action from the hematopoietic compartment, was confirmed in a mouse model of respiratory syncytial virus infection. Therefore, different from the long-standing perception of glycolysis as a proviral pathway, our findings establish an antiviral, immunometabolic aspect of glycolysis that may have therapeutic implications.

Targeted Gene Delivery to Macrophages by Biodegradable Star-Shaped Polymers.

In this report, two biodegradable star-shaped polyasparamide derivatives and four analogues modified with either mannose or folic acid moiety for preferential targeting of a difficult-to-transfect immune cell type, i.e., macrophage, have been synthesized. Each of the prepared star polymers complexes with plasmid DNA to form nanosized particles featuring a core-shell-like morphology. Mannose or folate functionalized star polymers can greatly improve the transfection performance on a macrophage cell line RAW 264.7. As a result, a combination of targeting ligand modification and topological structures of gene carriers is a promising strategy for immune cells-based gene therapy.

Rapamycin-sensitive mTORC1 signaling is involved in physiological primordial follicle activation in mouse ovary.

In mammals, resting female oocytes reside in primordial ovarian follicles. An individual primordial follicle may stay quiescent for a protracted period of time before initiating follicular growth, which is also termed "activation." Female reproductive capacity is sustained by the gradual, streamlined activation of the entire population of primordial follicles, but this process also results in reproductive senescence in older animals. Based on the recent findings that genetically triggered, excessive mammalian target of rapamycin complex 1 (mTORC1) activation in mouse oocytes leads to accelerated primordial follicle activation, we examined the necessity of mTORC1 signaling in physiological primordial follicle activation. We found that induction of oocyte mTORC1 activity is associated with early follicular growth in neonatal mouse ovaries. Pharmacological inhibition of mTORC1 activity in vivo by rapamycin treatment leads to a marked, but partial, suppression of primordial follicle activation. The suppressive effect of rapamycin on primordial follicle activation was reproduced in cultured ovaries. While rapamycin did not apparently affect several plausible cellular targets in neonatal mouse ovaries, such as mTORC2, AKT, or cyclin-dependent kinase (CDK) inhibitor p27-KIP1, its inhibitory effect on Cyclin A2 gene expression implies that mTORC1 signaling in oocytes may engage a Cyclin A/CDK regulatory network that promotes primordial follicle activation. The current work strengthens the concept that mTORC1-dependent events in the oocytes of primordial follicles may represent potential targets for intervention in humans to slow the depletion of the ovarian reserve.

Regulation of interleukin-10 receptor ubiquitination and stability by beta-TrCP-containing ubiquitin E3 ligase.

Interleukin-10 (IL-10) initiates potent anti-inflammatory effects via activating its cell surface receptor, composed of IL-10R1 and IL-10R2 subunits. The level of IL-10R1 is a major determinant of the cells' responsiveness to IL-10. Here, via a series of biochemical analyses using 293T cells reconstituted with IL-10R1, we identify the latter as a novel substrate of ßTrCP-containing ubiquitin E3 ligase. Within the intracellular tail of IL-10R1, a canonical ((318)DpSGFGpS) and a slightly deviated ((369)DpSGICLQEP) ßTrCP recognition motif can additively recruit ßTrCP in a phosphorylation-dependent manner. ßTrCP recruitment leads to ubiquitination, endocytosis and degradation of IL-10R1, subsequently reducing the cellular responsiveness to IL-10. Our study uncovers a novel negative regulatory mechanism that may potentially affect IL-10 function in target cells under physiological or pathological conditions.

Pathogen recognition receptor signaling accelerates phosphorylation-dependent degradation of IFNAR1.

An ability to sense pathogens by a number of specialized cell types including the dendritic cells plays a central role in host's defenses. Activation of these cells through the stimulation of the pathogen-recognition receptors induces the production of a number of cytokines including Type I interferons (IFNs) that mediate the diverse mechanisms of innate immunity. Type I IFNs interact with the Type I IFN receptor, composed of IFNAR1 and IFNAR2 chains, to mount the host defense responses. However, at the same time, Type I IFNs elicit potent anti-proliferative and pro-apoptotic effects that could be detrimental for IFN-producing cells. Here, we report that the activation of p38 kinase in response to pathogen-recognition receptors stimulation results in a series of phosphorylation events within the IFNAR1 chain of the Type I IFN receptor. This phosphorylation promotes IFNAR1 ubiquitination and accelerates the proteolytic turnover of this receptor leading to an attenuation of Type I IFN signaling and the protection of activated dendritic cells from the cytotoxic effects of autocrine or paracrine Type I IFN. In this paper we discuss a potential role of this mechanism in regulating the processes of innate immunity.

Inducible priming phosphorylation promotes ligand-independent degradation of the IFNAR1 chain of type I interferon receptor.

Phosphorylation-dependent ubiquitination and ensuing down-regulation and lysosomal degradation of the interferon alpha/beta receptor chain 1 (IFNAR1) of the receptor for Type I interferons play important roles in limiting the cellular responses to these cytokines. These events could be stimulated either by the ligands (in a Janus kinase-dependent manner) or by unfolded protein response (UPR) inducers including viral infection (in a manner dependent on the activity of pancreatic endoplasmic reticulum kinase). Both ligand-dependent and -independent pathways converge on phosphorylation of Ser(535) within the IFNAR1 degron leading to recruitment of beta-Trcp E3 ubiquitin ligase and concomitant ubiquitination and degradation. Casein kinase 1 alpha (CK1 alpha) was shown to directly phosphorylate Ser(535) within the ligand-independent pathway. Yet given the constitutive activity of CK1 alpha, it remained unclear how this pathway is stimulated by UPR. Here we report that induction of UPR promotes the phosphorylation of a proximal residue, Ser(532), in a pancreatic endoplasmic reticulum kinase-dependent manner. This serine serves as a priming site that promotes subsequent phosphorylation of IFNAR1 within its degron by CK1 alpha. These events play an important role in regulating ubiquitination and degradation of IFNAR1 as well as the extent of Type I interferon signaling.