Currently, biological membrane-derived nanoparticles (NPs) have shown enormous potential as drug delivery vehicles due to their outstanding biomimetic properties. To make these NPs more adaptive to complex biological systems, some methods have been developed to modify biomembranes and endow them with more functions while preserving their inherent natures. In this review, we introduce five common approaches used for biomembrane decoration: membrane hybridization, the postinsertion method, chemical methods, metabolism engineering and gene engineering. These methods can functionalize a series of biomembranes derived from red blood cells, white blood cells, tumor cells, platelets, exosomes and so on. Biomembrane engineering could markedly facilitate the targeted drug delivery, treatment and diagnosis of cancer, inflammation, immunological diseases, bone diseases and Alzheimer's disease. It is anticipated that these membrane modification techniques will advance biomembrane-derived NPs into broader applications in the future. Nanoparticles (NPs), made of nanoscale and biocompatible materials, can be utilized for targeted drug delivery and theranostic purposes To compensate for the deficiencies of artificially synthesized NPs, biomimetic NPs are becoming a research focus. In 2011, Zhang's team first synthesized red blood cell (RBC) membrane-camouflaged NPs However, with the development of membrane-derived NPs, it was found that coating with a single membrane leads to certain limitations in the function of the NPs. For example, RBC membrane-coated NPs can reduce macrophage uptake, leading to a prolonged circulation time. However, their low targeting ability limits their therapeutic effects The frequently used methods to fabricate biomembrane-derived NPs include the following three steps: membrane extraction, the preparation of NP cores and the coating of the NP cores with the membrane-derived vesicles Figure C 31. Unlvia mechanical forces such as sonication and homogenization. Then, the cell membranes are isolated from the mixture containing nuclei and organelles via high-speed differential centrifugation The approaches to separating and extracting biomembranes depend on whether the cell has a nucleus. Nucleus-free cells, such as RBCs, can be lysed with hypotonic solution or repeated freeze‒thaw processes. Then, the membrane debris is isolated from the cell contents by centrifugation. Regarding nucleated cells, such as cancer cells and macrophage cells, the membrane isolation and extraction process is more complicated. In addition to hypotonic treatment, the cell structures need to be further destroyed In addition, exosomes are usually obtained as complete vesicles by ultracentrifuging the supernatants of the cell culture medium in situ packaging methods. Physical extrusion relies on a mechanical force to destroy the membrane structure, thus enabling it to coat the core of NPs Biomembrane-derived NPs can be fabricated by different methods, such as coextrusion, sonication, microfluidic electroporation or sonication, and in situ. In this fabrication approach, when cells are incubated with gold NPs, iron-oxide NPs, or quantum dots, they can release vesicles containing these exogenous NPs In addition to utilizing purified membrane materials, there appears to be a unique technique that utilizes live cells to package core-shell NPs Membrane hybridization refers to hybridizing two different types of biological membranes or biological membranes and liposomes into a mixed membrane Figure B. Since Two kinds of membranes can be easily hybridized by fusion In addition to the hybridization of biomembranes and biomembranes, liposomes can also be fused with biomembranes. For example, the freeze-thaw method was used to fuse exosome membranes and liposomes, which can modulate the interplay between engineered exosomes and cells by altering the lipid components or the characteristics of exogenous lipids The postinsertion method is a nondestructive method for the physical insertion of functional ligands into biomembranes, which avoids damaging the membrane structure Figure B. This mTo insert the functional moieties, ligand-linker-hydrophobic anchor conjugates must first be synthesized. Hydrophobic tethered ligands are spontaneously embedded in the extracted biological membranes Chemical methods are convenient for modifying biological membranes with functional ligands Figure B. With dChemical methods to engineer biological membranes have been developed recently. However, the application of chemical methods is limited because their harsh reaction conditions may destroy cell viability or membrane structure; therefore, more detailed characterization and method improvement are needed Metabolic engineering is a flexible and versatile method that utilizes natural biosynthetic pathways to express ligands on biomembranes without destroying the membrane structure Figure B. This aAfter the membranes are extracted, bioorthogonal chemistry occurs in the next step to connect moieties. The reaction groups preintroduced onto the cell membranes can specifically and rapidly interact with the reaction groups on the functional ligands under mild physiological conditions. For example, azide groups can be modified onto cell membranes through glucose metabolism. By virtue of the biological orthogonal reaction of azide groups and bicyclo[6.1.0]nonyne (BCN), functional ligands containing BCN groups can be easily connected to cell membranes Gene engineering uses biological tools to selectively modulate cell genes to modify the needed proteins or antibodies on the cell membranes Figure B. Then, Currently, there are many developed methods to carry genes into cells. Adenoviruses and retroviruses are the most common vehicles, but they possess undesirable disadvantages, such as viral toxicity and host immune rejection. Physical methods include gene gun bombardment Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) is becoming a dominant gene engineering tool in multiple organisms. Cas9 is an RNA-guided DNA endonuclease that is easily designed to target different sites by changing its guide RNA sequence. When the guide RNA recognizes a specific sequence in the genome, the Cas9 protein subsequently cuts the DNA sequence. During the process of DNA repair, an insertion or site-directed mutation can be introduced. Therefore, it is easy for CRISPR/Cas9 combined with gene delivery systems to delete, integrate and replace genes and thus enable the cells to express the needed proteins on their membrane surface 2Red blood cells (RBCs) are the most abundant cells in the human body and are responsible for the transport of oxygen and COvia the postinsertion of SHp-PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (SHp-PEG-DSPE). The resultant SHp-modified RBC membrane-coated NPs could markedly prolong the systemic circulation of the encapsulated neuroprotective agent NR2B9C and improve its active targeting toward the ischemic area in rats with middle cerebral artery occlusion, thereby decreasing ischemic brain lesions Although RBC membrane-coated biomimetic NPs possess a long circulation ability, they lack sufficient targeting activity. Folic acid (FA) was first used to functionalize RBC membranes by postinserting FA-PEG-lipid conjugates into the membranes in vitro BBB and BBTB barriers . The T7 peptide possesses a strong affinity for transferrin receptors (TfRs), which are highly expressed on the surface of the BBB. The NGR peptide can selectively bind to CD13, which is overexpressed during angiogenesis. As such, RBC membrane-camouflaged solid lipid NPs (RBCSLNs) decorated with T7 and NGR peptide (T7/NGR-RBCSLNs) were designed Figure A. With ts Figure B and 2C DCDX (DCDX-RBCNPs) could also traverse the BBB and accumulate at the tumor site, as DCDX exhibits strong affinity for nicotinic acetylcholine receptors expressed on the brain endothelium surface. The strong binding affinity of streptavidin to biotin was leveraged to construct DCDX-RBCNPs. Briefly, after streptavidin was preimbedded into the RBC membrane surface, biotinylated DCDX was efficiently incorporated into the NP surface. This modification approach avoids positively charged peptide interactions with negatively charged RBC membranes. With enhanced brain targeting efficiency, DCDX-RBCNPs loaded with doxorubicin exerted excellent therapeutic efficacy in glioma-bearing mice Similarly, RBC membrane-coated NPs modified with White blood cells (WBCs) are immune cells that are responsible for protecting the body from infection, engulfing foreign bacteria and repairing tissue injury in vivo tumor cells were modified with BCN groups via intratumoral administration of AC4mann-BCN, whose metabolism would attach BCN to the membrane surface. Then, BCN can serve as an excellent targeting tag to chemically react with azide groups. Compared to unmodified T-cell membrane-coated NPs, the azide-modified T-cell membrane-coated NPs showed 1.5-fold higher tumor accumulation owing to specific binding to artificial BCN receptors on tumor cells Owing to the specific immune recognition proteins expressed on the T-cell surface , activated T cells are able to recognize associated molecules on tumor cells, presenting inherent and strong tumor affinity. However, the natural tumor targeting of T cells is compromised by the intra- and interheterogeneity of tumors via the postinsertion method  can trigger the clustering of intercellular adhesion molecule 1 (ICAM-1) on the inflamed endothelium, locally improve vascular permeability, and extravasate across the inflamed endothelial layer. Because of their inherited source cell function, neutrophil membrane-coated NPs readily accumulate in the tumor microenvironment  in vitro is time-consuming and shows poor reproducibility in vitro and visibly directed reinfused CTLs to the tumor site via magnetic resonance imaging and magnetic control. Ultimately, tumor growth in EG7 tumor-bearing mice was efficiently inhibited, indicating the great potential of this “one-but-all” aAPC platform for T-cell-based antitumor immunotherapy.Mature antigen presenting cells (APCs) can handle certain antigens on tumor cell surfaces and activate cytotoxic T lymphocyte (CTL)-mediated anticancer immune responses. However, using natural APCs to expand and stimulate T cellsSince cancer cell membranes express both “self-markers” and “self-recognition” molecules, they can endow encapsulated NPs with prolonged systemic circulation and homotypic targeting capability in vivoIn addition to inherent homotypic targeting, the cancer cell membranes could be decorated with ligands to further augment their affinity for target cells. Inflamed endothelial cells highly express vascular cell adhesion molecule-1 (VCAM-1), which can recruit immune cells expressing its cognate ligand, very late antigen-4 (VLA-4). VLA-4 is a heterodimer that consists of integrin α4 and integrin β1. Wild-type C1498 leukemia cells highly express integrin β1 but lack integrin α4. Following viral transduction of C1498 cells with the integrin α4 gene, the resultant engineered cells expressed both VLA-4 components. The membranes from the genetically engineered cells were wrapped on the polymeric NP cores, and the resultant biomimetic NPs improved the delivery of the loaded anti-inflammatory drug dexamethasone to inflamed lung tissue and exerted important treatment efficacy in vitro to a level comparable to that achieved by lipopolysaccharides  (NP-R@M-M), which can specifically bind to its receptors on DCs Figure A and 4B 4ManNAz. Then, the endo-BCN cRGD peptide was linked to azide groups on the membrane surface through the click reaction. The cRGD-decorated cell membrane was coated onto the nanocomposite core, comprising conjugated polymer (CP) and ultrasmall iron oxide (IO) nanoparticles (cRGD-CM-CPIO). With cRGD-mediated BBB crossing capabilities, cRGD-CM-CPIO exhibited significantly higher tumor accumulation than unmodified membrane-coated NPs (CM-CPIO) and was validated as an efficient imaging contrast agent In addition, cancer cell membranes can also be functionalized with additional ligands to facilitate their crossing of physiological barriers. For example, the cancer cell membrane could be modified with the cRGD peptide, which targets the integrin αvβ3 receptors highly expressed in the neovasculature, through metabolic engineering and a subsequent click reaction. Azide groups were introduced onto the glioma cell membrane by pretreating the cells with Acazo@CMSN). DAazo@CMSN could home to the bone marrow TI-niche through the interaction of C-X-C motif chemokine receptor 4  and stromal cell-derived factor-1 . Subsequently, aTGFβRII is detached from the NP surface via the hypoxic bone marrow microenvironment-mediated cleavage of the azobenzene linker and then inhibited GDF15-stimulated TGF-β signaling to block chemoresistance. Then, D@MSN could be taken up by ALL cells through homotypic targeting to enhance the chemotherapeutic efficacy of daunorubicin In addition to improving the targeting ability, cancer cell membranes could also be decorated with a specific antibody to inhibit niche-mediated chemoresistance. Acute lymphoblastic leukemia (ALL) cells secrete pro-growth differentiation factor-15 (pro-GDF15) during chemotherapy, which can be cleaved by therapy-induced niche (TI-niche) cell-provided furin and subsequently activate TGF-β signaling to promote chemoresistance. The mesoporous silica NPs encapsulating daunorubicin (D@MSN) were cloaked with NALM-6 ALL cell membrane vesicles, which were preloaded with TGFβRII neutralizing antibody (aTGFβRII) through the postinsertion of its lipid conjugates incorporating a hypoxia-responsive azobenzene linker (DAPlatelets (PLTs) show a circulation half-life of approximately 30 h because they also express the “self-marker” of CD47 As PLT membrane-camouflaged NPs can target CTCs through membrane surface adhesion molecules, additional ligands can be introduced onto the membrane to kill CTCs. Biocompatible silica NPs were cloaked with the activated PLT membranes, followed by the conjugation of TRAIL onto the membrane surface by leveraging the affinity between streptavidin and biotin. The resultant biomimetic NPs could adhere to the CTC-induced thrombosis in the vasculature to deliver high-concentration cancer-killing TRAIL, thereby producing targeted therapeutic effects Activated PLT membranes can also be engineered with additional ligands to strengthen their targeting efficiency to primary and metastatic tumors. For example, the recombinant VAR2CSA (rVAR2) peptide exhibited strong binding to oncofetal chondroitin sulfate (ofCS), which is selectively expressed on tumor cells. The rVAR2 peptide was introduced onto the activated PLT membranes through the postinsertion of DSPE-PEG-rVAR2, which were then coated on disulfide-containing PLGA-ss-hyaluronan (HA) NPs loaded with docetaxel (rVAR2-PM/PLGA-ss-HA) Figure A and 5B Exosomes are extracellular vesicles released by all cells and range in size from 40 to 100 nm However, there still exist many limitations for exosomes as drug delivery tools. For example, despite possessing specific lipid and protein components, exosomes undergo rapid elimination from the blood circulation after intravenous injection via electroporation. The exosome membranes were further modified with an alkyne group for subsequent binding of NRP-1-targeted peptide  with azido groups. The resulting exosomes engineered via a chemical method could effectively traverse the BBB and accumulate in the glioma for enhanced imaging and treatment efficacy. Moreover, SPION-induced magnetic flow hyperthermia and curcumin-induced tumor growth inhibition also exerted strong synergistic antitumor efficacy Certain types of exosomes with a strong capability of crossing the BBB have been extensively studied for brain-targeted drug delivery but lack reliable targeting ability to brain tumors. Neuropilin-1 (NRP-1) is selectively expressed in glioma cells and the tumor endothelium and serves as an ideal target of glioma. To construct brain-targeted exosomes, superparamagnetic iron oxide NPs (SPION) and curcumin were encapsulated in the exosomes via a heterobifunctional linker, which readily linked with azide-containing c(RGDyK) peptide via copper-free click chemistry . By exploiting the high affinity of the c(RGDyK) peptide for integrin αvβ3 on the reactive cerebral endothelium, the engineered exosome cRGD-Exo-cur displayed significantly higher accumulation in the lesion region than unmodified exosomes, especially with the fluorescence ratio of the ipsilateral to the contralateral region of 11 -specific nanobodies could be combined with phospholipid (DMPE)-PEG derivatives to prepare nanobody-PEG-micelles. After incubation with exosomes from Neuro2A cells or PLTs, nanobody-PEG-micelles transferred to exosome membranes in a temperature-dependent manner. After the introduction of EGFR-specific nanobodies, the exosomes showed significantly increased binding ability to EGFR-overexpressing tumor cells. Furthermore, compared with the unmodified exosomes that were eliminated from the blood circulation of the mice within 10 min, exosomes decorated with nanobody-PEG-micelles were still detectable in plasma after 1 h. Therefore, the insertion of ligand-conjugated PEGylated phospholipids could endow exosomes with prolonged blood retention and enhanced cell specificity, effectively promoting exosome enrichment in targeted tissues for improved cargo delivery Monotypic cell membranes hardly meet the complex needs of drug delivery for specific diseases. In contrast, the hybridization of various kinds of cell membranes that inherit the specific properties from the source cells can endow the NPs with a variety of biofunctions, thereby demonstrating superior efficacy and safety As mentioned above, the RBC membranes from natural long-circulating vesicles can prolong the blood circulation time of the modified NPs. On this basis, another type of cell membrane can be hybridized with the RBC membrane to add new functions, e.g., homotypic targeting ability. For example, for combination treatment of melanoma, the membranes from RBCs and melanoma cells (B16-F10) were hybridized and wrapped on doxorubicin-loaded hollow copper sulfide NPs (DCuS@[RBC-B16]). RBC membranes markedly prolonged the blood retention of DCuS@[RBC-B16], with 20.2% ID/g remaining at 24 h postinjection, relative to 14.5% ID/g of DCuS@[B16] remaining. Furthermore, DCuS@[RBC-B16] NPs that retained the characteristics of B16-F10 cells exhibited strong specific self-recognition to the B16-F10 cells. Ultimately, owing to enhanced tumor-targeted delivery, DCuS@[RBC-B16] NPs exerted striking synergistic photothermal/chemotherapeutic effects, with an almost 100% growth inhibition rate of melanoma tumors Likewise, membranes originating from RBCs and the MCF-7 cancer cell line were hybridized and coated on melanin NPs (Melanin@RBC-M) for PTT of tumors. Interestingly, a higher MCF-7 membrane ratio led to a stronger homotypic targeting ability of Melanin@RBC-M, while a higher RBC membrane ratio induced longer blood retention. In MCF-7 tumor-burdened nude mice, Melanin@RBC-M with a 1:1 membrane protein weight ratio of RBCs to MCF-7 showed the highest tumor delivery and superior PTT efficacy when compared with other Melanin@RBC-M NPs with different ratios of RBCs to MCF-7 or pristine melanin NPs. This occurred because of the optimal balance between prolonged blood retention and homotypic targeting Based on the same principle, RBC membranes were mixed with retinal endotheliocyte (REC) membranes for coating onto PLGA NPs ([RBC-REC]NPs) PLTs can bind to exposed collagen in injured blood vessels, which enables the PLT membrane to actively target damaged vasculature In addition to RBC membranes, leukocyte membrane camouflage can also be utilized to escape immune clearance for prolonged blood retention and can be mixed with tumor cell membranes for additional homotypic targeting. For example, the membranes from macrophages (murine J774A.1 cells) and tumor cells (head and neck tumor HN12 cells) were fused with exogenous phospholipids, yielding leutusome in vitro, FMs induced stronger T-cell activation and DC maturation than cancer cell membranes or DC membranes  on their surface s Figure B and 7C.Certainly, all the mentioned membrane engineering technologies are not mutually exclusive and thus can be used together to obtain multiple advantages. For example, membranes derived from PLTs and WBCs were hybridized to be wrapped onto magnetic beads, and then their surface was decorated with antiepithelial cell adhesion molecules (anti-EpCAMs) that could recognize EpCAM-positive CTCs Although membrane engineering technology has shown promising outcomes in various fields, this technology is still in its initial stage and confined to laboratory research. Numerous challenges prevent its industrial process scale-up or clinical translation.via the postinsertion method maintain the activity of membrane proteins, but the binding is less firm than when ligands are introduced through covalent reactions Each modification method has its inherent advantages and limits Table . For exaThe cell membranes are a part of living entities. Therefore, during the modification process, the reaction conditions, such as the reaction temperature, the special reagents used, or the substrate concentration, may damage the activity of the cell membranes. However, there is a lack of necessary criteria or a suitable judgment basis to choose the reaction conditions. Furthermore, the characterization methods to verify successful membrane modification provide only very limited information. For example, particle size measurement and morphology observation make it difficult to recognize modified small molecules. Western blot analysis provides information only on the distinction of protein components between the engineered and the source cell membrane. However, it cannot evaluate whether the cell membrane activity is retained after subjection to the engineering process. Therefore, there is a great need to develop more sophisticated methods to improve the visualization of membrane engineering processes.In addition, other existing challenges also prevent industrial process scale-up or clinical application. First, the extraction technologies should be optimized to acquire enough biomembranes, and tailored procedures are required to enhance the membrane purity from the nucleated cells. Second, strict quality control is required in every step involved in the preparation process of engineered biomembrane-derived NPs. For example, some conventional formulation parameters, such as drug encapsulation efficiency, drug content and drug release behavior, should be monitored. Furthermore, for the hybrid membrane-coated NPs, many points are worth considering, such as each membrane ratio, fusion efficiency and fused membrane stability. For NPs prepared from genetically engineered membranes, the expression level and practical effects of the exogenous components are significant in determining the final performance of NPs in disease therapy. As such, the identification of the moiety-presenting profile and level and evaluation system should be established in detail. Furthermore, the long-term storage stability of the biomimetic NPs should be fully studied and is worth further improvement.Modified biological membrane-derived NPs are becoming a research hotspot to resolve the limitations of single membrane-derived NPs. A variety of membrane modification methods, including membrane hybridization, the postinsertion method, chemical methods, metabolic engineering and gene engineering, endow biomimetic NPs with additional functions to meet the multiple requirements of drug delivery and disease therapy and diagnosis. Currently, numerous modified biological membrane-derived NPs have been developed and have shown encouraging outcomes in preclinical research.et al. synthesized gadolinium-Schiff base-doped quantum dot (GdQD)-based probes for the fast, facile, spatial detection of membrane injuries et al. used sucrose at 8.5 wt% as a lyoprotectant and improved the cryopreservation efficiency of exosomes, enabling the preservation of their physicochemical properties and functionality for a long time However, membrane engineering technologies are still in their infancy. To realize scale-up production and clinical translation, there is still a long way to go. For example, necessary criteria to select the reaction conditions and sophisticated characterization methods to improve the visualization of the membrane engineering process should be established. Recently, Amitava Moulick Despite much room for improvement, it cannot be overlooked that membrane engineering techniques can effectively functionalize NPs, thus enhancing their therapeutic or diagnostic effects. Overall, we strongly believe that these emerging biomembrane engineering approaches will acquire a wider application in solving pressing medical problems in the foreseeable future.


Nature, Hong et al. revealed that the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) innate immune pathway is critically required for the IL-6-dependent survival of chromosomally unstable cancer cells,1 implying that chronic inflammation can be therapeutically targeted to kill cancers displaying chromosomal instability (CIN).In a recent study published in 2 It is therefore of pivotal importance to identify novel strategies to treat cancers with CIN.CIN, usually defined as a persistently elevated rate of gain or loss of whole chromosomes during mitotic cell division, can also cause structural alterations of chromosome fragments. Representing a hallmark of cancer, CIN contributes to tumorigenesis through generating intratumoral genomic heterogeneity that drives cancer evolution and causes poor patient prognosis.CIN often results in the formation of micronuclei containing whole chromosomes or fragments of chromosomes which are not incorporated into the daughter nuclei. Micronuclei are susceptible to nuclear envelope collapse, which exposes chromosomal double-stranded DNA (dsDNA) to the cytoplasmic environment, initiating a cGAS-STING-dependent inflammatory immune response.5 whose activation normally facilitates proinflammatory signaling controlled by type I interferon (IFN), which is expected to promote apoptosis and immunosurveillance. Strikingly, Hong et al. found that IL-6-STAT3 signaling axis downstream of cGAS-STING instead enables the survival of cancer cells with CIN.1cGAS-STING was originally discovered as a sensor of viral DNA,The authors employed two well-established strategies to induce acute and chronical CIN phenotypes. Acute CIN was induced by treating cells with small-molecule inhibitors of the cell cycle kinases Mps1 and Wee1, which cause a strong increase in chromosome missegregation and the formation of micronuclei. Chronic CIN was induced in cells and mice by overexpressing a dominant-negative mutant of the microtubule depolymerase MCAK that causes chromosome missegregation.It was first showed in human BT549 triple-negative breast cancer (TNBC) cells and 4T1 mouse TNBC cells that acute CIN induction effectively activated the cGAS-STING signaling, resulting in the increased phosphorylation of STAT1, STAT3, and IRF3. Interestingly, CRISPR-Cas9-mediated knockout of cGAS or STING, or treatment with a small-molecule inhibitor of cGAS, strongly reduced the viability of cells with acute CIN, indicating that cGAS-STING signaling enables the tolerance of TNBC cells to acutely induced CIN. Moreover, knockout of cGAS or STING reduced cell proliferation following chronic induction of CIN, and decreased tumor growth in allografted mice with chronic CIN. Thus, functional cGAS-STING signaling pathway promotes survival of cancer cells with CIN in vitro and in vivo.Then Hong et al. investigated the key signaling pathways downstream of CIN-activated cGAS. Transcriptomic analysis in TNBC cells showed that cGAS knockout reduced the CIN-triggered increase of IFN-STAT1-STAT3 signaling and TNF-NF-кB signaling. Functional genetic analysis showed that knockout of STAT3, but not STAT1, sensitized cells to acute or chronic CIN induction, indicating the importance of STAT3 activation, but not STAT1 activation, for the survival of chromosomally unstable cells downstream of cGAS.The authors further found that knockout of STAT1, but not STAT3, largely rescued the sensitivity of cGAS knockout cells to acute and chronic CIN induction. Moreover, while overexpressing a constitutively active mutant of STAT1 increased the sensitivity to acute CIN induction, STAT1 knockout eliminated CIN-mediated death of STAT3 knockout cells. Thus, STAT1 activation/phosphorylation promotes the death of cells with CIN.Next, the authors examined the contribution of RELA-mediated canonical and RELB-mediated non-canonical NF-кB signaling to CIN sensitivity. The results showed that knockout of RELB or its activator NIK, but not RELA, sensitized cells to acute CIN induction. Thus, the inflammatory signaling induced by CIN has opposing effect on cell survival, i.e., the viability of cells with CIN requires cGAS-STING-STAT3 and non-canonical NF-кB signaling that antagonizes cell death caused by STAT1 activation.Further transcriptomic analysis showed that IL-6, an inflammatory cytokine required for the activation of STAT3 signaling, was upregulated upon acute CIN induction in a cGAS, STING and RELB-dependent manner. Interestingly, following CIN induction, supplementation with recombinant IL-6, or lentiviral overexpression of IL-6, restored the viability of cGAS knockout and STING knockout cells. The observation that IL-6 supplementation did not restore the viability of STAT3 knockout cells experiencing CIN indicated that IL-6 acts upstream of STAT3. Thus, the survival of TNBC cells with acute CIN relies on a cGAS-STING-mediated IL-6-STAT3 pathway.Strikingly, treatment with tocilizumab, a humanized monoclonal antibody that inhibits IL-6-STAT3 pathway through binding the IL-6 receptor (IL-6R), significantly reduced the viability and proliferation of TNBC cell lines with acute or chronic CIN, but had no effect on cells without CIN induction, or on untransformed breast epithelia cells. Then the authors xenografted TNBC cells with chronic CIN into immunocompromised mice, and found that tocilizumab treatment strongly reduced the growth of tumors with high rate of CIN. Similarly, tocilizumab treatment in syngeneic allografts largely delayed the progression of tumors with chronic CIN. Thus, pharmacological inhibition of IL-6R by tocilizumab shows selective toxicity to TNBC cells undergoing acute or chronic CIN, and selectively blocks the in vivo outgrowth of human and mouse TNBC cells with CIN.Hong et al. further performed correlation analysis of datasets from human primary breast cancers in The Cancer Genome Atlas, and found that tumors with high cGAS expression had high CIN, as well as elevated expression of IL-6/IL-6R. Further analysis showed that, compared to tumors with high overall inflammation, tumors with high CIN and activated IL-6 signaling had reduced patient survival. Thus, cGAS-IL-6-IL-6R signaling is frequently activated in human breast cancers, resulting in poor patient prognosis.Moreover, gene essentiality analysis using the DepMap database showed that high IL-6R level is associated with less dependency on genes involved in the regulation of DNA repair and chromosome segregation. Next, the authors induced chronic or acute CIN in a panel of non-TNBC cell lines of different types with different expression levels of IL-6 and IL-6R. They found that, following treatment with increasing doses of tocilizumab, only cancer cell lines expressing high levels of IL-6 and/or IL-6R were susceptible to tocilizumab-induced cell death. Moreover, in a lung cancer xenograft mouse model, treatment with tocilizumab significantly delayed the growth of tumor with high but not low rates of CIN. In addition, tocilizumab treatment largely reduced the viability of primary cell cultures with high IL-6/IL-6R expression, which were derived from genetically engineered mouse model of CIN-induced acute T-cell lymphoma.Taken together, Hong et al. revealed that the cGAS-STING axis drives IL-6 signaling to enable the survival of cancers with CIN Fig. . They shFuture studies are required to investigate, at the mechanistic level, how cancers with frequent chromosome missegregation cope with cGAS-STING signaling pathway. Clinical trials are also anticipated to repurpose tocilizumab, a clinically approved drug used for the treatment of autoimmune diseases such as arthritis, to improve the prognosis of patients with cancers displaying CIN and activated IL-6-STAT3 signaling. Moreover, since CIN-inducing spindle poisons such as paclitaxel and vincristine are widely used for chemotherapy, it will be of great interest to test whether they can synergize for cancer therapy with tocilizumab.


Near-infrared photoimmunotherapy (NIR-PIT) is a novel molecularly-targeted therapy that induces rapid cancer cell death by systemically administering an antibody-photoabsorber conjugate (APC) that binds to cancer cells and irradiating NIR light that drives photochemical transformations of the APC. APCs are constructed by using a monoclonal antibody targeting a cancer cell surface antigen and conjugating it to IRDye700DX silica-phthalocyanine dye. NIR-PIT can selectively kill cancer cells while leaving normal tissues unaffected. Moreover, NIR-PIT activates anti-cancer immunity through the induction of immunogenic cell death of cancer cells. Currently, NIR-PIT is being applied clinically in head and neck squamous cell carcinoma. In previous preclinical studies, NIR-PIT showed excellent efficacy against urologic cancers including bladder cancer and prostate cancer. The clinical application of NIR-PIT will expand to urologic cancers in the near future.Near-infrared photoimmunotherapy (NIR-PIT) is a novel molecularly-targeted therapy that selectively kills cancer cells by systemically injecting an antibody-photoabsorber conjugate (APC) that binds to cancer cells, followed by the application of NIR light that drives photochemical transformations of the APC. APCs are synthesized by selecting a monoclonal antibody that binds to a receptor on a cancer cell and conjugating it to IRDye700DX silica-phthalocyanine dye. Approximately 24 h after APC administration, NIR light is delivered to the tumor, resulting in nearly-immediate necrotic cell death of cancer cells while causing no harm to normal tissues. In addition, NIR-PIT induces a strong immunologic effect, activating anti-cancer immunity that can be further boosted when combined with either immune checkpoint inhibitors or immune suppressive cell-targeted  NIR-PIT. Currently, a global phase III study of NIR-PIT in recurrent head and neck squamous cell carcinoma is ongoing. The first APC and NIR laser systems were approved for clinical use in September 2020 in Japan. In the near future, the clinical applications of NIR-PIT will expand to other cancers, including urologic cancers. In this review, we provide an overview of NIR-PIT and its possible applications in urologic cancers. TM; Rakuten Medical Inc., San Diego, CA, USA) were approved for clinical use by the Pharmaceuticals and Medical Devices Agency (PMDA) in September 2020. In the US, a clinical trial of EGFR-targeted NIR-PIT in newly diagnosed HNSCCs and SCC of the skin recently got underway. These major advances presage the application of NIR-PIT to other cancers, including urologic cancers. In this review, we present an overview of NIR-PIT and discuss how it could be applied to treat urologic cancers in the clinic based on extensive preclinical data. We also highlight potential target molecules for NIR-PIT in urologic cancers.Near-infrared photoimmunotherapy (NIR-PIT) is a new molecularly-targeted anti-cancer therapy that uses NIR light to induce photochemical reactions within antibody-photoabsorber conjugates (APCs), thereby leading to rapid cell death ,2. To syIn 1983, the term “photoimmunotherapy” was used to imply a targeted photodynamic therapy using antibodies that kill target cells based on reactive oxygen species (ROS)-induced cytotoxicity using antibodies that are conjugated to conventional photosensitizers such as hematoporphyrin . However2 . P. P138]. Although CD44 expression is rare in adenocarcinomas of the prostate, it is highly expressed in all cases of neuroendocrine prostate cancer (NEPC) . Thus, CGlypican-1 (GPC1) is a heparan sulfate proteoglycan that is highly expressed in human prostate cancer cell lines . GPC1-taAlthough prostate cancer is considered an immunologically “cold” tumor, scattered Tregs and other T cells exist in the tumor microenvironment. Targeting such cells could activate the immune system, converting the cold environment to a more active immunologic environment. A preclinical study showed that CD25-targeted NIR-PIT depleted Tregs in murine prostate cancer cells, resulting in reduced bioluminescence in these luciferase-expressing cells in vivo . Thus, CKidney cancer is the ninth most common cancer worldwide. There are approximately 431,288 new cases and 179,368 deaths annually across the world . The mosApproximately 70–75% of RCC patients are localized at initial diagnosis and are cured by surgery . For smaFor locally advanced RCC, neoadjuvant NIR-PIT can be combined with surgery to reduce tumor volume and extension and stimulate anti-cancer host immunity ,36, whicTo date, there have been no preclinical studies of NIR-PIT in renal cell carcinoma mouse models. Since molecular events are distinct among the histologic subtypes, candidate target molecules for NIR-PIT would be explored according to the histologic subtype. Candidate target molecules for NIR-PIT against renal cell carcinoma are described below.EGFR overexpression was observed in 84% of ccRCC, in 68% of papillary RCC, and in 75% of chromophobe RCC . Thus, EPD-L1 is highly expressed in 24% of ccRCC compared with 11% in non-ccRCC . Given tThe presence of tumor-infiltrating Tregs is associated with poor survival in ccRCC ,154. ThuccRCC is a highly vascularized tumor. Tyrosine kinase inhibitors targeting VEGFR, such as sorafenib, sunitinib, axitinib, and cabozantinib, show anti-cancer efficacy against ccRCC. Cancer neovasculature-targeted NIR-PIT, targeting VEGFR-2, for instance, showed anti-cancer effects in xenograft mouse models of gastric cancer  Cancer nTesticular cancer is relatively rare, but it is the most commonly diagnosed malignancy in males aged 20 to 39 years . Germ ceSince germ cell tumors are extremely chemosensitive, chemotherapy is a mainstay of treatment for germ cell tumors, especially in patients with metastatic testicular cancer . Thus, NThe efficacy of NIR-PIT for germ cell tumors has never been studied scientifically. Since seminomas and non-seminomas are molecularly distinct, candidate target molecules for NIR-PIT can be different as well. Podoplanin (PDPN) is a type I transmembrane glycoprotein expressed in various normal cells, such as type I lung alveolar cells, kidney podocytes, and lymphatic endothelial cells . A previA previous study reported that 43% of chemorefractory embryonal carcinomas expressed EGFR . Thus, Ec-KIT is a type III receptor tyrosine kinase that plays a crucial role in hematopoiesis, pigmentation, and spermatogenesis . MoreovePenile cancer is rare cancer that accounts for less than 1% of all malignancies worldwide . HistoloAlthough currently most clinical data has come from patients with HNSCC, NIR-PIT holds great potential as a viable treatment for a range of urologic cancers. NIR-PIT has the advantage of being highly selective, and thus causes no or minimal damage to healthy tissues in the surrounding area. This is of particular importance in urologic cancers where minimal collateral damage equates to better functional status after treatment. Thus, NIR-PIT could achieve organ and function preservation while it eradicates cancer cells. Moreover, NIR-PIT substantially activates the anti-cancer host immunity through the induction of ICD. The combination of NIR-PIT with immune activation therapies such as ICIs is a promising strategy for treating urologic cancers. In the near future, NIR-PIT may create a paradigm shift in the clinical practice for urologic cancers.


The highly infectious novel coronavirus disease (COVID-19) emerged in Wuhan, China in late 2019 and soon became a global pandemic. COVID-19 is escalating medical staff psychological stress and creating an increasingly heavy professional burden. Fear of transmitting the virus to family, community perception of frontline workers as potential disease carriers, extreme workloads and moral dilemmas add additional stressors. In Novi Sad Clinical Centre of Vojvodina (CCV) for the past 2 years there has been a continuous struggle against the COVID-19 crisis. Both senior specialist doctors and newly hired young doctors, some without work experience, were hired immediately after completing their studies.To investigate the mental health of clinical first-line medical staff in COVID-19 pandemic.This is a cross-sectional study involving CCV staff who worked in the first line of patient treatment during the COVID-19 pandemic. They were given a self-administered questionnaire which included information on demographic and socio-economic characteristics and the validated Depression, Anxiety, and Stress Scales (DASS-21) and the Impact of Events Scale–Revised (IES-R) instrument. A total of 190 medical workers were involved.Sixty-two  participants screened positive for anxiety, 38 (20%) for depression, 68  for stress, and 22  for clinical concern of PTSD. The most endangered are young nurses and doctors with less than 6 months of previous work experience.In conclusion, our results suggest frontline medical staff involved in treatment of COVID-19 patients should be closely monitored as a high-risk group for depression and anxiety, and given proper training before deployment.No significant relationships.


The rapid development of genome editing technology has brought major breakthroughs in the fields of life science and medicine. In recent years, the clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing toolbox has been greatly expanded, not only with emerging CRISPR-associated protein (Cas) nucleases, but also novel applications through combination with diverse effectors. Recently, transposon-associated programmable RNA-guided genome editing systems have been uncovered, adding myriads of potential new tools to the genome editing toolbox. CRISPR-based genome editing technology has also revolutionized cardiovascular research. Here we first summarize the advances involving newly identified Cas orthologs, engineered variants and novel genome editing systems, and then discuss the applications of the CRISPR-Cas systems in precise genome editing, such as base editing and prime editing. We also highlight recent progress in cardiovascular research using CRISPR-based genome editing technologies, including the generation of genetically modified in vitro and animal models of cardiovascular diseases (CVD) as well as the applications in treating different types of CVD. Finally, the current limitations and future prospects of genome editing technologies are discussed. Genome editing technology refers to a series of technologies capable of manipulating cellular DNA sequences at desired genomic sites by generating altered DNA sequences through nuclease-mediated site-specific DNA breaks that are resolved through DNA repair pathways –3. Among2 phase during cell division [The exponential development of genome editing technology has dramatically changed the landscape of biological and medical research, heralding a new era of precision medicine based on genome editing . CRISPR-division . Precisedivision –18. DespCardiovascular disease (CVD) is a group of disorders of the heart and blood vessels that has consistently been ranked as the leading threat to human health worldwide. Many gene mutations have been linked to CVD, and the number is still increasing , 20. LosIn this review, we highlight the recent advances in CRISPR-based genome editing technology, mainly in the past three years, and discuss the tremendous innovation this epoch-making technology has brought to the field of cardiovascular research.Natural CRISPR-Cas systems are originally identified as adaptive immune systems in bacteria and archaea, and can be divided into two classes based on their composition and mechanisms. These systems are further divided into six types (I–VI) and dozens of subtypes based on the characteristics and accessory genes flanking the CRISPR array . The mosThe class 1 type I CRISPR system is the most prevalent CRISPR system, in which the multi-subunit CRISPR-associated complex for antiviral defense (Cascade) identifies DNA targets, and the helicase-nuclease enzyme Cas3 degrades DNA  . As a result, the exploitation of miniature Cas proteins with high efficiency is in sustained demand Fig. b. For in effects . In addi effects –37. The  effects . Notably effects . With apVery recently, CRISPR-Cas systems were found to be widely encoded in the genomes of diverse bacteriophages, where they are involved in competition with other viruses , 39. TheGenomic targeting by Cas nucleases requires a PAM sequence near the site where the Cas nuclease binds DNA sequences complementary to the single guide RNA (sgRNA). The requirement of PAM is the gatekeeper for CRISPR-Cas mediated genome targeting, as whether a genomic sequence possesses a PAM for a certain Cas nuclease determines whether the site can be targeted and edited by CRISPR. Efforts have been made to develop Cas nucleases with broader PAM compatibility to pursue true PAM-free nucleases. However, PAM-free nucleases might have potential drawbacks, such as self-targeting of gRNA-expressing DNA constructs and reduced efficiency as more time is required for interrogating the whole genome. Therefore, it is better to develop an arsenal of divergent PAM-dependent Cas nucleases that collectively cover all genomic sequences  Fig. 1cc1c.Streptococcus anginosus, together with the T1227K mutation, into the open reading frame (ORF) of ScCas9 generates ScCas9++ with NNG PAM compatibility [Using phage-assisted non-continuous and continuous evolution strategies, three new SpCas9 variants  were characterized to recognize most NR PAM sequences, together with SpCas9-NG  . To relatibility . A similtibility . The chitibility  and Cas1tibility .Off-target activity is a major challenge when using CRISPR tools for disease-related gene therapy. Over the years, continuous efforts have been made to construct high-fidelity Cas9 variants including eSpCas9(1.1) , SpCas9-DpbCas12e has been validated as a naturally occurring high-fidelity Cas nuclease with striking avoidance of off-target activity . In a rePrecise genome editing is essential for preclinical research and clinical gene therapy, and HDR-mediated gene editing has long been the only option. Efforts have been made to improve HDR efficiency, such as the use of rationally designed single-stranded oligodeoxynucleotide (ssODN) templates instead of double-stranded DNA  or the aBase editors can make precise base substitutions without requiring DSBs or donor DNA templates, and are independent of HDR, providing a promising therapeutic tool for human genetic diseases in which the most relevant variants are single nucleotide mutations , 74. CurThe cytosine base editor (CBE) and adenine base editor (ABE) are widely used base editors that enable the editing of all four types of base transitions  , 17 Figa, b. SinProgrammable C-to-G base editors (CGBEs) that can achieve targeted C-to-G and G-to-C base transversions have recently been developed , 90. CGBThe therapeutic applications of base editors have been hampered by their genome-wide off-target effects. Recent studies have shown that cytidine deaminases used in CBE induce genome-wide off-target editing independently of sgRNA or Cas9 , 94. In Prime editing is a newly developed precise genome editing technology that enables all types of base conversion, small deletions, and insertions, as desired. PEs consist of a prime editor protein and prime editing gRNA (pegRNA). The PE protein is constructed by fusing an engineered Cas9 nickase (H840A) with reverse transcriptase, which can be targeted to the genomic locus by pegRNA . The pegA series of PE systems, namely PE2, PE3b, PE4, and PE5b, have been developed and are most widely used. All these systems share a common PE2 protein with an engineered Moloney murine leukemia virus (M-MLV) reverse transcriptase instead of the wild-type M-MLV reverse transcriptase in PE1 to increase editing efficiency , 100. ThIn addition, a dual pegRNA strategy has been used to improve editing efficiency, which can also achieve programmable insertion, deletion, and replacement of large genomic sequences at specific genomic sites –110. UsiCAST systems, consisting of transposase subunits and CRISPR effectors, facilitate the RNA-guided transposition of mobile genetic elements, making it a promising system for targeted, precise, and efficient insertion of large DNA segments. Most identified CASTs are derived from Tn7-like transposons that retain the core genes of the transposition machinery, but have no genes for target selection , 112. InType I-F and type V-K CAST systems have been successfully reconstituted to achieve the integration of donor DNA into specific bacterial genome sites , 114 Fia. An impThe safe, effective, and tissue-specific delivery of CRISPR-Cas tools in vivo determines whether CRISPR-based gene therapy can be used for this tissue. Therapeutic in vivo delivery systems for CRISPR-Cas have recently been discussed , 119. CRAs a gene therapy delivery system approved by the Food and Drug Administration (FDA), lipid nanoparticles (LNP) have been demonstrated to safely deliver therapeutic small molecules and nucleic acid drugs to hepatocytes and antigen-presenting cells via systemic administration or intramuscular injection. The LNP system was used to deliver gene editing tools in the first clinical trial involving human gene editing in vivo . HoweverPcsk9) can achieve base editing in multiple tissues, reduce serum PCSK9 levels by 78%, and partially restore visual function when designed for retinal editing in a mouse model of blindness [Virus-like particle (VLP) systems, which combine the advantages of viral and non-viral delivery systems, are another promising in vivo gene editing delivery vehicle . VLPs calindness . The mamlindness . Based oCVD, including heart and vascular diseases, are leading causes of morbidity and mortality at different ages , 136. InGenetic studies have identified various pathogenic genetic variants associated with the occurrence of CVD , 152. ReDNMT3A) gene-deleted hiPSC-CMs generated using CRISPR-Cas9 gene editing showed altered contraction kinetics and impaired glucose/lipid metabolism, suggesting an important role of DNA methylation in cardiac diseases [SCN10A gene (encoding NaV1.8) knockout hiPSC-CMs help demonstrate that the voltage-gated sodium channel Nav1.8 contributes to late Na+ current (INaL) formation and displays a harmful proarrhythmogenic function [Human induced pluripotent stem cells (hiPSCs) are promising for modeling human cardiomyopathies in vitro because they can differentiate into cardiomyocytes . Throughdiseases . The homfunction .RRAD) gene mimic the single-cell electrophysiological characteristics of Brugada syndrome, a disorder predisposing the patient to ventricular arrhythmias, indicating that RRAD is possibly a novel susceptibility gene for Brugada syndrome [2+ signaling consequences and drug sensitivities [TNNT2) variants, the endogenous TNNT2 gene was knocked out in hiPSCs using CRISPR-Cas9, and 51 different TNNT2 variants were expressed using lentivirus in differentiated TNNT2 knockout hiPSC-CMs. The results revealed that various TNNT2 variants exhibit different pathogenic mechanisms, greatly expanding the knowledge of which and how TNNT2 variants cause hypertrophic cardiomyopathy (HCM) and DCM [The precise introduction of point mutations into hiPSC-CMs facilitates the determination of the causal relationship between genetic mutations and heart diseases. Striated muscle-enriched protein kinase (SPEG) E1680K homozygous mutant hiPSC-CMs recapitulate the hallmarks of dilated cardiomyopathy (DCM), confirming that SPEG E1680K is a novel DCM-causing mutation . Genome syndrome . CRISPR-tivities . Genome- and DCM .KCNH2 in short QT syndrome patience-specific hiPSC-CMs to elucidate the single-cell phenotype of short QT syndrome [Genome editing has been used to correct mutations to generate optimal isogenic controls for patient-derived iPSC-CMs, enabling the determination of genotype–phenotype relationships more precisely. Genome editing has been performed to correct the missense mutation T618I in the potassium channel gene syndrome . Using isyndrome . Isogenisyndrome . Base edMyh6 R404Q mutation was generated using SpCas9/ssODN-mediated directed genomic DNA editing, and heterozygous mice developed a typical HCM phenotype [Gaa) gene at the c.1826 locus and generate a novel mouse model of infantile-onset Pompe disease (IOPD), which recapitulates HCM and the skeletal muscle weakness of human IOPD [Kcnk3 using SpCas9/ssODN-mediated genome editing, creating a novel rat model of pulmonary arterial hypertension [ccm2 led to a lethal multi-cavernous lesion that histologically mimics the typical human hemorrhagic cerebral cavernous malformation [CRISPR-based germline genome editing tools have revolutionized the generation of genetically modified animal models of CVD. Compared to conventional gene targeting technologies using embryonic stem cells, CRISPR-based gene editing technologies are easier to operate, faster, and applicable to most species. One strategy for generating animal models of CVD is to introduce targeted point mutations, insertions, or deletions using HDR-mediated germline genome editing. A mouse model of HCM with a henotype . A similman IOPD . A CRISPman IOPD . Recentlrtension . Anotherrtension , 160, whormation .Myh6, Sav1, and Tbx20, in postnatal cardiomyocyte-Cas9 transgenic mice caused a similar degree of DNA disruption and subsequent mRNA downregulation, but only Myh6 disruption induced HCM and heart failure, suggesting that the effect of postnatal cardiac genome editing is target-dependent [Mef2d) leads to cardiac hypertrophy in mice, indicating that CRISPR-mediated genome editing can be used to generate CVD mouse models by controlling transcription in the postnatal heart [Alk1 gene and AAV1-VEGF successfully induced mutations in Alk1 in brain ECs and generated brain arteriovenous malformations in adult mice [Ctnnb1) gene resulted in a mutation of 36.1% of the Ctnnb1 alleles and dramatically decreased levels of CTNNB1 in brain ECs, leading to BBB breakdown in EC-restricted Tie2Cas9 mice [Cdh5 promoter resulted in efficient genome editing in the ECs of the peripheral vasculature in adult mice, which provides a powerful tool to construct animal models of peripheral vascular diseases [Compared with germline genome editing, somatic genome editing is a more flexible method for obtaining CVD models, which overcomes the challenges of germline modification, such as embryonic lethality and the cost and time required to establish, reproduce, and maintain these models. It is also suitable for rapid and relatively high-throughput studies on the functions of CVD-related genes. As early as 2016, a cardiomyocyte-specific SpCas9 transgenic mouse model was successfully generated to achieve somatic editing in the heart . Followiependent . Mouse mependent . CRISPR-al heart . Severalult mice . We receas9 mice . The AAVdiseases .Therapeutic genome editing can be used to treat monogenic CVD, and the technology could permanently correct mutations and eventually eradicate specific CVD. Programmed edits were introduced into the human germline genome –169. HowDMD) [DMD gene, most of which are located in the regions crossing exons 43 to 53 and disrupt the ORF, resulting in non-functional truncated proteins.DMD is an X-linked disorder characterized by proximal muscle weakness and cardiomyopathy caused by mutations in the largest human gene, dystrophin (DMD) . A varieDMD gene. The systemic application of AAV9 carrying an intein-split SpCas9 and a pair of sgRNAs targeting sequences flanking exon 51 in a pig model of DMD lacking exon 52 induced dystrophin expression in the heart and reduced arrhythmogenic vulnerability [A single-cut genome editing strategy was applied in both iPSC-CMs and mouse models of DMD bearing an exon 44 deletion mutation (Δ44), one of the most common causative mutations of DMD. The ORF can be restored by disrupting the exon splice site to skip the adjacent exon, inserting one nucleotide, or deleting two nucleotides in exon 44 . Systemirability . The lonrability , 150. AArability , 150.Dmd exon 51 deletion mutation in iPSC-CMs, and intramuscular delivery of AAV9 encoding ABE components amends the mutation in ∆E51 DMD mice [DmdE4* mice, which harbor a 4 bp deletion in exon 4 of the Dmd gene and recapitulate many characteristics of human DMD [Dmd gene and restored up to 90% of dystrophin proteins in the heart of DmdE4* mice, resulting in improved cardiac function and an increased life span [mdx4cv mouse model carrying a premature stop codon (CAA-to-TAA) in exon 53 of the Dmd gene. After 10 months of treatment, a near-complete rescue of dystrophin was found in the hearts of mdx4cv mice without obvious toxicity [Base editing and prime editing show great promise for treating DMD. Both ABE and PE can restore dystrophin protein expression by inducing exon skipping or exon reframing to correct the DMD mice . CBE hasuman DMD . A singlife span . Alternatoxicity .Myh6R404Q/+ embryos corrects the mutant allele at a rate of 62.5% to 70.8%, abolishing the HCM phenotype in postnatal mice and their progeny. Moreover, in utero delivery of intein-split ABEmax-NG induced a high correction rate without introducing indels or off-target editing in Myh6R404Q/+ fetuses [LZTR1) gene, indicating new possibilities for personalized therapeutic genome editing in HCM patients [MYBPC3, which causes familial HCM, in human embryos [MYBPC3 gene without mosaicism or off-target mutations [Inherited cardiomyopathies, including HCM and DCM, are candidate genetic disorders that are suitable for genome editing-related treatment. ABEmax-NG has been shown to correct a pathogenic R404Q/+ mutation in embryos of the HCM mouse model . Adminis fetuses . Introni fetuses . CRISPR-patients . Notably embryos . Co-injeutations .TTN mutations were corrected using SpCas9/ssODN. Engineered heart muscle generated from corrected hiPSC-CMs shows normalized titin protein levels and contractile function [TTNtv-specific sgRNA was also shown to restore the reading frame of TTN protein in hiPSC-CMs, leading to increased full-length TTN protein levels and normalized sarcomere function [RBM20 has been reported [R634Q and RBM20R636S mutant iPSCs were corrected by ABE and PE, with the efficiency of 92% and 40%, respectively. In addition, AAV9-mediated systemic delivery of ABE components corrected 66% of the RBM20 transcripts expressed in cardiomyocytes of postnatal RBMR636Q/R636Q mice. The corrected mice showed restored cardiac size and function, and prolonged life span [Genome editing has also been used to correct DCM-causing mutations. In addition to previous studies using hiPSC-CMs showing that truncated titin (TTNtv) mutations are the most common causes of DCM –185, recfunction . Genome function . More rereported . The RBMife span .Cardiac arrhythmia caused by autosomal-dominant mutations can be treated with CRISPR-mediated specific disruption of the mutant allele, which has been validated in several mouse models , 187. ReAtherosclerosis is a chronic disease that refers to the formation of fibrofatty lesions in the arterial wall, and causes ischemic stroke, ischemic cardiomyopathy, myocardial infarction, and peripheral arterial disease. Blood concentration of low-density lipoprotein cholesterol (LDL-C) is one of the best-established causal risk factors for atherosclerosis.Pcsk9, is specifically expressed in the liver and functions primarily as an antagonist to the LDL receptor. Disruption of PCSK9 activity can reduce circulating LDL-C levels, thereby lowering the risk of atherosclerosis [Pcsk9 gene can efficiently introduce loss-of-function mutations into endogenous Pcsk9 genes in vivo and chronically decrease plasma cholesterol levels in the blood [Pcsk9 gene in vivo, leading to significantly decreased serum PCSK9 and cholesterol levels [Pcsk9 gene into adult mice demonstrated 63% base editing in the liver, resulting in 78% reduction in serum PCSK9 levels [Pcsk9 gene editing has also been validated in nonhuman primates [Pcsk9 gene in the liver of macaques and cynomolgus monkeys. A single-dose treatment of LNPs carrying CRISPR base editors leads to stable Pcsk9 knockdown in the liver and a 60% reduction in blood LDL-C for at least 8 months [The lipid metabolism-related gene, clerosis . Severalhe blood . The AAVl levels . Single 9 levels . In addiprimates , 181. CR8 months . To ensuCRISPR-based genome editing technology has been rapidly applied in almost all fields, from basic biology to translational medicine. The development of novel systems and tools for more accurate, efficient, and faster genome editing and tighter control of the duration, efficiency, and specificity of genome editors will further benefit their translational applications. Newly uncovered thousands of phage-encoded CRISPR systems provide a valuable resource for searching novel miniature single-effector CRISPR-Cas systems . In addiGenome editing technologies have been successfully translated into human clinical trials for enhanced chimeric antigen receptor (CAR) T-cell therapy, cell-based regenerative medicine, and treatment of monogenic diseases, such as transfusion-dependent beta thalassemia (TDT) and sickle cell disease (SCD) , 193. ReLike any cutting-edge technology, gene editing technology could be a double-edged sword. Genome editing has been listed as a potential weapon of mass destruction in the 2016 annual worldwide threat assessment report of the U.S. intelligence community, indicating a high risk of extreme misuse. Recent rapid advances have made genome editing technologies more accessible and difficult to control, which may further lower the threshold for genome editing misuse and increase biosecurity threats. The possible misuse of genome editing technology and biosecurity risks may include, but are not limited to creating (1) pathogens with increased virulence, (2) new pathogens and biotoxins, and (3) gene-driven animals that may have irreversible effects on specific populations and the environment. Regulations and guidelines should be developed after extensive consultation to ensure that the development of gene editing technologies will not harm living organisms, including humans, or the environment.The emerging novel Cas nucleases and their extended applications have greatly expanded the CRISPR-based genome editing toolbox and promoted the development of life science and medicine. CRISPR-based genome editing technology has also revolutionized cardiovascular research, accelerating the generation of genetically modified models of CVD and its application in the treatment of different types of CVD. However, this technology may also bring huge potential biological threats, which should be strictly controlled to prevent its abuse.


At present, there is no validated marker to identify the subpopulation of patients with advanced gastric cancer (AGC) who might benefit from neoadjuvant chemotherapy (NACT). In view of this clinical challenge, the identification of non-invasive biomarkers for efficacy prediction of NACT in patients with AGC is imperative. Herein, we aimed to develop a non-invasive, liquid-biopsy-based assay by using an exosome-derived RNAs model based on multi-omics characteristics of RNAs. We firstly used a multi-omics strategy to characterize the mRNAs, microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) profiles of circulating exosome enriched fractions in responders to NACT paired with non-responders, using RNA sequencing. Finally, numerous miRNAs, mRNAs and lncRNAs were identified to be associated with the response to NACT in patients with AGC, and it was validated in an independent cohort with promising AUC values. Furthermore, we established a 6-exosome-RNA panel that could robustly identified responders from non-responders treated with fluorouracil-based neoadjuvant chemotherapy.The online version contains supplementary material available at 10.1186/s12943-022-01684-9. Gastric cancer (GC) is frequently diagnosed at an advanced (cTNM II-III) stage (AGC), accounting for 71% approximately . FluorouRecent clinical studies consistently highlight that molecular subtypes of GC are associated with chemotherapeutic efficacy and can be used to guide treatment, such as The Cancer Genome Atlas (TCGA) subtypes  and the In contrast to other methods, using exosomes for liquid biopsy offer some distinctive advantages. Exosomes are 30–150 nm extracellular vesicles (EVs) derived from multivesicular bodies (MVBs) that promote cell-cell communication by transmitting cellular molecular constituents, such as DNA, messenger RNAs (mRNAs), non-coding RNAs (ncRNAs), proteins and lipids . TherefoHerein, we firstly used a multi-omics strategy to characterize the mRNAs, microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) profiles of circulating exosome enriched fractions in responders to NACT paired with non-responders, using RNA sequencing. Finally, numerous miRNAs, mRNAs and lncRNAs were identified to be associated with the response to NACT in patients with AGC, and it was validated in an independent cohort with promising AUC values. Furthermore, we established a 6-exosome-RNA panel that could robustly identified responders from non-responders treated with fluorouracil-based neoadjuvant chemotherapy.A total of 73 individuals including 10 healthy donors, 29 patients responded to NACT and 34 patients who did not respond were enrolled. After plasma collection, all patients received both NACT and surgery  and median TPM expression value > 50 were chosen. Finally, a panel of 18 exosomal RNAs, including 5 mRNAs, 9 miRNAs and 4 lncRNAs , 2 mRNAs  and 2 lncRNAs , had a significant difference and consistent expression trend with the training set between responders and non-responders  and AUC value .To further explore more effective approaches based on these exosomal data, we tried to construct multi-biomarker models to elevate predictive potency. We used the least absolute shrinkage and selection operator (LASSO) Cox regression model to screen the most robust markers for treatment efficacy Fig. A-B. Ten-P < 0.05, Fig. Subsequently, the accuracy of this 6-exosome-RNA model was further validated in the new cohort with 43 patients. The results found that the scores of responders and non-responder based on our constructed model were significantly different , 2 mRNAs (LZIC and SRSF6) and 2 lncRNAs (lncFTH1–211 and lncPTMA-209) were identified and validated. Furthermore, we developed a 6-biomarker model that predicted responders with promising sensitivity, specificity and AUC values both in the training and testing cohorts. Our biomarkers and their combination may facilitate individually precision treatment of NACT for patients with AGC to improve treatment efficacy.Additional file 1: Supplement Fig. 1. Plasma collection, exosome separation procedure and identification. (A) Flow diagram to show plasma collection and following treatment. (B) Extraction and sequencing of RNAs from plasma-derived exosome enriched fractions. (C) Detection of exosomal markers by western blot. (D) Exosome morphological detection by transmission electron microscopy. (E) Size distribution of exosomes was assessed by nanoparticle tracking analysis. (F) The RNA concentration of exosomes enriched fraction in different groups. (G) The intersection of differentially expressed RNAs between healthy donors vs AGC patients and responders vs non-responders.


N = 23) as compared to BIOMED-2/EuroClonality (N = 6) as judged on identical IGH and IGK targets. The same clonal rearrangements were also identified in paired fresh frozen cHL samples. To validate the neoplastic origin of the detected clonotypes, IG-NGS clonality analysis was performed on isolated HRS cells, demonstrating identical clonotypes as detected in cHL whole-tissue specimens. Interestingly, IG-NGS and HRS single-cell analysis after DEPArray™ digital sorting revealed rearrangement patterns and copy number variation profiles indicating clonal diversity and intratumoral heterogeneity in cHL. Our data demonstrate improved performance of NGS-based detection of IG gene rearrangements in cHL whole-tissue specimens, providing a sensitive molecular diagnostic assay for clonality assessment in Hodgkin lymphoma.Clonality analysis in classic Hodgkin lymphoma (cHL) is of added value for correctly diagnosing patients with atypical presentation or histology reminiscent of T cell lymphoma, and for establishing the clonal relationship in patients with recurrent disease. However, such analysis has been hampered by the sparsity of malignant Hodgkin and Reed-Sternberg (HRS) cells in a background of reactive immune cells. Recently, the EuroClonality-NGS Working Group developed a novel next-generation sequencing (NGS)-based assay and bioinformatics platform (ARResT/Interrogate) to detect immunoglobulin (IG) gene rearrangements for clonality testing in B-cell lymphoproliferations. Here, we demonstrate the improved performance of IG-NGS compared to conventional BIOMED-2/EuroClonality analysis to detect clonal gene rearrangements in 16 well-characterized primary cHL cases within the IG heavy chain (IGH) and kappa light chain (IGK) loci. This was most obvious in formalin-fixed paraffin-embedded (FFPE) tissue specimens, where three times more clonal cases were detected with IG-NGS (9 cases) compared to BIOMED-2 (3 cases). In total, almost four times more clonal rearrangements were detected in FFPE with IG-NGS ( In cHL, the HRS cells represent functionally impaired B lymphocytes that have lost the capacity to express immunoglobulins and many of the B-cell markers, but show CD15 and CD30 expression3. Evidence for their B-cell origin has been obtained by the detection of clonal rearrangements of the immunoglobulin genes, i.e., the immunoglobulin heavy (IGH) and light chain gene loci7. Furthermore, the presence of somatic hypermutation (SHM) within the rearranged variable (V) genes demonstrates that HRS cells originate from (post-)germinal center B lymphocytes10. Although most cHL cases are diagnosed based on clinical presentation and histopathology, the molecular detection of clonal immunoglobulin (IG) gene rearrangements is helpful for diagnosing cases with a more atypical presentation and a histology similar to T cell lymphoma, or for clonal comparison in patients with recurrent disease12.Classic Hodgkin lymphoma (cHL) is a B-cell neoplasm, characterized by the presence of a small number of malignant cells, called Hodgkin and Reed-Sternberg (HRS) cells, residing in a background of inflammatory cells, including reactive B cells and plasma cells16. For assessing B-cell clonality, multiplex PCR reactions are performed for the detection of IGH and kappa light chain (IGK) gene rearrangements, followed by fragment analysis using GeneScan or heteroduplex analysis. The applicability of the BIOMED-2/EuroClonality assay in cHL has been evaluated in different studies, demonstrating B-cell clonality in about 50% of formalin-fixed paraffin-embedded (FFPE) cHL tissue specimens23. Although the combination of both IGH and IGK rearrangement analysis, including incomplete rearrangements not undergoing SHM, has certainly enhanced the detection rate of clonal rearrangements in cHL, more sensitive techniques can improve clonality assessment in cHL.Currently, the gold standard for clonality testing of lymphomas is the BIOMED-2/EuroClonality assay developed and validated by the EuroClonality Consortium24. In this assay, new primers have been designed for complementary IG gene targets that will result in shorter amplicon length, particularly for the incomplete IGH targets as well as IGK targets. This enables better clonality detection in genomic DNA from FFPE tissue specimens, especially samples with inferior DNA quality. Another advantage of IG-NGS is the immediate sequence availability of the identified clonotypes, both of the malignant clone as well as the non-neoplastic background B cells. This will provide more reliable information to detect minor clones in a background of polyclonal B cells and assessing the clonal relationship of multiple B-cell malignancies within the same patient.Recently, a next-generation sequencing (NGS)-based clonality assay for detection of IG gene rearrangements (IG-NGS) has been developed by the EuroClonality-NGS Working GroupIn this study, we compared the performance of NGS-based clonality assessment in cHL to the conventional BIOMED-2/EuroClonality assay to identify clonal IGH and IGK gene rearrangements in whole-tissue specimens, including both FFPE and fresh frozen (FF) tissue. We demonstrate improved performance of the IG-NGS assay to detect B-cell clonality in both types of tissue specimens. Furthermore, our data reveal evidence for biclonality in some cHL tumor samples, indicating that co-existence of multiple malignant clones and clonal diversity can occur in this type of lymphoma.25, for which both FFPE and FF tissue material was available18 .This study was performed with 16 lymph node biopsies of patients diagnosed with cHL according to the 2017 revised 4th edition of the World Health Organization classification18 Table . IG-NGS 18 Table 26. All c24. In short, three multiplex PCR reactions were performed with 40–50 ng input DNA for the detection of IG gene rearrangements, which involved variable (V), diversity (D), joining (J), kappa deleting element (KDE), and the recombination signal sequence (RSS) gene segments of the following five targets: IGHV-IGHD-IGHJ (FR3), IGHD-IGHJ, IGKV-IGKJ, IGKV-KDE, and Intron RSS-KDE. After bead purification, library preparation and amplification, the samples were pooled with equal DNA concentrations (corresponding to equimolar amounts) for sequencing on Ion Torrent PGM. The Ion 318™ Chip Kit v2 BC  was loaded with 24 or 32 samples per chip. The sequencing results were analyzed and visualized using the bioinformatics pipeline ARResT/Interrogate (http://arrest.tools/interrogate)27. The output data defines a clonotype , notated as a combination of the 5′ gene (V3-9), nucleotides deleted from the 5′ gene (-1), the added nucleotides at the junction including the D-segment if present (20), nucleotides deleted from the 3′ gene (-6), and the 3′ gene (J3)28. Data were considered reliable with at least a minimum amount of 1000 reads for IGHV-IGHD-IGHJ  and IGKV-IGKJ , and 500 reads for IGHD-IGHJ , IGKV-KDE (mean: 4201 reads), and Intron RSS-KDE (mean: 2869 reads). For the assessment of B-cell clonality, each DNA sample (extracted as independent isolates from FF and FFPE specimens of the same cHL tumor tissue) was analyzed in duplicate by multiplex PCR and NGS to rule out misinterpretation or artifacts, and validate less-abundant clonotypes. Only clonotypes that are detected with a dominant abundancy above threshold in duplicate are considered as clonal rearrangements.Detection of IGH and IGK gene rearrangements by next-generation sequencing (IG-NGS) was performed as recently describedAdditional information regarding ‘Materials and Methods’, including the strategy for clone identification, enrichment of HRS cells and single-cell analysis are provided in the 18. Eleven cases were of the nodular sclerosing subtype (NSHL), of which four cases (36%) were Epstein-Barr virus (EBV)-positive, and five cases were diagnosed as mixed cellularity HL (MSHL), with two EBV-positive cases (40%). The average percentage of malignant HRS cells in total tissue as judged by CD30 immunohistochemical staining varied between 1% and 15%, and the fraction of HRS cells related to total B-cell counts ranged from 3% to 43% (Table In this study 16 classic Hodgkin lymphoma (cHL) cases were included, of which both archival FF and FFPE tissue specimens were available Table 18. Eleve3% Table . First, 3% Table . The rem3% Table . NotablyTo define clonality in cHL by IG-NGS, we reasoned that it would be important to identify the clone frequencies of abundant clonotypes in non-malignant reactive lymphoproliferative samples, which would define thresholds above which potentially malignant clonotypes could be identified. Furthermore, it would be essential to demonstrate that identified clonotypes in cHL whole-tissue specimens corresponded to IG gene rearrangements of the corresponding HRS cells, and not reactive B-cell clones. To this end, we established a specific workflow procedure as summarized in Fig. N = 30; RLN1-RLN30)  IGHV-IGHD-IGHJ, IGHD-IGHJ, IGKV-IGKJ, IGKV-KDE, and Intron RSS-KDE) was established in a cohort of reactive lymphoid tissue specimens  and 9 out of 16 FFPE cases (56%) with IG-NGS Table . FF yiel+ HRS cells in FF tissue sections, and flow cytometric isolation of CD30+MUM1+ HRS cells after mild enzymatic digestion of the paired FFPE tissue specimen, as described by Juskevicius et al.29. Genomic DNA was isolated from the HRS-enriched cell fractions of both procedures, and subjected to IG-NGS analysis. In each of the two cHL cases, identical clonotypes were detected in higher abundance in the HRS-enriched cell fractions as compared to whole-tissue specimens, both for FF as well as FFPE tissues  using laser microdissection (LMD) of CD30ues Fig. . These r30. However, IG-NGS failed to detect an IGHV-IGHD-IGHJ gene rearrangement for case 16, which was identified with conventional BIOMED-2/EuroClonality testing for FR3 using a different IGHV-FR3 forward primer  showed clonal IG gene rearrangements with IG-NGS, while only 3 clonal cases (20%) were detected with BIOMED-2/EuroClonality, and clonality assessment with IG-NGS was often supported by multiple targets Table . SimilarSince NGS-based clonality assessment revealed more detailed information regarding the clonal composition of lymphomas, two out of 14 cases  showed multiple clonal IG gene rearrangements exceeding the allelic capacity of a single B-cell clone. Pathology review provided no indications for a composite lymphoma in both cHL FFPE tissue specimens. Case 8 displayed two unproductive IGHD-IGHJ gene rearrangements (IGHD3-9/IGHJ6 and IGHD1-26/IGHJ5) combined with one productive IGHV-IGHD-IGHJ gene rearrangement (IGHV3-9/IGHD6-13/IGHJ3), implying the presence of two B-cell clones  of single cells followed by shallow whole-genome sequencing (sWGS), and subsequent CNV analysis of the individual HRS cells. DNA integrity analysis with quantitative PCR on DNA isolated from the FFPE tissue specimens indicated that only case 9 (QC score: 0.35) fulfilled the quality criteria for the subsequent sWGS approach. Unsupervised hierarchical cluster analysis revealed two separate clusters with distinct CNV profiles of the individual HRS cells  profiling on CD30lls Fig.  and S10.Detection of IG gene rearrangements for clonality analysis in cHL has remained challenging due to the low frequency of malignant HRS cells in a background of reactive B cells and plasma cells. By direct comparison of NGS-based clonality assessment with the conventional BIOMED-2/GeneScan analysis to detect IG gene rearrangements in undissected cHL-tumor tissue, our study demonstrated a significantly improved performance of the IG-NGS assay. The clonotypes detected by IG-NGS were shown to be of neoplastic cell origin, based on the identification of identical IG gene rearrangements in HRS-enriched cell fractions as compared to whole tissue specimens. Furthermore, our data revealed evidence for biclonality in two cHL cases, which was confirmed by single-cell analysis in one of the cases.26. Here, we investigated the applicability of the IG-NGS assay to detect clonal IG gene rearrangements in cHL. To define threshold values for tumor clone identification in cHL in the context of this study, clonality data of reactive lymphoproliferative samples without clear indications for EBV positivity were used to establish relative B-cell clone frequencies in non-malignant tissues. Identical clonotypes could be identified in duplicate analysis of the reactive lymphoproliferative samples with slightly increased frequencies compared to less-abundant background clonotypes.The recently developed IG-NGS clonality assay of the EuroClonality-NGS Working Group set out to improve clonality detection in lymphoid malignancies and has been validated for different mature B-cell lymphoma subtypes, but not yet for cHL26, where clonal IG gene rearrangements were detected in 96% of the lymphoma cases. This demonstrates that the identification of clonal IG gene rearrangements in cHL with NGS is less efficient , most likely due to the lower fraction of malignant HRS cells within the tumor specimens. Studies in larger cohorts are required to firmly establish the clonality detection rate in cHL.On the basis of our defined thresholds, clonal IG gene rearrangements were detected with IG-NGS in 88% of the FF samples and 56% of the FFPE specimens, often at multiple targets for both IGH and IGK. Since all clonotypes were detected in duplicate in both FF and FFPE, these likely represent true clonal rearrangements and not artifacts. Furthermore, the identified dominant clonotypes showed no overlap between different samples, excluding cross-contamination or index-hopping. As expected, cHL tissues with relative higher frequencies of CD30-positive HRS cells as a proportion of total B cells showed more pronounced dominant clonotypes , which is most likely due to the absence of active IG gene-specific transcription9 and epigenetic silencing of the BCR locus36. Nonetheless, IG gene rearrangements in cHL can serve as informative molecular fingerprints for clonality testing of cHL. To confirm that the observed dominant clonotypes in undissected tumor-tissue samples were derived from neoplastic cell origin, HRS cells were enriched by flow cytometry (FFPE tissue) and LMD (FF tissue). IG-NGS clonality testing showed identical clonal gene rearrangements and corresponding clonotypes at a higher abundancy in the isolated HRS-cell fraction as compared to whole-tumor tissue DNA, substantiating their neoplastic cell origin.Our studies demonstrate the presence of SHM in IGHV and to a lesser extent in IGKV gene regions of the malignant HRS cells (Supplementary Fig. 38, biclonality has been reported mostly in the context of composite lymphomas41, and also within single lymphoma subtypes43 and in immunosuppressed patients45. It is known that, especially in patients with a history of infectious mononucleosis or immune deficiencies, EBV latent antigens can regulate or mimic different survival and proliferative signaling pathways, and thereby promote lymphomagenesis48. Since both of our cases with suspected biclonality showed EBV positivity, this might contribute to clonal diversity with the detection of two clonal IGHD-IGHJ and one IGHV-IGHD-IGHJ rearrangement in case 8, and multiple IGK gene rearrangements in case 9, which do not fit within a single B-cell clone (Supplementary Fig. 34, and is in concordance with a recent study of Mangano et al.31. Unfortunately, such single-cell analysis could not be performed on case 8, due to the inferior FFPE-DNA quality.Furthermore, our data revealed the presence of multiple clonal rearrangements, which suggests the occurrence of biclonality in cHL (Supplementary Fig. 49. Moreover, this approach allows more detailed analysis on the clonal composition and its diversity that underlies cHL pathogenesis.In conclusion, IG-NGS clonality testing is a more accurate and sensitive assay for clonality detection in whole-tumor tissue DNA of cHL samples compared to the conventional BIOMED-2/EuroClonality assay. Additionally, this new IG-NGS based clonality testing will be an ideal tool in clinical diagnostics for recurrent disease, since clonal comparison based on nucleotide sequences and assigned clonotypes is more reliable than comparing fragment lengths, as has been shown in non-Hodgkin lymphoma patientsSupplmentary information.Supplementary Table S4.


Experimental findings for SARS-CoV-2 related to the glycan biochemistry of coronaviruses indicate that attachments from spike protein to glycoconjugates on the surfaces of red blood cells (RBCs), other blood cells and endothelial cells are key to the infectivity and morbidity of COVID-19. To provide further insight into these glycan attachments and their potential clinical relevance, the classic hemagglutination (HA) assay was applied using spike protein from the Wuhan, Alpha, Delta and Omicron B.1.1.529 lineages of SARS-CoV-2 mixed with human RBCs. The electrostatic potential of the central region of spike protein from these four lineages was studied through molecular modeling simulations. Inhibition of spike protein-induced HA was tested using the macrocyclic lactone ivermectin (IVM), which is indicated to bind strongly to SARS-CoV-2 spike protein glycan sites. The results of these experiments were, first, that spike protein from these four lineages of SARS-CoV-2 induced HA. Omicron induced HA at a significantly lower threshold concentration of spike protein than the three prior lineages and was much more electropositive on its central spike protein region. IVM blocked HA when added to RBCs prior to spike protein and reversed HA when added afterward. These results validate and extend prior findings on the role of glycan bindings of viral spike protein in COVID-19. They furthermore suggest therapeutic options using competitive glycan-binding agents such as IVM and may help elucidate rare serious adverse effects (AEs) associated with COVID-19 mRNA vaccines, which use spike protein as the generated antigen. Key to the infectivity and morbidity of SARS-CoV-2 are glycans that protrude tangentially from 22 N-linked glycosylation sites on each monomer of its spike protein ,2,3,4,5.The RBC has an especially dense surface distribution of SA, 35 million SA molecules per cell, arrayed on its sialoglycoprotein coating mainly as terminal residues of glycophorin A (GPA) ,15. SA iThis hemagglutinating property of SARS-CoV-2 has important clinical consequences. First, with trillions of RBCs each circulating through narrow pulmonary capillaries about once per minute, even small, dynamically aggregating and disaggregating RBC clumps  can Although the blood cell types and processes entailed in clumping, clotting and vascular obstruction of COVID-19 are wide-ranging, virally induced hemagglutination (HA) is a central event that is amenable to in vitro study. The classic HA assay was used here to study this, using cell culture supernatants and SARS-CoV-2 trimeric spike protein. Indeed, SARS-CoV-2 spike protein mixed with human whole blood caused RBC aggregation , while sThe goals of this study were to determine whether principles of glycobiology as established for coronaviruses and in particular for SARS-CoV-2, can be validated using the classic HA assay, to test whether HA inhibition is achieved by an agent indicated to competitively bind to those glycans and to determine the comparative hemagglutinating potencies of the Wuhan virus and its Alpha, Delta and Omicron B.1.1.529 variants. The clinical relevance of testing HA induced by SARS-CoV-2 using spike protein rather than whole virus and the utility of additionally testing for HA inhibition via competitive glycan binding using IVM was suggested in an earlier study  [A complete structure of the reference spike protein was generated from the original 20B strain (pdb: 7bnm) as previously described . All gaper 2022] ,93. Thiser 2022] ,94. Trimer 2022] . The eleer 2022] .Spike protein from four lineages of SARS-CoV-2 induced HA in human RBCs, which supports other indications that spike protein-induced RBC clumping, as well as viral attachments to other blood cells and endothelial cells, may be key to the morbidities of COVID-19. IVM, a macrocyclic lactone indicated to bind strongly to multiple glycan sites on SARS-CoV-2 spike protein, blocked HA when added to RBCs prior to spike protein and reversed HA when added afterward, which suggests therapeutic options for COVID-19 treatment using this drug or other competitive glycan-binding agents. The Omicron B.1.1.529 variant had significantly greater HA-inducing activity than the three prior lineages tested, which may relate to the findings from molecular modeling that the electrostatic charge of the central region of its spike protein was considerably more positive than for those of the prior lineages and, from a prior study, that its SA-binding affinity is greater than for prior lineages . Whether