Precision Enhancement of CAR-NK Cells through Non-Viral Engineering and Highly Multiplexed Base Editing.

Natural killer (NK) cells' unique ability to kill transformed cells expressing stress ligands or lacking major histocompatibility complexes (MHC) has prompted their development for immunotherapy. However, NK cells have demonstrated only moderate responses against cancer in clinical trials and likely require advanced genome engineering to reach their full potential as a cancer therapeutic. Multiplex genome editing with CRISPR/Cas9 base editors (BE) has been used to enhance T cell function and has already entered clinical trials but has not been reported in human NK cells. Here, we report the first application of BE in primary NK cells to achieve both loss-of-function and gain-of-function mutations. We observed highly efficient single and multiplex base editing, resulting in significantly enhanced NK cell function. Next, we combined multiplex BE with non-viral TcBuster transposon-based integration to generate IL-15 armored CD19 CAR-NK cells with significantly improved functionality in a highly suppressive model of Burkitt's lymphoma both in vitro and in vivo. The use of concomitant non-viral transposon engineering with multiplex base editing thus represents a highly versatile and efficient platform to generate CAR-NK products for cell-based immunotherapy and affords the flexibility to tailor multiple gene edits to maximize the effectiveness of the therapy for the cancer type being treated.

Correction of Fanconi Anemia Mutations Using Digital Genome Engineering.

Fanconi anemia (FA) is a rare genetic disease in which genes essential for DNA repair are mutated. Both the interstrand crosslink (ICL) and double-strand break (DSB) repair pathways are disrupted in FA, leading to patient bone marrow failure (BMF) and cancer predisposition. The only curative therapy for the hematological manifestations of FA is an allogeneic hematopoietic cell transplant (HCT); however, many (>70%) patients lack a suitable human leukocyte antigen (HLA)-matched donor, often resulting in increased rates of graft-versus-host disease (GvHD) and, potentially, the exacerbation of cancer risk. Successful engraftment of gene-corrected autologous hematopoietic stem cells (HSC) circumvents the need for an allogeneic HCT and has been achieved in other genetic diseases using targeted nucleases to induce site specific DSBs and the correction of mutated genes through homology-directed repair (HDR). However, this process is extremely inefficient in FA cells, as they are inherently deficient in DNA repair. Here, we demonstrate the correction of FANCA mutations in primary patient cells using 'digital' genome editing with the cytosine and adenine base editors (BEs). These Cas9-based tools allow for C:G > T:A or A:T > C:G base transitions without the induction of a toxic DSB or the need for a DNA donor molecule. These genetic corrections or conservative codon substitution strategies lead to phenotypic rescue as illustrated by a resistance to the alkylating crosslinking agent Mitomycin C (MMC). Further, FANCA protein expression was restored, and an intact FA pathway was demonstrated by downstream FANCD2 monoubiquitination induction. This BE digital correction strategy will enable the use of gene-corrected FA patient hematopoietic stem and progenitor cells (HSPCs) for autologous HCT, obviating the risks associated with allogeneic HCT and DSB induction during autologous HSC gene therapy.

Mitochondria and Their Relationship with Common Genetic Abnormalities in Hematologic Malignancies.

Hematologic malignancies are known to be associated with numerous cytogenetic and molecular genetic changes. In addition to morphology, immunophenotype, cytochemistry and clinical characteristics, these genetic alterations are typically required to diagnose myeloid, lymphoid, and plasma cell neoplasms. According to the current World Health Organization (WHO) Classification of Tumors of Hematopoietic and Lymphoid Tissues, numerous genetic changes are highlighted, often defining a distinct subtype of a disease, or providing prognostic information. This review highlights how these molecular changes can alter mitochondrial bioenergetics, cell death pathways, mitochondrial dynamics and potentially be related to mitochondrial genetic changes. A better understanding of these processes emphasizes potential novel therapies.

Nonviral genome engineering of natural killer cells.

Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system capable of immune surveillance. Given their ability to rapidly and effectively recognize and kill aberrant cells, especially transformed cells, NK cells represent a unique cell type to genetically engineer to improve its potential as a cell-based therapy. NK cells do not express a T cell receptor and thus do not contribute to graft-versus-host disease, nor do they induce T cell-driven cytokine storms, making them highly suited as an off-the-shelf cellular therapy. The clinical efficacy of NK cell-based therapies has been hindered by limited in vivo persistence and the immunosuppressive tumor microenvironment characteristic of many cancers. Enhancing NK cell resistance to tumor inhibitory signaling through genome engineering has the potential to improve NK cell persistence in the tumor microenvironment and restore cytotoxic functions. Alongside silencing NK cell inhibitory receptors, NK cell killing can be redirected by the integration of chimeric antigen receptors (CARs). However, NK cells are associated with technical and biological challenges not observed in T cells, typically resulting in low genome editing efficiencies. Viral vectors have achieved the greatest gene transfer efficiencies but carry concerns of random, insertional mutagenesis given the high viral titers necessary. As such, this review focuses on nonviral methods of gene transfer within the context of improving cancer immunotherapy using engineered NK cells.

Protein regulation strategies of the mouse spleen in response to Babesia microti infection.

BACKGROUND: Babesia is a protozoan parasite that infects red blood cells in some vertebrates. Some species of Babesia can induce zoonoses and cause considerable harm. As the largest immune organ in mammals, the spleen plays an important role in defending against Babesia infection. When infected with Babesia, the spleen is seriously injured but still actively initiates immunomodulatory responses. METHODS: To explore the molecular mechanisms underlying the immune regulation and self-repair of the spleen in response to infection, this study used data-independent acquisition (DIA) quantitative proteomics to analyse changes in expression levels of global proteins and in phosphorylation modification in spleen tissue after Babesia microti infection in mice. RESULTS: After mice were infected with B. microti, their spleens were seriously damaged. Using bioinformatics methods to analyse dynamic changes in a large number of proteins, we found that the spleen still initiated immune responses to combat the infection, with immune-related proteins playing an important role, including cathepsin D (CTSD), interferon-induced protein 44 (IFI44), interleukin-2 enhancer-binding factor 2 (ILF2), interleukin enhancer-binding factor 3 (ILF3) and signal transducer and activator of transcription 5A (STAT5A). In addition, some proteins related to iron metabolism were also involved in the repair of the spleen after B. microti infection, including serotransferrin, lactoferrin, transferrin receptor protein 1 (TfR1) and glutamate-cysteine ligase (GCL). At the same time, the expression and phosphorylation of proteins related to the growth and development of the spleen also changed, including protein kinase C-δ (PKC-δ), mitogen-activated protein kinase (MAPK) 3/1, growth factor receptor-bound protein 2 (Grb2) and P21-activated kinase 2 (PAK2). CONCLUSIONS: Immune-related proteins, iron metabolism-related proteins and growth and development-related proteins play an important role in the regulation of spleen injury and maintenance of homeostasis. This study provides an important basis for the diagnosis and treatment of babesiosis.

Quantitative proteomics and phosphoproteomic analyses of mouse livers after tick-borne Babesia microti infection.

Babesia microti is a tick-borne protozoan parasite that infects the red blood cells of mice, humans, and other mammals. The liver tissues of BALB/c mice infected with B. microti exhibit severe injury. To further investigate the molecular mechanisms underlying liver injury and liver self-repair after B. microti infection, data-independent acquisition (DIA) quantitative proteomics was used to analyse changes in the expression and phosphorylation of proteins in liver tissues of BALB/c mice during a B. microti infection period and a recovery period. The expression of FABP1 and ACBP, which are related to fatty acid transport in the liver, was downregulated after infection with B. microti, as was the expression of Acox1, Ehhadh and Acaa1a, which are crucial rate-limiting enzymes in the process of fatty acid ß oxidation. The phosphorylation levels of AMP-activated protein kinase (AMPK) and Hormone-sensitive lipase (HSL) were also downregulated. In addition, the expression of PSMB9, CTSC, and other immune-related proteins was increased, reflecting an active immune regulation mechanism in the mice. The weights of mice infected with B. microti were significantly reduced, and the phosphorylation levels of IRS-1, c-Raf, mTOR, and other proteins related to growth and development were downregulated.

Comprehensive analysis of protein expression levels and phosphorylation levels in host skin in response to tick (Haemaphysalis longicornis) bite.

Ticks are parasitic arthropods that suck blood from the surface of most vertebrates. They can transmit a variety of pathogens. The blood sucking of ticks causes varying degrees of damage to the skin of the host. Proteins related to immune regulation, vascular repair, and wound healing in mammalian skin respond to tick bites by regulating their expression and post-translational modifications to protect the skin from injury. Phosphorylation of proteins, as the most common post-translational modification of proteins, plays an important role in the rapid regulation of cell signal transduction, gene expression and cell cycle. To systematically explore the molecular regulatory mechanisms employed by mammalian skin to resist tick bites, larval, nymphal, and adult Haemaphysalis longicornis were used to bite the skin tissues of healthy rabbits in the present study. The quantitative proteomic technology data-independent acquisition was then carried out to investigate in depth the changes in protein expression and phosphorylation in rabbit skin after tick bite. The results showed that among the 4034 proteins and 1795 phosphorylated proteins identified, a total of 202 proteins and 435 phosphorylation sites were changed after H. longicornis bite. In order to provide convenience for sucking blood, active substances in the saliva of H. longicornis injected into the rabbit's skin can cause the expression level of trichohyalin and peptidyl arginine deiminase 3 in the skin of the host downregulate, which can make the host hair loss and regeneration disorders. At the same time, the active substances in saliva of the H. longicornis led to the phosphorylation of microtubule actin cross-linking factor 1 in the host's skin and further inactivation, so as to delay the healing of the host wound. In response to tick bites, the host skin promotes coagulation through high expression of fibrinogen and fibronectin, and vascular repair through high expression of integrin linked kinase and tenascin C, as well as accelerated phosphorylation of the phosphorylated protein Nck adaptor protein 1, and wound healing through high expression of ezrin and integrin. The upregulation of proteins such as coronin, NADPH oxidase, calnexin, and calreticulin and phosphorylation level of IL-4R in the host skin after the H. longicornis bite indicated that the immune response was playing an important defensive role in response to tick bites. Meanwhile, we found that the upregulated two lectins, mannose receptor C-type 1 and DC-SIGN, may serve as molecular makers to identify and monitor whether the skin is bitten by ticks. SIGNIFICANCE: Haemaphysalis longicornis are parasitic arthropods that suck blood from the surface of most vertebrates. They can transmit a variety of pathogens and are harmful to humans and livestock. The present study is the first quantitative proteomic study on protein expression levels in the rabbit skin after infection by H. longicornis. It is also the first quantitative phosphoproteomic study in the host skin infected by ticks. In this study, we found that tick bites cause the host hair loss and regeneration disorders. For resisting tick bite, the host activates the immune response and initiates vascular repair and wound-healing systems. In addition, some phosphorylated proteins promote host immunity and vascular repair. These results can help us further understand the defence mechanism of the host against tick bites, provide a basis for the development of an anti-tick vaccine, the development of anti-tick drugs, and the diagnosis of tick-borne diseases.

Changes in Protein Phosphorylation during Salivary Gland Degeneration in Haemaphysalis longicornis.

The ticks feed large amount of blood from their hosts and transmit pathogens to the victims. The salivary gland plays an important role in the blood feeding. When the female ticks are near engorgement, the salivary gland gradually loses its functions and begins to rapidly degenerate. In this study, data-independent acquisition quantitative proteomics was used to study changes in the phosphorylation modification of proteins during salivary gland degeneration in Haemaphysalis longicornis. In this quantitative study, 400 phosphorylated proteins and 850 phosphorylation modification sites were identified. Trough RNA interference experiments, we found that among the proteins with changes in phosphorylation, apoptosis-promoting Hippo protein played a role in salivary gland degeneration.

PLX3397 treatment inhibits constitutive CSF1R-induced oncogenic ERK signaling, reduces tumor growth, and metastatic burden in osteosarcoma.

Osteosarcoma (OSA) is a heterogeneous and aggressive solid tumor of the bone. We recently identified the colony stimulating factor 1 receptor (Csf1r) gene as a novel driver of osteosarcomagenesis in mice using the Sleeping Beauty (SB) transposon mutagenesis system. Here, we report that a CSF1R-CSF1 autocrine/paracrine signaling mechanism is constitutively activated in a subset of human OSA cases and is critical for promoting tumor growth and contributes to metastasis. We examined CSF1R and CSF1 expression in OSAs. We utilized gain-of-function and loss-of-function studies (GOF/LOF) to evaluate properties of cellular transformation, downstream signaling, and mechanisms of CSF1R-CSF1 action. Genetic perturbation of CSF1R in immortalized osteoblasts and human OSA cell lines significantly altered oncogenic properties, which were dependent on the CSF1R-CSF1 autocrine/paracrine signaling. These functional alterations were associated with changes in the known CSF1R downstream ERK effector pathway and mitotic cell cycle arrest. We evaluated the recently FDA-approved CSF1R inhibitor Pexidartinib (PLX3397) in OSA cell lines in vitro and in vivo in cell line and patient-derived xenografts. Pharmacological inhibition of CSF1R signaling recapitulated the in vitro genetic alterations. Moreover, in orthotopic OSA cell line and subcutaneous patient-derived xenograft (PDX)-injected mouse models, PLX3397 treatment significantly inhibited local OSA tumor growth and lessened metastatic burden. In summary, CSF1R is utilized by OSA cells to promote tumorigenesis and may represent a new molecular target for therapy.

A proteomics analysis of the ovarian development in females of Haemaphysalis longicornis.

Haemaphysalis longicornis is an ixodid tick that can spread a wide variety of pathogens, affecting humans, livestock and wildlife health. The high reproductive capability of this species is initiated by the ingestion of a large amount of blood ingested by the engorged female tick. The degree of ovarian development is proportional to the number of eggs laid. Studying the regulatory mechanism of tick ovary development is relevant for the development of novel tick control methods. In this study, we used quantitative proteomics to study the dynamic changes in protein expression and protein phosphorylation during ovarian development of engorged female H. longicornis ticks. Synergistic action of many proteins (n = 3031) is required to achieve ovarian development and oocyte formation rapidly. Through bioinformatics analysis, changes in protein expressions and phosphorylation modifications in regulating the ovarian development of female ticks are described. Many proteins play an essential role during ovarian development. Also, protein phosphorylation appeared an important reproductive strategy to enable ticks to efficiently convert large amounts of blood in the ovaries into egg-producing components and ultimately produce many eggs.

Glucose and urea metabolic enzymes are differentially phosphorylated during freezing, anoxia, and dehydration exposures in a freeze tolerant frog.

Vertebrate freeze tolerance requires multiple adaptations underpinned by specialized biochemistry. Freezing of extracellular water leads to intracellular dehydration as pure water is incorporated into growing ice crystals and also results in the cessation of blood supply to tissues, creating an anoxic cellular environment. Hence, the freeze tolerant wood frog, Rana sylvatica, must endure both dehydration and anoxia stresses in addition to freezing. The metabolic responses to freezing, dehydration and anoxia involve both protein/enzyme adaptations and the production of metabolites with metabolic or osmotic functions, particularly glucose and urea. The present study uses a phosphoproteome analysis to examine the differential phosphorylation of metabolic enzymes involved in the production of these two metabolites in liver in response to freezing, anoxia, or dehydration exposures. Our results show stress-specific responses in the abundance of phosphopeptides retrieved from nine glycolytic enzymes and three urea cycle enzymes in liver of wood frogs exposed to 24 h freezing, 24 h anoxia, or dehydration to 40% of total body water loss, as compared with 5 °C acclimated controls. Data show changes in the abundance of phosphopeptides belonging to glycogen phosphorylase (GP) and phosphofructokinase 2 (PFK2) that were consistent with differential phosphorylation control of glycogenolysis and a metabolic block at PFK1 that can facilitate glucose synthesis as the cryoprotectant during freezing. Anoxia-exposed animals showed similar changes in GP phosphorylation but no changes to PFK2; changes that would facilitate mobilization of glycogen as a fermentative fuel for anaerobic glycolysis. Urea is commonly produced as a compatible osmolyte in response to amphibian dehydration. Selected urea cycle enzymes showed small changes in phosphopeptide abundance in response to dehydration, but during freezing differential phosphorylation occurred that may facilitate this ATP expensive process when energy resources are sparse. These results add to the growing body of literature demonstrating the importance and efficiency of reversible protein phosphorylation as a regulatory mechanism allowing animals to rapidly respond to environmental stress.

Salivary gland proteome analysis of developing adult female Haemaphysalis longicornis ticks: molecular motor and TCA cycle-related proteins play an important role throughout development.

BACKGROUND: Ticks are notorious blood-feeding arthropods that can spread a variety of deadly diseases. The salivary gland is an important organ for ticks to feed on blood, and this organ begins to develop rapidly when ixodid ticks suck blood. When these ticks reach a critical weight, the salivary glands stop developing and begin to degenerate. The expression levels of a large number of proteins during the development and degeneration of salivary glands change, which regulate the biological functions of the salivary glands. Furthermore, to the best of our knowledge, there are only a few reports on the role of molecular motor and TCA cycle-related proteins in the salivary glands of ticks. RESULTS: We used iTRAQ quantitative proteomics to study the dynamic changes in salivary gland proteins in female Haemaphysalis longicornis at four feeding stages: unfed, partially fed, semi-engorged and engorged. Using bioinformatics methods to analyze the dynamic changes of a large number of proteins, we found that molecular motor and TCA cycle-related proteins play an important role in the physiological changes of the salivary glands. The results of RNAi experiments showed that when dynein, kinesin, isocitrate dehydrogenase and citrate synthase were knocked down independently, the weight of the engorged female ticks decreased by 63.5%, 54.9%, 42.6% and 48.6%, respectively, and oviposition amounts decreased by 83.1%, 76.0%, 50.8%, and 55.9%, respectively, and the size of type III acini of females salivary glands decreased by 35.6%, 33.3%, 28.9%, and 20.0%, respectively. CONCLUSIONS: The results showed that the expression of different types of proteins change in different characteristics in salivary glands during the unfed to engorged process of female ticks. Corresponding expression changes of these proteins at different developmental stages of female ticks are very important to ensure the orderly development of the organ. By analyzing these changes, some proteins, such as molecular motor and TCA cycle-related proteins, were screened and RNAi carried out. When these mRNAs were knocked down, the female ticks cannot develop normally. The research results provide a new protein target for the control of ticks and tick-borne diseases.

Comprehensive Analysis of the Global Protein Changes That Occur During Salivary Gland Degeneration in Female Ixodid Ticks Haemaphysalis longicornis.

Ticks are notorious blood-sucking arthropods that can spread a variety of pathogens and cause great harm to the health of humans, wildlife and domestic animals. The salivary glands of female ticks degenerate rapidly when the ticks reach critical weight or become engorged, which can be caused by hormones and by the synergistic effects of multiple proteins. To explore the complex molecular mechanisms of salivary gland degeneration in ticks, this study applies iTRAQ quantitative proteomic technology for the first time to study changes in protein expression in the salivary glands of female Haemaphysalis longicornis during the process of degeneration and to search for proteins that play an important role in salivary gland degeneration. It was found that the expression of some proteins associated with energy production was continuously down-regulated during salivary gland degeneration, while some proteins associated with DNA or protein degradation were consistently up-regulated. Furthermore, the expression of some proteins related to cell apoptosis or autophagy was also changed. These proteins were knocked down by RNAi to observe the phenotypic and physiological changes in female ticks. The results showed that the time required for engorgement and the mortality rates of the female ticks increased after RNAi of F0F1-type ATP synthase, NADH-ubiquinone oxidoreductase, cytochrome C, or apoptosis-inducing factor (AIF). The corresponding engorged weights, oviposition amounts, and egg hatching rates of the female ticks decreased after RNAi. Interference of the expression of AIF in engorged ticks by RNAi showed that the degeneration of salivary glands of female ticks was slowed down.