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COVID-19 has forced rapid clinical translation of novel vaccine technologies, principally mRNA vaccines, that have resulted in meaningful efficacy and adequate safety in response to the global pandemic. Notwithstanding this success, there remains an opportunity for innovation in vaccine technology to address current limitations and meet the challenges of inevitable future pandemics. We describe a universal vaccine cell (UVC) rationally designed to mimic the natural physiologic immunity induced post viral infection of host cells. Induced pluripotent stem cells were CRISPR engineered to delete MHC-I expression and simultaneously overexpress a NK Ligand adjuvant to increase rapid cellular apoptosis which was hypothesized to enhance viral antigen presentation in the resulting immune microenvironment leading to a protective immune response. Cells were further engineered to express the parental variant WA1/2020 SARS-CoV-2 spike protein as a representative viral antigen prior to irradiation and cryopreservation. The cellular vaccine was then used to immunize non-human primates in a standard 2-dose, IM injected prime + boost vaccination with 1e8 cells per 1 ml dose resulting in robust neutralizing antibody responses (1e3 nAb titers) with decreasing levels at 6 months duration. Similar titers generated in this established NHP model have translated into protective human neutralizing antibody levels in SARS-Cov-2 vaccinated individuals. Animals vaccinated with WA1/2020 spike antigens were subsequently challenged with 1.0 × 105 TCID50 infectious Delta (B.1.617.2) SARS-CoV-2 in a heterologous challenge which resulted in an approximately 3-log order decrease in viral RNA load in the lungs. These heterologous viral challenge results reflect the ongoing real-world experience of original variant WA1/2020 spike antigen vaccinated populations exposed to rapidly emerging variants like Delta and now Omicron. This cellular vaccine is designed to be a rapidly scalable cell line with a modular poly-antigenic payload to allow for practical, large-scale clinical manufacturing and use in an evolving viral variant environment. Human clinical translation of the UVC is being actively explored for this and potential future pandemics.

Despite rapid clinical translation of COVID-19 vaccines in response to the global pandemic, an opportunity remains for vaccine technology innovation to address current limitations and meet challenges of inevitable future pandemics. We describe a universal vaccine cell (UVC) genetically engineered to mimic natural physiological immunity induced upon viral infection of host cells. Cells engineered to express the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike as a representative viral antigen induce robust neutralizing antibodies in immunized non-human primates. Similar titers generated in this established non-human primate (NHP) model have translated into protective human neutralizing antibody levels in SARS-CoV-2-vaccinated individuals. Animals vaccinated with ancestral spike antigens and subsequently challenged with SARS-CoV-2 Delta variant in a heterologous challenge have an approximately 3 log decrease in viral subgenomic RNA in the lungs. This cellular vaccine is designed as a scalable cell line with a modular poly-antigenic payload, allowing for rapid, large-scale clinical manufacturing and use in an evolving viral variant environment.

Adoptive cellular therapy using genetically engineered immune cells holds tremendous promise for the treatment of advanced cancers. While the number of available receptors targeting tumor specific antigens continues to grow, the current reliance on viral vectors for clinical production of engineered immune cells remains a significant bottleneck limiting translation of promising new therapies. Here, we describe an optimized methodology for efficient CRISPR-Cas9 based, non-viral engineering of primary human T cells that overcomes key limitations of previous approaches. By synergizing temporal optimization of reagent delivery, reagent composition, and integration mechanism, we achieve targeted integration of large DNA cargo at efficiencies nearing those of viral vector platforms with minimal toxicity. CAR-T cells generated using our approach are highly functional and elicit potent anti-tumor cytotoxicity in vitro and in vivo. Importantly, our method is readily adaptable to cGMP compliant manufacturing and clinical scale-up, offering a near-term alternative to the use of viral vectors for production of genetically engineered T cells for cancer immunotherapy.

Recombinant adeno-associated viruses (rAAVs) are one of the most commonly used vectors for a variety of gene therapy applications. In the last 2 decades, research focused primarily on the characterization and isolation of new cap, genes resulting in hundreds of natural and engineered AAV capsid variants, while the rep gene, the other major AAV open reading frame, has been less studied. This is due to the fact that the rep gene from AAV serotype 2 (AAV2) enables the single-stranded DNA packaging of recombinant genomes into most AAV serotype and engineered capsids. However, a major by-product of all vector productions is empty AAV capsids, lacking the encapsidated vector genome, especially for non-AAV2 vectors. Despite the packaging process being considered the rate-limiting step for rAAV production, none of the rep genes from the other AAV serotypes have been characterized for their packaging efficiency. Thus, in this study AAV2 rep was replaced with the rep gene of a select number of AAV serotypes. However, this led to a lowering of capsid protein expression, relative to the standard AAV2-rep system. In further experiments the 3′ end of the AAV2 rep gene was reintroduced to promote increased capsid expression and a series of chimeras between the different AAV Rep proteins were generated and characterized for their vector genome packaging ability. The utilization of these novel Rep hybrids increased the percentage of genome containing (full) capsids approximately 2- to -4-fold for all of the non-AAV2 serotypes tested. Thus, these Rep chimeras could revolutionize rAAV production. IMPORTANCE A major by-product of all adeno-associated virus (AAV) vector production systems are “empty” capsids, void of the desired therapeutic gene, and thus do not provide any curative benefit for the treatment of the targeted disease. In fact, empty capsids can potentially elicit additional immune responses in vivo gene therapies if not removed by additional purification steps. Thus, there is a need to increase the genome packaging efficiency and reduce the number of empty capsids from AAV biologics. The novel Rep hybrids from different AAV serotypes described in this study are capable of reducing the percentage of empty capsids in all tested AAV serotypes and improve overall yields of genome-containing AAV capsids at the same time. They can likely be integrated easily into existing AAV manufacturing protocols to optimize the production of the generated AAV gene therapy products.

Adoptive transfer of tumor-infiltrating lymphocytes (TIL) fails to consistently elicit tumor rejection. Manipulation of intrinsic factors that inhibit T cell effector function and neoantigen recognition may therefore improve TIL therapy outcomes. We previously identified the cytokine-induced SH2 protein (CISH) as a key regulator of T cell functional avidity in mice. Here, we investigate the mechanistic role of CISH in regulating human T cell effector function in solid tumors and demonstrate that CRISPR/Cas9 disruption of CISH enhances TIL neoantigen recognition and response to checkpoint blockade.Single-cell gene expression profiling was used to identify a negative correlation between high CISH expression and TIL activation in patient-derived TIL. A GMP-compliant CRISPR/Cas9 gene editing process was developed to assess the impact of CISH disruption on the molecular and functional phenotype of human peripheral blood T cells and TIL. Tumor-specific T cells with disrupted Cish function were adoptively transferred into tumor-bearing mice and evaluated for efficacy with or without checkpoint blockade.CISH expression was associated with T cell dysfunction. CISH deletion using CRISPR/Cas9 resulted in hyper-activation and improved functional avidity against tumor-derived neoantigens without perturbing T cell maturation. Cish knockout resulted in increased susceptibility to checkpoint blockade in vivo.CISH negatively regulates human T cell effector function, and its genetic disruption offers a novel avenue to improve the therapeutic efficacy of adoptive TIL therapy.This study was funded by Intima Bioscience, U.S. and in part through the Intramural program CCR at the National Cancer Institute.

PD-1 acts as a negative regulator of T cell-mediated immune responses in the setting of persistent antigen expression, including cancer and chronic pathogen infections. Antibody-mediated blockade of the PD-1/PD-L1 axis benefits a subset of patients with highly immunogenic malignancies; however, many patients fail to respond due to a requirement for expression of the cell surface ligand PD-L1 within the tumor microenvironment. CISH is a member of a new class of intra-cellular immune checkpoint molecules that function downstream of the T cell receptor to regulate antigen-specific effector functions, including reactivity to cancer neoantigens. Herein, we employed multiplex CRISPR editing of primary human T cells to systematically compare the function of CISH deletion relative to PDCD1 (the gene encoding PD-1) and/or VSIG9 (the gene encoding TIGIT) in a model of neoantigen-mediated cancer cell cytolysis. PD-1 and TIGIT disruption enhanced cytolytic activity exclusively in the setting of high PD-L1 expression. In contrast, CISH inactivation enhanced antigen-specific cytolysis of tumor cells regardless of PD-L1 expression, including outperforming PD-1 and TIGIT disruption even in the presence of high PD-L1 tumor cells. Furthermore, we observed a synergistic increase in tumor cell killing when CISH and PD-1 or TIGIT are inactivated in combination, supporting the notion that these immune checkpoints regulate non-redundant pathways of T cell activation. Together, these data demonstrate that the intra-cellular immune checkpoint protein CISH can potentially enhance anti-tumor responses against a broad range of cancer types regardless of PD-L1 biomarker status.

The a priori T cell repertoire and immune response against SARS-CoV-2 viral antigens may explain the varying clinical course and prognosis of patients having a mild COVID-19 infection as opposed to those developing more fulminant multisystem organ failure and associated mortality. Using a novel SARS-Cov-2-specific artificial antigen presenting cell (aAPC), coupled with a rapid expansion protocol (REP) as practiced in tumor infiltrating lymphocytes (TIL) therapy, we generate an immune catalytic quantity of Virus Induced Lymphocytes (VIL). Using T cell receptor (TCR)-specific aAPCs carrying co-stimulatory molecules and major histocompatibility complex (MHC) class-I immunodominant SARS-CoV-2 peptide-pentamer complexes, we expand virus-specific VIL derived from peripheral blood mononuclear cells (PBMC) of convalescent COVID-19 patients up to 1000-fold. This is achieved in a clinically relevant 7-day vein-to-vein time-course as a potential adoptive cell therapy (ACT) for COVID-19. We also evaluate this approach for other viral pathogens using Cytomegalovirus (CMV)-specific VIL from donors as a control. Rapidly expanded VIL are enriched in virus antigen-specificity and show an activated, polyfunctional cytokine profile and T effector memory phenotype which may contribute to a robust immune response. Virus-specific T cells can also be delivered allogeneically via MHC-typing and patient human leukocyte antigen (HLA)-matching to provide pragmatic treatment in a large-scale therapeutic setting. These data suggest that VIL may represent a novel therapeutic option that warrants further clinical investigation in the armamentarium against COVID-19 and other possible future pandemics.

Recombinant Adeno-associated viruses (rAAVs) are one of the most commonly used vectors for a variety of gene therapy applications. In the last two decades research focused primarily on the characterization and isolation of new cap genes resulting in hundreds of natural and engineered AAV capsid variants while the rep gene, the other major AAV open reading frame, has been less studied. This is due to the fact that the rep gene from AAV serotype 2 (AAV2) enables the ssDNA packaging of recombinant genomes into most AAV serotype and engineered capsids. However, a major byproduct of all vector productions is empty AAV capsids, lacking the encapsidated vector genome, especially for non-AAV2 vectors. Despite the packaging process being considered the rate-limiting step for rAAV production, none of the rep genes from the other AAV serotypes have been characterized for their packaging efficiency. Thus, in this study AAV2 rep was replaced with the rep gene of a select number of AAV serotypes. However, this led to a lowering of capsid protein expression, relative to the standard AAV2-rep system. In further experiments the 3’end of the AAV2 rep gene was reintroduced to promote increased capsid expression and a series of chimeras between the different AAV Rep proteins were generated and characterized for their vector genome packaging ability. The utilization of these novel Rep hybrids increased the percentage of genome containing (full) capsids ~2-4-fold for all of the non-AAV2 serotypes tested. Thus, these Rep chimeras could revolutionize rAAV production.ImportanceA major byproduct of all Adeno-associated virus (AAV) vector production systems are “empty” capsids, void of the desired therapeutic gene, and thus do not provide any curative benefit for the treatment of the targeted disease. In fact, empty capsids can potentially elicit additional immune responses in vivo gene therapies if not removed by additional purification steps. Thus, there is a need to increase the genome packaging efficiency and reduce the number of empty capsids from AAV biologics. The novel Rep hybrids from different AAV serotypes described in this study are capable of reducing the percentage of empty capsids in all tested AAV serotypes and improve overall yields of genome-containing AAV capsids at the same time. They can likely be integrated easily into existing AAV manufacturing protocols to optimize the production of the generated AAV gene therapy products.

Argonautes are nucleases that can be programmed by short oligonucleotides to cleave complementary sequences. Here, we performed an unbiased bioinformatic search to mine bacterial genomes for prokaryotic Argonautes (pAgos) harboring a PIWI domain. Our search identified 3,033 pAgos in total, of which 1,464 portend to the subgroup of long pAgos with more than 600 amino acids. We purified a subset of 49 pAgos which were found in proximity to helicases and tested their nuclease activity in vitro. Ten of these were active towards single-stranded DNA substrates and this activity could be programmed by exogenous guide DNAs or RNAs. Cleavage of double-stranded plasmid DNA was much less readily observed and was fostered by elevated temperatures or exogenous addition of a DNA single-strand binding protein (ET-SSB). The efficiency of pAgo-mediated plasmid cleavage was dependent on the DNA target sequence as well as the surrounding sequence, suggesting that unwinding of the DNA double helix was a limiting factor. Intriguingly, we identified a cluster of pAgos from the Clostridial clade which was active at 37°C and activity was enhanced by exogenous ET-SSB. This suggests that Clostridial pAgos may be particularly suited to catalyze DNA double-strand cleavage and implies that such pAgos may be repurposed as gene editing tools in future.

A major hurdle to the therapeutic potential of AAV in gene therapy lies in achieving clinically meaningful AAV doses, and secondarily, the ability to manufacture commercially viable titers of AAV to support this. By virtue of neutralizing antibodies against AAV that impede patient repeat dosing, the dose of AAV for in vivo gene delivery has been high, which has resulted in unfortunate recent safety concerns and deaths in patients given higher-dose AAV gene therapy., Adeno-associated viruses (AAVs) have attracted significant attention in the field of gene and cell therapy due to their use in highly effective delivery of therapeutic genes into human cells. The ability to generate recombinant AAV (rAAV) vectors comprising unique or substituted protein sequences has led to the development of capsid variants with improved therapeutic properties. Seeking novel AAV vectors capable of enhanced transduction for therapeutic applications, we have developed a series of unique capsid variants termed AAV X-Vivo (AAV-XV) derived from chimeras of AAV12 VP1/2 sequences and the VP3 sequence of AAV6. These AAV variants showed enhanced infection compared to the wild-type parental viruses of human primary T cells, hematopoietic stem cells (HSCs), and neuronal cell lines, and superiority over AAV6 for genomic integration of DNA sequences by AAV alone or in combination with CRISPR gene editing. AAV-XV variants demonstrate transduction efficiency equivalent to AAV6 but at 100-fold-lower multiplicities of infection (MOI), enabling T cell engineering at low AAV doses. The protein coding sequence of these novel AAV chimeras revealed disruptions within the assembly-activating protein (AAP), which likely accounted for the observed lower virus yield. A series of genome alterations, reverting the AAP sequence back to that of wild-type AAV6, had a negative impact on the enhanced transduction seen with AAV-VX, indicating overlapping functions within this sequence for both viral assembly and effective T cell transduction. Our findings show these AAV-XV variants are highly efficient at cell transduction at low doses and demonstrate the importance of the AAP coding region in both viral particle assembly and cell infection. IMPORTANCE A major hurdle to the therapeutic potential of AAV in gene therapy lies in achieving clinically meaningful AAV doses, and secondarily, the ability to manufacture commercially viable titers of AAV to support this. By virtue of neutralizing antibodies against AAV that impede patient repeat dosing, the dose of AAV for in vivo gene delivery has been high, which has resulted in unfortunate recent safety concerns and deaths in patients given higher-dose AAV gene therapy. We have generated new AAV variants possessing unique combinations of capsid proteins for gene and cell therapy applications termed AAV-XV, which have high levels of cell transduction and gene delivery at lower MOI. Furthermore, we demonstrate a novel finding, and an important consideration for recombinant AAV design, that a region of the AAV genome encoding the capsid viral protein and AAP is critical for both virus yield and the enhancement of infection/transduction.

Background Neoantigen-specific T cells isolated from tumors have shown promise clinically but fail to consistently elicit durable tumor regression. Expression of the intracellular checkpoint CISH is elevated in human tumor infiltrating lymphocytes (TIL) and has been shown to inhibit neoantigen reactivity in murine TIL. Methods To explore CISH function in human T cells we developed a CRISPR/Cas9-based strategy to knockout (KO) CISH in human T cells with high-efficiency (>90%) and without detectable off-target editing. Results CISH KO in peripheral blood T cells enhanced proliferation, cytokine polyfunctionality, and cytotoxicity in vitro. To determine if CISH KO similarly enhances TIL function, we developed a clinical-scale, GMP-compliant manufacturing process for CISH disruption in primary human TIL. In process validation runs we achieved CISH KO efficiencies >90% without detectable off-target editing while maintaining high viability and expansion. Compared to WT controls, CISH KO in patient-derived TIL demonstrated increased proliferation, T cell receptor (TCR) avidity, neoantigen recognition, and unmasked reactivity to common p53 mutations. Hyperactivation in CISH KO TIL did not increase differentiation, suggesting that CISH KO may uncouple activation and differentiation pathways. Single cell profiling identifies a pattern of CISH expression inverse to key regulators of activation, and CISH KO in human TIL increases PD1 expression. Adoptive transfer of Cish KO T cells synergistically combines with PD1 inhibition resulting in durable tumor regression in mice, highlighting orthogonal dual cell surface and intracellular checkpoint inhibition as a novel combinatorial approach for T cell immunotherapy. Conclusions These pre-clinical data offer new insight into neoantigen recognition and serve as the basis for a recently initiated human clinical trial at the University of Minnesota (NCT04426669) evaluating inhibition of the novel intracellular immune checkpoint CISH in a CRISPR-engineered, neoantigen-specific T cell therapy for solid tumors. Updates from the clinical trial will be highlighted. Trial Registration NCT04426669

Adeno-associated viruses (AAV) have attracted significant attention in the field of gene and cell therapy due to highly effective delivery of therapeutic genes into human cells. The ability to generate recombinant AAV vectors compromised of unique or substituted protein sequences has led to the development of capsid variants with improved therapeutic properties. Seeking a novel AAV capable of enhanced transduction of human T cells for applications in immunotherapy, we have developed a unique capsid variant termed AAV X-Vivo (AAV-XV) that is a chimera of AAV12 VP1/2 sequences and the VP3 sequence of AAV6. This AAV chimera showed enhanced infection of human primary T cells and hematopoietic stem cells, and superiority over wildtype AAV6 for the genomic integration of DNA sequences either by AAV alone or in combination with CRISPR gene editing. AAV-XV demonstrated transduction efficiency equivalent to AAV6 at multiplicities of infection 2 logs lower, enabling T cell engineering at low AAV doses. Analyzing the protein coding sequence of AAV-XV revealed disruptions within the assembly-activating protein (AAP) which likely accounted for observed lower virus yield. A series of genome alterations reverting the AAP sequence back to wildtype had a negative impact on the enhanced transduction seen with AAV-VX, indicating overlapping functions within this sequence for both viral assembly and effective T cell transduction. Our findings show that AAV-XV is highly efficient at T cell engineering at low AAV dose and demonstrates the importance of AAP coding region in both viral particle assembly and cell infection.IMPORTANCEA major hurdle to the therapeutic potential of AAV in gene therapy lies in achieving clinically meaningful AAV doses, and secondarily, ability to manufacture commercially viable titers of AAV to support this. By virtue of neutralizing antibodies against AAV that impede patient repeat-dosing, the dose of AAV for in vivo gene delivery has been high, which has resulted in unfortunate recent safety concerns and deaths in patients given higher-dose AAV gene therapy. We have generated a new AAV variant possessing a unique combination of capsid proteins for ex-vivo application termed AAV-XV, which delivers high levels of cell transduction and gene delivery at a lower MOI. Furthermore, we demonstrate a novel finding, and an important consideration for recombinant AAV design, that a region of the AAV genome encoding the capsid protein and AAP gene is critical for both virus yield and the enhancement of infection/transduction.

While neoantigen-specific tumor infiltrating lymphocytes (TIL) can be derived from in antigen-expressing tumors, their adoptive transfer fails to consistently elicit durable tumor regression. There has been much focus on the role of activation/exhaustion markers such as PD1, CD39 and TOX in TIL senescence. We found these markers were inversely expressed to Cytokine-Induced SH2 protein (CISH), a negative regulator of TCR signaling and tumor immunity in mice. To evaluate the physiological role of CISH in human TIL we developed a high-efficiency CRIPSR-based method to knock out CISH in fully mature TIL. CISH KO resulted in increased T cell receptor (TCR) avidity, tumor cytolysis and neoantigen recognition. CISH expression in the tumor resections correlated with TIL inactivity against p53 hotspot mutations and CISH KO in TIL unmasked reactivity against these universal neoantigens. While CISH KO resulted in T cell hyperactivation and expansion it did not alter maturation, perhaps by preferential PLCγ-1 and not AKT inhibition. Lastly, CISH KO in T cells increased PD1 expression and the adoptive transfer of Cish KO T cells synergistically combines with PD1 antibody blockade resulting in durable tumor regression and survival in a preclinical animal model. These data offer new insights into the regulation of neoantigen recognition, expression of activation/exhaustion markers, and functional/maturation signals in tumor-specific T cells.

While neoantigen-specific tumor infiltrating lymphocytes (TIL) can be derived from in antigen-expressing tumors, their adoptive transfer fails to consistently elicit durable tumor regression. There has been much focus on the role of activation/exhaustion markers such as PD1, CD39 and TOX in TIL senescence. We found these markers were inversely expressed to Cytokine-Induced SH2 protein (CISH), a negative regulator of TCR signaling and tumor immunity in mice. To evaluate the physiological role of CISH in human TIL we developed a high-efficiency CRIPSR-based method to knock out CISH in fully mature TIL. CISH KO resulted in increased T cell receptor (TCR) avidity, tumor cytolysis and neoantigen recognition. CISH expression in the tumor resections correlated with TIL inactivity against p53 hotspot mutations and CISH KO in TIL unmasked reactivity against these universal neoantigens. While CISH KO resulted in T cell hyperactivation and expansion it did not alter maturation, perhaps by preferential PLCγ-1 and not AKT inhibition. Lastly, CISH KO in T cells increased PD1 expression and the adoptive transfer of Cish KO T cells synergistically combines with PD1 antibody blockade resulting in durable tumor regression and survival in a preclinical animal model. These data offer new insights into the regulation of neoantigen recognition, expression of activation/exhaustion markers, and functional/maturation signals in tumor-specific T cells.

Lu et al. disrupt the interaction between miR-155 and the transcription factor PU.1 by specifically removing miR-155–binding site from PU.1 mRNA in mice. They show that this interaction is required for plasma cell formation and extrafollicular response to immunization in vivo., A single microRNA (miRNA) can regulate the expression of many genes, though the level of repression imparted on any given target is generally low. How then is the selective pressure for a single miRNA/target interaction maintained across long evolutionary distances? We addressed this problem by disrupting in vivo the interaction between miR-155 and PU.1 in mice. Remarkably, this interaction proved to be key to promoting optimal T cell–dependent B cell responses, a previously unrecognized role for PU.1. Mechanistically, miR-155 inhibits PU.1 expression, leading to Pax5 down-regulation and the initiation of the plasma cell differentiation pathway. Additional PU.1 targets include a network of genes whose products are involved in adhesion, with direct links to B–T cell interactions. We conclude that the evolutionary adaptive selection of the miR-155–PU.1 interaction is exercised through the effectiveness of terminal B cell differentiation.

Tracking antigen-specific B cells in vivo since the onset of antigen stimulation shows that miR-155 regulates the early expansion of B-blasts and later on the optimal proliferation and survival of plasmablasts in a B-cell–intrinsic manner., A fast antibody response can be critical to contain rapidly dividing pathogens. This can be achieved by the expansion of antigen-specific B cells in response to T-cell help followed by differentiation into plasmablasts. MicroRNA-155 (miR-155) is required for optimal T-cell–dependent extrafollicular responses via regulation of PU.1, although the cellular processes underlying this defect are largely unknown. Here, we show that miR-155 regulates the early expansion of B-blasts and later on the survival and proliferation of plasmablasts in a B-cell–intrinsic manner, by tracking antigen-specific B cells in vivo since the onset of antigen stimulation. In agreement, comparative analysis of the transcriptome of miR-155–sufficient and miR-155–deficient plasmablasts at the peak of the response showed that the main processes regulated by miR-155 were DNA metabolic process, DNA replication, and cell cycle. Thus, miR-155 controls the extent of the extrafollicular response by regulating the survival and proliferation of B-blasts, plasmablasts and, consequently, antibody production.

Voltage-gated proton currents regulate generation of reactive oxygen species (ROS) in phagocytic cells. In B cells, stimulation of the B cell antigen receptor (BCR) results in the production of ROS that participate in B cell activation, but the involvement of proton channels is unknown. We report here that the voltage-gated proton channel HVCN1 associated with the BCR complex and was internalized together with the BCR after activation. BCR-induced generation of ROS was lower in HVCN1-deficient B cells, which resulted in attenuated BCR signaling via impaired BCR-dependent oxidation of the tyrosine phosphatase SHP-1. This resulted in less activation of the kinases Syk and Akt, impaired mitochondrial respiration and glycolysis, and diminished antibody responses in vivo. Our findings identify unanticipated functions for proton channels in B cells and demonstrate the importance of ROS in BCR signaling and downstream metabolism.

Genome engineering has been greatly enhanced by the availability of Cas9 endonuclease that can be targeted to almost any genomic locus using so called guide RNAs (gRNAs). However, the introduction of foreign DNA sequences to tag an endogenous gene is still cumbersome as it requires the synthesis or cloning of homology templates. Here we present a strategy that enables the tagging of endogenous loci using one generic donor plasmid. It contains the tag of interest flanked by two gRNA recognition sites that allow excision of the tag from the plasmid. Co-transfection of cells with Cas9, a gRNA specifying the genomic locus of interest, the donor plasmid and a cassette-specific gRNA triggers the insertion of the tag by a homology-independent mechanism. The strategy is efficient and delivers clones that display a predictable integration pattern. As showcases we generated NanoLuc luciferase- and TurboGFP-tagged reporter cell lines., CRISPR-Cas9 has greatly enhanced genome engineering however gene tagging can still be cumbersome due to a requirement for homology donors. Here the authors introduce a generic system for gene tagging that does not require homology between the donor and the genomic target site.

Rac1 and Rac2 GTPases transduce signals from multiple receptors leading to cell migration, adhesion, proliferation, and survival. In the absence of Rac1 and Rac2, B cell development is arrested at an IgD− transitional B cell stage that we term transitional type 0 (T0). We show that T0 cells cannot enter the white pulp of the spleen until they mature into the T1 and T2 stages, and that this entry into the white pulp requires integrin and chemokine receptor signaling and is required for cell survival. In the absence of Rac1 and Rac2, transitional B cells are unable to migrate in response to chemokines and cannot enter the splenic white pulp. We propose that loss of Rac1 and Rac2 causes arrest at the T0 stage at least in part because transitional B cells need to migrate into the white pulp to receive survival signals. Finally, we show that in the absence of Syk, a kinase that transduces B cell antigen receptor signals required for positive selection, development is arrested at the same T0 stage, with transitional B cells excluded from the white pulp. Thus, these studies identify a novel developmental checkpoint that coincides with B cell positive selection.

Neomorphic mutations targeting R132 of the TCA cycle enzyme, IDH1, have been identified in multiple cancer types and lead to a build up of (R)-2-hydroxyglutarate ((R)-2HG). Several mechanisms have been proposed to account for mutant-IDH1-mediated transformation: due to its structure similarity with alpha-ketoglutarate (α-KG), (R)-2HG can inhibit α-KG-dependent enzymes that act as tumour suppressors such as TET2 or EGLN, (R)-2HG might inhibit electron transport chain function, or rapid (R)-2HG generation may deplete the cellular pool of α-KG leading to depletion of NADPH. Recently, it has also emerged that IDH1 may be required to catalyse a series of back reactions through TCA cycle components (reductive decarboxylation) to facilitate fatty acid synthesis from media glutamine, particularly under hypoxic conditions. According to some reports in the literature, heterodimer formation between mutant an wild-type alleles of IDH1 is important for the production of high levels of (R)-2HG. We were interested in exploring novel ways to target tumour cells bearing mutant IDH1 alleles that were distinct from the obvious opportunity available to identify mutant-specific IDH1 inhibitors. The potential metabolic vulnerabilities of mutant IDH1 cancers raised the possibility that wild-type IDH1 might be essential for tumourigenesis or tumour maintenance in this context. We therefore employed Horizon's rAAV-mediated homologous recombination gene engineering technology to generate conditional knockouts of the IDH1+ or IDH1R132C alleles in the fibrosarcoma cell line, HT1080. Despite several attempts, we did not recover a correctly targeted conditional knock-out of the IDH1R132C allele. However, we did recover a derivative cell line where the wild-type allele had been converted into a loxP-flanked IDH1R132C allele, and two independent clones where the wild-type allele had been flanked with loxP sites. Interestingly, in these latter two clones, no expression of wild-type IDH1 was detectable. Here we present data characterising the growth of these clones under a variety of culture conditions, including the hypoxic state where IDH1 has been proposed to be important for enabling lipid synthesis from glutamine. We also demonstrate that HT1080 cells that express no detectable levels of wild-type IDH1 retain the ability to be tumorigenic in immuno-compromised mice. These data suggest that the wild-type allele of IDH1 is dispensable for cancer formation. Citation Format: Julie A. Wickenden, Paul Russell, Amy Smith, Tom Henley, Jane Elliott, Dan Gitterman, Mark Stockdale, Christine Schofield, Chris Torrance, Jonathan D. Moore. Wild-type IDH1: A molecular target in IDH1 mutant cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4620. doi:10.1158/1538-7445.AM2014-4620