An evolved AAV variant enables efficient genetic engineering of murine T cells.

Precise targeting of large transgenes to T cells using homology-directed repair has been transformative for adoptive cell therapies and T cell biology. Delivery of DNA templates via adeno-associated virus (AAV) has greatly improved knockin efficiencies, but the tropism of current AAV serotypes restricts their use to human T cells employed in immunodeficient mouse models. To enable targeted knockins in murine T cells, we evolved Ark313, a synthetic AAV that exhibits high transduction efficiency in murine T cells. We performed a genome-wide knockout screen and identified QA2 as an essential factor for Ark313 infection. We demonstrate that Ark313 can be used for nucleofection-free DNA delivery, CRISPR-Cas9-mediated knockouts, and targeted integration of large transgenes. Ark313 enables preclinical modeling of Trac-targeted CAR-T and transgenic TCR-T cells in immunocompetent models. Efficient gene targeting in murine T cells holds great potential for improved cell therapies and opens avenues in experimental T cell immunology.

Modular pooled discovery of synthetic knockin sequences to program durable cell therapies.

Chronic stimulation can cause T cell dysfunction and limit the efficacy of cellular immunotherapies. Improved methods are required to compare large numbers of synthetic knockin (KI) sequences to reprogram cell functions. Here, we developed modular pooled KI screening (ModPoKI), an adaptable platform for modular construction of DNA KI libraries using barcoded multicistronic adaptors. We built two ModPoKI libraries of 100 transcription factors (TFs) and 129 natural and synthetic surface receptors (SRs). Over 30 ModPoKI screens across human TCR- and CAR-T cells in diverse conditions identified a transcription factor AP4 (TFAP4) construct that enhanced fitness of chronically stimulated CAR-T cells and anti-cancer function in vitro and in vivo. ModPoKI's modularity allowed us to generate an ∼10,000-member library of TF combinations. Non-viral KI of a combined BATF-TFAP4 polycistronic construct enhanced fitness. Overexpressed BATF and TFAP4 co-occupy and regulate key gene targets to reprogram T cell function. ModPoKI facilitates the discovery of complex gene constructs to program cellular functions.

RASA2 ablation in T cells boosts antigen sensitivity and long-term function.

The efficacy of adoptive T cell therapies for cancer treatment can be limited by suppressive signals from both extrinsic factors and intrinsic inhibitory checkpoints1,2. Targeted gene editing has the potential to overcome these limitations and enhance T cell therapeutic function3-10. Here we performed multiple genome-wide CRISPR knock-out screens under different immunosuppressive conditions to identify genes that can be targeted to prevent T cell dysfunction. These screens converged on RASA2, a RAS GTPase-activating protein (RasGAP) that we identify as a signalling checkpoint in human T cells, which is downregulated upon acute T cell receptor stimulation and can increase gradually with chronic antigen exposure. RASA2 ablation enhanced MAPK signalling and chimeric antigen receptor (CAR) T cell cytolytic activity in response to target antigen. Repeated tumour antigen stimulations in vitro revealed that RASA2-deficient T cells show increased activation, cytokine production and metabolic activity compared with control cells, and show a marked advantage in persistent cancer cell killing. RASA2-knockout CAR T cells had a competitive fitness advantage over control cells in the bone marrow in a mouse model of leukaemia. Ablation of RASA2 in multiple preclinical models of T cell receptor and CAR T cell therapies prolonged survival in mice xenografted with either liquid or solid tumours. Together, our findings highlight RASA2 as a promising target to enhance both persistence and effector function in T cell therapies for cancer treatment.

The bromodomain protein TRIM28 controls the balance between growth and invasiveness in melanoma.

Melanoma tumors are highly metastatic partly due to the ability of melanoma cells to transition between invasive and proliferative states. However, the mechanisms underlying this plasticity are still not fully understood. To identify new epigenetic regulators of melanoma plasticity, we combined data mining, tumor models, proximity proteomics, and CUT&RUN sequencing. We focus on the druggable family of bromodomain epigenetic readers and identify TRIM28 as a new regulator of melanoma plasticity. We find that TRIM28 promotes the expression of pro-invasive genes and that TRIM28 controls the balance between invasiveness and growth of melanoma cells. We demonstrate that TRIM28 acts via the transcription factor JUNB that directly regulates the expression of pro-invasive and pro-growth genes. Mechanistically, TRIM28 controls the expression of JUNB by negatively regulating its transcriptional elongation by RNA polymerase II. In conclusion, our results demonstrate that a TRIM28-JUNB axis controls the balance between invasiveness and growth in melanoma tumors and suggest that the bromodomain protein TRIM28 could be targeted to reduce tumor spread.

Auxilin is a novel susceptibility gene for congenital heart block which directly impacts fetal heart function.

OBJECTIVE: Neonatal lupus erythematosus (NLE) may develop after transplacental transfer of maternal autoantibodies with cardiac manifestations (congenital heart block, CHB) including atrioventricular block, atrial and ventricular arrhythmias, and cardiomyopathies. The association with anti-Ro/SSA antibodies is well established, but a recurrence rate of only 12%-16% despite persisting maternal autoantibodies suggests that additional factors are required for CHB development. Here, we identify fetal genetic variants conferring risk of CHB and elucidate their effects on cardiac function. METHODS: A genome-wide association study was performed in families with at least one case of CHB. Gene expression was analysed by microarrays, RNA sequencing and PCR and protein expression by western blot, immunohistochemistry, immunofluorescence and flow cytometry. Calcium regulation and connectivity were analysed in primary cardiomyocytes and cells induced from pleuripotent stem cells. Fetal heart performance was analysed by Doppler/echocardiography. RESULTS: We identified DNAJC6 as a novel fetal susceptibility gene, with decreased cardiac expression of DNAJC6 associated with the disease risk genotype. We further demonstrate that fetal cardiomyocytes deficient in auxilin, the protein encoded by DNAJC6, have abnormal connectivity and Ca2+ homoeostasis in culture, as well as decreased cell surface expression of the Cav1.3 calcium channel. Doppler echocardiography of auxilin-deficient fetal mice revealed cardiac NLE abnormalities in utero, including abnormal heart rhythm with atrial and ventricular ectopias, as well as a prolonged atrioventricular time intervals. CONCLUSIONS: Our study identifies auxilin as the first genetic susceptibility factor in NLE modulating cardiac function, opening new avenues for the development of screening and therapeutic strategies in CHB.

TRIM21 controls Toll-like receptor 2 responses in bone-marrow-derived macrophages.

TRIM21 is an interferon-stimulated E3 ligase that controls the activity of pattern-recognition signaling via ubiquitination of interferon regulatory factors and DDX41. Previous studies on the role of TRIM21 in innate immune responses have yielded contradictory results, suggesting that the role of TRIM21 is cell specific. Here, we report that bone-marrow-derived macrophages (BMDMs) generated from Trim21-/- mice have reduced expression of mature macrophage markers. Reflecting their reduced differentiation in response to macrophage colony-stimulating factor (M-CSF), Trim21-/- BMDMs had decreased expression of M-CSF signature genes. Although Trim21-/- BMDMs responded normally to Toll-like receptor 9 (TLR9) activation, they produced lower levels of pro-inflammatory cytokines in response to the TLR2 agonist PAM3CSK4. In line with this, the response to infection with the Bacillus Calmette-Guérin strain of Mycobacterium bovis was also diminished in Trim21-/- BMDMs. Our results indicate that TRIM21 controls responses to TLR2 agonists.

miR-31 regulates energy metabolism and is suppressed in T cells from patients with Sjögren's syndrome.

Systemic autoimmune diseases are characterized by the overexpression of type I IFN stimulated genes, and accumulating evidence indicate a role for type I IFNs in these diseases. However, the underlying mechanisms for this are still poorly understood. To explore the role of type I IFN regulated miRNAs in systemic autoimmune disease, we characterized cellular expression of miRNAs during both acute and chronic type I IFN responses. We identified a T cell-specific reduction of miR-31-5p levels, both after intramuscular injection of IFNß and in patients with Sjögren's syndrome (SjS). To interrogate the role of miR-31-51p in T cells we transfected human CD4+ T cells with a miR-31-5p inhibitor and performed metabolic measurements. This identified an increase in basal levels of glucose metabolism after inhibition of miR-31-5p. Furthermore, treatment with IFN-α also increased the basal levels of human CD4+ T-cell metabolism. In all, our results suggest that reduced levels of miR-31-5p in T cells of SjS patients support autoimmune T-cell responses during chronic type I IFN exposure.

Imiquimod induces ER stress and Ca(2+) influx independently of TLR7 and TLR8.

Endoplasmic reticulum (ER) stress is a physiological response to protein overload or misfolded proteins in the ER. Certain anti-cancer drugs, e.g. bortezomib and nelfinavir, induce ER stress implying that this could be a successful therapeutic strategy against several forms of cancer. To find novel ER-stress inducers we screened a panel of natural and synthetic Toll-like receptor (TLR) agonists against human keratinocytes and identified the anti-cancer drug imiquimod (IMQ) as a potent inducer of ER stress. Other TLR7 and TLR8 agonists, including resiquimod and gardiquimod, did not induce ER stress, demonstrating that IMQ induces ER stress independently of TLR7 and TLR8. We further confirmed this by showing that IMQ could still induce ER stress in mouse Tlr7(-/-) cells. IMQ also induced a rapid and transient influx of extracellular Ca(2+) together with the release of Ca(2+) from internal stores. Depletion of Ca(2+) from the ER is a known cause of ER stress suggesting that IMQ induces ER stress via depletion of ER Ca(2+). The ER-stress inducing property of IMQ is possibly of importance for its efficacy in treating basal cell carcinoma, in situ melanoma, and squamous cell carcinoma. Our data could potentially be harnessed for rational design of even more potent ER-stress inducers and new anti-cancer drugs.

EphB4 localises to the nucleus of prostate cancer cells.

The EphB4 receptor tyrosine kinase is over-expressed in a variety of different epithelial cancers including prostate where it has been shown to be involved in survival, migration and angiogenesis. We report here that EphB4 also resides in the nucleus of prostate cancer cell lines. We used in silico methods to identify a bipartite nuclear localisation signal (NLS) in the extracellular domain and a monopartite NLS sequence in the intracellular kinase domain of EphB4. To determine whether both putative NLS sequences were functional, fragments of the EphB4 sequence containing each NLS were cloned to create EphB4NLS-GFP fusion proteins. Localisation of both NLS-GFP proteins to the nuclei of transfected cells was observed, demonstrating that EphB4 contains two functional NLS sequences. Mutation of the key amino residues in both NLS sequences resulted in diminished nuclear accumulation. As nuclear translocation is often dependent on importins we confirmed that EphB4 and importin-α can interact. To assess if nuclear EphB4 could be implicated in gene regulatory functions potential EphB4-binding genomic loci were identified using chromatin immunoprecipitation and Lef1 was confirmed as a potential target of EphB4-mediated gene regulation. These novel findings add further complexity to the biology of this important cancer-associated receptor.

Localized in vivo gene editing of murine cancer-associated fibroblasts.

Discovering the role of fibroblasts residing in the tumor microenvironment (TME) requires controlled, localized perturbations because fibroblasts play critical roles in regulating immunity and tumor biology at multiple sites. Systemic perturbations can lead to unintended, confounding secondary effects, and methods to locally genetically engineer fibroblasts are lacking. To specifically investigate murine stromal cell perturbations restricted to the TME, we developed an adeno-associated virus (AAV)-based method to target any gene-of-interest in fibroblasts at high efficiency (>80%). As proof of concept, we generated single (sKO) and double gene KOs (dKO) of Osmr, Tgfbr2, and Il1r1 in cancer-associated fibroblasts (CAFs) and investigated how their cell states and those of other cells of the TME subsequently change in mouse models of melanoma and pancreatic ductal adenocarcinoma (PDAC). Furthermore, we developed an in vivo knockin-knockout (KIKO) strategy to achieve long-term tracking of CAFs with target gene KO via knocked-in reporter gene expression. This validated in vivo gene editing toolbox is fast, affordable, and modular, and thus holds great potential for further exploration of gene function in stromal cells residing in tumors and beyond.

Ultra-high efficiency T cell reprogramming at multiple loci with SEED-Selection.

Multiplexed reprogramming of T cell specificity and function can generate powerful next-generation cellular therapies. However, current manufacturing methods produce heterogenous mixtures of partially engineered cells. Here, we develop a one-step process to enrich for unlabeled cells with knock-ins at multiple target loci using a family of repair templates named Synthetic Exon/Expression Disruptors (SEEDs). SEED engineering associates transgene integration with the disruption of a paired endogenous surface protein, allowing non-modified and partially edited cells to be immunomagnetically depleted (SEED-Selection). We design SEEDs to fully reprogram three critical loci encoding T cell specificity, co-receptor expression, and MHC expression, with up to 98% purity after selection for individual modifications and up to 90% purity for six simultaneous edits (three knock-ins and three knockouts). These methods are simple, compatible with existing clinical manufacturing workflows, and can be readily adapted to other loci to facilitate production of complex gene-edited cell therapies.

Scalable intracellular delivery via microfluidic vortex shedding enhances the function of chimeric antigen receptor T-cells.

Adoptive chimeric antigen receptor T-cell (CAR-T) therapy is transformative and approved for hematologic malignancies. It is also being developed for the treatment of solid tumors, autoimmune disorders, heart disease, and aging. Despite unprecedented clinical outcomes, CAR-T and other engineered cell therapies face a variety of manufacturing and safety challenges. Traditional methods, such as lentivirus transduction and electroporation, result in random integration or cause significant cellular damage, which can limit the safety and efficacy of engineered cell therapies. We present hydroporation as a gentle and effective alternative for intracellular delivery. Hydroporation resulted in 1.7- to 2-fold higher CAR-T yields compared to electroporation with superior cell viability and recovery. Hydroporated cells exhibited rapid proliferation, robust target cell lysis, and increased pro-inflammatory and regulatory cytokine secretion in addition to improved CAR-T yield by day 5 post-transfection. We demonstrate that scaled-up hydroporation can process 5 × 108 cells in less than 10 s, showcasing the platform as a viable solution for high-yield CAR-T manufacturing with the potential for improved therapeutic outcomes.

Scalable intracellular delivery via microfluidic vortex shedding enhances the function of chimeric antigen receptor T-cells.

Adoptive chimeric antigen receptor T-cell (CAR-T) therapy is transformative and approved for hematologic malignancies. It is also being developed for the treatment of solid tumors, autoimmune disorders, heart disease, and aging. Despite unprecedented clinical outcomes, CAR-T and other engineered cell therapies face a variety of manufacturing and safety challenges. Traditional methods, such as lentivirus transduction and electroporation, result in random integration or cause significant cellular damage, which can limit the safety and efficacy of engineered cell therapies. We present hydroporation as a gentle and effective alternative for intracellular delivery. Hydroporation resulted in 1.7- to 2-fold higher CAR-T yields compared to electroporation with superior cell viability and recovery. Hydroporated cells exhibited rapid proliferation, robust target cell lysis, and increased pro-inflammatory and regulatory cytokine secretion in addition to improved CAR-T yield by day 5 post-transfection. We demonstrate that scaled-up hydroporation can process 5 x 108 cells in less than 10 s, showcasing the platform as a viable solution for high-yield CAR-T manufacturing with the potential for improved therapeutic outcomes.

Light induced expression of gRNA allows for optogenetic gene editing of T lymphocytes in vivo.

There is currently a lack of tools capable of perturbing genes in both a precise and spatiotemporal fashion. CRISPR's ease of use and flexibility, coupled with light's unparalleled spatiotemporal resolution deliverable from a controllable source, makes optogenetic CRISPR a well-suited solution for precise spatiotemporal gene perturbations. Here we present a new optogenetic CRISPR tool, BLU-VIPR, that diverges from prevailing split-Cas design strategies and instead focuses on optogenetic regulation of gRNA production. This simplifies spatiotemporal gene perturbation and works in vivo with cells previously intractable to optogenetic gene editing. We engineered BLU-VIPR around a new potent blue-light activated transcription factor and ribozyme-flanked gRNA. The BLU-VIPR design is genetically encoded and ensures precise excision of multiple gRNAs from a single mRNA transcript, allowing for optogenetic gene editing in T lymphocytes in vivo.

Use of Crossed Polarizers to Enhance Images of the Eyelids.

PURPOSE: To describe imaging of the external eye with Crossed Polarizers to enhance clinically important features in digital photographs of the eyelids. METHODS: External photographs with and without crossed polarizing filters were taken of patients with blepharitis and controls with no clinical eye pathology. RESULTS: Photographing eyelid skin through Crossed Polarizers decreased reflections on the skin surface and improved visualization of eyelid telangiectasias and blood vessels in patients with a broad range of skin pigmentation and ethnicities. CONCLUSIONS: The use of Crossed Polarizers in imaging the external eye reduces reflections and glare from the eyelid skin and margins, thereby allowing for a more detailed evaluation of underlying structures and analysis of images. These findings suggest that including Crossed Polarizers in clinical photography has informative applications for assessing eyelid disease.

Assessment of signs of anterior blepharitis using standardized color photographs.

PURPOSE: To describe a standardized technique for acquiring and viewing photographic images of eyelids, assess the reproducibility and validity of a grading protocol for signs of anterior blepharitis, and to explore whether the signs depend on the eyelid or the area of the eyelid assessed. METHODS: Subjects with anterior blepharitis ranging from none to severe were examined by ophthalmologists at clinical sites. Digital images of the eyelids of subjects were acquired using a protocol that allowed for the calibration of color and luminance. Three ophthalmologists at a centralized reading center applied a novel protocol for grading features of anterior blepharitis from the digital images viewed on color-calibrated monitors. The agreement among graders was assessed using percent agreement and weighted kappa statistics (Kw), and the correlation of photographic and clinical gradings was assessed using Spearman correlation coefficients. RESULTS: Agreement among graders was excellent (Kw > 0.80) on the number of eyelid margin vessels and was substantial (Kw between 0.61 and 0.80) for erythema, collarettes, number of engorged vessels, and number of lashes. Grading of the photographic images and the clinical assessments of erythema and lid debris were moderately correlated (r = 0.27-0.45). The grades for different features depended on whether the upper or lower eyelid, eyelid skin or lid margin, and central or lateral lid were assessed. CONCLUSIONS: The application of a protocol to obtain and display calibrated digital images of eyelids supports the standardized assessment of anterior blepharitis in clinical care and research studies.

Utility of digital stereo images for optic disc evaluation.

PURPOSE: To assess the suitability of digital stereo images for optic disc evaluations in glaucoma. METHODS: Stereo color optic disc images in both digital and 35-mm slide film formats were acquired contemporaneously from 29 subjects with various cup-to-disc ratios (range, 0.26-0.76; median, 0.475). Using a grading scale designed to assess image quality, the ease of visualizing optic disc features important for glaucoma diagnosis, and the comparative diameters of the optic disc cup, experienced observers separately compared the primary digital stereo images to each subject's 35-mm slides, to scanned images of the same 35-mm slides, and to grayscale conversions of the digital images. Statistical analysis accounted for multiple gradings and comparisons and also assessed image formats under monoscopic viewing. RESULTS: Overall, the quality of primary digital color images was judged superior to that of 35-mm slides (P < 0.001), including improved stereo (P < 0.001), but the primary digital color images were mostly equivalent to the scanned digitized images of the same slides. Color seemingly added little to grayscale optic disc images, except that peripapillary atrophy was best seen in color (P < 0.0001); both the nerve fiber layer (P < 0.0001) and the paths of blood vessels on the optic disc (P < 0.0001) were best seen in grayscale. The preference for digital over film images was maintained under monoscopic viewing conditions. CONCLUSIONS: Digital stereo optic disc images are useful for evaluating the optic disc in glaucoma and allow the application of advanced image processing applications. Grayscale images, by providing luminance distinct from color, may be informative for assessing certain features.