Pooled Knockin Targeting for Genome Engineering of Cellular Immunotherapies.

Adoptive transfer of genetically modified immune cells holds great promise for cancer immunotherapy. CRISPR knockin targeting can improve cell therapies, but more high-throughput methods are needed to test which knockin gene constructs most potently enhance primary cell functions in vivo. We developed a widely adaptable technology to barcode and track targeted integrations of large non-viral DNA templates and applied it to perform pooled knockin screens in primary human T cells. Pooled knockin of dozens of unique barcoded templates into the T cell receptor (TCR)-locus revealed gene constructs that enhanced fitness in vitro and in vivo. We further developed pooled knockin sequencing (PoKI-seq), combining single-cell transcriptome analysis and pooled knockin screening to measure cell abundance and cell state ex vivo and in vivo. This platform nominated a novel transforming growth factor ß (TGF-ß) R2-41BB chimeric receptor that improved solid tumor clearance. Pooled knockin screening enables parallelized re-writing of endogenous genetic sequences to accelerate discovery of knockin programs for cell therapies.

Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function.

Human T cells are central effectors of immunity and cancer immunotherapy. CRISPR-based functional studies in T cells could prioritize novel targets for drug development and improve the design of genetically reprogrammed cell-based therapies. However, large-scale CRISPR screens have been challenging in primary human cells. We developed a new method, single guide RNA (sgRNA) lentiviral infection with Cas9 protein electroporation (SLICE), to identify regulators of stimulation responses in primary human T cells. Genome-wide loss-of-function screens identified essential T cell receptor signaling components and genes that negatively tune proliferation following stimulation. Targeted ablation of individual candidate genes characterized hits and identified perturbations that enhanced cancer cell killing. SLICE coupled with single-cell RNA sequencing (RNA-seq) revealed signature stimulation-response gene programs altered by key genetic perturbations. SLICE genome-wide screening was also adaptable to identify mediators of immunosuppression, revealing genes controlling responses to adenosine signaling. The SLICE platform enables unbiased discovery and characterization of functional gene targets in primary cells.

Observation of a correlated free four-neutron system.

A long-standing question in nuclear physics is whether chargeless nuclear systems can exist. To our knowledge, only neutron stars represent near-pure neutron systems, where neutrons are squeezed together by the gravitational force to very high densities. The experimental search for isolated multi-neutron systems has been an ongoing quest for several decades1, with a particular focus on the four-neutron system called the tetraneutron, resulting in only a few indications of its existence so far2-4, leaving the tetraneutron an elusive nuclear system for six decades. Here we report on the observation of a resonance-like structure near threshold in the four-neutron system that is consistent with a quasi-bound tetraneutron state existing for a very short time. The measured energy and width of this state provide a key benchmark for our understanding of the nuclear force. The use of an experimental approach based on a knockout reaction at large momentum transfer with a radioactive high-energy 8He beam was key.

Validation of the International Myositis Assessment and Clinical Studies Group guideline on cancer risk stratification.

OBJECTIVES: Adult-onset Idiopathic inflammatory myopathies (IIMs) are associated with cancer. Guideline on cancer risk stratifications and screening in IIM patients was recently published, but their external validity remains verified. We evaluated its applicability and reliability among a Hong Kong IIM cohort. METHODS: The longitudinal observational cohort collected data from IIM patients fulfilling relevant classification criteria from 8 rheumatology centres in Hong Kong. Demographic, clinical and laboratory data were reviewed from 2004-2023. IIM patients were stratified into standard, intermediate or high-risk subgroups according to the IMACS guideline. The occurrence of malignancy at or after IIM diagnosis was analyzed. Independent risk factors for cancer were evaluated. RESULTS: 479 patients were included with 327 females (68.3%) and mean age of IIM diagnosis at 54.5 ± 13.6 years. 214 (44.7%) and 238 (49.7%) patients were stratified to high and intermediate risk groups, respectively. Only 5.6% belonged to the standard-risk group. 60 patients (12.5%) had cancer within 3 years of IIM diagnosis. Nasopharyngeal (25%), lung (21.1%) and breast (10.5%) were the top 3 cancers. Significantly more patients (44, 20.6%) in the high-risk group developed cancer within 3 years, compared with intermediate (6.7%, p< 0.001) and standard-risk (0%, p= 0.009) groups. Risk factors for cancer included older age (OR : 1.048, 95%CI : 1.019-1.078), Gottron's rash (OR : 2.453, 95%CI : 1.123-5.356), absence of ILD (OR 2.695, 95% CI : 1.154-6.295), anti-TIF1g positivity (OR : 4.627, 95% CI : 2.046-10.461) and anti-SAE1 positivity (OR : 5.325, 95% CI : 1.271-22.300). CONCLUSIONS: Our real-world study supported the accuracy of cancer risk stratification. Vast majority of IIM patients would be subjected to extensive cancer screening when the guideline was applied.

Recent Advances in Liver Transplantation for Hepatocellular Carcinoma.

OPINION STATEMENT: Liver transplantation for hepatocellular carcinoma (HCC) remains an evolving field. Major challenges HCC transplant patients face today include liver organ donor shortages and the need for both better pre-transplant bridging/downstaging therapies and post-transplant HCC recurrence treatment options. The advent of immunotherapy and the demonstrated efficacy of immune checkpoint inhibitors in multiple solid tumors including advanced/unresectable HCC hold promise in expanding both the neoadjuvant and adjuvant HCC transplant treatment regimen, though caution is needed with these immune modulating agents leading up to and following transplant. New options for pre-transplant HCC management will expand access to this curative option as well as ensure patients have adequate control of their HCC prior to transplant to maximize the utility of a liver donor. Machine perfusion has been an active area of investigation in recent years and could expand the organ donor pool, helping address current liver donor shortages. Finally, additional HCC biomarkers such as AFP-L3 and DCP have shown promise in improving risk stratification of HCC patients. Together, these three recent advancements will likely alter HCC transplant guidelines in the coming years.

Reprogramming human T cell function and specificity with non-viral genome targeting.

Decades of work have aimed to genetically reprogram T cells for therapeutic purposes1,2 using recombinant viral vectors, which do not target transgenes to specific genomic sites3,4. The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair5,6. Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.

Deep-learning features based on F18 fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) to predict preoperative colorectal cancer lymph node metastasis.

AIM: The objective of this study was to create and authenticate a prognostic model for lymph node metastasis (LNM) in colorectal cancer (CRC) that integrates clinical, radiomics, and deep transfer learning features. MATERIALS AND METHODS: In this study, we analyzed data from 119 CRC patients who underwent F18 fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) scanning. The patient cohort was divided into training and validation subsets in an 8:2 ratio, with an additional 33 external data points for testing. Initially, we conducted univariate analysis to screen clinical parameters. Radiomics features were extracted from manually drawn images using pyradiomics, and deep-learning features, radiomics features, and clinical features were selected using Least Absolute Shrinkage and Selection Operator (LASSO) regression and Spearman correlation coefficient. We then constructed a model by training a support vector machine (SVM), and evaluated the performance of the prediction model by comparing the area under the curve (AUC), sensitivity, and specificity. Finally, we developed nomograms combining clinical and radiological features for interpretation and analysis. RESULTS: The deep learning radiomics (DLR) nomogram model, which was developed by integrating deep learning, radiomics, and clinical features, exhibited excellent performance. The area under the curve was (AUC = 0.934, 95% confidence interval [CI]: 0.884-0.983) in the training cohort, (AUC = 0.902, 95% CI: 0.769-1.000) in the validation cohort, and (AUC = 0.836, 95% CI: 0.673-0.998) in the test cohort. CONCLUSION: We developed a preoperative predictive machine-learning model using deep transfer learning, radiomics, and clinical features to differentiate LNM status in CRC, aiding in treatment decision-making for patients.

Association of serum 25-hydroxyvitamin D with cardiovascular mortality and kidney outcome in patients with early stages of CKD.

PURPOSE: While serum 25-hydroxyvitamin D (25[OH]D) deficiency is prevalent in chronic kidney disease (CKD), the effects of 25(OH)D deficiency on cardiovascular mortality and kidney outcomes in patients with early-stage CKD remain incompletely understood. METHODS: This multicenter retrospective cohort study included adult patients with stages 1-3 CKD from 19 medical centers across China between January 2000 and May 2021. The primary outcome was cardiovascular mortality. The secondary study outcome included CKD progression (defined as a sustained > 40% eGFR decrease from baseline or progress to end-stage kidney disease), and annual percentage change of eGFR. RESULTS: Of 9229 adults with stages 1-3 CKD, 27.0% and 38.9% had severe (< 10 ng/mL) and moderate (10 to < 20 ng/mL) serum 25(OH)D deficiency, respectively. Compared with patients having 25(OH)D ≥ 20 ng/mL, a significantly higher risk of cardiovascular mortality (hazard ratio [HR] 1.90, 95% CI 1.37-2.63), CKD progression (HR 2.20, 95% CI 1.68-2.88), and a steeper annual decline in eGFR (estimate - 7.87%; 95% CI - 10.24% to - 5.51% per year) was found in those with serum 25(OH)D < 10 ng/mL. Similar results were obtained in subgroups and by sensitivity analyses. CONCLUSIONS: 25(OH)D deficiency is associated with increased risks of cardiovascular mortality and CKD progression in patients with early-stage CKD. Studies are needed to determine whether early intervention for 25(OH)D deficiency could improve the prognosis of patients with early-stage CKD.

[Analysis of advanced fibrosis in metabolic dysfunction-associated fatty liver disease patients with chronic hepatitis B].

Objective: To investigate the clinical and pathological characteristics of chronic hepatitis B (CHB) with metabolic dysfunction-associated fatty liver disease (MAFLD), as well as associations with advanced fibrosis. Methods: CHB patients who underwent liver biopsy at Tianjin Second People's Hospital from June 2016 to September 2019 were included in the study. The patients were divided into two groups based on whether they had concomitant MAFLD; a CHB group and a MAFLD-CHB group. t-tests and Chi-square tests were used to compare pathological characteristics and basic features in the two groups. Logistic regression analysis was used to analyze factors associated with advanced fibrosis. Results: The CHB group included 110 patients, and the MAFLD-CHB group included 272 patients. There were significant differences in smoking, alcohol consumption, hypertension incidence, body metabolic index, alanine aminotransferase, gamma-glutamyl transferase (GGT), high-density lipoprotein, low-density lipoprotein, fasting plasma glucose, and platelets (PLT) between the two groups (all P<0.05). The MAFLD-CHB group had a higher incidence of advanced fibrosis than the CHB group (P<0.05). In logistic regression analysis MAFLD [odds ratio (OR)=2.204, 95% confidence interval (CI) 1.018-4.774, P=0.045], GGT (OR=1.008, 95%CI 1.002-1.013, P=0.005), and PLT (OR=0.995, 95%CI 0.991-0.999, P=0.019) were associated with advanced fibrosis (all P<0.05). In the MAFLD-CHB group type 2 diabetes (OR=3.281, 95%CI 1.375-7.832, P=0.007), GGT (OR=1.011, 95%CI 1.003-1.018, P=0.005), and PLT (OR=0.993, 95%CI 0.988-0.998, P=0.004) were associated with advanced fibrosis (P<0.05). Conclusion: Patients with MAFLD-CHB are more likely to develop advanced fibrosis than patients with CHB alone. In the MAFLD-CHB group type 2 diabetes mellitus was associated with advanced fibrosis. It is important to strictly control relevant risk factors in MAFLD-CHB patients, especially in patients with type 2 diabetes.

[Study on the mechanism of cross-linked hyaluronic acid-dexamethasone hydrogelin post-traumatic osteoarthritis].

Objective: To explore the mechanism of cross-linked hyaluronic acid-dexamethasone hydrogel (cHA-Dex) in inhibiting chondrocyte apoptosis and alleviating early post-traumatic osteoarthritis (PTOA). Methods: To generate PTOA model, anterior cruciate ligament transection (ACLT)was performed on SD rats (n=70), and the sham surgery group (n=70) was set as control. The changes in inflammatory indicators such as interleukin-1ß (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), matrix metalloproteinase-3 (MMP-3), and matrix metalloproteinase-13 (MMP-13) in the joint lavage fluid were measured at different time points (1-14 days, 5 rats at each time point) after surgery. The cHA-Dex (0.5 mg/ml) hydrogel (experimental group, n=70) and ordinary low-molecular-weight hyaluronic acid (HA) hydrogel premixed with Dex, that was, HA-Dex (0.5 mg/ml) hydrogel (control group, n=70) were injected into the joint cavity of PTOA rats, and the release amount and cumulative release amount of Dex in the joint fluid of rats at each time point(1-14 days, 5 rats at each time point) were detected to reveal the release mechanism of cHA-Dex hydrogel. The cartilage of knee joint of patients with osteoarthritis (OA) who underwent knee arthroplasty in the Second Hospital of Shanxi Medical University from January 2020 to December 2022 was taken for in vitro tissue block culture (Outbridge score=1 or 2,n=18). After the cartilage tissue block was treated with cHA-Dex hydrogel premixed with 0.1, 0.2, and 0.5 mg/ml Dex, the mRNA expression levels of IL-1ß, IL-6, TNF-α, MMP-3, and MMP-13 in the articular cartilage tissue block were detected. OA chondrocytes were isolated from cartilage samples using enzymatic hydrolysis and cultured in vitro (n=18). Chondrocytes were divided into 4 groups: saline, cHA hydrogel, Dex (0.5 mg/ml), and cHA-Dex (0.5 mg/ml) hydrogel group. The effects of different interventions on chondrocyte proliferation and apoptosis were tested. Results: The Osteoarthritis Research Society International (OARSI) score of safranine O-solid green staining in PTOA group was 3.34±0.35, and it was 1.17±0.21 in Sham group(P=0.010). The Meachim score of knee joint osteophytes in PTOA rats was significantly higher than that in the Sham group (2.66±0.41 vs 0.22±0.17, P=0.010), indicating PTOA model in rat was established successfully. The cHA-Dex hydrogel, which corresponded to the peak changes of inflammatory factors in the joints of PTOA rats in the early stage, was also released in the early stage and sustained-released in the late stage. After the OA articular cartilage tissue block was treated with cHA-Dex hydrogel premixed with 0.1, 0.2, and 0.5 mg/ml Dex, the mRNA expression levels of IL-1 ß, IL-6, TNF-α, MMP-3, and MMP-13 in the tissue block were reduced significantly (all P<0.05) and in a dose-dependent manner. Compared with Dex (0.5 mg/ml) alone group, the apoptosis rate of cHA-Dex (0.5 mg/ml) hydrogel group was significantly reduced (0.60±0.07 vs 6.63±0.98, P=0.010).Compared with the normal saline or the cHA hydrogel alone group, the cHA-Dex (0.5 mg/ml) hydrogel group had significant cell proliferation, and the difference at each time point were all significant statistically (all P<0.05). Conclusion: For the early inflammation of PTOA, cHA-Dex hydrogel can not only inhibit cartilage inflammation, but also reverse the increased apoptosis and decreased proliferation rate of chondrocytes caused by Dex, and finally alleviate the progress of PTOA by releasing Dex.