Allogeneic CD19-targeted CAR-T therapy in patients with severe myositis and systemic sclerosis.

Allogeneic chimeric antigen receptor (CAR)-T cells hold great promise for expanding the accessibility of CAR-T therapy, whereas the risks of allograft rejection have hampered its application. Here, we genetically engineered healthy-donor-derived, CD19-targeting CAR-T cells using CRISPR-Cas9 to address the issue of immune rejection and treated one patient with refractory immune-mediated necrotizing myopathy and two patients with diffuse cutaneous systemic sclerosis with these cells. This study was registered at ClinicalTrials.gov (NCT05859997). The infused cells persisted for over 3 months, achieving complete B cell depletion within 2 weeks of treatment. During the 6-month follow-up, we observed deep remission without cytokine release syndrome or other serious adverse events in all three patients, primarily shown by the significant improvement in the clinical response index scores for the two diseases, respectively, and supported by the observations of reversal of inflammation and fibrosis. Our results demonstrate the high safety and promising immune modulatory effect of the off-the-shelf CAR-T cells in treating severe refractory autoimmune diseases.

Engineering IscB to develop highly efficient miniature editing tools in mammalian cells and embryos.

The IscB proteins, as the ancestors of Cas9 endonuclease, hold great promise due to their small size and potential for diverse genome editing. However, their activity in mammalian cells is unsatisfactory. By introducing three residual substitutions in IscB, we observed an average 7.5-fold increase in activity. Through fusing a sequence-non-specific DNA-binding protein domain, the eIscB-D variant achieved higher editing efficiency, with a maximum of 91.3%. Moreover, engineered ωRNA was generated with a 20% reduction in length and slightly increased efficiency. The engineered eIscB-D/eωRNA system showed an average 20.2-fold increase in activity compared with the original IscB. Furthermore, we successfully adapted eIscB-D for highly efficient cytosine and adenine base editing. Notably, eIscB-D is highly active in mouse cell lines and embryos, enabling the efficient generation of disease models through mRNA/ωRNA injection. Our study suggests that these miniature genome-editing tools have great potential for diverse applications.

Dual Antiplatelet Treatment up to 72 Hours after Ischemic Stroke.

BACKGROUND: Dual antiplatelet treatment has been shown to lower the risk of recurrent stroke as compared with aspirin alone when treatment is initiated early (≤24 hours) after an acute mild stroke. The effect of clopidogrel plus aspirin as compared with aspirin alone administered within 72 hours after the onset of acute cerebral ischemia from atherosclerosis has not been well studied. METHODS: In 222 hospitals in China, we conducted a double-blind, randomized, placebo-controlled, two-by-two factorial trial involving patients with mild ischemic stroke or high-risk transient ischemic attack (TIA) of presumed atherosclerotic cause who had not undergone thrombolysis or thrombectomy. Patients were randomly assigned, in a 1:1 ratio, within 72 hours after symptom onset to receive clopidogrel (300 mg on day 1 and 75 mg daily on days 2 to 90) plus aspirin (100 to 300 mg on day 1 and 100 mg daily on days 2 to 21) or matching clopidogrel placebo plus aspirin (100 to 300 mg on day 1 and 100 mg daily on days 2 to 90). There was no interaction between this component of the factorial trial design and a second part that compared immediate with delayed statin treatment (not reported here). The primary efficacy outcome was new stroke, and the primary safety outcome was moderate-to-severe bleeding - both assessed within 90 days. RESULTS: A total of 6100 patients were enrolled, with 3050 assigned to each trial group. TIA was the qualifying event for enrollment in 13.1% of the patients. A total of 12.8% of the patients were assigned to a treatment group no more than 24 hours after stroke onset, and 87.2% were assigned after 24 hours and no more than 72 hours after stroke onset. A new stroke occurred in 222 patients (7.3%) in the clopidogrel-aspirin group and in 279 (9.2%) in the aspirin group (hazard ratio, 0.79; 95% confidence interval [CI], 0.66 to 0.94; P = 0.008). Moderate-to-severe bleeding occurred in 27 patients (0.9%) in the clopidogrel-aspirin group and in 13 (0.4%) in the aspirin group (hazard ratio, 2.08; 95% CI, 1.07 to 4.04; P = 0.03). CONCLUSIONS: Among patients with mild ischemic stroke or high-risk TIA of presumed atherosclerotic cause, combined clopidogrel-aspirin therapy initiated within 72 hours after stroke onset led to a lower risk of new stroke at 90 days than aspirin therapy alone but was associated with a low but higher risk of moderate-to-severe bleeding. (Funded by the National Natural Science Foundation of China and others; INSPIRES ClinicalTrials.gov number, NCT03635749.).

Engineering APOBEC3A deaminase for highly accurate and efficient base editing.

Cytosine base editors (CBEs) are effective tools for introducing C-to-T base conversions, but their clinical applications are limited by off-target and bystander effects. Through structure-guided engineering of human APOBEC3A (A3A) deaminase, we developed highly accurate A3A-CBE (haA3A-CBE) variants that efficiently generate C-to-T conversion with a narrow editing window and near-background level of DNA and RNA off-target activity, irrespective of methylation status and sequence context. The engineered deaminase domains are compatible with PAM-relaxed SpCas9-NG variant, enabling accurate correction of pathogenic mutations in homopolymeric cytosine sites through flexible positioning of the single-guide RNAs. Dual adeno-associated virus delivery of one haA3A-CBE variant to a mouse model of tyrosinemia induced up to 58.1% editing in liver tissues with minimal bystander editing, which was further reduced through single dose of lipid nanoparticle-based messenger RNA delivery of haA3A-CBEs. These results highlight the tremendous promise of haA3A-CBEs for precise genome editing to treat human diseases.

Allele-Specific Suppression of Variant MHC With High-Precision RNA Nuclease CRISPR-Cas13d Prevents Hypertrophic Cardiomyopathy.

BACKGROUND: Familial hypertrophic cardiomyopathy has severe clinical complications of heart failure, arrhythmia, and sudden cardiac death. Heterozygous single nucleotide variants (SNVs) of sarcomere genes such as MYH7 are the leading cause of this type of disease. CRISPR-Cas13 (clustered regularly interspaced short palindromic repeats and their associated protein 13) is an emerging gene therapy approach for treating genetic disorders, but its therapeutic potential in genetic cardiomyopathy remains unexplored. METHODS: We developed a sensitive allelic point mutation reporter system to screen the mutagenic variants of Cas13d. On the basis of Cas13d homology structure, we rationally designed a series of Cas13d variants and obtained a high-precision Cas13d variant (hpCas13d) that specifically cleaves the MYH7 variant RNAs containing 1 allelic SNV. We validated the high precision and low collateral cleavage activity of hpCas13d through various in vitro assays. We generated 2 HCM mouse models bearing distinct MYH7 SNVs and used adenovirus-associated virus serotype 9 to deliver hpCas13d specifically to the cardiomyocytes. We performed a large-scale library screening to assess the potency of hpCas13d in resolving 45 human MYH7 allelic pathogenic SNVs. RESULTS: Wild-type Cas13d cannot distinguish and specifically cleave the heterozygous MYH7 allele with SNV. hpCas13d, with 3 amino acid substitutions, had minimized collateral RNase activity and was able to resolve various human MYH7 pathological sequence variations that cause hypertrophic cardiomyopathy. In vivo application of hpCas13d to 2 hypertrophic cardiomyopathy models caused by distinct human MYH7 analogous sequence variations specifically suppressed the altered allele and prevented cardiac hypertrophy. CONCLUSIONS: Our study unveils the great potential of CRISPR-Cas nucleases with high precision in treating inheritable cardiomyopathy and opens a new avenue for therapeutic management of inherited cardiac diseases.

Therapeutic Effects of Hematopoietic Stem Cell Derived From Gene-Edited Mice on ß654-Thalassemia.

ß-thalassemia is an inherited blood disease caused by reduced or inadequate ß-globin synthesis due to ß-globin gene mutation. Our previous study developed a gene-edited mice model (ß654-ER mice) by CRISPR/Cas9-mediated genome editing, targeting both the ßIVS2-654 (C > T) mutation site and the 3' splicing acceptor site at 579 and corrected abnormal ß-globin mRNA splicing in the ß654-thalassemia mice. Herein, we further explored the therapeutic effect of the hematopoietic stem cells (HSCs) from ß654-ER mice on ß-thalassemia by consecutive HSC transplantation. The results indicated that HSC transplantation derived from gene-edited mice can significantly improve the survival rate of mice after lethal radiation doses and effectively achieve hematopoietic reconstruction and long-term hematopoiesis. Clinical symptoms, including hematologic parameters and tissue pathology of transplanted recipients, were significantly improved compared to the non-transplanted ß654 mice. The therapeutic effect of gene-edited HSC transplantation demonstrated no significant difference in hematological parameters and tissue pathology compared with wild-type mouse-derived HSCs. Our data revealed that HSC transplantation from gene-edited mice completely recovered the ß-thalassemia phenotype. Our study systematically investigated the therapeutic effect of HSCs derived from ß654-ER mice on ß-thalassemia and further confirmed the efficacy of our gene-editing approach. Altogether, it provided a reference and primary experimental data for the clinical usage of such gene-edited HSCs in the future.

In vivo base editing rescues primary hyperoxaluria type 1 in rats.

Primary hyperoxaluria type 1 (PH1) is a childhood-onset autosomal recessive disease, characterized by nephrocalcinosis, multiple recurrent urinary calcium oxalate stones, and a high risk of progressive kidney damage. PH1 is caused by inherent genetic defects of the alanine glyoxylate aminotransferase (AGXT) gene. The in vivo repair of disease-causing genes was exceedingly inefficient before the invention of base editors which can efficiently introduce precisely targeted base alterations without double-strand DNA breaks. Adenine base editor (ABE) can precisely convert A·T to G·C with the assistance of specific guide RNA. Here, we demonstrated that systemic delivery of dual adeno-associated virus encoding a split-ABE8e could artificially repair 13% of the pathogenic allele in AgxtQ84X rats, a model of PH1, alleviating the disease phenotype. Specifically, ABE treatment partially restored the expression of alanine-glyoxylate-aminotransferase (AGT), reduced endogenous oxalate synthesis and alleviated calcium oxalate crystal deposition. Western blot and immunohistochemistry confirmed that ABE8e treatment restored AGT protein expression in hepatocytes. Moreover, the precise editing efficiency in the liver remained stable six months after treatment. Thus, our findings provided a prospect of in vivo base editing as a personalized and precise medicine for PH1 by directly correcting the mutant Agxt gene.

Targeted Gene Insertion: The Cutting Edge of CRISPR Drug Development with Hemophilia as a Highlight.

The remarkable advance in gene editing technology presents unparalleled opportunities for transforming medicine and finding cures for hereditary diseases. Human trials of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9)-based therapeutics have demonstrated promising results in disrupting or deleting target sequences to treat specific diseases. However, the potential of targeted gene insertion approaches, which offer distinct advantages over disruption/deletion methods, remains largely unexplored in human trials due to intricate technical obstacles and safety concerns. This paper reviews the recent advances in preclinical studies demonstrating in vivo targeted gene insertion for therapeutic benefits, targeting somatic solid tissues through systemic delivery. With a specific emphasis on hemophilia as a prominent disease model, we highlight advancements in insertion strategies, including considerations of DNA repair pathways, targeting site selection, and donor design. Furthermore, we discuss the complex challenges and recent breakthroughs that offer valuable insights for progressing towards clinical trials.

Numerical simulation and experimental verification of the velocity field in asymmetric circular bends.

To address the measurement accuracy challenges posed by the internal flow complexity in atypical circular bend pipes with short turning sections and without extended straight pipe segments, this study designed an experimental circular "S"-shaped bent pipe with a diameter of 0.4 m and a bending angle of 135°. Numerical analysis was used to determine the stable region for velocity distribution within the experimental segment. Furthermore, a novel evaluation method based on the coefficient of variation was proposed to accurately locate the optimal position for installing thermal mass flow meters on the test cross section. Additionally, a formula for calculating the pipeline flow rate based on velocity differences was derived. This formula considers pipeline flow as the dependent variable and uses the velocity at two points in the test cross section as the independent variable. Experimental validation on a primary standard test bench demonstrated that the flow rate calculated by this method had an error controlled within 0.625% compared to the standard flow rate, thus effectively verifying the method's high accuracy and engineering applicability. This research provides a new testing methodology and practical basis for flow measurement in complex pipeline systems, offering significant guidance for research and applications in related fields.

Dual-responsive nanocarriers for efficient cytosolic protein delivery and CRISPR-Cas9 gene therapy of inflammatory skin disorders.

Developing protein drugs that can target intracellular sites remains a challenge due to their inadequate membrane permeability. Efficient carriers for cytosolic protein delivery are required for protein-based drugs, cancer vaccines, and CRISPR-Cas9 gene therapies. Here, we report a screening process to identify highly efficient materials for cytosolic protein delivery from a library of dual-functionalized polymers bearing both boronate and lipoic acid moieties. Both ligands were found to be crucial for protein binding, endosomal escape, and intracellular protein release. Polymers with higher grafting ratios exhibit remarkable efficacies in cytosolic protein delivery including enzymes, monoclonal antibodies, and Cas9 ribonucleoprotein while preserving their activity. Optimal polymer successfully delivered Cas9 ribonucleoprotein targeting NLRP3 to disrupt NLRP3 inflammasomes in vivo and ameliorate inflammation in a mouse model of psoriasis. Our study presents a promising option for the discovery of highly efficient materials tailored for cytosolic delivery of specific proteins and complexes such as Cas9 ribonucleoprotein.

Gene Editing of the Endogenous Cryptic 3' Splice Site Corrects the RNA Splicing Defect in the ß654-Thalassemia Mouse Model.

ß654-thalassemia is caused by a point mutation in the second intron (IVS-II) of the ß-globin gene that activates a cryptic 3' splice site, leading to incorrect RNA splicing. Our previous study demonstrated that when direct deletion of the ß654 mutation sequence or the cryptic 3' splice site in the IVS-II occurs, correct splicing of ß-globin mRNA can be restored. Herein, we conducted an in-depth analysis to explore a more precise gene-editing method for treating ß654-thalassemia. A single-base substitution of the cryptic 3' acceptor splice site was introduced in the genome of a ß654-thalassemia mouse model using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9(Cas9)-mediated homology-directed repair (HDR). All of the HDR-edited mice allow the detection of correctly spliced ß-globin mRNA. Pathological changes were improved compared with the nonedited ß654 mice. This resulted in a more than twofold increase in the survival rate beyond the weaning age of the mice carrying the ß654 allele. The therapeutic effects of this gene-editing strategy showed that the typical ß-thalassemia phenotype can be improved in a dose-dependent manner when the frequency of HDR is over 20%. Our research provides a unique and effective method for correcting the splicing defect by gene editing the reactive splicing acceptor site in a ß654 mouse model.

Luteinizing Hormone Receptor Mutation (LHRN316S) Causes Abnormal Follicular Development Revealed by Follicle Single-Cell Analysis and CRISPR/Cas9.

Abnormal interaction between granulosa cells and oocytes causes disordered development of ovarian follicles. However, the interactions between oocytes and cumulus granulosa cells (CGs), oocytes and mural granulosa cells (MGs), and CGs and MGs remain to be fully explored. Using single-cell RNA-sequencing (scRNA-seq), we determined the transcriptional profiles of oocytes, CGs and MGs in antral follicles. Analysis of scRNA-seq data revealed that CGs may regulate follicular development through the BMP15-KITL-KIT-PI3K-ARF6 pathway with elevated expression of luteinizing hormone receptor (LHR). Because internalization of the LHR is regulated by Arf6, we constructed LHRN316S mice by CRISPR/Cas9 to further explore mechanisms of follicular development and novel treatment strategies for female infertility. Ovaries of LHRN316S mice exhibited reduced numbers of corpora lutea and ovulation. The LHRN316S mice had a reduced rate of oocyte maturation in vitro and decreased serum progesterone levels. Mating LHRN316S female mice with ICR wild type male mice revealed that the infertility rate of LHRN316S mice was 21.4% (3/14). Litter sizes from LHRN316S mice were smaller than those from control wild type female mice. The oocytes from LHRN316S mice had an increased rate of maturation in vitro after progesterone administration in vitro. Furthermore, progesterone treated LHRN316S mice produced offspring numbers per litter equivalent to WT mice. These findings provide key insights into cellular interactions in ovarian follicles and provide important clues for infertility treatment.

FFAR2 expressing myeloid-derived suppressor cells drive cancer immunoevasion.

BACKGROUND: Emerging evidences suggest that aberrant metabolites contributes to the immunosuppressive microenvironment that leads to cancer immune evasion. Among tumor immunosuppressive cells, myeloid-derived suppressor cells (MDSCs) are pathologically activated and extremely immunosuppressive, which are closely associated with poor clinical outcomes of cancer patients. However, the correlation between MDSCs mediated immunosuppression and particular cancer metabolism remained elusive. METHODS: Spontaneous lung adenocarcinoma and subcutaneous mouse tumor models, gas chromatography-mass spectrometry (GC-MS) and immunofluorescence assay of patient-derived lung adenocarcinoma tissues, and flow cytometry, RNA sequencing and Western blotting of immune cells, were utilized. RESULTS: Metabolite profiling revealed a significant accumulation of acetic acids in tumor tissues from both patients and mouse model, which contribute to immune suppression and cancer progression significantly through free fatty acid receptor 2 (FFAR2). Furthermore, FFAR2 is highly expressed in the myeloid-derived suppressor cells (MDSCs) from the tumor of lung adenocarcinoma (LUAD) patients which is greatly associated with poor prognosis. Surprisingly, whole or myeloid Ffar2 gene deletion markedly inhibited urethane-induced lung carcinogenesis and syngeneic tumor growth with reduced MDSCs and increased CD8+ T cell infiltration. Mechanistically, FFAR2 deficiency in MDSCs significantly reduced the expression of Arg1 through Gαq/Calcium/PPAR-γ axis, which eliminated T cell dysfunction through relieving L-Arginine consumption in tumor microenvironment. Therefore, replenishment of L-Arginine or inhibition to PPAR-γ restored acetic acids/FFAR2 mediated suppression to T cells significantly. Finally, FFAR2 inhibition overcame resistance to immune checkpoint blockade through enhancing the recruitment and cytotoxicity of tumor-infiltrating T cells. CONCLUSION: Altogether, our results demonstrate that the acetic acids/FFAR2 axis enhances MDSCs mediated immunosuppression through Gαq/calcium/PPAR-γ/Arg1 signaling pathway, thus contributing to cancer progression. Therefore, FFAR2 may serve as a potential new target to eliminate pathologically activated MDSCs and reverse immunosuppressive tumor microenvironment, which has great potential in improving clinical outcomes of cancer immunotherapy.

Efficient expansion and CRISPR-Cas9-mediated gene correction of patient-derived hepatocytes for treatment of inherited liver diseases.

Cell-based ex vivo gene therapy in solid organs, especially the liver, has proven technically challenging. Here, we report a feasible strategy for the clinical application of hepatocyte therapy. We first generated high-quality autologous hepatocytes through the large-scale expansion of patient-derived hepatocytes. Moreover, the proliferating patient-derived hepatocytes, together with the AAV2.7m8 variant identified through screening, enabled CRISPR-Cas9-mediated targeted integration efficiently, achieving functional correction of pathogenic mutations in FAH or OTC. Importantly, these edited hepatocytes repopulated the injured mouse liver at high repopulation levels and underwent maturation, successfully treating mice with tyrosinemia following transplantation. Our study combines ex vivo large-scale cell expansion and gene editing in patient-derived transplantable hepatocytes, which holds potential for treating human liver diseases.

Immediate- or Delayed-Intensive Statin in Acute Cerebral Ischemia: The INSPIRES Randomized Clinical Trial.

Importance: Comparisons are limited for immediate-intensive and delayed-intensive statin for secondary stroke prevention and neuroprotection in patients with acute mild ischemic stroke or transient ischemic attack (TIA) from atherosclerosis. Objective: To estimate whether immediate-intensive statin therapy is safe and can lower the risk of recurrent stroke compared with delayed-intensive statin in patients with acute mild ischemic stroke or high-risk TIA from atherosclerosis. Design, Setting, and Participants: The Intensive Statin and Antiplatelet Therapy for High-Risk Intracranial or Extracranial Atherosclerosis (INSPIRES) trial, a double-blind, placebo-controlled, 2 × 2 factorial, randomized clinical trial enrolled patients from September 2018 to October 2022. The trial was conducted at 222 hospitals in China. Patients aged 35 to 80 years with mild ischemic stroke or high-risk TIA of presumed atherosclerosis within 72 hours of symptom onset were assessed. Interventions: Patients were randomly assigned to receive immediate-intensive atorvastatin (80 mg daily on days 1-21; 40 mg daily on days 22-90) or 3-day delayed treatment (placebo for days 1-3, followed by placebo and atorvastatin, 40 mg daily on days 4-21, and then atorvastatin, 40 mg daily on days 22-90). Main Outcomes and Measures: The primary efficacy outcome was new stroke within 90 days, and a secondary efficacy outcome was poor functional outcome. Moderate to severe bleeding was the primary safety outcome. Results: A total of 11 431 patients were assessed for eligibility, and 6100 patients (median [IQR] age, 65 [57-71] years; 3915 men [64.2%]) were enrolled, with 3050 assigned to each treatment group. Within 90 days, new stroke occurred in 245 patients (8.1%) in the immediate-intensive statin group and 256 patients (8.4%) in the delayed group (hazard ratio, 0.95; 95% CI, 0.80-1.13). Poor functional outcome occurred in 299 patients (9.8%) and 348 patients (11.4%) in the immediate-intensive and delayed-intensive statin groups, respectively (odds ratio, 0.83; 95% CI, 0.71-0.98). Moderate to severe bleeding occurred in 23 of 3050 patients (0.8%) and 17 of 3050 patients (0.6%), in the immediate-intensive and delayed-intensive statin groups, respectively. Conclusions and Relevance: Immediate-intensive statin initiated within 72 hours did not reduce the risk of stroke within 90 days and may be associated with improved functional outcomes without significant difference in moderate to severe bleeding, compared with 3-day delayed-intensive statin in Chinese patients with acute mild ischemic stroke or TIA from atherosclerosis. Trial Registration: ClinicalTrials.gov Identifier: NCT03635749.

Assessment of Patient-Derived Xenograft Growth and Antitumor Activity: The NCI PDXNet Consensus Recommendations.

Although patient-derived xenografts (PDX) are commonly used for preclinical modeling in cancer research, a standard approach to in vivo tumor growth analysis and assessment of antitumor activity is lacking, complicating the comparison of different studies and determination of whether a PDX experiment has produced evidence needed to consider a new therapy promising. We present consensus recommendations for assessment of PDX growth and antitumor activity, providing public access to a suite of tools for in vivo growth analyses. We expect that harmonizing PDX study design and analysis and assessing a suite of analytical tools will enhance information exchange and facilitate identification of promising novel therapies and biomarkers for guiding cancer therapy.

Long-term amelioration of an early-onset familial atrial fibrillation model with AAV-mediated in vivo gene therapy.

Atrial fibrillation (AF) is a common cardiac disease with high prevalence in the general population. Despite a mild manifestation at the onset stage, it causes serious consequences, including sudden death, when the disease progresses to the late stage. Most available treatments of AF focus on symptom management or alleviation, due to a lack of fundamental knowledge and the fact that considerable variations of AF exist. With the popularisation of the next-generation sequencing technology, several causal genetic factors, including MYL4, have been discovered to contribute to AF, giving hope to developing its gene therapies. In this study, we attempted to treat a previously established rat AF model, which carried Myl4E11K/E11K loss of function mutation, via overexpression of exogenous wild-type Myl4 by AAV9 vectors. Our results showed that delivery of Myl4 expressing AAV9 to postnatal rat models rescued the symptoms of AF, indicating the therapeutic potential that early gene therapy intervention can achieve long-term effects in treating cardiac arrhythmias caused by gene mutations.