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CRISPR,CAR-T,and NK:Current applications and future perspectives

genes & Diseases 2024年第4期11卷 246-257页

作者： Mohadeseh Khoshandam Hossein Soltaninejad Amir Ali Hamidieh Saman Hosseinkhani Department of Reproductive Biology Academic Center for EducationCultureand Research(ACECR)Qom branch***Iran National Institute of genetic Engineering and Biotechnology(NIGEB) Tehran 14965/161Iran Department of stem cells technology and Tissue Regeneration Faculty of Interdisciplinary Science and TechnologiesTarbiat Modares UniversityTehran 15614Iran Pediatric cell therapy and gene therapy Research Center GeneCell&Tissue Research InstituteTehran University of Medical SciencesTehran ***Iran Department of Biochemistry Faculty of Biological SciencesTarbiat Modares UniversityTehran 15614Iran

Chimeric antigen receptor T(CAR-T)cell therapy represents a breakthrough in personalized cancer treatments.In this regard,synthetic receptors comprised of antigen recognition domains,signaling,and stimulatory domains are used to reprogram T-cells to target tum or cells and destroy them.Despite the success of this approach in refractory B-cell malignancies,the optimal potency of CAR T-cell therapy for many other cancers,particularly solid tumors,has not been validated.Natural killer cells are powerful cytotoxic lymphocytes specialized in recognizing and dispensing the tumor cells in coordination with other anti-tumor immunity cells.Based on these studies,many investigations are focused on the accurate designing of CAR T-cells with clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system or other novel gene editing tools that can induce hereditary changes with or without the presence of a double-stranded break into the genome.These methodologies can be specifically focused on negative controllers of T-cells,induce modifications to a particular gene,and produce reproducible,safe,and powerful allogeneic CAR T-cells for on-demand cancer immunotherapy.The improvement of the CRISPR/Cas9 innovation offers an adaptable and proficient gene-editing capability in activating different pathways to help natural killer cells interact with novel CARs to particularly target tumor cells.Novel achievements and future challenges of combining next-generation CRISPR-Cas9 gene editing tools to optimize CAR T-cell and natural killer cell treatment for future clinical trials toward the foundation of modern cancer treatments have been assessed inthis review.

关键词： Cancer CAR T-cell CRISPR gene editing Immunotherapy NK cell

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programmable synthetic receptors:the next-generation of cell and gene therapies

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Signal Transduction and Targeted therapy 2024年第2期9卷 376-400页

作者： Fei Teng Tongtong Cui Li Zhou Qingqin Gao Qi Zhou Wei Li University of Chinese Academy of Sciences Beijing 101408China State Key Laboratory of stem cell and Regenerative Biology Institute of ZoologyChinese Academy of SciencesBeijing 100101China Institute for stem cell and Regeneration Chinese Academy of SciencesBeijing 100101China Beijing Institute for stem cell and Regenerative Medicine Beijing 100101China

cell and gene therapies hold tremendous promise for treating a range of difficult-to-treat diseases.However,concerns over the safety and efficacy require to be further addressed in order to realize their full potential.Synthetic receptors,a synthetic biology tool that can precisely control the function of therapeutic cells and genetic modules,have been rapidly developed and applied as a powerful solution.Delicately designed and engineered,they can be applied to finetune the therapeutic activities,i.e.,to regulate production of dosed,bioactive payloads by sensing and processing user-defined signals or biomarkers.This review provides an overview of diverse synthetic receptor systems being used to reprogram therapeutic cells and their wide applications in biomedical research.With a special focus on four synthetic receptor systems at the forefront,including chimeric antigen receptors(CARs)and synthetic Notch(synNotch)receptors,we address the generalized strategies to design,construct and improve synthetic receptors.Meanwhile,we also highlight the expanding landscape of therapeutic applications of the synthetic receptor systems as well as current challenges in their clinical translation.

关键词： synthetic landscape payload

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One-step cell biomanufacturing platform:porous gelatin microcarrier beads promote human embryonic stem cell-derived midbrain dopaminergic progenitor cell differentiation in vitro and survival after transplantation in vivo

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Neural Regeneration Research 2024年第2期19卷 458-464页

作者： Lin Feng Da Li Yao Tian Chengshun Zhao Yun Sun Xiaolong Kou Jun Wu Liu Wang Qi Gu Wei Li Jie Hao Baoyang Hu Yukai Wang State Key Laboratory of stem cell and Reproductive Biology Institute of ZoologyChinese Academy of SciencesBeijingChina Institute for stem cell and Regenerative Medicine Chinese Academy of SciencesBeijingChina National stem cell Resource Center Chinese Academy of SciencesBeijingChina Savaid Medical School University of Chinese Academy of SciencesBeijingChina Beijing Institute for stem cell and Regenerative Medicine BeijingChina State Key Laboratory of Membrane Biology Institute of ZoologyChinese Academy of SciencesBeijingChina

Numerous studies have shown that cell replacement therapy can replenish lost cells and rebuild neural circuitry in animal models of Parkinson’s disease.Transplantation of midbrain dopaminergic progenitor cells is a promising treatment for Parkinson’s disease.However,transplanted cells can be injured by mechanical damage during handling and by changes in the transplantation niche.Here,we developed a one-step biomanufacturing platform that uses small-aperture gelatin microcarriers to produce beads carrying midbrain dopaminergic progenitor cells.These beads allow midbrain dopaminergic progenitor cell differentiation and cryopreservation without digestion,effectively maintaining axonal integrity in vitro.Importantly,midbrain dopaminergic progenitor cell bead grafts showed increased survival and only mild immunoreactivity in vivo compared with suspended midbrain dopaminergic progenitor cell grafts.Overall,our findings show that these midbrain dopaminergic progenitor cell beads enhance the effectiveness of neuronal cell transplantation.

关键词： axonal integrity cell cryopreservation cellular environment cellular niche cell replacement therapy dopaminergic progenitors human pluripotent stem cell mechanical damage neuronal cell delivery Parkinson’s disease small-aperture gelatin microcarriers

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Allogeneic mesenchymal stem cells may be a viable treatment modality in cerebral palsy

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World Journal of Clinical Cases 2024年第9期12卷 1585-1596页

作者： Osman Boyalı Serdar Kabatas ErdinçCivelek Omer Ozdemir Yeliz Bahar-Ozdemir Necati Kaplan Eyüp Can Savrunlu Erdal Karaöz Department of Neurosurgery University of Health Sciences TurkeyGaziosmanpaşa Training and Research HospitalIstanbul 34360Turkey Center for stem cell&gene therapy Research and Practice University of Health Sciences TurkeyIstanbul 34360Turkey Department of Physical Medicine and Rehabilitation Health Sciences University Sultan Abdulhamid Han Training and Research HospitalIstanbul 34668Turkey Department of Neurosurgery Istanbul Rumeli UniversityÇorlu Reyap HospitalTekirdağ59860Turkey Department of Neurosurgery Nevşehir State HospitalNevşehir 50300Turkey Center for Regenerative Medicine and stem cell Research&Manufacturing(LivMedcell) Liv HospitalIstanbul 34340Turkey Department of Histology and Embryology Istinye UniversityFaculty of Medicineİstanbul 34010Turkey Center for stem cell and Tissue Engineering Research and Practice Istinye UniversityIstanbul 34340Turkey

BACKGROUND Cerebral palsy(CP)describes a group of disorders affecting movement,balance,and posture.Disturbances in motor functions constitute the main body of CP symptoms.These symptoms surface in early childhood and patients are affected for the rest of their lives.Currently,treatment involves various pharmacotherapies for different types of CP,including antiepileptics for epilepsy and Botox A for focal spasticity.However,none of these methods can provide full symptom relief.This has prompted researchers to look for new treatment modalities,one of which is mesenchymal stem cell therapy(MSCT).Despite being a promising tool and offering a wide array of possibilities,mesenchymal stem cells(MSCs)still need to be investigated for their efficacy and safety.AIM To analyze the efficacy and safety of MSCT in CP patients.METHODS Our sample consists of four CP patients who cannot stand or walk without external support.All of these cases received allogeneic MSCT six times as 1×106/kg intrathecally,intravenously,and intramuscularly using umbilical cord-derived MSCs(UC-MSC).We monitored and assessed the patients pre-and post-treatment using the Wee Functional Independence Measure(WeeFIM),Gross Motor Function Classification System(GMFCS),and Manual Ability Classification Scale(MACS)instruments.We utilized the Modified Ashworth Scale(MAS)to measure spasticity.RESULTS We found significant improvements in MAS scores after the intervention on both sides.Two months:Rightχ^(2)=4000,P=0.046,leftχ^(2)=4000,P=0.046;four months:Rightχ^(2)=4000,P=0.046,leftχ^(2)=4000,P=0.046;12 months:Rightχ^(2)=4000,P=0.046,leftχ^(2)=4000,P=0.046.However,there was no significant difference in motor functions based on WeeFIM results(P>0.05).GMFCS and MACS scores differed significantly at 12 months after the intervention(P=0.046,P=0.046).Finally,there was no significant change in cognitive functions(P>0.05).CONCLUSION In light of our findings,we believe that UC-MSC therapy has a positive effect on spasticity,and it partially improves motor functions.

关键词： Cerebral palsy Mesenchymal stem cell Transplantation Wharton’s jelly Muscle spasticity

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Effects of exosomes from mesenchymal stem cells on functional recovery of a patient with total radial nerve injury: A pilot study

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World Journal of stem cells 2024年第1期16卷 19-32页

作者： ErdinçCivelek Serdar Kabatas Eyüp Can Savrunlu Furkan Diren Necati Kaplan Demet Ofluoğlu Erdal Karaöz Department of Neurosurgery University of Health SciencesGaziosmanpaşa Training and Research HospitalGaziosmanpaşa 34255IstanbulTurkey Department of Neurosurgery University of Health Sciences TurkeyGaziosmanpaşa Training and Research HospitalGaziosmanpaşa 34255IstanbulTurkey Center for stem cell&gene therapy Research and Practice University of Health Sciences TurkeyGaziosmanpaşa 34255IstanbulTurkey Department of Neurosurgery Nevşehir State HospitalNevşehir 50300Turkey Department of Neurosurgery Istanbul Rumeli UniversityÇorlu Reyap HospitalÇorlu 59860TekirdağTurkey Department of Physical Medicine and Rehabilitation Ofluoğlu KlinikGöztepe 34728IstanbulTurkey Liv Hospital Center for Regenerative Medicine and Stem Cell Research&Manufacturing(LivMedCell)Beşiktaş34340IstanbulTurkey Department of Histology and Embryology Istinye UniversityFaculty of MedicineZeytinburnu 34010IstanbulTurkey Istinye University Center for Stem Cell and Tissue Engineering Research and PracticeBeşiktaş34340IstanbulTurkey

BACKGROUND Peripheral nerve injury can result in significant clinical complications that have uncertain prognoses.Currently,there is a lack of effective pharmacological interventions for nerve damage,despite the existence of several small compounds,Despite the objective of achieving full functional restoration by surgical intervention,the persistent challenge of inadequate functional recovery remains a significant concern in the context of peripheral nerve injuries.AIM To examine the impact of exosomes on the process of functional recovery following a complete radial nerve damage.METHODS A male individual,aged 24,who is right-hand dominant and an immigrant,arrived with an injury caused by a knife assault.The cut is located on the left arm,specifically below the elbow.The neurological examination and electrodiagnostic testing reveal evidence of left radial nerve damage.The sural autograft was utilized for repair,followed by the application of 1 mL of mesenchymal stem cell-derived exosome,comprising 5 billion microvesicles.This exosome was split into four equal volumes of 0.25 mL each and delivered microsurgically to both the proximal and distal stumps using the subepineural pathway.The patient was subjected to a period of 180 d during which they had neurological examination and electrodiagnostic testing.RESULTS The duration of the patient’s follow-up period was 180 d.An increasing Tinel’s sign and sensory-motor recovery were detected even at the 10th wk following nerve grafting.Upon the conclusion of the 6-mo post-treatment period,an evaluation was conducted to measure the extent of improvement in motor and sensory functions of the nerve.This assessment was based on the British Medical Research Council scale and the Mackinnon-Dellon scale.The results indicated that the level of improvement in motor function was classified as M5,denoting an excellent outcome.Additionally,the level of improvement in sensory function was classified as S3+,indicating a good outcome.It is noteworthy that these assessments were conducte

关键词： Mesenchymal stem cell Exosomes Radial nerve Sural nerve

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Efficacy and safety of human umbilical cord-derived mesenchymal stem cells in the treatment of refractory immune thrombocytopenia:a prospective,single arm,phase I trial

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Signal Transduction and Targeted therapy 2024年第5期9卷 2250-2259页

作者： Yunfei Chen Yanmei Xu Ying Chi Ting Sun Yuchen Gao Xueqing Dou Zhibo Han Feng Xue Huiyuan Li Wei Liu Xiaofan Liu Huan Dong Rongfeng Fu Mankai Ju Xinyue Dai Wentian Wang Yueshen Ma Zhen Song Jundong Gu Wei Gong Renchi Yang Lei Zhang State Key Laboratory of Experimental Hematology National Clinical Research Centre for Blood DiseasesHaihe Laboratory of Cell EcosystemTianjin Key Laboratory of Gene Therapy for Blood DiseasesCAMS Key Laboratory of Gene Therapy for Blood DiseasesInstitute of Hematology&Blood Diseases HospitalChinese Academy of Medical Sciences&Peking Union Medical CollegeTianjin 300020China Tianjin Institutes of Health Science Tianjin 301600China National Clinical Research Center for Hematologic Diseases Jiangsu Institute of HematologyThe First Affiliated Hospital of Soochow UniversitySuzhou 215006China National Engineering Research Centre of cell Products Tianjin Key Laboratory of Engineering Technologies for Cell PharmaceuticalAmCellGene Engineering Co.LtdTianjin 300457China School of Population Medicine and Public Health Chinese Academy of Medical Sciences and Peking Union Medical College100730 BeijingChina

Patients with refractory immune thrombocytopenia(ITP)frequently encounter substantial bleeding risks and demonstrate limited responsiveness to existing therapies.Umbilical cord-derived mesenchymal stem cells(UC-MSCs)present a promising alternative,capitalizing on their low immunogenicity and potent immunomodulatory effects for treating diverse autoimmune disorders.This prospective phase I trial enrolled eighteen eligible patients to explore the safety and efficacy of UC-MSCs in treating refractory ITP.The research design included administering UC-MSCs at escalating doses of 0.5×10^(6)cells/kg,1.0×10^(6)cells/kg,and 2.0×10^(6)cells/kg weekly for four consecutive weeks across three cohorts during the dose-escalation phase,followed by a dose of 2.0×10^(6)cells/kg weekly for the dose-expansion phase.Adverse events,platelet counts,and changes in peripheral blood immunity were monitored and recorded throughout the administration and follow-up period.Ultimately,12(with an addition of three patients in the 2.0×10^(6)cells/kg group due to dose-limiting toxicity)and six patients were enrolled in the dose-escalation and dose-expansion phase,respectively.Thirteen patients(13/18,72.2%)experienced one or more treatment emergent adverse events.Serious adverse events occurred in four patients(4/18,22.2%),including gastrointestinal hemorrhage(2/4),profuse menstruation(1/4),and acute myocardial infarction(1/4).The response rates were 41.7%in the dose-escalation phase(5/12,two received 1.0×10^(6)cells/kg per week,and three received 2.0×10^(6)cells/kg per week)and 50.0%(3/6)in the dose-expansion phase.The overall response rate was 44.4%(8/18)among all enrolled patients.To sum up,UC-MSCs are effective and well tolerated in treating refractory ITP(ClinicalTrials.gov ID:NCT04014166).

关键词： doses immunity treatment

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Current assessment and management of measurable residual disease in patients with acute lymphoblastic leukemia in the setting of CAR-T-cell therapy

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Chinese Medical Journal 2024年第2期137卷 140-151页

作者： Minghao Lin Xiaosu Zhao Yingjun Chang Xiangyu Zhao Peking University People’s Hospital Peking University Institute of HematologyNational Clinical Research Center for Hematologic DiseaseBeijing Key Laboratory of Hematopoietic Stem Cell TransplantationBeijing 100044China

Chimeric antigen receptor(CAR)-modified T-cell therapy has achieved remarkable success in the treatment of acute lymphoblastic leukemia(ALL).Measurable/minimal residual disease(MRD)monitoring plays a significant role in the prognostication and management of patients undergoing CAR-T-cell therapy.Common MRD detection methods include flow cytometry(FCM),polymerase chain reaction(PCR),and next-generation sequencing(NGS),and each method has advantages and limitations.It has been well documented that MRD positivity predicts a poor prognosis and even disease relapse.Thus,how to perform prognostic evaluations,stratify risk based on MRD status,and apply MRD monitoring to guide individual therapeutic decisions have important implications in clinical practice.This review assesses the common and novel MRD assessment methods.In addition,we emphasize the critical role of MRD as a prognostic biomarker and summarize the latest studies regarding MRD-directed combination therapy with CAR-T-cell therapy and allogeneic hematopoietic stem cell transplantation(allo-HSCT),as well as other therapeutic strategies to improve treatment effect.Furthermore,this review discusses current challenges and strategies for MRD detection in the setting of disease relapse after targeted therapy.

关键词： Measurable/minimal residual disease Acute lymphoblastic leukemia Chimeric antigen receptor-modified T-cell therapy Allogeneic hematopoietic stem cell transplantation Relapse

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The evolving views of hematopoiesis:from embryo to adulthood and from in vivo to in vitro

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Journal of genetics and Genomics 2024年第1期51卷 3-15页

作者： Yifan Zhang Feng Liu Shandong Provincial Key Laboratory of Animal cell and Developmental Biology School of Life SciencesShandong UniversityQingdaoShandong 266237China State Key Laboratory of Membrane Biology Institute of ZoologyInstitute for Stem Cell and RegenerationUniversity of Chinese Academy of SciencesChinese Academy of SciencesBeijing 100101China

The hematopoietic system composed of hematopoietic stem and progenitor cells(HSPCs)and their differentiated lineages serves as an ideal model to uncover generic principles of cell fate transitions.From gastrulation onwards,there successively emerge primitive hematopoiesis(that produces specialized he-matopoietic cells),pro-definitive hematopoiesis(that produces lineage-restricted progenitor cells),and definitive hematopoiesis(that produces multipotent HSPCs).These nascent lineages develop in several transient hematopoietic sites and finally colonize into lifelong hematopoietic sites.The development and maintenance of hematopoietic lineages are orchestrated by cell-intrinsic gene regulatory networks and cell-extrinsic microenvironmental cues.Owing to the progressive methodology(e.g.,high-throughput lineage tracing and single-cell functional and omics analyses),our understanding of the developmental origin of hematopoietic lineages and functional properties of certain hematopoietic organs has been updated;meanwhile,new paradigms to characterize rare cell types,cell heterogeneity and its causes,and comprehensive regulatory landscapes have been provided.Here,we review the evolving views of HSPC biology during developmental and postnatal hematopoiesis.Moreover,we discuss recent advances in the in vitro induction and expansion of HSPCs,with a focus on the implications for clinical applications.

关键词： Hematopoietic stem and progenitor cell Developmental hematopoiesis Postnatal hematopoiesis In vitro hematopoiesis

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Improve meat production and virus resistance by simultaneously editing multiple genes in livestock using Cas12i^(Max)

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Science China(Life Sciences) 2024年第3期67卷 555-564页

作者： Jilong Ren Tang Hai Yangcan Chen Ke Sun Zhiqiang Han Jing Wang Chongyang Li Qingwei Wang Leyun Wang Huabing Zhu Dawei Yu Wei Li Shanjiang Zhao State Key Laboratory of Animal Nutrition Key Laboratory of Animal GeneticsBreeding and Reproduction of Ministry of Agriculture and Rural AffairsInstitute of Animal ScienceChinese Academy of Agricultural SciencesBeijing 100193China State Key Laboratoryf stem cell and Reproductive Biology Instituteof ZooogyChinese Academyof SciencesBeijing 100101China Beijing Institute for stem cell and Regenerative Medicine Beijing 100101China Beijing Farm Animal Research Center Institute of ZoologyChinese Academy of SciencesBeijing 100101China

The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated gene(Cas) system is continually optimized to achieve the most efficient gene editing effect. The Cas12i^(Max), a Cas12i variant, exhibits powerful DNA editing activity and enriches the gene editing toolbox. However, the application of Cas12i^(Max)in large domestic animals has not yet been reported. To verify the efficiency and feasibility of multiple gene editing in large animals, we generated porcine fibroblasts with simultaneous knockouts of IGF2, ANPEP, CD163,and MSTN via Cas12i^(Max)in one step. Phenotypically stable pigs were created through somatic cell nuclear transfer technology. They exhibited improved growth performance and muscle quality. Furthermore, we simultaneously edited three genes in bovine fibroblasts. A knockout of MSTN and PRNP was created and the amino acid Q-G in CD18 was precisely substituted. Meanwhile, no off-target phenomenon was observed by sum-type analysis or off-target detection. These results verified the effectiveness of Cas12i^(Max)for gene editing in livestock animals and demonstrated the potential application of Cas12i^(Max)in the field of animal trait improvement for agricultural production.

关键词： Cas12i^(Max) gene editing livestock multiple trait improvement

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A single-nucleus transcriptomic atlas of primate liver aging uncovers the pro-senescence role of SREBP2 in hepatocytes

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Protein & cell 2024年第2期15卷 98-120页

作者： Shanshan Yang Chengyu Liu Mengmeng Jiang Xiaoqian Liu Lingling Geng Yiyuan Zhang Shuhui Sun Kang Wang jian Yin Shuai Ma Si Wang Juan Carlos Izpisua Belmonte Weiqi Zhang Jing Qu Guang-Hui Liu Advanced Innovation Center for Human Brain Protection and National Clinical Research Center for Geriatric Disorders Xuanwu Hospital Capital Medical UniversityBeijing 100053China State Key Laboratory of Membrane Biology Institute of ZoologyChinese Academy of SciencesBeijing 100101China Aging Translational Medicine Center International Center for Aging and CancerBeijing Municipal Geriatric Medical Research CenterXuanwu HospitalCapital Medical UniversityBeijing 100053China State Key Laboratory of stem cell and Reproductive Biology Institute of ZoologyChinese Academy of SciencesBeijing 100101China CAS Key Laboratory of Genomic and Precision Medicine Beijing Institute of GenomicsChinese Academy of Sciences and China National Center for BioinformationBeijing 100101China Xuanwu Hospital Capital Medical University Beijing 100053China University of Chinese Academy of Sciences Beijing 100049China Institute for stem cell and Regeneration Chinese Academy of SciencesBeijing 100101China Beijing Institute for stem cell and Regenerative Medicine Beijing 100101China Aging Biomarker Consortium Beijing 100101China Altos Labs Inc.San DiegoCA 94022USA

Aging increases the risk ofliver diseases and systemic susceptibility to aging-related diseases.However,cell type-specific changes and the underlying mechanism of liver aging in higher vertebrates remain incompletely characterized.Here,we constructed the first single-nucleus transcriptomic landscape of primate liver aging,in which we resolved cell type-specific gene expression fluctuation in hepatocytes across three liver zonations and detected aberrant cell-cell interactions between hepatocytes and niche cells.Upon in-depth dissection of this rich dataset,we identifed impaired lipid metabolism and upregulation of chronic inflammation-related genes prominently associated with declined liver functions during aging.In particular,hyperactivated sterol regulatory element-binding protein(SREBP)signaling was a hallmark of the aged liver,and consequently,forced activation of SREBP2 in human primary hepatocytes recapitulated in vivo aging phenotypes,manifesting as impaired detoxification and accelerated cellular senescence.This study expands our knowledge of primate liver aging and informs the development of diagnostics and therapeutic interventions for liver aging and associated diseases.

关键词： single-nucleus RNA sequencing liver hepatocytes aging senescence SREBP2

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