The microcephaly-capillary malformation (MIC-CAP) syndrome is a life-threatening disease caused by biallelic mutations of the STAMBP gene, which encodes an endosomal deubiquitinating enzyme. To establish a suitable preclinical animal model for clinical therapeutic practice, we generated a central nervous system (CNS)-specific Stambp knockout mouse model (Stambp Sox1-cKO) that phenocopies Stambp null mice including progressive microcephaly, postnatal growth retardation and complete penetrance of preweaning death. In this MIC-CAP syndrome mouse model, early-onset neuronal death occurs specifically in the hippocampus and cortex, accompanied by aggregation of ubiquitinated proteins, and massive neuroinflammation. Importantly, neonatal AAV9-mediated gene supplementation of Stambp in the brain could significantly improve neurological defects, sustain growth, and prolong the lifespan of StambpSox1-cKO mice. Together, our findings reveal a central role of brain defects in the pathogenesis of STAMBP deficiency and provide preclinical evidence that postnatal gene replacement is an effective approach to cure the disease.
{"title":"AAV-mediated Stambp gene replacement therapy rescues neurological defects in a mouse model of microcephaly-capillary malformation syndrome.","authors":"Meixin Hu, Jun Li, Jingxin Deng, Chunxue Liu, Yingying Liu, Huiping Li, Weijun Feng, Xiu Xu","doi":"10.1016/j.ymthe.2024.08.017","DOIUrl":"10.1016/j.ymthe.2024.08.017","url":null,"abstract":"<p><p>The microcephaly-capillary malformation (MIC-CAP) syndrome is a life-threatening disease caused by biallelic mutations of the STAMBP gene, which encodes an endosomal deubiquitinating enzyme. To establish a suitable preclinical animal model for clinical therapeutic practice, we generated a central nervous system (CNS)-specific Stambp knockout mouse model (Stambp <sup>Sox1-cKO</sup>) that phenocopies Stambp null mice including progressive microcephaly, postnatal growth retardation and complete penetrance of preweaning death. In this MIC-CAP syndrome mouse model, early-onset neuronal death occurs specifically in the hippocampus and cortex, accompanied by aggregation of ubiquitinated proteins, and massive neuroinflammation. Importantly, neonatal AAV9-mediated gene supplementation of Stambp in the brain could significantly improve neurological defects, sustain growth, and prolong the lifespan of Stambp<sup>Sox1-cKO</sup> mice. Together, our findings reveal a central role of brain defects in the pathogenesis of STAMBP deficiency and provide preclinical evidence that postnatal gene replacement is an effective approach to cure the disease.</p>","PeriodicalId":19020,"journal":{"name":"Molecular Therapy","volume":null,"pages":null},"PeriodicalIF":12.1,"publicationDate":"2024-08-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142018089","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-21DOI: 10.1016/j.ymthe.2024.08.018
Sonal Dharani, Hana Cho, Jorge Postigo Fernandez, Alexandre Juillerat, Julien Valton, Philippe Duchateau, Laurent Poirot, Shipra Das
Adoptive cell therapy using chimeric antigen receptor (CAR) T cells has proven to be lifesaving for many cancer patients. However, its therapeutic efficacy has been limited in solid tumors. One key factor for this is cancer-associated fibroblasts (CAFs) that modulate the tumor microenvironment (TME) to inhibit T cell infiltration and induce "T cell dysfunction." Additionally, the sparsity of tumor-specific antigens (TSA) and expression of CAR-directed tumor-associated antigens (TAA) on normal tissues often results in "on-target off-tumor" cytotoxicity, raising safety concerns. Using TALEN-mediated gene editing, we present here an innovative CAR T cell engineering strategy to overcome these challenges. Our allogeneic "Smart CAR T cells" are designed to express a constitutive CAR, targeting FAP+ CAFs in solid tumors. Additionally, a second CAR targeting a TAA such as mesothelin is specifically integrated at a TCR signaling-inducible locus like PDCD1. FAPCAR-mediated CAF targeting induces expression of the mesothelin CAR, establishing an IF/THEN-gated circuit sensitive to dual antigen sensing. Using this approach, we observe enhanced anti-tumor cytotoxicity, while limiting "on-target off-tumor" toxicity. Our study thus demonstrates TALEN-mediated gene editing capabilities for design of allogeneic IF/THEN-gated dual CAR T cells that efficiently target immunotherapy-recalcitrant solid tumors while mitigating potential safety risks, encouraging clinical development of this strategy.
事实证明,使用嵌合抗原受体(CAR)T 细胞的适应性细胞疗法可以挽救许多癌症患者的生命。然而,它对实体瘤的疗效有限。其中一个关键因素是癌症相关成纤维细胞(CAFs),它们会调节肿瘤微环境(TME),抑制 T 细胞浸润并诱发 "T 细胞功能障碍"。此外,肿瘤特异性抗原(TSA)的稀缺性和CAR定向肿瘤相关抗原(TAA)在正常组织上的表达往往会导致 "靶外 "细胞毒性,从而引发安全性问题。利用 TALEN 介导的基因编辑,我们在此介绍一种创新的 CAR-T 细胞工程策略,以克服这些挑战。我们的异体 "智能 CAR T 细胞 "被设计为表达组成型 CAR,靶向实体瘤中的 FAP+ CAFs。此外,第二种靶向肿瘤相关抗原(TAA)(如间皮素)的CAR特异性地整合在TCR信号诱导位点(如PDCD1)上。FAPCAR 介导的 CAF 靶向会诱导间皮素 CAR 的表达,从而建立一个对双重抗原感应敏感的 IF/THEN 门控回路。利用这种方法,我们观察到了增强的抗肿瘤细胞毒性,同时限制了 "靶标外 "毒性。因此,我们的研究展示了 TALEN 介导的基因编辑能力,可用于设计异体 IF/THEN 门控双 CAR T 细胞,有效靶向免疫疗法再现性实体瘤,同时降低潜在的安全风险,从而鼓励这一策略的临床开发。
{"title":"TALEN-edited allogeneic inducible dual CAR T cells enable effective targeting of solid tumors while mitigating off-tumor toxicity.","authors":"Sonal Dharani, Hana Cho, Jorge Postigo Fernandez, Alexandre Juillerat, Julien Valton, Philippe Duchateau, Laurent Poirot, Shipra Das","doi":"10.1016/j.ymthe.2024.08.018","DOIUrl":"10.1016/j.ymthe.2024.08.018","url":null,"abstract":"<p><p>Adoptive cell therapy using chimeric antigen receptor (CAR) T cells has proven to be lifesaving for many cancer patients. However, its therapeutic efficacy has been limited in solid tumors. One key factor for this is cancer-associated fibroblasts (CAFs) that modulate the tumor microenvironment (TME) to inhibit T cell infiltration and induce \"T cell dysfunction.\" Additionally, the sparsity of tumor-specific antigens (TSA) and expression of CAR-directed tumor-associated antigens (TAA) on normal tissues often results in \"on-target off-tumor\" cytotoxicity, raising safety concerns. Using TALEN-mediated gene editing, we present here an innovative CAR T cell engineering strategy to overcome these challenges. Our allogeneic \"Smart CAR T cells\" are designed to express a constitutive CAR, targeting FAP<sup>+</sup> CAFs in solid tumors. Additionally, a second CAR targeting a TAA such as mesothelin is specifically integrated at a TCR signaling-inducible locus like PDCD1. FAPCAR-mediated CAF targeting induces expression of the mesothelin CAR, establishing an IF/THEN-gated circuit sensitive to dual antigen sensing. Using this approach, we observe enhanced anti-tumor cytotoxicity, while limiting \"on-target off-tumor\" toxicity. Our study thus demonstrates TALEN-mediated gene editing capabilities for design of allogeneic IF/THEN-gated dual CAR T cells that efficiently target immunotherapy-recalcitrant solid tumors while mitigating potential safety risks, encouraging clinical development of this strategy.</p>","PeriodicalId":19020,"journal":{"name":"Molecular Therapy","volume":null,"pages":null},"PeriodicalIF":12.1,"publicationDate":"2024-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142018092","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-10DOI: 10.1016/j.ymthe.2024.08.009
Devin J Jones, Divya Soundararajan, Noah K Taylor, Osasumwen V Aimiuwu, Pranav Mathkar, Amy Shore, Jia Jie Teoh, Wanqi Wang, Tristan T Sands, Matthew C Weston, Scott Q Harper, Wayne N Frankel
Effective gene therapy for gain-of-function or dominant-negative disease mutations may require eliminating expression of the mutant copy together with wild-type replacement. We evaluated such a knockdown-replace strategy in a mouse model of DNM1 disease, a debilitating and intractable neurodevelopmental epilepsy. To challenge the approach robustly, we expressed a patient-based variant in GABAergic neurons-which resulted in growth delay and lethal seizures evident by postnatal week three-and delivered to newborn pups an AAV9-based vector encoding a ubiquitously expressed, Dnm1-specific interfering RNA (RNAi) bivalently in tail-to-tail configuration with a neuron-specific, RNAi-resistant, codon-optimized Dnm1 cDNA. Pups receiving RNAi or cDNA alone fared no better than untreated pups, whereas the vast majority of mutants receiving modest doses survived with almost full growth recovery. Synaptic recordings of cortical neurons derived from treated pups revealed that significant alterations in transmission from inhibitory to excitatory neurons were rectified by bivalent vector application. To examine the mutant transcriptome and impact of treatment, we used RNA sequencing and functional annotation clustering. Mutants displayed abnormal expression of more than 1,000 genes in highly significant and relevant functional clusters, clusters that were abrogated by treatment. Together these results suggest knockdown-replace as a potentially effective strategy for treating DNM1 and related genetic neurodevelopmental disease.
{"title":"Effective knockdown-replace gene therapy in a novel mouse model of DNM1 developmental and epileptic encephalopathy.","authors":"Devin J Jones, Divya Soundararajan, Noah K Taylor, Osasumwen V Aimiuwu, Pranav Mathkar, Amy Shore, Jia Jie Teoh, Wanqi Wang, Tristan T Sands, Matthew C Weston, Scott Q Harper, Wayne N Frankel","doi":"10.1016/j.ymthe.2024.08.009","DOIUrl":"10.1016/j.ymthe.2024.08.009","url":null,"abstract":"<p><p>Effective gene therapy for gain-of-function or dominant-negative disease mutations may require eliminating expression of the mutant copy together with wild-type replacement. We evaluated such a knockdown-replace strategy in a mouse model of DNM1 disease, a debilitating and intractable neurodevelopmental epilepsy. To challenge the approach robustly, we expressed a patient-based variant in GABAergic neurons-which resulted in growth delay and lethal seizures evident by postnatal week three-and delivered to newborn pups an AAV9-based vector encoding a ubiquitously expressed, Dnm1-specific interfering RNA (RNAi) bivalently in tail-to-tail configuration with a neuron-specific, RNAi-resistant, codon-optimized Dnm1 cDNA. Pups receiving RNAi or cDNA alone fared no better than untreated pups, whereas the vast majority of mutants receiving modest doses survived with almost full growth recovery. Synaptic recordings of cortical neurons derived from treated pups revealed that significant alterations in transmission from inhibitory to excitatory neurons were rectified by bivalent vector application. To examine the mutant transcriptome and impact of treatment, we used RNA sequencing and functional annotation clustering. Mutants displayed abnormal expression of more than 1,000 genes in highly significant and relevant functional clusters, clusters that were abrogated by treatment. Together these results suggest knockdown-replace as a potentially effective strategy for treating DNM1 and related genetic neurodevelopmental disease.</p>","PeriodicalId":19020,"journal":{"name":"Molecular Therapy","volume":null,"pages":null},"PeriodicalIF":12.1,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141913352","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Liver cancer is one of the most prevalent malignant tumors worldwide. According to the Barcelona Clinic Liver Cancer staging criteria, clinical guidelines provide tutorials to clinical management of liver cancer at their individual stages. However, most patients diagnosed with liver cancer are at advanced stage; therefore, many researchers conduct investigations on targeted therapy, aiming to improve the overall survival of these patients. To date, small-molecule-based targeted therapies are highly recommended (first line: sorafenib and lenvatinib; second line: regorafenib and cabozantinib) by current the clinical guidelines of the American Society of Clinical Oncology, European Society for Medical Oncology, and National Comprehensive Cancer Network. Herein, we summarize the small-molecule-based targeted therapies in liver cancer, including the approved and preclinical therapies as well as the therapies under clinical trials, and introduce their history of discovery, clinical trials, indications, and molecular mechanisms. For drug resistance, the revealed mechanisms of action and the combination therapies are also discussed. In fact, the known small-molecule-based therapies still have limited clinical benefits to liver cancer patients. Therefore, we analyze the current status and give our ideas for the urgent issues and future directions in this field, suggesting clues for novel techniques in liver cancer treatment.
{"title":"Small-molecule-based targeted therapy in liver cancer.","authors":"Yue Ming, Yanqiu Gong, Xuewen Fu, Xinyu Ouyang, Yong Peng, Wenchen Pu","doi":"10.1016/j.ymthe.2024.08.001","DOIUrl":"10.1016/j.ymthe.2024.08.001","url":null,"abstract":"<p><p>Liver cancer is one of the most prevalent malignant tumors worldwide. According to the Barcelona Clinic Liver Cancer staging criteria, clinical guidelines provide tutorials to clinical management of liver cancer at their individual stages. However, most patients diagnosed with liver cancer are at advanced stage; therefore, many researchers conduct investigations on targeted therapy, aiming to improve the overall survival of these patients. To date, small-molecule-based targeted therapies are highly recommended (first line: sorafenib and lenvatinib; second line: regorafenib and cabozantinib) by current the clinical guidelines of the American Society of Clinical Oncology, European Society for Medical Oncology, and National Comprehensive Cancer Network. Herein, we summarize the small-molecule-based targeted therapies in liver cancer, including the approved and preclinical therapies as well as the therapies under clinical trials, and introduce their history of discovery, clinical trials, indications, and molecular mechanisms. For drug resistance, the revealed mechanisms of action and the combination therapies are also discussed. In fact, the known small-molecule-based therapies still have limited clinical benefits to liver cancer patients. Therefore, we analyze the current status and give our ideas for the urgent issues and future directions in this field, suggesting clues for novel techniques in liver cancer treatment.</p>","PeriodicalId":19020,"journal":{"name":"Molecular Therapy","volume":null,"pages":null},"PeriodicalIF":12.1,"publicationDate":"2024-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141902422","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-08DOI: 10.1016/j.ymthe.2024.08.002
Muhammed Burak Demircan, Luca J Zinser, Alexander Michels, Mar Guaza-Lasheras, Fabian John, Johanna M Gorol, Samuel A Theuerkauf, Dorothee M Günther, Dirk Grimm, Florian R Greten, Petr Chlanda, Frederic B Thalheimer, Christian J Buchholz
One of the biggest challenges for in vivo gene therapy are vectors mediating highly selective gene transfer into a defined population of therapy-relevant cells. Here we present DARPin-targeted AAVs (DART-AAVs) displaying DARPins specific for human and murine CD8. Insertion of DARPins into the GH2/GH3 loop of the capsid protein 1 (VP1) of AAV2 and AAV6 resulted in high selectivity for CD8-positive T cells with unimpaired gene delivery activity. Remarkably, the capsid core structure was unaltered with protruding DARPins detectable. In complex primary cell mixtures, including donor blood or systemic injections into mice, the CD8-targeted AAVs were by far superior to unmodified AAV2 and AAV6 in terms of selectivity, target cell viability, and gene transfer rates. In vivo, up to 80% of activated CD8+ T cells were hit upon a single vector injection into conditioned humanized or immunocompetent mice. While gene transfer rates decreased significantly under non-activated conditions, genomic modification selectively in CD8+ T cells was still detectable upon Cre delivery into indicator mice. In both mouse models, selectivity for CD8+ T cells was close to absolute with exceptional detargeting from liver. The CD8-AAVs described here expand strategies for immunological research and in vivo gene therapy options.
体内基因治疗面临的最大挑战之一是如何将高选择性基因转移到确定的治疗相关细胞群中。在这里,我们展示了DARPin靶向AAVs(DART-AAVs),它显示了特异于人类和小鼠CD8的DARPin。将 DARPins 插入 AAV2 和 AAV6 的囊膜蛋白 1 (VP1) 的 GH2/GH3 环后,CD8 阳性 T 细胞具有高度选择性,基因递送活性不受影响。值得注意的是,AAV2 和 AAV6 的噬菌体核心结构没有改变,而且还能检测到突出的 DARPins。在复杂的原代细胞混合物(包括供体血液或小鼠全身注射)中,CD8 靶向 AAV 在选择性、靶细胞活力和基因转移率方面远远优于未修饰的 AAV2 和 AAV6。在体内,向条件化人源化小鼠或免疫功能健全的小鼠注射一次载体后,高达 80% 的活化 CD8+ T 细胞被击中。虽然基因转移率在非活化条件下明显下降,但将 Cre 运送到指示性小鼠体内后,仍可检测到 CD8+ T 细胞中选择性的基因组修饰。在这两种小鼠模型中,CD8+ T 细胞的选择性接近绝对值,而且能从肝脏中脱靶。本文描述的 CD8-AAVs 扩展了免疫学研究和体内基因治疗的策略。
{"title":"T-cell specific in vivo gene delivery with DART-AAVs targeted to CD8.","authors":"Muhammed Burak Demircan, Luca J Zinser, Alexander Michels, Mar Guaza-Lasheras, Fabian John, Johanna M Gorol, Samuel A Theuerkauf, Dorothee M Günther, Dirk Grimm, Florian R Greten, Petr Chlanda, Frederic B Thalheimer, Christian J Buchholz","doi":"10.1016/j.ymthe.2024.08.002","DOIUrl":"10.1016/j.ymthe.2024.08.002","url":null,"abstract":"<p><p>One of the biggest challenges for in vivo gene therapy are vectors mediating highly selective gene transfer into a defined population of therapy-relevant cells. Here we present DARPin-targeted AAVs (DART-AAVs) displaying DARPins specific for human and murine CD8. Insertion of DARPins into the GH2/GH3 loop of the capsid protein 1 (VP1) of AAV2 and AAV6 resulted in high selectivity for CD8-positive T cells with unimpaired gene delivery activity. Remarkably, the capsid core structure was unaltered with protruding DARPins detectable. In complex primary cell mixtures, including donor blood or systemic injections into mice, the CD8-targeted AAVs were by far superior to unmodified AAV2 and AAV6 in terms of selectivity, target cell viability, and gene transfer rates. In vivo, up to 80% of activated CD8+ T cells were hit upon a single vector injection into conditioned humanized or immunocompetent mice. While gene transfer rates decreased significantly under non-activated conditions, genomic modification selectively in CD8+ T cells was still detectable upon Cre delivery into indicator mice. In both mouse models, selectivity for CD8+ T cells was close to absolute with exceptional detargeting from liver. The CD8-AAVs described here expand strategies for immunological research and in vivo gene therapy options.</p>","PeriodicalId":19020,"journal":{"name":"Molecular Therapy","volume":null,"pages":null},"PeriodicalIF":12.1,"publicationDate":"2024-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141902423","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Dysregulated T cell activation underpins the immunopathology of rheumatoid arthritis (RA), yet the machineries that orchestrate T cell effector program remain incompletely understood. Herein, we leveraged bulk and single-cell RNA sequencing data from RA patients and validated protein disulfide isomerase family A member 3 (PDIA3) as a potential therapeutic target. PDIA3 is remarkably upregulated in pathogenic CD4 T cells derived from RA patients and positively correlates with C-reactive protein level and disease activity score 28. Pharmacological inhibition or genetic ablation of PDIA3 alleviates RA-associated articular pathology and autoimmune responses. Mechanistically, T cell receptor signaling triggers intracellular calcium flux to activate NFAT1, a process that is further potentiated by Wnt5a under RA settings. Activated NFAT1 then directly binds to the Pdia3 promoter to enhance the expression of PDIA3, which complexes with STAT1 or PKM2 to facilitate their nuclear import for transcribing T helper 1 (Th1) and Th17 lineage-related genes, respectively. This non-canonical regulatory mechanism likely occurs under pathological conditions, as PDIA3 could only be highly induced following aberrant external stimuli. Together, our data support that targeting PDIA3 is a vital strategy to mitigate autoimmune diseases, such as RA, in clinical settings.
{"title":"PDIA3 orchestrates effector T cell program by serving as a chaperone to facilitate the non-canonical nuclear import of STAT1 and PKM2.","authors":"Chun-Liang Yang, Fa-Xi Wang, Jia-Hui Luo, Shan-Jie Rong, Wan-Ying Lu, Qi-Jie Chen, Jun Xiao, Ting Wang, Dan-Ni Song, Jing Liu, Qian Mo, Shuo Li, Yu Chen, Ya-Nan Wang, Yan-Jun Liu, Tong Yan, Wei-Kuan Gu, Shu Zhang, Fei Xiong, Qi-Lin Yu, Zi-Yun Zhang, Ping Yang, Shi-Wei Liu, Decio Eizirik, Ling-Li Dong, Fei Sun, Cong-Yi Wang","doi":"10.1016/j.ymthe.2024.05.038","DOIUrl":"10.1016/j.ymthe.2024.05.038","url":null,"abstract":"<p><p>Dysregulated T cell activation underpins the immunopathology of rheumatoid arthritis (RA), yet the machineries that orchestrate T cell effector program remain incompletely understood. Herein, we leveraged bulk and single-cell RNA sequencing data from RA patients and validated protein disulfide isomerase family A member 3 (PDIA3) as a potential therapeutic target. PDIA3 is remarkably upregulated in pathogenic CD4 T cells derived from RA patients and positively correlates with C-reactive protein level and disease activity score 28. Pharmacological inhibition or genetic ablation of PDIA3 alleviates RA-associated articular pathology and autoimmune responses. Mechanistically, T cell receptor signaling triggers intracellular calcium flux to activate NFAT1, a process that is further potentiated by Wnt5a under RA settings. Activated NFAT1 then directly binds to the Pdia3 promoter to enhance the expression of PDIA3, which complexes with STAT1 or PKM2 to facilitate their nuclear import for transcribing T helper 1 (Th1) and Th17 lineage-related genes, respectively. This non-canonical regulatory mechanism likely occurs under pathological conditions, as PDIA3 could only be highly induced following aberrant external stimuli. Together, our data support that targeting PDIA3 is a vital strategy to mitigate autoimmune diseases, such as RA, in clinical settings.</p>","PeriodicalId":19020,"journal":{"name":"Molecular Therapy","volume":null,"pages":null},"PeriodicalIF":12.1,"publicationDate":"2024-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141186813","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Despite the remarkable success of chimeric antigen receptor (CAR) T therapy in hematological malignancies, its efficacy in solid tumors remains limited. Cytokine-engineered CAR T cells offer a promising avenue, yet their clinical translation is hindered by the risks associated with constitutive cytokine expression. In this proof-of-concept study, we leverage the endogenous interferon (IFN)-γ promoter for transgenic interleukin (IL)-15 expression. We demonstrate that IFN-γ expression is tightly regulated by T cell receptor signaling. By introducing an internal ribosome entry site IL15 into the 3' UTR of the IFN-γ gene via homology directed repair-mediated knock-in, we confirm that IL-15 expression can co-express with IFN-γ in an antigen stimulation-dependent manner. Importantly, the insertion of transgenes does not compromise endogenous IFN-γ expression. In vitro and in vivo data demonstrate that IL-15 driven by the IFN-γ promoter dramatically improves CAR T cells' antitumor activity, suggesting the effectiveness of IL-15 expression. Last, as a part of our efforts toward clinical translation, we have developed an innovative two-gene knock-in approach. This approach enables the simultaneous integration of CAR and IL-15 genes into TRAC and IFN-γ gene loci using a single AAV vector. CAR T cells engineered to express IL-15 using this approach demonstrate enhanced antitumor efficacy. Overall, our study underscores the feasibility of utilizing endogenous promoters for transgenic cytokines expression in CAR T cells.
尽管 CAR T 疗法在血液恶性肿瘤中取得了巨大成功,但其在实体瘤中的疗效仍然有限。细胞因子工程化的 CAR T 细胞提供了一条前景广阔的途径,但其临床转化却受到组成型细胞因子表达相关风险的阻碍。在这项概念验证研究中,我们利用内源性 IFN-γ 启动子进行转基因 IL-15 表达。我们证明 IFN-γ 的表达受到 TCR 信号的严格调控。通过 HDR 介导的基因敲入将 IRES-IL15 导入 IFN-γ 基因的 3'-UTR 中,我们证实 IL-15 的表达可与 IFN-γ 以抗原刺激依赖性的方式共同表达。重要的是,转基因的插入不会影响内源性 IFN-γ 的表达。体外和体内数据表明,IFN-γ 启动子驱动的 IL-15 能显著提高 CAR T 细胞的抗肿瘤活性,这表明 IL-15 的表达是有效的。最后,作为临床转化努力的一部分,我们开发了一种创新的双基因敲入方法。这种方法使用单一 AAV 载体将 CAR 和 IL-15 基因同时整合到 TRAC 和 IFN-γ 基因座中。使用这种方法设计的表达 IL-15 的 CAR T 细胞显示出更强的抗肿瘤功效。总之,我们的研究强调了利用内源性启动子在 CAR T 细胞中表达转基因细胞因子的可行性。
{"title":"Building a novel TRUCK by harnessing the endogenous IFN-gamma promoter for cytokine expression.","authors":"Liya Ma, Kaiwen Zhang, Jian Xu, Jian Wang, Ting Jiang, Xiaolong Du, Jiaxin Zhang, Jing Huang, Fengyi Ren, Dong Liu, Weiwei Xue, Dongxu Kan, Mengjiao Yao, Yutian Liang, Hongxing Jason-Sun","doi":"10.1016/j.ymthe.2024.06.017","DOIUrl":"10.1016/j.ymthe.2024.06.017","url":null,"abstract":"<p><p>Despite the remarkable success of chimeric antigen receptor (CAR) T therapy in hematological malignancies, its efficacy in solid tumors remains limited. Cytokine-engineered CAR T cells offer a promising avenue, yet their clinical translation is hindered by the risks associated with constitutive cytokine expression. In this proof-of-concept study, we leverage the endogenous interferon (IFN)-γ promoter for transgenic interleukin (IL)-15 expression. We demonstrate that IFN-γ expression is tightly regulated by T cell receptor signaling. By introducing an internal ribosome entry site IL15 into the 3' UTR of the IFN-γ gene via homology directed repair-mediated knock-in, we confirm that IL-15 expression can co-express with IFN-γ in an antigen stimulation-dependent manner. Importantly, the insertion of transgenes does not compromise endogenous IFN-γ expression. In vitro and in vivo data demonstrate that IL-15 driven by the IFN-γ promoter dramatically improves CAR T cells' antitumor activity, suggesting the effectiveness of IL-15 expression. Last, as a part of our efforts toward clinical translation, we have developed an innovative two-gene knock-in approach. This approach enables the simultaneous integration of CAR and IL-15 genes into TRAC and IFN-γ gene loci using a single AAV vector. CAR T cells engineered to express IL-15 using this approach demonstrate enhanced antitumor efficacy. Overall, our study underscores the feasibility of utilizing endogenous promoters for transgenic cytokines expression in CAR T cells.</p>","PeriodicalId":19020,"journal":{"name":"Molecular Therapy","volume":null,"pages":null},"PeriodicalIF":12.1,"publicationDate":"2024-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141327697","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-07Epub Date: 2024-06-18DOI: 10.1016/j.ymthe.2024.06.020
Molly A McPheron, Marcia V Felker
Spinal muscular atrophy is a rare and progressive neuromuscular disease that, without treatment, leads to progressive weakness and often death. A plethora of studies have led to the approval of three high-cost and effective treatments since 2016. These treatments, nusinersen, onasemnogene abeparvovec, and risdiplam, have not been directly compared and have varying challenges in administration. In this review, we discuss the evidence supporting the use of these medications, the process of treatment selection, monitoring after treatment, the limited data comparing treatments, as well as future directions for investigation and therapy.
{"title":"Clinical perspectives: Treating spinal muscular atrophy.","authors":"Molly A McPheron, Marcia V Felker","doi":"10.1016/j.ymthe.2024.06.020","DOIUrl":"10.1016/j.ymthe.2024.06.020","url":null,"abstract":"<p><p>Spinal muscular atrophy is a rare and progressive neuromuscular disease that, without treatment, leads to progressive weakness and often death. A plethora of studies have led to the approval of three high-cost and effective treatments since 2016. These treatments, nusinersen, onasemnogene abeparvovec, and risdiplam, have not been directly compared and have varying challenges in administration. In this review, we discuss the evidence supporting the use of these medications, the process of treatment selection, monitoring after treatment, the limited data comparing treatments, as well as future directions for investigation and therapy.</p>","PeriodicalId":19020,"journal":{"name":"Molecular Therapy","volume":null,"pages":null},"PeriodicalIF":12.1,"publicationDate":"2024-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141419957","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-07Epub Date: 2024-06-26DOI: 10.1016/j.ymthe.2024.06.033
Immacolata Porreca, Robert Blassberg, Jennifer Harbottle, Bronwyn Joubert, Olga Mielczarek, Jesse Stombaugh, Kevin Hemphill, Jonathan Sumner, Deividas Pazeraitis, Julia Liz Touza, Margherita Francescatto, Mike Firth, Tommaso Selmi, Juan Carlos Collantes, Zaklina Strezoska, Benjamin Taylor, Shengkan Jin, Ceri M Wiggins, Anja van Brabant Smith, John J Lambourne
Gene editing technologies hold promise for enabling the next generation of adoptive cellular therapies. In conventional gene editing platforms that rely on nuclease activity, such as clustered regularly interspaced short palindromic repeats CRISPR-associated protein 9 (CRISPR-Cas9), allow efficient introduction of genetic modifications; however, these modifications occur via the generation of DNA double-strand breaks (DSBs) and can lead to unwanted genomic alterations and genotoxicity. Here, we apply a novel modular RNA aptamer-mediated Pin-point base editing platform to simultaneously introduce multiple gene knockouts and site-specific integration of a transgene in human primary T cells. We demonstrate high editing efficiency and purity at all target sites and significantly reduced frequency of chromosomal translocations compared with the conventional CRISPR-Cas9 system. Site-specific knockin of a chimeric antigen receptor and multiplex gene knockout are achieved within a single intervention and without the requirement for additional sequence-targeting components. The ability to perform complex genome editing efficiently and precisely highlights the potential of the Pin-point platform for application in a range of advanced cell therapies.
{"title":"An aptamer-mediated base editing platform for simultaneous knockin and multiple gene knockout for allogeneic CAR-T cells generation.","authors":"Immacolata Porreca, Robert Blassberg, Jennifer Harbottle, Bronwyn Joubert, Olga Mielczarek, Jesse Stombaugh, Kevin Hemphill, Jonathan Sumner, Deividas Pazeraitis, Julia Liz Touza, Margherita Francescatto, Mike Firth, Tommaso Selmi, Juan Carlos Collantes, Zaklina Strezoska, Benjamin Taylor, Shengkan Jin, Ceri M Wiggins, Anja van Brabant Smith, John J Lambourne","doi":"10.1016/j.ymthe.2024.06.033","DOIUrl":"10.1016/j.ymthe.2024.06.033","url":null,"abstract":"<p><p>Gene editing technologies hold promise for enabling the next generation of adoptive cellular therapies. In conventional gene editing platforms that rely on nuclease activity, such as clustered regularly interspaced short palindromic repeats CRISPR-associated protein 9 (CRISPR-Cas9), allow efficient introduction of genetic modifications; however, these modifications occur via the generation of DNA double-strand breaks (DSBs) and can lead to unwanted genomic alterations and genotoxicity. Here, we apply a novel modular RNA aptamer-mediated Pin-point base editing platform to simultaneously introduce multiple gene knockouts and site-specific integration of a transgene in human primary T cells. We demonstrate high editing efficiency and purity at all target sites and significantly reduced frequency of chromosomal translocations compared with the conventional CRISPR-Cas9 system. Site-specific knockin of a chimeric antigen receptor and multiplex gene knockout are achieved within a single intervention and without the requirement for additional sequence-targeting components. The ability to perform complex genome editing efficiently and precisely highlights the potential of the Pin-point platform for application in a range of advanced cell therapies.</p>","PeriodicalId":19020,"journal":{"name":"Molecular Therapy","volume":null,"pages":null},"PeriodicalIF":12.1,"publicationDate":"2024-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141469643","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-07Epub Date: 2024-07-02DOI: 10.1016/j.ymthe.2024.06.004
Tyler F Hill, Parnal Narvekar, Gregory D Asher, Jasmine N Edelstein, Nathan D Camp, Annaiz Grimm, Kerri R Thomas, Michael D Leiken, Katherine M Molloy, Peter J Cook, Sean P Arlauckas, Richard A Morgan, Sarah K Tasian, David J Rawlings, Richard G James
Bispecific antibodies are an important tool for the management and treatment of acute leukemias. As a next step toward clinical translation of engineered plasma cells, we describe approaches for secretion of bispecific antibodies by human plasma cells. We show that human plasma cells expressing either fragment crystallizable domain-deficient anti-CD19 × anti-CD3 (blinatumomab) or anti-CD33 × anti-CD3 bispecific antibodies mediate T cell activation and direct T cell killing of B acute lymphoblastic leukemia or acute myeloid leukemia cell lines in vitro. We demonstrate that knockout of the self-expressed antigen, CD19, boosts anti-CD19-bispecific secretion by plasma cells and prevents self-targeting. Plasma cells secreting anti-CD19-bispecific antibodies elicited in vivo control of acute lymphoblastic leukemia patient-derived xenografts in immunodeficient mice co-engrafted with autologous T cells. In these studies, we found that leukemic control elicited by engineered plasma cells was similar to CD19-targeted chimeric antigen receptor-expressing T cells. Finally, the steady-state concentration of anti-CD19 bispecifics in serum 1 month after cell delivery and tumor eradication was comparable with that observed in patients treated with a steady-state infusion of blinatumomab. These findings support further development of ePCs for use as a durable delivery system for the treatment of acute leukemias, and potentially other cancers.
双特异性抗体是管理和治疗急性白血病的重要工具。作为工程浆细胞临床转化的下一步,我们描述了人类浆细胞分泌双特异性抗体的方法。我们发现,表达片段可结晶结构域缺陷的抗 CD19 × 抗 CD3(blinatumomab)或抗 CD33 × 抗 CD3 双特异性抗体的人浆细胞能在体外介导 T 细胞活化并直接杀伤 B 型急性淋巴细胞白血病或急性髓性白血病细胞系。我们证明,敲除自身表达的抗原 CD19 会促进浆细胞分泌抗 CD19 双特异性抗体,并阻止自身靶向。分泌抗 CD19 双特异性抗体的浆细胞在免疫缺陷小鼠体内控制了与自体 T 细胞共同移植的急性淋巴细胞白血病患者衍生的异种移植物。在这些研究中,我们发现工程浆细胞诱导的白血病控制与 CD19 靶向嵌合抗原受体表达 T 细胞相似。最后,细胞递送和肿瘤根除1个月后,血清中抗CD19双特异性的稳态浓度与稳态输注blinatumomab治疗患者的浓度相当。这些研究结果支持进一步开发ePCs,将其用作治疗急性白血病和其他潜在癌症的持久递送系统。
{"title":"Human plasma cells engineered to secrete bispecifics drive effective in vivo leukemia killing.","authors":"Tyler F Hill, Parnal Narvekar, Gregory D Asher, Jasmine N Edelstein, Nathan D Camp, Annaiz Grimm, Kerri R Thomas, Michael D Leiken, Katherine M Molloy, Peter J Cook, Sean P Arlauckas, Richard A Morgan, Sarah K Tasian, David J Rawlings, Richard G James","doi":"10.1016/j.ymthe.2024.06.004","DOIUrl":"10.1016/j.ymthe.2024.06.004","url":null,"abstract":"<p><p>Bispecific antibodies are an important tool for the management and treatment of acute leukemias. As a next step toward clinical translation of engineered plasma cells, we describe approaches for secretion of bispecific antibodies by human plasma cells. We show that human plasma cells expressing either fragment crystallizable domain-deficient anti-CD19 × anti-CD3 (blinatumomab) or anti-CD33 × anti-CD3 bispecific antibodies mediate T cell activation and direct T cell killing of B acute lymphoblastic leukemia or acute myeloid leukemia cell lines in vitro. We demonstrate that knockout of the self-expressed antigen, CD19, boosts anti-CD19-bispecific secretion by plasma cells and prevents self-targeting. Plasma cells secreting anti-CD19-bispecific antibodies elicited in vivo control of acute lymphoblastic leukemia patient-derived xenografts in immunodeficient mice co-engrafted with autologous T cells. In these studies, we found that leukemic control elicited by engineered plasma cells was similar to CD19-targeted chimeric antigen receptor-expressing T cells. Finally, the steady-state concentration of anti-CD19 bispecifics in serum 1 month after cell delivery and tumor eradication was comparable with that observed in patients treated with a steady-state infusion of blinatumomab. These findings support further development of ePCs for use as a durable delivery system for the treatment of acute leukemias, and potentially other cancers.</p>","PeriodicalId":19020,"journal":{"name":"Molecular Therapy","volume":null,"pages":null},"PeriodicalIF":12.1,"publicationDate":"2024-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141498521","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}