Pub Date : 2024-08-07Epub Date: 2024-07-19DOI: 10.1016/j.ymthe.2024.07.009
Saru Basnet, Mirte Van der Heijden, Dafne C A Quixabeira, Elise Jirovec, Susanna A M Grönberg-Vähä-Koskela, James H A Clubb, Anna Kanerva, Santeri Pakola, Lyna Haybout, Victor Arias, Otto Hemminki, Tatiana Kudling, Sadia Zafar, Victor Cervera-Carrascon, Joao M Santos, Akseli Hemminki
{"title":"Overcoming effector T cell exhaustion in ovarian cancer ascites with a novel adenovirus encoding for a MUC1 bispecific antibody engager and IL-2 cytokine.","authors":"Saru Basnet, Mirte Van der Heijden, Dafne C A Quixabeira, Elise Jirovec, Susanna A M Grönberg-Vähä-Koskela, James H A Clubb, Anna Kanerva, Santeri Pakola, Lyna Haybout, Victor Arias, Otto Hemminki, Tatiana Kudling, Sadia Zafar, Victor Cervera-Carrascon, Joao M Santos, Akseli Hemminki","doi":"10.1016/j.ymthe.2024.07.009","DOIUrl":"10.1016/j.ymthe.2024.07.009","url":null,"abstract":"","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":"141727546","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}
Vagus nerve regulates viral infection and inflammation via the alpha 7 nicotinic acetylcholine receptor (α7 nAChR); however, the role of α7 nAChR in ZIKA virus (ZIKV) infection, which can cause severe neurological diseases such as microcephaly and Guillain-Barré syndrome, remains unknown. Here, we first examined the role of α7 nAChR in ZIKV infection in vitro. A broad effect of α7 nAChR activation was identified in limiting ZIKV infection in multiple cell lines. Combined with transcriptomics analysis, we further demonstrated that α7 nAChR activation promoted autophagy and ferroptosis pathways to limit cellular ZIKV viral loads. Additionally, activation of α7 nAChR prevented ZIKV-induced p62 nucleus accumulation, which mediated an enhanced autophagy pathway. By regulating proteasome complex and an E3 ligase NEDD4, activation of α7 nAChR resulted in increased amount of cellular p62, which further enhanced the ferroptosis pathway to reduce ZIKV infection. Moreover, utilizing in vivo neonatal mouse models, we showed that α7 nAChR is essential in controlling the disease severity of ZIKV infection. Taken together, our findings identify an α7 nAChR-mediated effect that critically contributes to limiting ZIKV infection, and α7 nAChR activation offers a novel strategy for combating ZIKV infection and its complications.
{"title":"Activation of nicotinic acetylcholine receptor α7 subunit limits Zika viral infection via promoting autophagy and ferroptosis.","authors":"Caiqi Zhao, Jie Chen, Zhihua Liu, Huabin Liang, Xiaoyan Chen, Lianping Cheng, Shitao Xie, Zhekai Lin, Renlan Wu, Qi Zhao, Yue Xue, Xiaoyun Lai, Xia Jin, Jin-Fu Xu, Xiao Su","doi":"10.1016/j.ymthe.2024.05.037","DOIUrl":"10.1016/j.ymthe.2024.05.037","url":null,"abstract":"<p><p>Vagus nerve regulates viral infection and inflammation via the alpha 7 nicotinic acetylcholine receptor (α7 nAChR); however, the role of α7 nAChR in ZIKA virus (ZIKV) infection, which can cause severe neurological diseases such as microcephaly and Guillain-Barré syndrome, remains unknown. Here, we first examined the role of α7 nAChR in ZIKV infection in vitro. A broad effect of α7 nAChR activation was identified in limiting ZIKV infection in multiple cell lines. Combined with transcriptomics analysis, we further demonstrated that α7 nAChR activation promoted autophagy and ferroptosis pathways to limit cellular ZIKV viral loads. Additionally, activation of α7 nAChR prevented ZIKV-induced p62 nucleus accumulation, which mediated an enhanced autophagy pathway. By regulating proteasome complex and an E3 ligase NEDD4, activation of α7 nAChR resulted in increased amount of cellular p62, which further enhanced the ferroptosis pathway to reduce ZIKV infection. Moreover, utilizing in vivo neonatal mouse models, we showed that α7 nAChR is essential in controlling the disease severity of ZIKV infection. Taken together, our findings identify an α7 nAChR-mediated effect that critically contributes to limiting ZIKV infection, and α7 nAChR activation offers a novel strategy for combating ZIKV infection and its complications.</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":"141186857","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: 2024-06-17DOI: 10.1016/j.ymthe.2024.06.023
Marco Cortese, Erica Torchiaro, Alice D'Andrea, Consalvo Petti, Federica Invrea, Letizia Franco, Chiara Donini, Valeria Leuci, Simonetta Maria Leto, Valentina Vurchio, Francesca Cottino, Claudio Isella, Sabrina Arena, Elisa Vigna, Andrea Bertotti, Livio Trusolino, Dario Sangiolo, Enzo Medico
HER2 amplification occurs in approximately 5% of colorectal cancer (CRC) cases and is associated only partially with clinical response to combined human epidermal growth factor receptor 2 (HER2)/epidermal growth factor receptor (EGFR)-targeted treatment. An alternative approach based on adoptive cell therapy using T cells engineered with anti-HER2 chimeric antigen receptor (CAR) proved to be toxic due to on-target/off-tumor activity. Here we describe a combinatorial strategy to safely target HER2 amplification and carcinoembryonic antigen (CEA) expression in CRC using a synNotch-CAR-based artificial regulatory network. The natural killer (NK) cell line NK-92 was engineered with an anti-HER2 synNotch receptor driving the expression of a CAR against CEA only when engaged. After being transduced and sorted for HER2-driven CAR expression, cells were cloned. The clone with optimal performances in terms of specificity and amplitude of CAR induction demonstrated significant activity in vitro and in vivo specifically against HER2-amplified (HER2amp)/CEA+ CRC models, with no effects on cells with physiological HER2 levels. The HER2-synNotch/CEA-CAR-NK system provides an innovative, scalable, and safe off-the-shelf cell therapy approach with potential against HER2amp CRC resistant or partially responsive to HER2/EGFR blockade.
约有5%的结直肠癌(CRC)病例存在HER2扩增,而且这种扩增只与HER2/EGFR联合靶向治疗的临床反应部分相关。事实证明,基于使用抗 HER2 嵌合抗原受体(CAR)设计的 T 细胞的采用性细胞疗法(ACT)的替代方法因其 "靶向非肿瘤 "活性而具有毒性。在这里,我们描述了一种组合策略,利用基于 synNotch-CAR 的人工调控网络,安全地靶向 CRC 中的 HER2 扩增和 CEA 表达。天然杀伤细胞系 NK-92 被设计成具有抗 HER2 synNotch 受体,只有在接合时才会驱动抗 CEA 的 CAR 表达。经过转导和分选 HER2 驱动的 CAR 表达后,细胞被克隆。在特异性和CAR诱导幅度方面表现最佳的克隆细胞在体外和体内对HER2amp/CEA+ CRC模型表现出显著的特异性活性,而对生理HER2水平的细胞没有影响。HER2-synNotch/CEA-CAR-NK系统提供了一种创新的、可扩展的、安全的现成细胞治疗方法,有望用于治疗对HER2/EGFR阻断有耐药性或部分耐药性的HER2amp CRC。
{"title":"Preclinical efficacy of a HER2 synNotch/CEA-CAR combinatorial immunotherapy against colorectal cancer with HER2 amplification.","authors":"Marco Cortese, Erica Torchiaro, Alice D'Andrea, Consalvo Petti, Federica Invrea, Letizia Franco, Chiara Donini, Valeria Leuci, Simonetta Maria Leto, Valentina Vurchio, Francesca Cottino, Claudio Isella, Sabrina Arena, Elisa Vigna, Andrea Bertotti, Livio Trusolino, Dario Sangiolo, Enzo Medico","doi":"10.1016/j.ymthe.2024.06.023","DOIUrl":"10.1016/j.ymthe.2024.06.023","url":null,"abstract":"<p><p>HER2 amplification occurs in approximately 5% of colorectal cancer (CRC) cases and is associated only partially with clinical response to combined human epidermal growth factor receptor 2 (HER2)/epidermal growth factor receptor (EGFR)-targeted treatment. An alternative approach based on adoptive cell therapy using T cells engineered with anti-HER2 chimeric antigen receptor (CAR) proved to be toxic due to on-target/off-tumor activity. Here we describe a combinatorial strategy to safely target HER2 amplification and carcinoembryonic antigen (CEA) expression in CRC using a synNotch-CAR-based artificial regulatory network. The natural killer (NK) cell line NK-92 was engineered with an anti-HER2 synNotch receptor driving the expression of a CAR against CEA only when engaged. After being transduced and sorted for HER2-driven CAR expression, cells were cloned. The clone with optimal performances in terms of specificity and amplitude of CAR induction demonstrated significant activity in vitro and in vivo specifically against HER2-amplified (HER2amp)/CEA<sup>+</sup> CRC models, with no effects on cells with physiological HER2 levels. The HER2-synNotch/CEA-CAR-NK system provides an innovative, scalable, and safe off-the-shelf cell therapy approach with potential against HER2amp CRC resistant or partially responsive to HER2/EGFR blockade.</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":"141419958","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: 2024-07-30DOI: 10.1016/j.ymthe.2024.07.001
Rob W J Collin, Bart P Leroy
{"title":"In vivo genome editing for inherited retinal disease: Opportunities and challenges.","authors":"Rob W J Collin, Bart P Leroy","doi":"10.1016/j.ymthe.2024.07.001","DOIUrl":"10.1016/j.ymthe.2024.07.001","url":null,"abstract":"","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":"141860362","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 : 2024-08-05DOI: 10.1016/j.ymthe.2024.08.004
Jitka Rybova, Teresa Sundararajan, Ladislav Kuchar, Theresa A Dlugi, Petr Ruzicka, William M McKillop, Jeffrey A Medin
Farber disease (FD) and spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME) are ultra-rare lysosomal storage disorders caused by deficient acid ceramidase (ACDase) activity. Although both conditions are caused by mutations in the ASAH1 gene, clinical presentations differ considerably. FD patients usually die in childhood, while SMA-PME patients can live until adulthood. There is no treatment for FD or SMA-PME. Hematopoietic stem cell transplantation (HSCT) and gene therapy strategies for the treatment of ACDase deficiency are being investigated. We have previously generated and characterized mouse models of both FD and SMA-PME that recapitulate the symptoms described in patients. Here, we show that HSCT improves lifespan, behavior, hematopoietic system anomalies, and plasma cytokine levels and significantly reduces histiocytic infiltration and ceramide accumulation throughout the tissues investigated, including the CNS, in both models of ACDase-deficient mice. HSCT was also successful in preventing lesion development and significant demyelination of the spinal cord seen in SMA-PME mice. Importantly, we note that only early and generally pre-symptomatic treatment was effective, and kidney impairment was not improved in either model.
{"title":"Hematopoietic stem cell transplantation leads to biochemical and functional correction in two mouse models of acid ceramidase deficiency.","authors":"Jitka Rybova, Teresa Sundararajan, Ladislav Kuchar, Theresa A Dlugi, Petr Ruzicka, William M McKillop, Jeffrey A Medin","doi":"10.1016/j.ymthe.2024.08.004","DOIUrl":"10.1016/j.ymthe.2024.08.004","url":null,"abstract":"<p><p>Farber disease (FD) and spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME) are ultra-rare lysosomal storage disorders caused by deficient acid ceramidase (ACDase) activity. Although both conditions are caused by mutations in the ASAH1 gene, clinical presentations differ considerably. FD patients usually die in childhood, while SMA-PME patients can live until adulthood. There is no treatment for FD or SMA-PME. Hematopoietic stem cell transplantation (HSCT) and gene therapy strategies for the treatment of ACDase deficiency are being investigated. We have previously generated and characterized mouse models of both FD and SMA-PME that recapitulate the symptoms described in patients. Here, we show that HSCT improves lifespan, behavior, hematopoietic system anomalies, and plasma cytokine levels and significantly reduces histiocytic infiltration and ceramide accumulation throughout the tissues investigated, including the CNS, in both models of ACDase-deficient mice. HSCT was also successful in preventing lesion development and significant demyelination of the spinal cord seen in SMA-PME mice. Importantly, we note that only early and generally pre-symptomatic treatment was effective, and kidney impairment was not improved in either model.</p>","PeriodicalId":19020,"journal":{"name":"Molecular Therapy","volume":null,"pages":null},"PeriodicalIF":12.1,"publicationDate":"2024-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141897843","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 : 2024-08-05DOI: 10.1016/j.ymthe.2024.08.005
Troy C Lund, Paul J Orchard, David R Nascene, Carina J King, Jennifer Braun, Stuti Thakkar, Willa Durose, Ilya Shestopalov, Himal Thakar, Ashish O Gupta
A 9-year-old boy with adrenoleukodystrophy due to ABCD1 whole-gene deletion was diagnosed with active cerebral adrenoleukodystrophy characterized by demyelination and gadolinium enhancement on brain MRI. He underwent hematopoietic cell transplant (HCT) with autologous CD34+ cells transduced with an ABCD1-expressing lentiviral vector (eli-cel [elivaldogene autotemcel]) as part of the ALD-104 clinical trial. Fifty days after HCT, the patient's MRI showed gadolinium resolution; the whole-blood vector copy number (VCN) was 0.666 copies/mL. Six months following HCT, an MRI showed re-emergence of gadolinium enhancement; the VCN had decreased to 0.029 copies/mL. Polyclonal antibodies to the ABCD1 gene product were detectable 9 months after transplant, showing reactivity to peroxisomes, suggesting an immune response; however, no antibody binding to human CD34+ cells could be shown. The patient underwent a successful allogeneic HCT 12 months after gene therapy with resultant gadolinium resolution, cerebral disease stabilization, and the disappearance of antibodies. The coincident VCN loss and appearance of antibody to the ABCD1 gene product is of interest, and we postulate that it is related to the patient's whole ABCD1 gene deletion. We suggest close monitoring of loss of gene therapy efficacy due to immune response in patients with full deletions who are considering gene therapy.
{"title":"Secondary failure of lentiviral vector gene therapy in a cerebral adrenoleukodystrophy patient with an ABCD1 whole-gene deletion.","authors":"Troy C Lund, Paul J Orchard, David R Nascene, Carina J King, Jennifer Braun, Stuti Thakkar, Willa Durose, Ilya Shestopalov, Himal Thakar, Ashish O Gupta","doi":"10.1016/j.ymthe.2024.08.005","DOIUrl":"10.1016/j.ymthe.2024.08.005","url":null,"abstract":"<p><p>A 9-year-old boy with adrenoleukodystrophy due to ABCD1 whole-gene deletion was diagnosed with active cerebral adrenoleukodystrophy characterized by demyelination and gadolinium enhancement on brain MRI. He underwent hematopoietic cell transplant (HCT) with autologous CD34<sup>+</sup> cells transduced with an ABCD1-expressing lentiviral vector (eli-cel [elivaldogene autotemcel]) as part of the ALD-104 clinical trial. Fifty days after HCT, the patient's MRI showed gadolinium resolution; the whole-blood vector copy number (VCN) was 0.666 copies/mL. Six months following HCT, an MRI showed re-emergence of gadolinium enhancement; the VCN had decreased to 0.029 copies/mL. Polyclonal antibodies to the ABCD1 gene product were detectable 9 months after transplant, showing reactivity to peroxisomes, suggesting an immune response; however, no antibody binding to human CD34<sup>+</sup> cells could be shown. The patient underwent a successful allogeneic HCT 12 months after gene therapy with resultant gadolinium resolution, cerebral disease stabilization, and the disappearance of antibodies. The coincident VCN loss and appearance of antibody to the ABCD1 gene product is of interest, and we postulate that it is related to the patient's whole ABCD1 gene deletion. We suggest close monitoring of loss of gene therapy efficacy due to immune response in patients with full deletions who are considering gene therapy.</p>","PeriodicalId":19020,"journal":{"name":"Molecular Therapy","volume":null,"pages":null},"PeriodicalIF":12.1,"publicationDate":"2024-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141897844","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 : 2024-08-05DOI: 10.1016/j.ymthe.2024.08.003
Courteney Tunstead, Evelina Volkova, Hazel Dunbar, Ian J Hawthorne, Alison Bell, Louise Crowe, Joanne C Masterson, Claudia C Dos Santos, Bairbre McNicholas, John G Laffey, Karen English
Clinical trials investigating the potential of mesenchymal stromal cells (MSCs) for the treatment of inflammatory diseases, such as acute respiratory distress syndrome (ARDS), have been disappointing, with less than 50% of patients responding to treatment. Licensed MSCs show enhanced therapeutic efficacy in response to cytokine-mediated activation signals. There are two distinct sub-phenotypes of ARDS: hypo- and hyper-inflammatory. We hypothesized that pre-licensing MSCs in a hyper-inflammatory ARDS environment would enhance their therapeutic efficacy in acute lung inflammation (ALI). Serum samples from patients with ARDS were segregated into hypo- and hyper-inflammatory categories based on interleukin (IL)-6 levels. MSCs were licensed with pooled serum from patients with hypo- or hyper-inflammatory ARDS or healthy serum controls. Our findings show that hyper-inflammatory ARDS pre-licensed MSC conditioned medium (MSC-CMHyper) led to a significant enrichment in tight junction expression and enhanced barrier integrity in lung epithelial cells in vitro and in vivo in a vascular endothelial growth factor (VEGF)-dependent manner. Importantly, while both MSC-CMHypo and MSC-CMHyper significantly reduced IL-6 and tumor necrosis factor alpha (TNF-α) levels in the bronchoalveolar lavage fluid (BALF) of lipopolysaccharide (LPS)-induced ALI mice, only MSC-CMHyper significantly reduced lung permeability and overall clinical outcomes including weight loss and clinical score. Thus, the hypo- and hyper-inflammatory ARDS environments may differentially influence MSC cytoprotective and immunomodulatory functions.
{"title":"The ARDS microenvironment enhances MSC-induced repair via VEGF in experimental acute lung inflammation.","authors":"Courteney Tunstead, Evelina Volkova, Hazel Dunbar, Ian J Hawthorne, Alison Bell, Louise Crowe, Joanne C Masterson, Claudia C Dos Santos, Bairbre McNicholas, John G Laffey, Karen English","doi":"10.1016/j.ymthe.2024.08.003","DOIUrl":"10.1016/j.ymthe.2024.08.003","url":null,"abstract":"<p><p>Clinical trials investigating the potential of mesenchymal stromal cells (MSCs) for the treatment of inflammatory diseases, such as acute respiratory distress syndrome (ARDS), have been disappointing, with less than 50% of patients responding to treatment. Licensed MSCs show enhanced therapeutic efficacy in response to cytokine-mediated activation signals. There are two distinct sub-phenotypes of ARDS: hypo- and hyper-inflammatory. We hypothesized that pre-licensing MSCs in a hyper-inflammatory ARDS environment would enhance their therapeutic efficacy in acute lung inflammation (ALI). Serum samples from patients with ARDS were segregated into hypo- and hyper-inflammatory categories based on interleukin (IL)-6 levels. MSCs were licensed with pooled serum from patients with hypo- or hyper-inflammatory ARDS or healthy serum controls. Our findings show that hyper-inflammatory ARDS pre-licensed MSC conditioned medium (MSC-CM<sub>Hyper</sub>) led to a significant enrichment in tight junction expression and enhanced barrier integrity in lung epithelial cells in vitro and in vivo in a vascular endothelial growth factor (VEGF)-dependent manner. Importantly, while both MSC-CM<sub>Hypo</sub> and MSC-CM<sub>Hyper</sub> significantly reduced IL-6 and tumor necrosis factor alpha (TNF-α) levels in the bronchoalveolar lavage fluid (BALF) of lipopolysaccharide (LPS)-induced ALI mice, only MSC-CM<sub>Hyper</sub> significantly reduced lung permeability and overall clinical outcomes including weight loss and clinical score. Thus, the hypo- and hyper-inflammatory ARDS environments may differentially influence MSC cytoprotective and immunomodulatory functions.</p>","PeriodicalId":19020,"journal":{"name":"Molecular Therapy","volume":null,"pages":null},"PeriodicalIF":12.1,"publicationDate":"2024-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141897845","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 : 2024-07-31DOI: 10.1016/j.ymthe.2024.07.025
Yi-Jia Li, Sheng-Hsuan Chien, Rui Huang, Andreas Herrmann, Qianqian Zhao, Pei-Chuan Li, Chunyan Zhang, Antons Martincuks, Nicole Lugo Santiago, Katherine Zong, Piotr Swiderski, Ross A Okimoto, Mihae Song, Lorna Rodriguez, Stephen J Forman, Xiuli Wang, Hua Yu
Although CRISPR-Cas9 technology is poised to revolutionize the treatment of diseases with underlying genetic mutations, it faces some significant issues limiting clinical entry. They include low-efficiency in vivo systemic delivery and undesired off-target effects. Here, we demonstrate, by modifying Cas9 with phosphorothioate-DNA oligos (PSs), that one can efficiently deliver single and bi-specific CRISPR-Cas9/guide RNA (gRNA) dimers in vitro and in vivo with reduced off-target effects. We show that PS-Cas9/gRNA-mediated gene knockout preserves chimeric antigen receptor T cell viability and expansion in vitro and in vivo. PS-Cas9/gRNA mediates gene perturbation in patient-derived tumor organoids and mouse xenograft tumors, leading to potent tumor antitumor effects. Further, HER2 antibody-PS-Cas9/gRNA conjugate selectively perturbs targeted genes in HER2+ ovarian cancer xenografts in vivo. Moreover, we created bi-specific PS-Cas9 with two gRNAs to target two adjacent sequences of the same gene, leading to efficient targeted gene disruption ex vivo and in vivo with markedly reduced unintended gene perturbation. Thus, the cell-penetrating PS-Cas9/gRNA can achieve efficient systemic delivery and precision in gene disruption.
尽管 CRISPR/Cas9 技术有望彻底改变对潜在基因突变疾病的治疗,但它面临着一些限制其进入临床的重大问题。这些问题包括体内系统递送效率低和非预期的脱靶效应。在这里,我们证明了通过用硫代磷酸 DNA 寡聚物(PS)修饰 Cas9,可以在体外和体内高效递送单特异性和双特异性 CRISPR/Cas9/指导 RNA(gRNA)二聚体,并减少脱靶效应。我们的研究表明,PS-Cas9/gRNA 介导的基因敲除可保持嵌合抗原受体 T 细胞在体外和体内的活力和扩增。PS-Cas9/gRNA可介导患者衍生肿瘤器官组织和小鼠异种移植肿瘤中的基因扰乱,从而产生强大的肿瘤抗肿瘤效应。此外,HER2 抗体-PS-Cas9/gRNA 结合物可选择性地扰乱体内 HER2+ 卵巢癌异种移植瘤中的靶基因。此外,我们还创造了带有两个 gRNA 的双特异性 PS-Cas9,可靶向同一基因的两个相邻序列,从而实现高效的体内外靶向基因破坏,并显著减少了意外的基因扰乱。因此,细胞穿透PS-Cas9/gRNA可以实现高效的全身性递送和精准的基因破坏。
{"title":"A platform to deliver single and bi-specific Cas9/guide RNA to perturb genes in vitro and in vivo.","authors":"Yi-Jia Li, Sheng-Hsuan Chien, Rui Huang, Andreas Herrmann, Qianqian Zhao, Pei-Chuan Li, Chunyan Zhang, Antons Martincuks, Nicole Lugo Santiago, Katherine Zong, Piotr Swiderski, Ross A Okimoto, Mihae Song, Lorna Rodriguez, Stephen J Forman, Xiuli Wang, Hua Yu","doi":"10.1016/j.ymthe.2024.07.025","DOIUrl":"10.1016/j.ymthe.2024.07.025","url":null,"abstract":"<p><p>Although CRISPR-Cas9 technology is poised to revolutionize the treatment of diseases with underlying genetic mutations, it faces some significant issues limiting clinical entry. They include low-efficiency in vivo systemic delivery and undesired off-target effects. Here, we demonstrate, by modifying Cas9 with phosphorothioate-DNA oligos (PSs), that one can efficiently deliver single and bi-specific CRISPR-Cas9/guide RNA (gRNA) dimers in vitro and in vivo with reduced off-target effects. We show that PS-Cas9/gRNA-mediated gene knockout preserves chimeric antigen receptor T cell viability and expansion in vitro and in vivo. PS-Cas9/gRNA mediates gene perturbation in patient-derived tumor organoids and mouse xenograft tumors, leading to potent tumor antitumor effects. Further, HER2 antibody-PS-Cas9/gRNA conjugate selectively perturbs targeted genes in HER2<sup>+</sup> ovarian cancer xenografts in vivo. Moreover, we created bi-specific PS-Cas9 with two gRNAs to target two adjacent sequences of the same gene, leading to efficient targeted gene disruption ex vivo and in vivo with markedly reduced unintended gene perturbation. Thus, the cell-penetrating PS-Cas9/gRNA can achieve efficient systemic delivery and precision in gene disruption.</p>","PeriodicalId":19020,"journal":{"name":"Molecular Therapy","volume":null,"pages":null},"PeriodicalIF":12.1,"publicationDate":"2024-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141875383","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 : 2024-07-31DOI: 10.1016/j.ymthe.2024.07.022
Varun Katta, Kiera O'Keefe, Yichao Li, Thiyagaraj Mayuranathan, Cicera R Lazzarotto, Rachael K Wood, Rachel M Levine, Alicia Powers, Kalin Mayberry, Garret Manquen, Yu Yao, Jingjing Zhang, Yoonjeong Jang, Nikitha Nimmagadda, Erin A Dempsey, GaHyun Lee, Naoya Uchida, Yong Cheng, Frank Fazio, Tim Lockey, Mike Meagher, Akshay Sharma, John F Tisdale, Sheng Zhou, Jonathan S Yen, Mitchell J Weiss, Shengdar Q Tsai
Sickle cell disease (SCD) is a common, severe genetic blood disorder. Current pharmacotherapies are partially effective and allogeneic hematopoietic stem cell transplantation is associated with immune toxicities. Genome editing of patient hematopoietic stem cells (HSCs) to reactivate fetal hemoglobin (HbF) in erythroid progeny offers an alternative potentially curative approach to treat SCD. Although the FDA released guidelines for evaluating genome editing risks, it remains unclear how best to approach pre-clinical assessment of genome-edited cell products. Here, we describe rigorous pre-clinical development of a therapeutic γ-globin gene promoter editing strategy that supported an investigational new drug application cleared by the FDA. We compared γ-globin promoter and BCL11A enhancer targets, identified a potent HbF-inducing lead candidate, and tested our approach in mobilized CD34+ hematopoietic stem progenitor cells (HSPCs) from SCD patients. We observed efficient editing, HbF induction to predicted therapeutic levels, and reduced sickling. With single-cell analyses, we defined the heterogeneity of HbF induction and HBG1/HBG2 transcription. With CHANGE-seq for sensitive and unbiased off-target discovery followed by targeted sequencing, we did not detect off-target activity in edited HSPCs. Our study provides a blueprint for translating new ex vivo HSC genome editing strategies toward clinical trials for treating SCD and other blood disorders.
{"title":"Development and IND-enabling studies of a novel Cas9 genome-edited autologous CD34<sup>+</sup> cell therapy to induce fetal hemoglobin for sickle cell disease.","authors":"Varun Katta, Kiera O'Keefe, Yichao Li, Thiyagaraj Mayuranathan, Cicera R Lazzarotto, Rachael K Wood, Rachel M Levine, Alicia Powers, Kalin Mayberry, Garret Manquen, Yu Yao, Jingjing Zhang, Yoonjeong Jang, Nikitha Nimmagadda, Erin A Dempsey, GaHyun Lee, Naoya Uchida, Yong Cheng, Frank Fazio, Tim Lockey, Mike Meagher, Akshay Sharma, John F Tisdale, Sheng Zhou, Jonathan S Yen, Mitchell J Weiss, Shengdar Q Tsai","doi":"10.1016/j.ymthe.2024.07.022","DOIUrl":"10.1016/j.ymthe.2024.07.022","url":null,"abstract":"<p><p>Sickle cell disease (SCD) is a common, severe genetic blood disorder. Current pharmacotherapies are partially effective and allogeneic hematopoietic stem cell transplantation is associated with immune toxicities. Genome editing of patient hematopoietic stem cells (HSCs) to reactivate fetal hemoglobin (HbF) in erythroid progeny offers an alternative potentially curative approach to treat SCD. Although the FDA released guidelines for evaluating genome editing risks, it remains unclear how best to approach pre-clinical assessment of genome-edited cell products. Here, we describe rigorous pre-clinical development of a therapeutic γ-globin gene promoter editing strategy that supported an investigational new drug application cleared by the FDA. We compared γ-globin promoter and BCL11A enhancer targets, identified a potent HbF-inducing lead candidate, and tested our approach in mobilized CD34<sup>+</sup> hematopoietic stem progenitor cells (HSPCs) from SCD patients. We observed efficient editing, HbF induction to predicted therapeutic levels, and reduced sickling. With single-cell analyses, we defined the heterogeneity of HbF induction and HBG1/HBG2 transcription. With CHANGE-seq for sensitive and unbiased off-target discovery followed by targeted sequencing, we did not detect off-target activity in edited HSPCs. Our study provides a blueprint for translating new ex vivo HSC genome editing strategies toward clinical trials for treating SCD and other blood disorders.</p>","PeriodicalId":19020,"journal":{"name":"Molecular Therapy","volume":null,"pages":null},"PeriodicalIF":12.1,"publicationDate":"2024-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141860360","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}