Molecular TherapyPub Date : 2025-03-05Epub Date: 2024-12-30DOI: 10.1016/j.ymthe.2024.12.043
Francesca Di Leva, Michele Arnoldi, Stefania Santarelli, Mathieu Massonot, Marianne Victoria Lemée, Carlotta Bon, Miguel Pellegrini, Maria Elena Castellini, Giulia Zarantonello, Andrea Messina, Yuri Bozzi, Raphael Bernier, Silvia Zucchelli, Simona Casarosa, Erik Dassi, Giuseppe Ronzitti, Christelle Golzio, Jasmin Morandell, Stefano Gustincich, Stefano Espinoza, Marta Biagioli
{"title":"SINEUP RNA rescues molecular phenotypes associated with CHD8 suppression in autism spectrum disorder model systems.","authors":"Francesca Di Leva, Michele Arnoldi, Stefania Santarelli, Mathieu Massonot, Marianne Victoria Lemée, Carlotta Bon, Miguel Pellegrini, Maria Elena Castellini, Giulia Zarantonello, Andrea Messina, Yuri Bozzi, Raphael Bernier, Silvia Zucchelli, Simona Casarosa, Erik Dassi, Giuseppe Ronzitti, Christelle Golzio, Jasmin Morandell, Stefano Gustincich, Stefano Espinoza, Marta Biagioli","doi":"10.1016/j.ymthe.2024.12.043","DOIUrl":"10.1016/j.ymthe.2024.12.043","url":null,"abstract":"<p><p>Loss-of-function mutations in the chromodomain helicase DNA-binding 8 (CHD8) gene are strongly associated with autism spectrum disorders (ASDs). Indeed, the reduction of CHD8 causes transcriptional, epigenetic, and cellular phenotypic changes correlated to disease, which can be monitored in assessing new therapeutic approaches. SINEUPs are a functional class of natural and synthetic antisense long non-coding RNAs able to stimulate the translation of sense target mRNA, with no effect on transcription. Here, we employed synthetic SINEUP-CHD8 targeting the first and third AUG of the CHD8 coding sequence to efficiently stimulate endogenous CHD8 protein production. SINEUP-CHD8 were effective in cells with reduced levels of the target protein and in patient-derived fibroblasts with CHD8 mutations. Functionally, SINEUP-CHD8 were able to revert molecular phenotypes associated with CHD8 suppression, i.e., genome-wide transcriptional dysregulation, and the reduction of H3K36me3 levels. Strikingly, in chd8-morpholino-treated and ENU mutant zebrafish embryos, SINEUP-chd8 injection confirmed the ability of SINEUP RNA to rescue the chd8-suppression-induced macrocephaly phenotype and neuronal hyperproliferation. Thus, SINEUP-CHD8 molecule(s) represent a proof-of-concept toward the development of an RNA-based therapy for neurodevelopmental syndromes with implications for, and beyond ASD, and relevant to genetic disorders caused by protein haploinsufficiency.</p>","PeriodicalId":19020,"journal":{"name":"Molecular Therapy","volume":" ","pages":"1180-1196"},"PeriodicalIF":12.1,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11897779/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142915326","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"TRAF1 promotes osteoclastogenesis by enhancing metabolic adaptation to oxidative phosphorylation in an AKT-dependent manner.","authors":"Honglei Kang, Renpeng Peng, Yimin Dong, Fuben Liao, Meipeng Zhu, Pengju Wang, Shi-An Hu, Peixuan Hu, Jia Wang, Zheming Liu, Kehan Song, Feng Li","doi":"10.1016/j.ymthe.2025.01.040","DOIUrl":"10.1016/j.ymthe.2025.01.040","url":null,"abstract":"<p><p>Tumor necrosis factor receptor-associated factor 1 (TRAF1) is a crucial signaling adaptor involved in multiple cellular events. However, its role in regulating osteoclastogenesis and energy metabolism remains unclear. Here, we report that TRAF1 promotes osteoclastogenesis and oxidative phosphorylation (OXPHOS). Employing RNA sequencing, we found that TRAF1 is markedly upregulated during osteoclastogenesis and is positively associated with osteoporosis. TRAF1 knockout inhibits osteoclastogenesis and increases bone mass in both normal and ovariectomized adult mice without affecting bone mass in childhood. Furthermore, TRAF1 promotes osteoclast OXPHOS by increasing the phosphorylation level of AKT. Mechanistically, TRAF1 functions to inhibit TRAF2-induced ubiquitination of Gβl, a known activator of AKT, and further upregulates AKT phosphorylation. Rescue experiments revealed that the inhibitory effects of TRAF1 knockout on osteoclastogenesis, OXPHOS, and bone mass are dependent on AKT. Collectively, our findings uncover a previously unrecognized function of TRAF1 in regulating osteoclastogenesis and energy metabolism, and establish a novel TRAF1-AKT-OXPHOS axis in osteoclasts.</p>","PeriodicalId":19020,"journal":{"name":"Molecular Therapy","volume":" ","pages":"933-949"},"PeriodicalIF":12.1,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11897774/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143040300","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Molecular TherapyPub Date : 2025-03-05Epub Date: 2025-02-02DOI: 10.1016/j.ymthe.2025.01.027
Brian Spencer, Sharareh Emadi, Paula Desplats, Simona Eleuteri, Sarah Michael, Kori Kosberg, Jay Shen, Edward Rockenstein, Christina Patrick, Anthony Adame, Tania Gonzalez, Michael Sierks, Eliezer Masliah
{"title":"Retraction Notice to: ESCRT-mediated Uptake and Degradation of Brain-targeted α-synuclein Single Chain Antibody Attenuates Neuronal Degeneration In Vivo.","authors":"Brian Spencer, Sharareh Emadi, Paula Desplats, Simona Eleuteri, Sarah Michael, Kori Kosberg, Jay Shen, Edward Rockenstein, Christina Patrick, Anthony Adame, Tania Gonzalez, Michael Sierks, Eliezer Masliah","doi":"10.1016/j.ymthe.2025.01.027","DOIUrl":"10.1016/j.ymthe.2025.01.027","url":null,"abstract":"","PeriodicalId":19020,"journal":{"name":"Molecular Therapy","volume":" ","pages":"1302"},"PeriodicalIF":12.1,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11897743/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143123149","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Molecular TherapyPub Date : 2025-03-05Epub Date: 2025-02-03DOI: 10.1016/j.ymthe.2025.01.049
Marta Garcia-Gomara, Naroa Legarra-Marcos, Maria Serena, Elvira Rojas-de-Miguel, Maria Espelosin, Irene Marcilla, Alberto Perez-Mediavilla, Maria Rosario Luquin, Jose Luis Lanciego, Maria Angeles Burrell, Mar Cuadrado-Tejedor, Ana Garcia-Osta
{"title":"FKBP51 inhibition ameliorates neurodegeneration and motor dysfunction in the neuromelanin-SNCA mouse model of Parkinson's disease.","authors":"Marta Garcia-Gomara, Naroa Legarra-Marcos, Maria Serena, Elvira Rojas-de-Miguel, Maria Espelosin, Irene Marcilla, Alberto Perez-Mediavilla, Maria Rosario Luquin, Jose Luis Lanciego, Maria Angeles Burrell, Mar Cuadrado-Tejedor, Ana Garcia-Osta","doi":"10.1016/j.ymthe.2025.01.049","DOIUrl":"10.1016/j.ymthe.2025.01.049","url":null,"abstract":"<p><p>Parkinson's disease (PD) is characterized by the loss of neuromelanin (NM)-containing dopaminergic (DA) neurons in the substantia nigra (SN) pars compacta (SNpc) and the buildup of α-synuclein (α-syn) inclusions, called Lewy bodies. To investigate the roles of NM and α-syn in DA neuron degeneration, we modeled PD by inducing NM accumulation in a humanized α-syn mouse model (Snca<sup>-</sup>; PAC-Tg(SNCA<sup>WT</sup>)) via the expression of human tyrosinase in the SN. We found that this mouse strain develops naturally progressive motor dysfunction and dopaminergic neuronal loss in the SN with aging. Upon tyrosinase injection, NM-containing neurons developed p62 and ubiquitin inclusions. Furthermore, the upregulation of genes associated with microglial activation in the midbrain indicated a role of pro-inflammatory factors in neurodegeneration. Midbrain RNA sequencing confirmed the microglial response and identified Fkbp5 as one of the more dysregulated genes. Next, we showed that FKBP51(51 kDa) was significantly upregulated with aging and in PD human brains. Pharmacological treatment with SAFit2, a potent FKBP51 inhibitor, led to a reduction in ubiquitin-positive inclusions, prevention of neurodegeneration in the SNpc, and improved motor function in NM-SNCAWT mice. These results highlight the critical role of FKBP51 in PD and propose SAFit2 as a promising therapeutic candidate for reducing neurodegeneration in PD.</p>","PeriodicalId":19020,"journal":{"name":"Molecular Therapy","volume":" ","pages":"895-916"},"PeriodicalIF":12.1,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11897814/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143190038","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Molecular TherapyPub Date : 2025-03-05Epub Date: 2025-02-03DOI: 10.1016/j.ymthe.2025.01.045
Weijie Du, Fatih Noyan, Oliver McCallion, Vanessa Drosdek, Jonas Kath, Viktor Glaser, Carla Fuster-Garcia, Mingxing Yang, Maik Stein, Clemens Franke, Yaolin Pu, Olaf Weber, Julia K Polansky, Toni Cathomen, Elmar Jaeckel, Joanna Hester, Fadi Issa, Hans-Dieter Volk, Michael Schmueck-Henneresse, Petra Reinke, Dimitrios L Wagner
{"title":"Gene editing of CD3 epsilon to redirect regulatory T cells for adoptive T cell transfer.","authors":"Weijie Du, Fatih Noyan, Oliver McCallion, Vanessa Drosdek, Jonas Kath, Viktor Glaser, Carla Fuster-Garcia, Mingxing Yang, Maik Stein, Clemens Franke, Yaolin Pu, Olaf Weber, Julia K Polansky, Toni Cathomen, Elmar Jaeckel, Joanna Hester, Fadi Issa, Hans-Dieter Volk, Michael Schmueck-Henneresse, Petra Reinke, Dimitrios L Wagner","doi":"10.1016/j.ymthe.2025.01.045","DOIUrl":"10.1016/j.ymthe.2025.01.045","url":null,"abstract":"<p><p>Adoptive transfer of antigen-specific regulatory T cells (Tregs) is a promising strategy to combat immunopathologies in transplantation and autoimmune diseases. However, their low frequency in peripheral blood poses challenges for both manufacturing and clinical application. Chimeric antigen receptors have been used to redirect the specificity of Tregs, using retroviral vectors. However, retroviral gene transfer is costly, time consuming, and raises safety issues. Here, we explored non-viral CRISPR-Cas12a gene editing to redirect Tregs, using human leukocyte antigen (HLA)-A2-specific constructs for proof-of-concept studies in transplantation models. Knock-in of an antigen-binding domain into the N terminus of CD3 epsilon (CD3ε) gene generates Tregs expressing a chimeric CD3ε-T cell receptor fusion construct (TRuC) protein that integrates into the endogenous TCR/CD3 complex. These CD3ε-TRuC Tregs exhibit potent antigen-dependent activation while maintaining responsiveness to TCR/CD3 stimulation. This enables preferential enrichment of TRuC-redirected Tregs over CD3ε knockout Tregs via repetitive CD3/CD28 stimulation in a good manufacturing practice-compatible expansion system. CD3ε-TRuC Tregs retained their phenotypic, epigenetic, and functional identity. In a humanized mouse model, HLA-A2-specific CD3ε-TRuC Tregs demonstrate superior protection of allogeneic HLA-A2<sup>+</sup> skin grafts from rejection compared with polyclonal Tregs. This approach provides a pathway for developing clinical-grade CD3ε-TRuC-based Treg cell products for transplantation immunotherapy and other immunopathologies.</p>","PeriodicalId":19020,"journal":{"name":"Molecular Therapy","volume":" ","pages":"997-1013"},"PeriodicalIF":12.1,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11897813/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143190042","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Molecular TherapyPub Date : 2025-03-05DOI: 10.1016/j.ymthe.2025.02.045
Vittorio DeFranciscis, Giovanni Amabile, Marcin Kortylewski
{"title":"Clinical applications of oligonucleotides for cancer therapy.","authors":"Vittorio DeFranciscis, Giovanni Amabile, Marcin Kortylewski","doi":"10.1016/j.ymthe.2025.02.045","DOIUrl":"10.1016/j.ymthe.2025.02.045","url":null,"abstract":"<p><p>Oligonucleotide therapeutics (ONTs) represent a rapidly evolving modality for cancer treatment, capitalizing on their ability to modulate gene expression with high specificity. With more than 20 nucleic acid-based therapies that gained regulatory approval, advances in chemical modifications, sequence optimization, and novel delivery systems have propelled ONTs from research tools to clinical realities. ONTs, including siRNAs, antisense oligonucleotides, saRNA, miRNA, aptamers, and decoys, offer promising solutions for targeting previously \"undruggable\" molecules, such as transcription factors, and enhancing cancer immunotherapy by overcoming tumor immune evasion. The promise of ONT application in cancer treatment is exemplified by the recent FDA approval of the first oligonucleotide-based treatment to myeloproliferative disease. At the same time, there are challenges in delivering ONTs to specific tissues, mitigating off-target effects, and improving cellular uptake and endosomal release. This review provides a comprehensive overview of ONTs in clinical trials, emerging delivery strategies, and innovative therapeutic approaches, emphasizing the role of ONTs in immunotherapy and addressing hurdles that hinder their clinical translation. By examining advances and remaining challenges, we highlight opportunities for ONTs to revolutionize oncology and enhance patient outcomes.</p>","PeriodicalId":19020,"journal":{"name":"Molecular Therapy","volume":" ","pages":""},"PeriodicalIF":12.1,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143567755","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}
Molecular TherapyPub Date : 2025-03-05Epub Date: 2025-01-21DOI: 10.1016/j.ymthe.2025.01.032
Gang Liu, Alan C Hsu, Silke Geirnaert, Christine Cong, Prema M Nair, Sj Shen, Jacqueline E Marshall, Tatt Jhong Haw, Michael Fricker, Ashleigh M Philp, Nicole G Hansbro, Stelios Pavlidis, Yike Guo, Janette K Burgess, Leandro Castellano, Antonio Ieni, Gaetano Caramori, Brain G G Oliver, K Fan Chung, Ian M Adcock, Darryl A Knight, Francesca Polverino, Ken Bracke, Peter A Wark, Philip M Hansbro
{"title":"Vitronectin regulates lung tissue remodeling and emphysema in chronic obstructive pulmonary disease.","authors":"Gang Liu, Alan C Hsu, Silke Geirnaert, Christine Cong, Prema M Nair, Sj Shen, Jacqueline E Marshall, Tatt Jhong Haw, Michael Fricker, Ashleigh M Philp, Nicole G Hansbro, Stelios Pavlidis, Yike Guo, Janette K Burgess, Leandro Castellano, Antonio Ieni, Gaetano Caramori, Brain G G Oliver, K Fan Chung, Ian M Adcock, Darryl A Knight, Francesca Polverino, Ken Bracke, Peter A Wark, Philip M Hansbro","doi":"10.1016/j.ymthe.2025.01.032","DOIUrl":"10.1016/j.ymthe.2025.01.032","url":null,"abstract":"<p><p>Vitronectin (VTN) is an important extracellular matrix protein in tissue remodeling, but its role in chronic obstructive pulmonary disease (COPD) is unknown. We show that VTN regulates tissue remodeling through urokinase plasminogen activator (uPA) signaling pathway in COPD. In human COPD airways and bronchoepithelial cells and the airways of mice with cigarette smoke (CS)-induced experimental COPD, VTN protein was not changed, but downstream uPA signaling was altered (increased plasminogen activator inhibitor-1) that induced collagen and airway remodeling. In the parenchyma, VTN levels were decreased, uPA signaling pathway differentially altered and collagen reduced in lung fibroblasts from human and lung parenchyma in experimental COPD. Vtn inhibition with siRNA in mouse fibroblasts altered uPA signaling increased matrix metalloproteinase-12, and reduced collagen, whereas over-expression restored collagen production after CS extract challenge. Vtn<sup>-/-</sup> and Vtn small interfering RNA-treated mice had exaggerated inflammation, emphysema, and impaired lung function compared with controls with CS-induced COPD. Restoration of VTN in the parenchyma may be a therapeutic option for emphysema and COPD.</p>","PeriodicalId":19020,"journal":{"name":"Molecular Therapy","volume":" ","pages":"917-932"},"PeriodicalIF":12.1,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11897773/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143007659","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Molecular TherapyPub Date : 2025-03-05Epub Date: 2025-01-25DOI: 10.1016/j.ymthe.2025.01.038
Anders Schlosser, Bartosz Pilecki, Claire Allen, Andrew V Benest, Amy P Lynch, Jing Hua, Nikita Ved, Zoe Blackley, Thomas L Andersen, Dorle Hennig, Jonas H Graversen, Sören Möller, Sofie Skallerup, Maria Ormhøj, Clemens Lange, Hansjürgen T Agostini, Jakob Grauslund, Steffen Heegaard, Ivanka Dacheva, Michael Koss, Wenzheng Hu, Bibiana Iglesias, Matthew S Lawrence, Hans Christian Beck, Lasse Bach Steffensen, Nick S Laursen, Gregers R Andersen, Uffe Holmskov, David O Bates, Grith L Sorensen
{"title":"Pharmacological blocking of microfibrillar-associated protein 4 reduces retinal neoangiogenesis and vascular leakage.","authors":"Anders Schlosser, Bartosz Pilecki, Claire Allen, Andrew V Benest, Amy P Lynch, Jing Hua, Nikita Ved, Zoe Blackley, Thomas L Andersen, Dorle Hennig, Jonas H Graversen, Sören Möller, Sofie Skallerup, Maria Ormhøj, Clemens Lange, Hansjürgen T Agostini, Jakob Grauslund, Steffen Heegaard, Ivanka Dacheva, Michael Koss, Wenzheng Hu, Bibiana Iglesias, Matthew S Lawrence, Hans Christian Beck, Lasse Bach Steffensen, Nick S Laursen, Gregers R Andersen, Uffe Holmskov, David O Bates, Grith L Sorensen","doi":"10.1016/j.ymthe.2025.01.038","DOIUrl":"10.1016/j.ymthe.2025.01.038","url":null,"abstract":"<p><p>Neovascular age-related macular degeneration and diabetic macular edema are leading causes of vision loss evoked by retinal neovascularization and vascular leakage. The glycoprotein microfibrillar-associated protein 4 (MFAP4) is an integrin α<sub>V</sub>β<sub>3/5/6</sub> ligand present in the extracellular matrix. Single-cell transcriptomics reveal MFAP4 expression in cell types in close proximity to vascular endothelial cells, including choroidal vascular mural cells, retinal astrocytes, and Müller cells. Binding of the anti-MFAP4 antibody, hAS0326, makes MFAP4 inaccessible for integrin receptor interaction, and thereby hAS0326 blocked endothelial cell motility in vitro. Intravitreal hAS0326 inhibited retinal vascular lesion area and neovessel volume in a laser-induced choroidal neovascularization mouse model, vascular permeability in streptozotocin-induced retinopathy, and vascular leakage area in a chronic non-human primate model of DL-2-aminoadipic acid-induced retinopathy. One dose of hAS0326 showed duration of efficacy of at least 12 weeks in the latter model. Moreover, hAS0326 treatment significantly enriched Gene Ontology terms involving reduction of integrin binding. Our data suggest that hAS0326 constitutes a promising treatment of neovascularization and vascular leakage in retinal diseases.</p>","PeriodicalId":19020,"journal":{"name":"Molecular Therapy","volume":" ","pages":"1048-1072"},"PeriodicalIF":12.1,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11897753/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143040292","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Molecular TherapyPub Date : 2025-03-05Epub Date: 2025-01-14DOI: 10.1016/j.ymthe.2025.01.024
Keerthana Shankar, Isabelle Zingler-Hoslet, Diana M Tabima, Seth Zima, Lei Shi, Kirstan Gimse, Matthew H Forsberg, Varun Katta, Sage Z Davis, Daniel Maldonado, Brittany E Russell, Muhammed Murtaza, Shengdar Q Tsai, Jose M Ayuso, Christian M Capitini, Krishanu Saha
{"title":"Virus-free CRISPR knockin of a chimeric antigen receptor into KLRC1 generates potent GD2-specific natural killer cells.","authors":"Keerthana Shankar, Isabelle Zingler-Hoslet, Diana M Tabima, Seth Zima, Lei Shi, Kirstan Gimse, Matthew H Forsberg, Varun Katta, Sage Z Davis, Daniel Maldonado, Brittany E Russell, Muhammed Murtaza, Shengdar Q Tsai, Jose M Ayuso, Christian M Capitini, Krishanu Saha","doi":"10.1016/j.ymthe.2025.01.024","DOIUrl":"10.1016/j.ymthe.2025.01.024","url":null,"abstract":"<p><p>Natural killer (NK) cells are an appealing off-the-shelf, allogeneic cellular therapy due to their cytotoxic profile. However, their activity against solid tumors remains suboptimal in part due to the upregulation of NK-inhibitory ligands, such as HLA-E, within the tumor microenvironment. Here, we utilize CRISPR-Cas9 to disrupt the KLRC1 gene (encoding the HLA-E-binding NKG2A receptor) and perform non-viral insertion of a GD2-targeting chimeric antigen receptor (CAR) within NK cells isolated from human peripheral blood. Genome editing with CRISPR-Cas9 ribonucleoprotein complexes yields efficient genomic disruption of the KLRC1 gene with 98% knockout efficiency and specific knockin of the GD2 CAR transgene as high as 23%, with minimal off-target activity as shown by CHANGE-seq, in-out PCR, amplicon sequencing, and long-read whole-genome sequencing. KLRC1-GD2 CAR NK cells display high viability and proliferation, as well as precise cellular targeting and potency against GD2<sup>+</sup> human tumor cells. Notably, KLRC1-GD2 CAR NK cells overcome HLA-E-based inhibition in vitro against HLA-E-expressing, GD2<sup>+</sup> melanoma cells. Using a single-step, virus-free genome editing workflow, this study demonstrates the feasibility of precisely disrupting inhibitory signaling within NK cells via CRISPR-Cas9 while expressing a CAR to generate potent allogeneic cell therapies against HLA-E<sup>+</sup> solid tumors.</p>","PeriodicalId":19020,"journal":{"name":"Molecular Therapy","volume":" ","pages":"1014-1030"},"PeriodicalIF":12.1,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11897758/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008787","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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