Pub Date : 2025-07-08DOI: 10.1038/s41434-025-00548-3
Tatevik Jalatyan, Erik Aznauryan, Rokib Hasan, Valeri Vardanyan, Stepan Nersisyan, David B. Thompson, Noah Davidsohn, Sanya Thomas, Simon van Haren, Jenny Tam, Denitsa Milanova, George M. Church, Lilit Nersisyan
Naturally occurring adeno-associated viruses (AAVs) are an integral part of gene therapy, yet engineering novel AAV variants is necessary to expand targetable tissues and treatable diseases. Directed evolution, particularly through DNA shuffling of the capsid genes of wild-type AAV serotypes, is a widely employed strategy to generate novel chimeric variants with desired properties. Yet, the computational analysis of such chimeric sequences presents challenges. We introduce hafoe, a novel computational tool designed for the exploratory analysis of chimeric AAV libraries, which does not require extensive bioinformatics expertise. hafoe accurately deciphers the serotype composition and enrichment patterns of chimeric AAV variants across different tissues. Validation against synthetic datasets demonstrates that hafoe identifies parental serotype compositions with an accuracy of 96.3% to 97.5%. Additionally, we engineered chimeric AAV capsid libraries and screened novel AAV variants for tropism to human dermal fibroblasts and dendritic cells, as well as canine muscle, and liver tissues. Using hafoe we identified and characterized enriched AAV variants in these tissues for potential use in gene therapy and vaccine development. Overall, hafoe can provide valuable insights that may further support the rational design of AAV vectors based on parental serotype and sequence preferences of the capsid genes in target tissues.
{"title":"hafoe: an interactive tool for the analysis of chimeric AAV libraries after random mutagenesis","authors":"Tatevik Jalatyan, Erik Aznauryan, Rokib Hasan, Valeri Vardanyan, Stepan Nersisyan, David B. Thompson, Noah Davidsohn, Sanya Thomas, Simon van Haren, Jenny Tam, Denitsa Milanova, George M. Church, Lilit Nersisyan","doi":"10.1038/s41434-025-00548-3","DOIUrl":"10.1038/s41434-025-00548-3","url":null,"abstract":"Naturally occurring adeno-associated viruses (AAVs) are an integral part of gene therapy, yet engineering novel AAV variants is necessary to expand targetable tissues and treatable diseases. Directed evolution, particularly through DNA shuffling of the capsid genes of wild-type AAV serotypes, is a widely employed strategy to generate novel chimeric variants with desired properties. Yet, the computational analysis of such chimeric sequences presents challenges. We introduce hafoe, a novel computational tool designed for the exploratory analysis of chimeric AAV libraries, which does not require extensive bioinformatics expertise. hafoe accurately deciphers the serotype composition and enrichment patterns of chimeric AAV variants across different tissues. Validation against synthetic datasets demonstrates that hafoe identifies parental serotype compositions with an accuracy of 96.3% to 97.5%. Additionally, we engineered chimeric AAV capsid libraries and screened novel AAV variants for tropism to human dermal fibroblasts and dendritic cells, as well as canine muscle, and liver tissues. Using hafoe we identified and characterized enriched AAV variants in these tissues for potential use in gene therapy and vaccine development. Overall, hafoe can provide valuable insights that may further support the rational design of AAV vectors based on parental serotype and sequence preferences of the capsid genes in target tissues.","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":"32 5","pages":"475-486"},"PeriodicalIF":4.5,"publicationDate":"2025-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.nature.comhttps://www.nature.com/articles/s41434-025-00548-3.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144591097","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-06-20DOI: 10.1038/s41434-025-00546-5
Konstantina Tzimou, Pol Hulsbus-Andreu, Ece Bahar Yildirim, Lars K Nielsen, Jesús Lavado-García
Optimization of recombinant adeno-associated virus (rAAV) production is essential for effective gene therapy applications. However, multiple factors affect the rAAV productivity in mammalian cells, and often they interact with each other, making the optimization process highly challenging. In our previous work, we showed how coupling mixture design (MD) with face-centered central composite design (FCCD) was the most suitable design of experiments (DOE) approach for optimizing rAAV2 productivity and cell viability. In this study, we built on this method and demonstrate that combining MD with FCCD can be used to optimize the percentage of full capsids in rAAV2 upstream preparation. Additionally, we investigate the influence of the gene of interest (GOI) on the optimal conditions for viral particle production and packaging efficiency. By integrating MD and FCCD methodologies, we achieved an improvement of almost 100-fold in Log(Vp) in the case of egfp-expressing rAAV, and a 12-fold increase in bdnf-expressing full rAAV capsids, suggesting that this combined approach is a versatile and effective strategy for optimizing rAAV production processes. These findings emphasize the need for a comprehensive understanding of the factors influencing rAAV production to enhance the efficiency and efficacy of viral vector applications in gene therapy.
{"title":"Mixture design as a tool for improving full-to-empty particle ratios across various GOIs in rAAV production.","authors":"Konstantina Tzimou, Pol Hulsbus-Andreu, Ece Bahar Yildirim, Lars K Nielsen, Jesús Lavado-García","doi":"10.1038/s41434-025-00546-5","DOIUrl":"10.1038/s41434-025-00546-5","url":null,"abstract":"<p><p>Optimization of recombinant adeno-associated virus (rAAV) production is essential for effective gene therapy applications. However, multiple factors affect the rAAV productivity in mammalian cells, and often they interact with each other, making the optimization process highly challenging. In our previous work, we showed how coupling mixture design (MD) with face-centered central composite design (FCCD) was the most suitable design of experiments (DOE) approach for optimizing rAAV2 productivity and cell viability. In this study, we built on this method and demonstrate that combining MD with FCCD can be used to optimize the percentage of full capsids in rAAV2 upstream preparation. Additionally, we investigate the influence of the gene of interest (GOI) on the optimal conditions for viral particle production and packaging efficiency. By integrating MD and FCCD methodologies, we achieved an improvement of almost 100-fold in Log(Vp) in the case of egfp-expressing rAAV, and a 12-fold increase in bdnf-expressing full rAAV capsids, suggesting that this combined approach is a versatile and effective strategy for optimizing rAAV production processes. These findings emphasize the need for a comprehensive understanding of the factors influencing rAAV production to enhance the efficiency and efficacy of viral vector applications in gene therapy.</p>","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":" ","pages":""},"PeriodicalIF":4.6,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144336457","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-06-14DOI: 10.1038/s41434-025-00544-7
Aisha H. A. Alsenousy, Sara A. Sharker, Mennatallah A. Gowayed, Samar S. Elblehi, Maher A. Kamel
The treatment of chronic disease (CKD) is a great challenge in healthcare that requires an innovative approach to address its complex nature. RNA nanotechnology has emerged rapidly and received attention in the last few years because of its significant aptitude for therapies. Hence, the present study aimed to design, construct, and characterize a multifunctional (anti-miR-34a DNA aptamer-kidney targeted) RNA nanoparticle (RNPs) based on bacteriophage phi29 packaging RNA three-way junction (pRNA-3WJ), and then explore their in vivo toxicity and therapeutic potentials in mice model of CKD. After confirming the safety and specific targeting capability of the prepared core 3WJ (3WJ) and the therapeutic 3WJ (3WJ-Kapt/anti-miR-34a) RNPs to renal tissue using healthy mice, CKD was induced in C57BL/6 mice using adenine. CKD mice were treated with a single intravenous injection of 3WJ or 3WJ-Kapt/anti-miR-34a. Every week, 5 mice of each group were selected randomly for sample collection for 4 weeks post-treatment. The anti-miR-34a 3WJ-RNPs have shown stability, safety, and efficacy in renal targeting using DNA aptamer, by targeting miR-34a in renal tissue, 3WJ-Kapt/anti-miR-34a suppressed profibrotic gene expression and induced anti-fibrotic pathways’ expression. Our present study provides preliminary and pioneering evidence for the promising treatment of renal fibrosis and CKD through targeting miR-34a in the renal tissue by 3WJ-RNPs.
{"title":"Aptamer-targeted anti-miR RNA construct based on 3WJ as a new approach for the treatment of chronic kidney disease in an experimental model","authors":"Aisha H. A. Alsenousy, Sara A. Sharker, Mennatallah A. Gowayed, Samar S. Elblehi, Maher A. Kamel","doi":"10.1038/s41434-025-00544-7","DOIUrl":"10.1038/s41434-025-00544-7","url":null,"abstract":"The treatment of chronic disease (CKD) is a great challenge in healthcare that requires an innovative approach to address its complex nature. RNA nanotechnology has emerged rapidly and received attention in the last few years because of its significant aptitude for therapies. Hence, the present study aimed to design, construct, and characterize a multifunctional (anti-miR-34a DNA aptamer-kidney targeted) RNA nanoparticle (RNPs) based on bacteriophage phi29 packaging RNA three-way junction (pRNA-3WJ), and then explore their in vivo toxicity and therapeutic potentials in mice model of CKD. After confirming the safety and specific targeting capability of the prepared core 3WJ (3WJ) and the therapeutic 3WJ (3WJ-Kapt/anti-miR-34a) RNPs to renal tissue using healthy mice, CKD was induced in C57BL/6 mice using adenine. CKD mice were treated with a single intravenous injection of 3WJ or 3WJ-Kapt/anti-miR-34a. Every week, 5 mice of each group were selected randomly for sample collection for 4 weeks post-treatment. The anti-miR-34a 3WJ-RNPs have shown stability, safety, and efficacy in renal targeting using DNA aptamer, by targeting miR-34a in renal tissue, 3WJ-Kapt/anti-miR-34a suppressed profibrotic gene expression and induced anti-fibrotic pathways’ expression. Our present study provides preliminary and pioneering evidence for the promising treatment of renal fibrosis and CKD through targeting miR-34a in the renal tissue by 3WJ-RNPs.","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":"32 4","pages":"359-375"},"PeriodicalIF":4.5,"publicationDate":"2025-06-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12310521/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144293652","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-06-10DOI: 10.1038/s41434-025-00542-9
Edwina Abou Haidar, Shilpa Prabhakar, Pike See Cheah, Killian S. Hanlon, Paula Espinoza, Adam V. Crain, Nikita Patel, Greta W. Radcliff, Ming Cheng, Iván Coto Hernández, Steven Minderler, Demitri de la Cruz, Carrie Ng, Cintia Carla da Hora, Alain Charest, Anat Stemmer-Rachamimov, Nate Jowett, Xandra O. Breakefield, Casey A. Maguire
Genetic diseases such as Neurofibromatosis type 1 (NF1) and Charcot-Marie Tooth disease involve Schwann cells (SCs) associated with peripheral nerves. Gene therapy using adeno-associated virus (AAV) vector mediated gene delivery is a promising strategy to treat these diseases. However, AAV-mediated transduction of SCs in vivo after intravascular delivery is relatively inefficient, with a lack of extensive characterization of different capsids to date. Here, we performed an in vivo selection with an AAV9 capsid peptide display library in a mouse model of NF1. We chose one capsid variant, AAV-SC3, which was present in NF1 nerves for comparison to two benchmark capsids after systemic injection. AAV-SC3 significantly outperformed one of the two benchmark capsids at levels of transgene mRNA in the neurofibroma. Immunofluorescence microscopy revealed transgene expressing Sox10-positive SCs throughout the neurofibroma with AAV-SC3 injection. Next, we performed a pooled screen with four of the top capsids from our initial selection and AAV9 and identified one capsid, AAV-SC4, with enhanced biodistribution to and transduction of normal sciatic nerve in mice. This capsid displayed a peptide with a known laminin-binding motif, which may provide a conduit for future laminin-targeting strategies. Our results provide a baseline for future AAV-based gene therapies developed for NF1 or other diseases that affect SCs.
{"title":"Engineered AAV capsids mediate transduction of murine neurofibroma and sciatic nerve","authors":"Edwina Abou Haidar, Shilpa Prabhakar, Pike See Cheah, Killian S. Hanlon, Paula Espinoza, Adam V. Crain, Nikita Patel, Greta W. Radcliff, Ming Cheng, Iván Coto Hernández, Steven Minderler, Demitri de la Cruz, Carrie Ng, Cintia Carla da Hora, Alain Charest, Anat Stemmer-Rachamimov, Nate Jowett, Xandra O. Breakefield, Casey A. Maguire","doi":"10.1038/s41434-025-00542-9","DOIUrl":"10.1038/s41434-025-00542-9","url":null,"abstract":"Genetic diseases such as Neurofibromatosis type 1 (NF1) and Charcot-Marie Tooth disease involve Schwann cells (SCs) associated with peripheral nerves. Gene therapy using adeno-associated virus (AAV) vector mediated gene delivery is a promising strategy to treat these diseases. However, AAV-mediated transduction of SCs in vivo after intravascular delivery is relatively inefficient, with a lack of extensive characterization of different capsids to date. Here, we performed an in vivo selection with an AAV9 capsid peptide display library in a mouse model of NF1. We chose one capsid variant, AAV-SC3, which was present in NF1 nerves for comparison to two benchmark capsids after systemic injection. AAV-SC3 significantly outperformed one of the two benchmark capsids at levels of transgene mRNA in the neurofibroma. Immunofluorescence microscopy revealed transgene expressing Sox10-positive SCs throughout the neurofibroma with AAV-SC3 injection. Next, we performed a pooled screen with four of the top capsids from our initial selection and AAV9 and identified one capsid, AAV-SC4, with enhanced biodistribution to and transduction of normal sciatic nerve in mice. This capsid displayed a peptide with a known laminin-binding motif, which may provide a conduit for future laminin-targeting strategies. Our results provide a baseline for future AAV-based gene therapies developed for NF1 or other diseases that affect SCs.","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":"32 4","pages":"385-397"},"PeriodicalIF":4.5,"publicationDate":"2025-06-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144266087","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Heparin cofactor II (HCII) is a critical anticoagulant protein that inactivates thrombin. In our previous mouse studies, we demonstrated that GalNAc-HCII, a small interfering RNA (siRNA) targeting HCII conjugated with N-acetylgalactosamine (GalNAc), exhibited promising therapeutic effects in hemophilia A mouse models. Further evaluation in large animal models, especially with FVIII inhibitors, is essential before GalNAc-HCII can proceed to clinical trials. In this study, we successfully established, for the first time, an acquired hemophilia A canine model by multiple intravenous injections of a rabbit-dog chimeric neutralizing anti-canine FVIII antibody. In the control group, the Beagle dogs exhibited spontaneous bleeding symptoms accompanied by prolonged activated partial thromboplastin time (APTT). After administration, GalNAc-HCII (0.8 and 1.6 mg/kg) demonstrated potent, dose-dependent, and durable HCII inhibitory effects. After 5 days, in normal dogs, GalNAc-HCII reduced HCII levels to 32.67% ± 3.07% and 10.62% ± 1.74% with 0.8 and 1.6 mg/kg GalNAc-HCII, respectively. In hemophilic dogs, GalNAc-HCII treatment significantly improved hemostatic function. Specifically, in the carotid artery thrombosis model, the thrombus formation time was shortened [29.7 ± 2.08 min (0.8 mg/kg) and 18.0 ± 1.0 min (1.6 mg/kg) vs. 40 min (control), P < 0.01]; in the knee joint puncture-induced bleeding model, joint bleeding and synovitis were alleviated; and in the saphenous vein bleeding model, the number of hemostatic events increased. Furthermore, repeated administration of GalNAc-HCII effectively reduced the prolonged APTT. This study demonstrates the efficacy of GalNAc-HCII in hemophilic dogs, suggesting it as a promising novel therapeutic option for patients with hemophilia, including those with FVIII inhibitors.
肝素辅助因子II （HCII）是一种重要的抗凝血蛋白，可使凝血酶失活。在我们之前的小鼠研究中，我们证明了GalNAc-HCII，一种靶向HCII的小干扰RNA （siRNA）与n -乙酰半乳糖胺（GalNAc）结合，在血友病a小鼠模型中显示出有希望的治疗效果。在GalNAc-HCII进入临床试验之前，必须在大型动物模型中进行进一步评估，特别是FVIII抑制剂。本研究首次通过多次静脉注射兔-犬嵌合中和抗犬FVIII抗体成功建立犬获得性血友病A模型。在对照组中，Beagle犬表现出自发性出血症状，并伴有活化的部分凝血活素时间（APTT）延长。给药后，GalNAc-HCII（0.8和1.6 mg/kg）显示出有效的、剂量依赖性的、持久的HCII抑制作用。5天后，在正常犬中，以0.8和1.6 mg/kg GalNAc-HCII分别使HCII水平降低至32.67%±3.07%和10.62%±1.74%。在血友病犬中，GalNAc-HCII治疗可显著改善止血功能。其中，颈动脉血栓形成模型的血栓形成时间缩短，分别为29.7±2.08 min （0.8 mg/kg）和18.0±1.0 min (1.6 mg/kg)，对照组为40 min
{"title":"RNAi targeting heparin cofactor II promotes hemostasis in a canine model of acquired hemophilia A","authors":"Yuyang Zhang, Tingting Liu, Haiming Kou, Huafang Wang, Yu Hu, Liang V. Tang","doi":"10.1038/s41434-025-00541-w","DOIUrl":"10.1038/s41434-025-00541-w","url":null,"abstract":"Heparin cofactor II (HCII) is a critical anticoagulant protein that inactivates thrombin. In our previous mouse studies, we demonstrated that GalNAc-HCII, a small interfering RNA (siRNA) targeting HCII conjugated with N-acetylgalactosamine (GalNAc), exhibited promising therapeutic effects in hemophilia A mouse models. Further evaluation in large animal models, especially with FVIII inhibitors, is essential before GalNAc-HCII can proceed to clinical trials. In this study, we successfully established, for the first time, an acquired hemophilia A canine model by multiple intravenous injections of a rabbit-dog chimeric neutralizing anti-canine FVIII antibody. In the control group, the Beagle dogs exhibited spontaneous bleeding symptoms accompanied by prolonged activated partial thromboplastin time (APTT). After administration, GalNAc-HCII (0.8 and 1.6 mg/kg) demonstrated potent, dose-dependent, and durable HCII inhibitory effects. After 5 days, in normal dogs, GalNAc-HCII reduced HCII levels to 32.67% ± 3.07% and 10.62% ± 1.74% with 0.8 and 1.6 mg/kg GalNAc-HCII, respectively. In hemophilic dogs, GalNAc-HCII treatment significantly improved hemostatic function. Specifically, in the carotid artery thrombosis model, the thrombus formation time was shortened [29.7 ± 2.08 min (0.8 mg/kg) and 18.0 ± 1.0 min (1.6 mg/kg) vs. 40 min (control), P < 0.01]; in the knee joint puncture-induced bleeding model, joint bleeding and synovitis were alleviated; and in the saphenous vein bleeding model, the number of hemostatic events increased. Furthermore, repeated administration of GalNAc-HCII effectively reduced the prolonged APTT. This study demonstrates the efficacy of GalNAc-HCII in hemophilic dogs, suggesting it as a promising novel therapeutic option for patients with hemophilia, including those with FVIII inhibitors.","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":"32 4","pages":"398-409"},"PeriodicalIF":4.5,"publicationDate":"2025-05-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144142373","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Resistance to radiotherapy is a significant challenge in the clinical management of non-small cell lung cancer (NSCLC). This study investigates a novel multimodal therapeutic strategy that combines oncolytic Newcastle disease virus (NDV) with an anti-VEGFR2 single-chain variable fragment (NDV-anti-VEGFR2) to enhance radiosensitivity in NSCLC. We engineered NDV-anti-VEGFR2 and assessed its efficacy in sensitizing Calu-1 cells to radiation. In vitro results demonstrated that NDV-anti-VEGFR2 significantly inhibited tumor cell proliferation when combined with radiotherapy. In vivo experiments revealed that NDV-anti-VEGFR2, combined with radiation, achieved a tumor growth inhibition rate of 86.48%, surpassing the effects of NDV or radiation alone. Mechanistic investigations indicated that NDV-anti-VEGFR2 mitigated hypoxia by downregulating HIF-1α and impaired DNA repair pathways, as evidenced by reduced levels of RAD51 and γ-H2AX. These findings suggest that NDV-anti-VEGFR2 not only normalizes tumor vasculature but also enhances the cytotoxic effects of radiation by compromising DNA repair mechanisms. Collectively, our results support the clinical potential of NDV-anti-VEGFR2 combined with radiotherapy as a promising strategy to overcome radiotherapy resistance in NSCLC. Future studies in immunocompetent models are warranted to elucidate the immune-mediated effects of this innovative therapeutic approach.
{"title":"Recombinant oncolytic virus NDV-anti-VEGFR2 enhances radiotherapy sensitivity in NSCLC by targeting VEGF signaling and impairing DNA repair","authors":"Liang Liu, Liying Song, Tianyan Liu, Kaiyuan Hui, Chenxi Hu, Jiarui Yang, Xuelei Pi, Yuanyuan Yan, Shishi Liu, Yating Zhang, Hongna Chen, Yukai Cao, Lihua Zhou, yun Qiao, Dan Yu, Chengkai Yin, Xu Li, Chenfeng Zhang, Deshan Li, Zhenzhong Wang, Zhihang Liu, Xiaodong Jiang","doi":"10.1038/s41434-025-00540-x","DOIUrl":"10.1038/s41434-025-00540-x","url":null,"abstract":"Resistance to radiotherapy is a significant challenge in the clinical management of non-small cell lung cancer (NSCLC). This study investigates a novel multimodal therapeutic strategy that combines oncolytic Newcastle disease virus (NDV) with an anti-VEGFR2 single-chain variable fragment (NDV-anti-VEGFR2) to enhance radiosensitivity in NSCLC. We engineered NDV-anti-VEGFR2 and assessed its efficacy in sensitizing Calu-1 cells to radiation. In vitro results demonstrated that NDV-anti-VEGFR2 significantly inhibited tumor cell proliferation when combined with radiotherapy. In vivo experiments revealed that NDV-anti-VEGFR2, combined with radiation, achieved a tumor growth inhibition rate of 86.48%, surpassing the effects of NDV or radiation alone. Mechanistic investigations indicated that NDV-anti-VEGFR2 mitigated hypoxia by downregulating HIF-1α and impaired DNA repair pathways, as evidenced by reduced levels of RAD51 and γ-H2AX. These findings suggest that NDV-anti-VEGFR2 not only normalizes tumor vasculature but also enhances the cytotoxic effects of radiation by compromising DNA repair mechanisms. Collectively, our results support the clinical potential of NDV-anti-VEGFR2 combined with radiotherapy as a promising strategy to overcome radiotherapy resistance in NSCLC. Future studies in immunocompetent models are warranted to elucidate the immune-mediated effects of this innovative therapeutic approach.","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":"32 5","pages":"517-528"},"PeriodicalIF":4.5,"publicationDate":"2025-05-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144092958","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-05-06DOI: 10.1038/s41434-025-00530-z
Deepshikha Bhardwaj, Ibrahim Youssef, Darren Imphean, Sydni K. Holmes, Venugopal Krishnan, Sandi Jo Estill-Terpack, Marc Diamond, Rajiv Chopra, Rachel M. Bailey, Bhavya R. Shah
Transcranial Magnetic Resonance Guided Focused Ultrasound can oscillate intravenously delivered microbubbles and transiently open the blood brain barrier (BBB) in a targeted brain region. However, high microbubble doses or Focused ultrasound pressures (FUS) leads to injury. So, we administered nitrous oxide (N2O), an anesthetic gas to determine reduced need of FUS pressure and microbubble dose for opening BBB. Swiss Webster mice were treated with N2O or medical air (MA) at varying FUS pressures, while the microbubble dose was kept constant and the vice-versa. Consequently, BBB opening was quantified by acoustic emissions and enhancement rate on T1-weighted MR. To compare the effect of N2O on gene delivery, following BBB opening with either MA or N2O, a viral vector expressing GFP was subsequently delivered. Additionally, Immunohistochemical studies quantified viral transfection efficacy and assessed acute cell injury. We observed that N2O significantly potentiates acoustic emissions and enhancement rate on post-contrast MRI images, compared to MA at all measured pressures (0.39, 0.45, 0.67 MPa). Furthermore, N2O reduces the microbubble dose to 0.02μl/kg and FUS pressures to 0.28 and 0.39 MPa for BBB disruption and enhanced viral gene delivery, respectively. Hence, N2O potentiates microbubble oscillations, allowing reduced microbubble dose and FUS pressures and improved viral gene delivery.
{"title":"Nitrous oxide enhances MR-guided focused ultrasound delivery of gene therapy to the murine hippocampus","authors":"Deepshikha Bhardwaj, Ibrahim Youssef, Darren Imphean, Sydni K. Holmes, Venugopal Krishnan, Sandi Jo Estill-Terpack, Marc Diamond, Rajiv Chopra, Rachel M. Bailey, Bhavya R. Shah","doi":"10.1038/s41434-025-00530-z","DOIUrl":"10.1038/s41434-025-00530-z","url":null,"abstract":"Transcranial Magnetic Resonance Guided Focused Ultrasound can oscillate intravenously delivered microbubbles and transiently open the blood brain barrier (BBB) in a targeted brain region. However, high microbubble doses or Focused ultrasound pressures (FUS) leads to injury. So, we administered nitrous oxide (N2O), an anesthetic gas to determine reduced need of FUS pressure and microbubble dose for opening BBB. Swiss Webster mice were treated with N2O or medical air (MA) at varying FUS pressures, while the microbubble dose was kept constant and the vice-versa. Consequently, BBB opening was quantified by acoustic emissions and enhancement rate on T1-weighted MR. To compare the effect of N2O on gene delivery, following BBB opening with either MA or N2O, a viral vector expressing GFP was subsequently delivered. Additionally, Immunohistochemical studies quantified viral transfection efficacy and assessed acute cell injury. We observed that N2O significantly potentiates acoustic emissions and enhancement rate on post-contrast MRI images, compared to MA at all measured pressures (0.39, 0.45, 0.67 MPa). Furthermore, N2O reduces the microbubble dose to 0.02μl/kg and FUS pressures to 0.28 and 0.39 MPa for BBB disruption and enhanced viral gene delivery, respectively. Hence, N2O potentiates microbubble oscillations, allowing reduced microbubble dose and FUS pressures and improved viral gene delivery.","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":"32 4","pages":"376-384"},"PeriodicalIF":4.5,"publicationDate":"2025-05-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144007622","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Gene therapy with AAV vectors is a promising approach for treating numerous genetic disorders but is often hindered by preexisting antibodies that neutralize the vectors. Given that females may exhibit stronger immune responses than males, this study hypothesizes that females may have higher preexisting antibody titers against AAV. Serum samples from two U.S. cohorts were analyzed for antibody titers, antibody subtypes, and transduction inhibition activity against AAV serotypes AAV1, AAV2, AAV5, AAV8, and AAV9. We found that among seropositive samples, females had higher preexisting antibody levels and neutralizing activities against AAV9 and other serotypes. Immunoglobulin subclass analysis showed IgG1 dominance in both sexes, but females had higher IgA levels, whereas males had higher levels of IgG2. We further evaluated the cellular level of this differential immune response to AAV by stimulation of male and female human PBMCs. We observed dose-dependent increase in cytokines and chemokines in female PBMCs which suggests a differential inflammatory response. Altogether, our findings suggest that the enhanced immune response in females could lead to neutralization and faster clearance of AAV vectors with potential to impact the efficacy of gene therapy.
{"title":"Unveiling the sex bias: higher preexisting and neutralizing titers against AAV in females and implications for gene therapy","authors":"Stephanee Warrington, Trish T. Hoang, Morten Seirup, Leila Abdelhamid, Hrittal Saha, Sojin Bing, Sima Saleh, Je-Nie Phue, Ronit Mazor","doi":"10.1038/s41434-025-00528-7","DOIUrl":"10.1038/s41434-025-00528-7","url":null,"abstract":"Gene therapy with AAV vectors is a promising approach for treating numerous genetic disorders but is often hindered by preexisting antibodies that neutralize the vectors. Given that females may exhibit stronger immune responses than males, this study hypothesizes that females may have higher preexisting antibody titers against AAV. Serum samples from two U.S. cohorts were analyzed for antibody titers, antibody subtypes, and transduction inhibition activity against AAV serotypes AAV1, AAV2, AAV5, AAV8, and AAV9. We found that among seropositive samples, females had higher preexisting antibody levels and neutralizing activities against AAV9 and other serotypes. Immunoglobulin subclass analysis showed IgG1 dominance in both sexes, but females had higher IgA levels, whereas males had higher levels of IgG2. We further evaluated the cellular level of this differential immune response to AAV by stimulation of male and female human PBMCs. We observed dose-dependent increase in cytokines and chemokines in female PBMCs which suggests a differential inflammatory response. Altogether, our findings suggest that the enhanced immune response in females could lead to neutralization and faster clearance of AAV vectors with potential to impact the efficacy of gene therapy.","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":"32 4","pages":"339-348"},"PeriodicalIF":4.5,"publicationDate":"2025-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144005557","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-04-30DOI: 10.1038/s41434-025-00538-5
Mahdiyar Dehshiri, Shokouh Rezaei, Saman Hosseinkhani
Chimeric peptides hold promising potential to be introduced as an ideal gene delivery platform based on their advantages over viral carriers, including but not limited to the safety profile and specific targeting. However, their gene transfer efficiency needs improvement. Here, we designed a new multi-functional chimeric peptide for enhanced gene delivery by adding a cyclic TAT motif to a previously designed MPG2H peptide to enable the targeting of cells with independent/dependent endocytosis cell entry mechanisms. CTATMPG2H was expressed and purified using affinity chromatography; then it was characterized through a gel retardation assay, circular dichroism (CD) spectropolarimetry, transmission electron microscopy (TEM) dynamic light scattering (DLS), and zeta potential analysis. CTATMPG2H was compared with MiRGD as a chimeric peptide control in all steps. After assessing the platform stability in various conditions, its gene transfer efficiency was evaluated in the HEK293T cell line with reporter genes. Additionally, mouse bone marrow-derived dendritic cells (BMDCs) were transfected to test CTATMPG2H potential in immunotherapy. The results illustrated a safe gene transfer profile for CTATMPG2H comparable to MiRGD and Polyethyleneimine (PEI). Flow cytometry results showed up to 48% gene transfer rate for CTATMPG2H to dendritic cells with minimal toxicity (viability rate ~80%). Moreover, the in silico investigation showed that the synergistic effects of electrostatic, hydrogen, and hydrophobic interactions enhance the stability and binding affinity of peptide-pDNA complexes, ensuring robust and specific targeting of nucleic acids. This research sets a foundation for future in vivo studies and potential clinical applications, aiming for safer and more effective gene therapy strategies.
{"title":"A novel multi-functional chimeric peptide for enhanced safe gene delivery in immunotherapy","authors":"Mahdiyar Dehshiri, Shokouh Rezaei, Saman Hosseinkhani","doi":"10.1038/s41434-025-00538-5","DOIUrl":"10.1038/s41434-025-00538-5","url":null,"abstract":"Chimeric peptides hold promising potential to be introduced as an ideal gene delivery platform based on their advantages over viral carriers, including but not limited to the safety profile and specific targeting. However, their gene transfer efficiency needs improvement. Here, we designed a new multi-functional chimeric peptide for enhanced gene delivery by adding a cyclic TAT motif to a previously designed MPG2H peptide to enable the targeting of cells with independent/dependent endocytosis cell entry mechanisms. CTATMPG2H was expressed and purified using affinity chromatography; then it was characterized through a gel retardation assay, circular dichroism (CD) spectropolarimetry, transmission electron microscopy (TEM) dynamic light scattering (DLS), and zeta potential analysis. CTATMPG2H was compared with MiRGD as a chimeric peptide control in all steps. After assessing the platform stability in various conditions, its gene transfer efficiency was evaluated in the HEK293T cell line with reporter genes. Additionally, mouse bone marrow-derived dendritic cells (BMDCs) were transfected to test CTATMPG2H potential in immunotherapy. The results illustrated a safe gene transfer profile for CTATMPG2H comparable to MiRGD and Polyethyleneimine (PEI). Flow cytometry results showed up to 48% gene transfer rate for CTATMPG2H to dendritic cells with minimal toxicity (viability rate ~80%). Moreover, the in silico investigation showed that the synergistic effects of electrostatic, hydrogen, and hydrophobic interactions enhance the stability and binding affinity of peptide-pDNA complexes, ensuring robust and specific targeting of nucleic acids. This research sets a foundation for future in vivo studies and potential clinical applications, aiming for safer and more effective gene therapy strategies.","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":"32 5","pages":"497-507"},"PeriodicalIF":4.5,"publicationDate":"2025-04-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144002900","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-04-26DOI: 10.1038/s41434-025-00537-6
Kumitaa Theva Das
{"title":"From bench to bedside: the future of stable lentiviral packaging cell lines in gene therapy","authors":"Kumitaa Theva Das","doi":"10.1038/s41434-025-00537-6","DOIUrl":"10.1038/s41434-025-00537-6","url":null,"abstract":"","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":"32 5","pages":"569-571"},"PeriodicalIF":4.5,"publicationDate":"2025-04-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143975284","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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