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}
Supported by encouraging trial outcomes, onasemnogene abeparvovec (OA) was authorized for spinal muscular atrophy (SMA). Nevertheless, efficacy of OA in advanced SMA patients remains underexplored. This investigation assessed clinical effectiveness and adverse effects of OA in a cohort including advanced SMA, and compared to historical survival data for SMA type 1 patients in Thailand. We conducted observational cohort study at Siriraj Hospital, Thailand, from May 2019 to April 2022. The study enrolled eight SMA patients receiving OA therapy. The cohort comprised five SMA type 1 patients treated at 16.7 months (6.5–24.9 months) and three SMA type 2 patients treated at 20.3 months (19–31.5 months). Before receiving OA, all Type 1 patients required 24-hour invasive ventilation and feeding support. Post-treatment, Three of five showed gradual improvement in motor scores, but none achieved new motor milestones. Survival rate was not improved, with all experiencing fatalities. Conversely, Type 2 patients exhibited motor score improvement without serious adverse events. OA did not significantly improve clinical outcomes or survival rates in advanced Type 1 SMA. These findings highlight need for additional caution when administering OA to severe SMA Type 1 and more specific guidelines in selecting subgroups for treatment.
{"title":"Fatal outcomes following onasemnogene abeparvovec in advanced-stage spinal muscular atrophy","authors":"Peerada Pongsakornkullachart, Pimchanok Kulsirichawaroj, Ratcharin Kongkasuwan, Prakarn Tovichien, Settapong Jitwongwai, Supaluck Kanjanauthai, Nutnicha Preeprem, Sivaporn Limpaninlachat, Nisasri Sermpon, Oranee Sanmaneechai","doi":"10.1038/s41434-025-00535-8","DOIUrl":"10.1038/s41434-025-00535-8","url":null,"abstract":"Supported by encouraging trial outcomes, onasemnogene abeparvovec (OA) was authorized for spinal muscular atrophy (SMA). Nevertheless, efficacy of OA in advanced SMA patients remains underexplored. This investigation assessed clinical effectiveness and adverse effects of OA in a cohort including advanced SMA, and compared to historical survival data for SMA type 1 patients in Thailand. We conducted observational cohort study at Siriraj Hospital, Thailand, from May 2019 to April 2022. The study enrolled eight SMA patients receiving OA therapy. The cohort comprised five SMA type 1 patients treated at 16.7 months (6.5–24.9 months) and three SMA type 2 patients treated at 20.3 months (19–31.5 months). Before receiving OA, all Type 1 patients required 24-hour invasive ventilation and feeding support. Post-treatment, Three of five showed gradual improvement in motor scores, but none achieved new motor milestones. Survival rate was not improved, with all experiencing fatalities. Conversely, Type 2 patients exhibited motor score improvement without serious adverse events. OA did not significantly improve clinical outcomes or survival rates in advanced Type 1 SMA. These findings highlight need for additional caution when administering OA to severe SMA Type 1 and more specific guidelines in selecting subgroups for treatment.","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":"32 5","pages":"553-560"},"PeriodicalIF":4.5,"publicationDate":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.nature.comhttps://www.nature.com/articles/s41434-025-00535-8.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144011513","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 : 2025-04-22DOI: 10.1038/s41434-025-00536-7
Irina Filz von Reiterdank, Mohammadreza Mojoudi, Raphaela Bento, McLean S. Taggart, Antonia T. Dinicu, Gregory Wojtkiewicz, J. H. Coert, Aebele B. Mink van der Molen, Ralph Weissleder, Biju Parekkadan, Korkut Uygun
Developing new strategies for local monitoring and delivery of immunosuppression is critical to making allografts safer and more accessible. Ex vivo genetic modification of grafts using machine perfusion presents a promising approach to improve graft function and modulate immune responses while minimizing risks of off-target effects and systemic immunogenicity in vivo. This proof-of-concept study demonstrates the feasibility of using normothermic machine perfusion (NMP) to mimic in vitro conditions for effective gene delivery. In this study, lentiviral vectors encoding the secreted biomarker Gaussia Luciferase (GLuc) and red fluorescent protein (RFP) were introduced ex vivo to rodent livers during a 72-h machine perfusion protocol. After an initial 24-h exposure to viral vectors, the organs were maintained in perfusion for an additional 48 h to monitor gene expression, aligning with in vitro benchmarks. Control livers were perfused in similar fashion, but without viral injections. Virally perfused livers exhibited nearly a 10-fold increase in luminescence compared to controls (p < 0.0001), indicating successful genetic modification of the organs. These findings validate the use of machine perfusion systems and viral vectors to genetically engineer whole organs ex vivo, laying the groundwork for a broad range of applications in transplantation through genetic manipulation of organ systems. Future studies will focus on refining this technology to enhance precision in gene expression and explore its implications for clinical translation.
{"title":"Ex vivo machine perfusion as a platform for lentiviral gene delivery in rat livers","authors":"Irina Filz von Reiterdank, Mohammadreza Mojoudi, Raphaela Bento, McLean S. Taggart, Antonia T. Dinicu, Gregory Wojtkiewicz, J. H. Coert, Aebele B. Mink van der Molen, Ralph Weissleder, Biju Parekkadan, Korkut Uygun","doi":"10.1038/s41434-025-00536-7","DOIUrl":"10.1038/s41434-025-00536-7","url":null,"abstract":"Developing new strategies for local monitoring and delivery of immunosuppression is critical to making allografts safer and more accessible. Ex vivo genetic modification of grafts using machine perfusion presents a promising approach to improve graft function and modulate immune responses while minimizing risks of off-target effects and systemic immunogenicity in vivo. This proof-of-concept study demonstrates the feasibility of using normothermic machine perfusion (NMP) to mimic in vitro conditions for effective gene delivery. In this study, lentiviral vectors encoding the secreted biomarker Gaussia Luciferase (GLuc) and red fluorescent protein (RFP) were introduced ex vivo to rodent livers during a 72-h machine perfusion protocol. After an initial 24-h exposure to viral vectors, the organs were maintained in perfusion for an additional 48 h to monitor gene expression, aligning with in vitro benchmarks. Control livers were perfused in similar fashion, but without viral injections. Virally perfused livers exhibited nearly a 10-fold increase in luminescence compared to controls (p < 0.0001), indicating successful genetic modification of the organs. These findings validate the use of machine perfusion systems and viral vectors to genetically engineer whole organs ex vivo, laying the groundwork for a broad range of applications in transplantation through genetic manipulation of organ systems. Future studies will focus on refining this technology to enhance precision in gene expression and explore its implications for clinical translation.","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":"32 4","pages":"421-429"},"PeriodicalIF":4.5,"publicationDate":"2025-04-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143983866","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 : 2025-04-15DOI: 10.1038/s41434-025-00533-w
Jona Röscheise, Maximilian Klimpel, Parameswari Govindarajan, Kerstin Otte, Holger Laux
Lentiviral vectors (LVVs) are widely used in gene therapy due to their ability to infect both dividing and non-dividing cells. For LVV production, the creation of stable packaging cell lines with integrated genes necessary for viral replication offer a more consistent and scalable alternative to transient plasmid transfection approach. Although the development of such stable LVV packaging cell lines has been reported, the molecular changes induced by stable and inducible viral gene expression and the impact of genome integrated viral genes on cellular pathways remain poorly characterized. For better insight, we investigated the molecular characteristics of a stable LVV packaging cell line and its host cell line (HEK293T/17) by comparing differential expressed genes. This pathway analysis revealed significant changes in pathway usage between packaging and host cell lines, influenced by different viral transgenes. Gag-pol expression was found to suppress host translational machinery, while rev and VSV-G expression modulated mitochondrial pathways, including oxidative phosphorylation. HIV-1 tat expression, on the other hand, activated histone-related genes. These regulatory shifts suggest a strategic reprogramming of host cellular states to favor viral replication, curbing protein synthesis and energy production to levels that support viral assembly but impair the host’s immune defense and the production of immune-related proteins. Our findings provide a deeper understanding of the molecular changes associated with stable viral gene expression, which can inform the optimization of LVV production in gene therapy applications.
{"title":"Unveiling molecular secrets: Analysis of stable lentiviral packaging cell lines enables identification of novel viral gene functions","authors":"Jona Röscheise, Maximilian Klimpel, Parameswari Govindarajan, Kerstin Otte, Holger Laux","doi":"10.1038/s41434-025-00533-w","DOIUrl":"10.1038/s41434-025-00533-w","url":null,"abstract":"Lentiviral vectors (LVVs) are widely used in gene therapy due to their ability to infect both dividing and non-dividing cells. For LVV production, the creation of stable packaging cell lines with integrated genes necessary for viral replication offer a more consistent and scalable alternative to transient plasmid transfection approach. Although the development of such stable LVV packaging cell lines has been reported, the molecular changes induced by stable and inducible viral gene expression and the impact of genome integrated viral genes on cellular pathways remain poorly characterized. For better insight, we investigated the molecular characteristics of a stable LVV packaging cell line and its host cell line (HEK293T/17) by comparing differential expressed genes. This pathway analysis revealed significant changes in pathway usage between packaging and host cell lines, influenced by different viral transgenes. Gag-pol expression was found to suppress host translational machinery, while rev and VSV-G expression modulated mitochondrial pathways, including oxidative phosphorylation. HIV-1 tat expression, on the other hand, activated histone-related genes. These regulatory shifts suggest a strategic reprogramming of host cellular states to favor viral replication, curbing protein synthesis and energy production to levels that support viral assembly but impair the host’s immune defense and the production of immune-related proteins. Our findings provide a deeper understanding of the molecular changes associated with stable viral gene expression, which can inform the optimization of LVV production in gene therapy applications.","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":"32 3","pages":"266-276"},"PeriodicalIF":4.5,"publicationDate":"2025-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143981535","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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