Pub Date : 2024-07-07DOI: 10.1038/s41434-024-00462-0
Jillian Maniego, Caitlin Harding, Jocelyn Habershon-Butcher, Pamela Hincks, Edward Ryder
Gene doping, which includes the non-therapeutic use of genes or genetic elements that have the capacity to enhance athletic performance, is prohibited in horseracing and equestrian sports. To provide a comprehensive assessment of matrix and detection techniques, a custom adeno-associated virus serotype 8 vector was designed to include PCR binding sites for multiple target genes and assay types. The vector was injected via an intramuscular route into two Thoroughbred horses and matrices collected at defined timepoints. DNA was analysed using 3 detection methods: qPCR, digital PCR, and NGS. Overall, there was a strong correlation across the different detection methods employed, although digital PCR was less sensitive at lower concentrations. High concentrations of vector were detected at early timepoints in plasma and whole blood, which rapidly dropped after 0.5 d to trace levels by 4 d and 9 d post-administration respectively, following a similar pattern to previous studies. Vector was detected in dried blood spots at lower levels than whole blood, but with a similar detection time. Detection in hair root bulbs in one horse was observed at over a month post-administration, which opens new avenues for future gene doping testing in humans and animals.
{"title":"Administration and detection of a multi-target rAAV gene doping vector in horses using multiple matrices and molecular techniques","authors":"Jillian Maniego, Caitlin Harding, Jocelyn Habershon-Butcher, Pamela Hincks, Edward Ryder","doi":"10.1038/s41434-024-00462-0","DOIUrl":"10.1038/s41434-024-00462-0","url":null,"abstract":"Gene doping, which includes the non-therapeutic use of genes or genetic elements that have the capacity to enhance athletic performance, is prohibited in horseracing and equestrian sports. To provide a comprehensive assessment of matrix and detection techniques, a custom adeno-associated virus serotype 8 vector was designed to include PCR binding sites for multiple target genes and assay types. The vector was injected via an intramuscular route into two Thoroughbred horses and matrices collected at defined timepoints. DNA was analysed using 3 detection methods: qPCR, digital PCR, and NGS. Overall, there was a strong correlation across the different detection methods employed, although digital PCR was less sensitive at lower concentrations. High concentrations of vector were detected at early timepoints in plasma and whole blood, which rapidly dropped after 0.5 d to trace levels by 4 d and 9 d post-administration respectively, following a similar pattern to previous studies. Vector was detected in dried blood spots at lower levels than whole blood, but with a similar detection time. Detection in hair root bulbs in one horse was observed at over a month post-administration, which opens new avenues for future gene doping testing in humans and animals.","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":"31 9-10","pages":"477-488"},"PeriodicalIF":4.6,"publicationDate":"2024-07-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141554697","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 : 2024-07-03DOI: 10.1038/s41434-024-00461-1
Tinghui Bai, Bohao Cui, Man Xing, Siyue Chen, Yanfang Zhu, Dongxue Lin, Yingying Guo, Mei Du, Xiaohong Wang, Dongming Zhou, Hua Yan
Neovascular age-related macular degeneration (nAMD) causes severe visual impairment. Pigment epithelium-derived factor (PEDF), soluble CD59 (sCD59), and soluble fms-like tyrosine kinase-1 (sFLT-1) are potential therapeutic agents for nAMD, which target angiogenesis and the complement system. Using the AAV2/8 vector, two bi-target gene therapy agents, AAV2/8-PEDF-P2A-sCD59 and AAV2/8-sFLT-1-P2A-sCD59, were generated, and their therapeutic efficacy was investigated in laser-induced choroidal neovascularization (CNV) and Vldlr−/− mouse models. After a single injection, AAV2/8-mediated gene expression was maintained at high levels in the retina for two months. Both AAV2/8-PEDF-P2A-sCD59 and AAV2/8-sFLT-1-P2A-sCD59 significantly reduced CNV development for an extended period without side effects and provided efficacy similar to two injections of current anti-vascular endothelial growth factor monotherapy. Mechanistically, these agents suppressed the extracellular signal-regulated kinase and nuclear factor-κB pathways, resulting in anti-angiogenic activity. This study demonstrated the safety and long-lasting effects of AAV2/8-PEDF-P2A-sCD59 and AAV2/8-sFLT-1-P2A-sCD59 in CNV treatment, providing a promising therapeutic strategy for nAMD.
{"title":"Stable inhibition of choroidal neovascularization by adeno-associated virus 2/8-vectored bispecific molecules","authors":"Tinghui Bai, Bohao Cui, Man Xing, Siyue Chen, Yanfang Zhu, Dongxue Lin, Yingying Guo, Mei Du, Xiaohong Wang, Dongming Zhou, Hua Yan","doi":"10.1038/s41434-024-00461-1","DOIUrl":"10.1038/s41434-024-00461-1","url":null,"abstract":"Neovascular age-related macular degeneration (nAMD) causes severe visual impairment. Pigment epithelium-derived factor (PEDF), soluble CD59 (sCD59), and soluble fms-like tyrosine kinase-1 (sFLT-1) are potential therapeutic agents for nAMD, which target angiogenesis and the complement system. Using the AAV2/8 vector, two bi-target gene therapy agents, AAV2/8-PEDF-P2A-sCD59 and AAV2/8-sFLT-1-P2A-sCD59, were generated, and their therapeutic efficacy was investigated in laser-induced choroidal neovascularization (CNV) and Vldlr−/− mouse models. After a single injection, AAV2/8-mediated gene expression was maintained at high levels in the retina for two months. Both AAV2/8-PEDF-P2A-sCD59 and AAV2/8-sFLT-1-P2A-sCD59 significantly reduced CNV development for an extended period without side effects and provided efficacy similar to two injections of current anti-vascular endothelial growth factor monotherapy. Mechanistically, these agents suppressed the extracellular signal-regulated kinase and nuclear factor-κB pathways, resulting in anti-angiogenic activity. This study demonstrated the safety and long-lasting effects of AAV2/8-PEDF-P2A-sCD59 and AAV2/8-sFLT-1-P2A-sCD59 in CNV treatment, providing a promising therapeutic strategy for nAMD.","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":"31 9-10","pages":"511-523"},"PeriodicalIF":4.6,"publicationDate":"2024-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.nature.com/articles/s41434-024-00461-1.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141497848","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 : 2024-06-25DOI: 10.1038/s41434-024-00458-w
Yeounsun Oh, Lee Wha Gwon, Hyomin K. Lee, Junho K. Hur, Kwang-Hyun Park, Kee-Pyo Kim, Seung Hwan Lee
The recently developed CRISPR activator (CRISPRa) system uses a CRISPR-Cas effector-based transcriptional activator to effectively control the expression of target genes without causing DNA damage. However, existing CRISPRa systems based on Cas9/Cas12a necessitate improvement in terms of efficacy and accuracy due to limitations associated with the CRISPR-Cas module itself. To overcome these limitations and effectively and accurately regulate gene expression, we developed an efficient CRISPRa system based on the small CRISPR-Cas effector Candidatus Woesearchaeota Cas12f (CWCas12f). By engineering the CRISPR-Cas module, linking activation domains, and using various combinations of linkers and nuclear localization signal sequences, the optimized eCWCas12f-VPR system enabled effective and target-specific regulation of gene expression compared with that using the existing CRISPRa system. The eCWCas12f-VPR system developed in this study has substantial potential for controlling the transcription of endogenous genes in living organisms and serves as a foundation for future gene therapy and biological research.
{"title":"Highly efficient and specific regulation of gene expression using enhanced CRISPR-Cas12f system","authors":"Yeounsun Oh, Lee Wha Gwon, Hyomin K. Lee, Junho K. Hur, Kwang-Hyun Park, Kee-Pyo Kim, Seung Hwan Lee","doi":"10.1038/s41434-024-00458-w","DOIUrl":"10.1038/s41434-024-00458-w","url":null,"abstract":"The recently developed CRISPR activator (CRISPRa) system uses a CRISPR-Cas effector-based transcriptional activator to effectively control the expression of target genes without causing DNA damage. However, existing CRISPRa systems based on Cas9/Cas12a necessitate improvement in terms of efficacy and accuracy due to limitations associated with the CRISPR-Cas module itself. To overcome these limitations and effectively and accurately regulate gene expression, we developed an efficient CRISPRa system based on the small CRISPR-Cas effector Candidatus Woesearchaeota Cas12f (CWCas12f). By engineering the CRISPR-Cas module, linking activation domains, and using various combinations of linkers and nuclear localization signal sequences, the optimized eCWCas12f-VPR system enabled effective and target-specific regulation of gene expression compared with that using the existing CRISPRa system. The eCWCas12f-VPR system developed in this study has substantial potential for controlling the transcription of endogenous genes in living organisms and serves as a foundation for future gene therapy and biological research.","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":"31 7-8","pages":"358-365"},"PeriodicalIF":4.6,"publicationDate":"2024-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.nature.com/articles/s41434-024-00458-w.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141450316","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 : 2024-06-05DOI: 10.1038/s41434-024-00456-y
Rodrigo Holanda Mendonça, Adriana Banzzatto Ortega, Ciro Matsui Jr, Vanessa van der Linden, Marcelo Kerstenetzky, Luis Fernando Grossklauss, Elizabeth L. Silveira-Lucas, Graziela Jorge Polido, Edmar Zanoteli
Spinal muscular atrophy (SMA) is a motor neuron disease associated with progressive muscle weakness, ventilatory failure, and reduced survival. Onasemnogene abeparvovec is the first gene replacement therapy (GT) approved to treat this condition. An observational retrospective study was conducted to assess adverse events and efficacy of GT in SMA patients. Forty-one patients with SMA (58.5% females and 80.1% SMA type 1) were included. The mean age at GT dosing was 18 (±6.4) months. Thirty-six patients (87.8%) were under previous treatment with nusinersen, and 10 (24.4%) continued nusinersen after GT. Mean CHOP-INTEND increased 13 points after 6 months and this finding did not differ between groups according to nusinersen maintenance after GT (p = 0.949). Among SMA type 1 patients, 14 (46.6%) reached the ability to sit alone. Liver transaminases elevation at least two times higher than the upper limit of normal value occurred in 29 (70.7%) patients. Thrombocytopenia occurred in 13 (31.7%) patients, and one presented thrombotic microangiopathy. Older age (>2 years) was associated with more prolonged use of corticosteroids (p = 0.021). GT is effective in SMA patients, combined nusinersen after GT did not appear to add gain in motor function and older age is associated with prolonged corticosteroid use.
脊髓性肌萎缩症（SMA）是一种运动神经元疾病，与进行性肌无力、呼吸衰竭和存活率降低有关。Onasemnogene abeparvovec是首个获准用于治疗这种疾病的基因替代疗法（GT）。我们开展了一项观察性回顾研究，以评估基因替代疗法对 SMA 患者的不良反应和疗效。研究纳入了 41 名 SMA 患者（58.5% 为女性，80.1% 为 SMA 1 型）。服用 GT 时的平均年龄为 18 (±6.4) 个月。36名患者（87.8%）曾接受过纽西奈森治疗，10名患者（24.4%）在GT治疗后继续接受纽西奈森治疗。6 个月后，CHOP-INTEND 的平均值增加了 13 分，根据 GT 后继续使用纽西奈森的情况，各组之间的结果并无差异（p = 0.949）。在 1 型 SMA 患者中，有 14 人（46.6%）能够单独坐立。29例（70.7%）患者的肝脏转氨酶高于正常值上限至少两倍。13例（31.7%）患者出现血小板减少，1例出现血栓性微血管病。年龄越大（大于 2 岁），使用皮质类固醇的时间越长（p = 0.021）。GT对SMA患者有效，但GT后联合使用纽西奈森似乎不会增加患者的运动功能，年龄越大，使用皮质类固醇的时间越长。
{"title":"Gene replacement therapy for spinal muscular atrophy: safety and preliminary efficacy in a Brazilian cohort","authors":"Rodrigo Holanda Mendonça, Adriana Banzzatto Ortega, Ciro Matsui Jr, Vanessa van der Linden, Marcelo Kerstenetzky, Luis Fernando Grossklauss, Elizabeth L. Silveira-Lucas, Graziela Jorge Polido, Edmar Zanoteli","doi":"10.1038/s41434-024-00456-y","DOIUrl":"10.1038/s41434-024-00456-y","url":null,"abstract":"Spinal muscular atrophy (SMA) is a motor neuron disease associated with progressive muscle weakness, ventilatory failure, and reduced survival. Onasemnogene abeparvovec is the first gene replacement therapy (GT) approved to treat this condition. An observational retrospective study was conducted to assess adverse events and efficacy of GT in SMA patients. Forty-one patients with SMA (58.5% females and 80.1% SMA type 1) were included. The mean age at GT dosing was 18 (±6.4) months. Thirty-six patients (87.8%) were under previous treatment with nusinersen, and 10 (24.4%) continued nusinersen after GT. Mean CHOP-INTEND increased 13 points after 6 months and this finding did not differ between groups according to nusinersen maintenance after GT (p = 0.949). Among SMA type 1 patients, 14 (46.6%) reached the ability to sit alone. Liver transaminases elevation at least two times higher than the upper limit of normal value occurred in 29 (70.7%) patients. Thrombocytopenia occurred in 13 (31.7%) patients, and one presented thrombotic microangiopathy. Older age (>2 years) was associated with more prolonged use of corticosteroids (p = 0.021). GT is effective in SMA patients, combined nusinersen after GT did not appear to add gain in motor function and older age is associated with prolonged corticosteroid use.","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":"31 7-8","pages":"391-399"},"PeriodicalIF":4.6,"publicationDate":"2024-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141261623","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 : 2024-06-04DOI: 10.1038/s41434-024-00457-x
Zhuang Mao, Changyao Wang, Juanli Liu, Xue Li, Han Duan, Yi Ye, Huifang Liu, Lin Lv, Guanzhen Xue, Zhichao He, Tana Wuren, Hua Wang
High-altitude pulmonary edema (HAPE) is a deadly form of altitude sickness, and there is no effective treatment for HAPE. Dental pulp stem cells (DPSCs) are a type of mesenchymal stem cell isolated from dental pulp tissues and possess various functions, such as anti-inflammatory and anti-oxidative stress. DPSCs have been used to treat a variety of diseases, but there are no studies on treating HAPE. In this study, Sprague-Dawley rats were exposed to acute low-pressure hypoxia to establish the HAPE model, and SOD1-modified DPSCs (DPSCsHiSOD1) were administered through the tail vein. Pulmonary arterial pressure, lung water content (LWC), total lung protein content of bronchoalveolar lavage fluid (BALF) and lung homogenates, oxidative stress, and inflammatory indicators were detected to evaluate the effects of DPSCsHiSOD1 on HAPE. Rat type II alveolar epithelial cells (RLE-6TN) were used to investigate the effects and mechanism of DPSCsHiSOD1 on hypoxia injury. We found that DPSCs could treat HAPE, and the effect was better than that of dexamethasone treatment. SOD1 modification could enhance the function of DPSCs in improving the structure of lung tissue, decreasing pulmonary arterial pressure and LWC, and reducing the total lung protein content of BALF and lung homogenates, through anti-oxidative stress and anti-inflammatory effects. Furthermore, we found that DPSCsHiSOD1 could protect RLE-6TN from hypoxic injury by reducing the accumulation of reactive oxygen species (ROS) and activating the Nrf2/HO-1 pathway. Our findings confirm that SOD1 modification could enhance the anti-oxidative stress ability of DPSCs through the Nrf2/HO-1 signalling pathway. DPSCs, especially DPSCsHiSOD1, could be a potential treatment for HAPE.
{"title":"Superoxide dismutase 1-modified dental pulp stem cells alleviate high-altitude pulmonary edema by inhibiting oxidative stress through the Nrf2/HO-1 pathway","authors":"Zhuang Mao, Changyao Wang, Juanli Liu, Xue Li, Han Duan, Yi Ye, Huifang Liu, Lin Lv, Guanzhen Xue, Zhichao He, Tana Wuren, Hua Wang","doi":"10.1038/s41434-024-00457-x","DOIUrl":"10.1038/s41434-024-00457-x","url":null,"abstract":"High-altitude pulmonary edema (HAPE) is a deadly form of altitude sickness, and there is no effective treatment for HAPE. Dental pulp stem cells (DPSCs) are a type of mesenchymal stem cell isolated from dental pulp tissues and possess various functions, such as anti-inflammatory and anti-oxidative stress. DPSCs have been used to treat a variety of diseases, but there are no studies on treating HAPE. In this study, Sprague-Dawley rats were exposed to acute low-pressure hypoxia to establish the HAPE model, and SOD1-modified DPSCs (DPSCsHiSOD1) were administered through the tail vein. Pulmonary arterial pressure, lung water content (LWC), total lung protein content of bronchoalveolar lavage fluid (BALF) and lung homogenates, oxidative stress, and inflammatory indicators were detected to evaluate the effects of DPSCsHiSOD1 on HAPE. Rat type II alveolar epithelial cells (RLE-6TN) were used to investigate the effects and mechanism of DPSCsHiSOD1 on hypoxia injury. We found that DPSCs could treat HAPE, and the effect was better than that of dexamethasone treatment. SOD1 modification could enhance the function of DPSCs in improving the structure of lung tissue, decreasing pulmonary arterial pressure and LWC, and reducing the total lung protein content of BALF and lung homogenates, through anti-oxidative stress and anti-inflammatory effects. Furthermore, we found that DPSCsHiSOD1 could protect RLE-6TN from hypoxic injury by reducing the accumulation of reactive oxygen species (ROS) and activating the Nrf2/HO-1 pathway. Our findings confirm that SOD1 modification could enhance the anti-oxidative stress ability of DPSCs through the Nrf2/HO-1 signalling pathway. DPSCs, especially DPSCsHiSOD1, could be a potential treatment for HAPE.","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":"31 7-8","pages":"422-433"},"PeriodicalIF":4.6,"publicationDate":"2024-06-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141247875","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 : 2024-05-16DOI: 10.1038/s41434-024-00455-z
M. Akula, S. M. McNamee, Z. Love, N. Nasraty, N. P. M. Chan, M. Whalen, M. O. Avola, A. M. Olivares, B. D. Leehy, A. S. Jelcick, P. Singh, A. K. Upadhyay, D. F. Chen, N. B. Haider
Degeneration of the macula is associated with several overlapping diseases including age-related macular degeneration (AMD) and Stargardt Disease (STGD). Mutations in ATP Binding Cassette Subfamily A Member 4 (ABCA4) are associated with late-onset dry AMD and early-onset STGD. Additionally, both forms of macular degeneration exhibit deposition of subretinal material and photoreceptor degeneration. Retinoic acid related orphan receptor α (RORA) regulates the AMD inflammation pathway that includes ABCA4, CD59, C3 and C5. In this translational study, we examined the efficacy of RORA at attenuating retinal degeneration and improving the inflammatory response in Abca4 knockout (Abca4−/−) mice. AAV5-hRORA-treated mice showed reduced deposits, restored CD59 expression and attenuated amyloid precursor protein (APP) expression compared with untreated eyes. This molecular rescue correlated with statistically significant improvement in photoreceptor function. This is the first study evaluating the impact of RORA modifier gene therapy on rescuing retinal degeneration. Our studies demonstrate efficacy of RORA in improving STGD and dry AMD-like disease.
{"title":"Retinoic acid related orphan receptor α is a genetic modifier that rescues retinal degeneration in a mouse model of Stargardt disease and Dry AMD","authors":"M. Akula, S. M. McNamee, Z. Love, N. Nasraty, N. P. M. Chan, M. Whalen, M. O. Avola, A. M. Olivares, B. D. Leehy, A. S. Jelcick, P. Singh, A. K. Upadhyay, D. F. Chen, N. B. Haider","doi":"10.1038/s41434-024-00455-z","DOIUrl":"10.1038/s41434-024-00455-z","url":null,"abstract":"Degeneration of the macula is associated with several overlapping diseases including age-related macular degeneration (AMD) and Stargardt Disease (STGD). Mutations in ATP Binding Cassette Subfamily A Member 4 (ABCA4) are associated with late-onset dry AMD and early-onset STGD. Additionally, both forms of macular degeneration exhibit deposition of subretinal material and photoreceptor degeneration. Retinoic acid related orphan receptor α (RORA) regulates the AMD inflammation pathway that includes ABCA4, CD59, C3 and C5. In this translational study, we examined the efficacy of RORA at attenuating retinal degeneration and improving the inflammatory response in Abca4 knockout (Abca4−/−) mice. AAV5-hRORA-treated mice showed reduced deposits, restored CD59 expression and attenuated amyloid precursor protein (APP) expression compared with untreated eyes. This molecular rescue correlated with statistically significant improvement in photoreceptor function. This is the first study evaluating the impact of RORA modifier gene therapy on rescuing retinal degeneration. Our studies demonstrate efficacy of RORA in improving STGD and dry AMD-like disease.","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":"31 7-8","pages":"413-421"},"PeriodicalIF":4.6,"publicationDate":"2024-05-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.nature.com/articles/s41434-024-00455-z.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140957146","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 : 2024-04-29DOI: 10.1038/s41434-024-00454-0
Alexander Renner, Anika Stahringer, Katharina Eva Ruppel, Stephan Fricke, Ulrike Koehl, Dominik Schmiedel
Allogeneic cell therapies, such as those involving macrophages or Natural Killer (NK) cells, are of increasing interest for cancer immunotherapy. However, the current techniques for genetically modifying these cell types using lenti- or gamma-retroviral vectors present challenges, such as required cell pre-activation and inefficiency in transduction, which hinder the assessment of preclinical efficacy and clinical translation. In our study, we describe a novel lentiviral pseudotype based on the Koala Retrovirus (KoRV) envelope protein, which we identified based on homology to existing pseudotypes used in cell therapy. Unlike other pseudotyped viral vectors, this KoRV-based envelope demonstrates remarkable efficiency in transducing freshly isolated primary human NK cells directly from blood, as well as freshly obtained monocytes, which were differentiated to M1 macrophages as well as B cells from multiple donors, achieving up to 80% reporter gene expression within three days post-transduction. Importantly, KoRV-based transduction does not compromise the expression of crucial immune cell receptors, nor does it impair immune cell functionality, including NK cell viability, proliferation, cytotoxicity as well as phagocytosis of differentiated macrophages. Preserving immune cell functionality is pivotal for the success of cell-based therapeutics in treating various malignancies. By achieving high transduction rates of freshly isolated immune cells before expansion, our approach enables a streamlined and cost-effective automated production of off-the-shelf cell therapeutics, requiring fewer viral particles and less manufacturing steps. This breakthrough holds the potential to significantly reduce the time and resources required for producing e.g. NK cell therapeutics, expediting their availability to patients in need.
异体细胞疗法，如涉及巨噬细胞或自然杀伤（NK）细胞的疗法，在癌症免疫疗法中越来越受到关注。然而，目前使用慢病毒或伽马逆转录病毒载体对这些细胞类型进行基因修饰的技术存在一些挑战，如需要细胞预激活和转导效率低，这阻碍了临床前疗效评估和临床转化。在我们的研究中，我们描述了一种基于考拉逆转录病毒（KoRV）包膜蛋白的新型慢病毒伪型。与其他伪型病毒载体不同的是，这种基于 KoRV 的包膜在转导直接从血液中新鲜分离的原代人类 NK 细胞、新鲜获得的单核细胞（已分化为 M1 巨噬细胞）以及来自多个供体的 B 细胞时表现出了显著的效率，在转导后三天内实现了高达 80% 的报告基因表达。重要的是，基于 KoRV 的转导不会影响关键免疫细胞受体的表达，也不会损害免疫细胞的功能，包括 NK 细胞的活力、增殖、细胞毒性以及分化巨噬细胞的吞噬功能。保持免疫细胞功能是细胞疗法成功治疗各种恶性肿瘤的关键。通过在扩增前实现新鲜分离免疫细胞的高转导率，我们的方法实现了现成细胞疗法的简化和高成本效益自动化生产，需要的病毒颗粒更少，生产步骤更少。这一突破有望大幅减少生产 NK 细胞疗法等所需的时间和资源，加快向有需要的患者提供这些疗法。
{"title":"Development of KoRV-pseudotyped lentiviral vectors for efficient gene transfer into freshly isolated immune cells","authors":"Alexander Renner, Anika Stahringer, Katharina Eva Ruppel, Stephan Fricke, Ulrike Koehl, Dominik Schmiedel","doi":"10.1038/s41434-024-00454-0","DOIUrl":"10.1038/s41434-024-00454-0","url":null,"abstract":"Allogeneic cell therapies, such as those involving macrophages or Natural Killer (NK) cells, are of increasing interest for cancer immunotherapy. However, the current techniques for genetically modifying these cell types using lenti- or gamma-retroviral vectors present challenges, such as required cell pre-activation and inefficiency in transduction, which hinder the assessment of preclinical efficacy and clinical translation. In our study, we describe a novel lentiviral pseudotype based on the Koala Retrovirus (KoRV) envelope protein, which we identified based on homology to existing pseudotypes used in cell therapy. Unlike other pseudotyped viral vectors, this KoRV-based envelope demonstrates remarkable efficiency in transducing freshly isolated primary human NK cells directly from blood, as well as freshly obtained monocytes, which were differentiated to M1 macrophages as well as B cells from multiple donors, achieving up to 80% reporter gene expression within three days post-transduction. Importantly, KoRV-based transduction does not compromise the expression of crucial immune cell receptors, nor does it impair immune cell functionality, including NK cell viability, proliferation, cytotoxicity as well as phagocytosis of differentiated macrophages. Preserving immune cell functionality is pivotal for the success of cell-based therapeutics in treating various malignancies. By achieving high transduction rates of freshly isolated immune cells before expansion, our approach enables a streamlined and cost-effective automated production of off-the-shelf cell therapeutics, requiring fewer viral particles and less manufacturing steps. This breakthrough holds the potential to significantly reduce the time and resources required for producing e.g. NK cell therapeutics, expediting their availability to patients in need.","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":"31 7-8","pages":"378-390"},"PeriodicalIF":4.6,"publicationDate":"2024-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.nature.com/articles/s41434-024-00454-0.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140832819","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 : 2024-04-27DOI: 10.1038/s41434-024-00453-1
Jordyn A. Lopes, Nicole E. Garnier, Yanlong Pei, Jacob G. E. Yates, Elena S. B. Campbell, Melanie M. Goens, Madison E. Hughes, Amira D. Rghei, Brenna A. Y. Stevens, Matthew M. Guilleman, Brad Thompson, Cezar M. Khursigara, Leonardo Susta, Sarah K. Wootton
Pseudomonas aeruginosa poses a significant threat to immunocompromised individuals and those with cystic fibrosis. Treatment relies on antibiotics, but persistent infections occur due to intrinsic and acquired resistance of P. aeruginosa towards multiple classes of antibiotics. To date, there are no licensed vaccines for this pathogen, prompting the urgent need for novel treatment approaches to combat P. aeruginosa infection and persistence. Here we validated AAV vectored immunoprophylaxis as a strategy to generate long-term plasma and mucosal expression of highly protective monoclonal antibodies (mAbs) targeting the exopolysaccharide Psl (Cam-003) and the PcrV (V2L2MD) component of the type-III secretion system injectosome either as single mAbs or together as a bispecific mAb (MEDI3902) in a mouse model. When administered intramuscularly, AAV-αPcrV, AAV-αPsl, and AAV-MEDI3902 significantly protected mice challenged intranasally with a lethal dose of P. aeruginosa strains PAO1 and PA14 and reduced bacterial burden and dissemination to other organs. While all AAV-mAbs provided protection, AAV-αPcrV and AAV-MEDI3902 provided 100% and 87.5% protection from a lethal challenge with 4.47 × 107 CFU PAO1 and 87.5% and 75% protection from a lethal challenge with 3 × 107 CFU PA14, respectively. Serum concentrations of MEDI3902 were ~10× lower than that of αPcrV, but mice treated with this vector showed a greater reduction in bacterial dissemination to the liver, lung, spleen, and blood compared to other AAV-mAbs. These results support further investigation into the use of AAV vectored immunoprophylaxis to prevent and treat P. aeruginosa infections and other bacterial pathogens of public health concern for which current treatment strategies are limited.
{"title":"AAV-vectored expression of monospecific or bispecific monoclonal antibodies protects mice from lethal Pseudomonas aeruginosa pneumonia","authors":"Jordyn A. Lopes, Nicole E. Garnier, Yanlong Pei, Jacob G. E. Yates, Elena S. B. Campbell, Melanie M. Goens, Madison E. Hughes, Amira D. Rghei, Brenna A. Y. Stevens, Matthew M. Guilleman, Brad Thompson, Cezar M. Khursigara, Leonardo Susta, Sarah K. Wootton","doi":"10.1038/s41434-024-00453-1","DOIUrl":"10.1038/s41434-024-00453-1","url":null,"abstract":"Pseudomonas aeruginosa poses a significant threat to immunocompromised individuals and those with cystic fibrosis. Treatment relies on antibiotics, but persistent infections occur due to intrinsic and acquired resistance of P. aeruginosa towards multiple classes of antibiotics. To date, there are no licensed vaccines for this pathogen, prompting the urgent need for novel treatment approaches to combat P. aeruginosa infection and persistence. Here we validated AAV vectored immunoprophylaxis as a strategy to generate long-term plasma and mucosal expression of highly protective monoclonal antibodies (mAbs) targeting the exopolysaccharide Psl (Cam-003) and the PcrV (V2L2MD) component of the type-III secretion system injectosome either as single mAbs or together as a bispecific mAb (MEDI3902) in a mouse model. When administered intramuscularly, AAV-αPcrV, AAV-αPsl, and AAV-MEDI3902 significantly protected mice challenged intranasally with a lethal dose of P. aeruginosa strains PAO1 and PA14 and reduced bacterial burden and dissemination to other organs. While all AAV-mAbs provided protection, AAV-αPcrV and AAV-MEDI3902 provided 100% and 87.5% protection from a lethal challenge with 4.47 × 107 CFU PAO1 and 87.5% and 75% protection from a lethal challenge with 3 × 107 CFU PA14, respectively. Serum concentrations of MEDI3902 were ~10× lower than that of αPcrV, but mice treated with this vector showed a greater reduction in bacterial dissemination to the liver, lung, spleen, and blood compared to other AAV-mAbs. These results support further investigation into the use of AAV vectored immunoprophylaxis to prevent and treat P. aeruginosa infections and other bacterial pathogens of public health concern for which current treatment strategies are limited.","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":"31 7-8","pages":"400-412"},"PeriodicalIF":4.6,"publicationDate":"2024-04-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.nature.com/articles/s41434-024-00453-1.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140802601","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 : 2024-04-24DOI: 10.1038/s41434-024-00452-2
Johanna Risse, Merlin Krzemien, Jan Schnalke, Thomas Heinemann
An increasing number of novel genomic therapies are expected to become available for patients with rare or ultra-rare diseases. However, the primary obstacle to equal patient access to these orphan genomic therapies are currently very high prices charged by manufacturers in the context of limited healthcare budgets. Taking into account ethical pricing theories, the paper proposes the implementation of a pricing infrastructure covering all European member states, which has the potential to promote distributive justice while maintaining the attractiveness of genomic therapy development.
{"title":"Towards ethical drug pricing: the European Orphan Genomic Therapies Fund","authors":"Johanna Risse, Merlin Krzemien, Jan Schnalke, Thomas Heinemann","doi":"10.1038/s41434-024-00452-2","DOIUrl":"10.1038/s41434-024-00452-2","url":null,"abstract":"An increasing number of novel genomic therapies are expected to become available for patients with rare or ultra-rare diseases. However, the primary obstacle to equal patient access to these orphan genomic therapies are currently very high prices charged by manufacturers in the context of limited healthcare budgets. Taking into account ethical pricing theories, the paper proposes the implementation of a pricing infrastructure covering all European member states, which has the potential to promote distributive justice while maintaining the attractiveness of genomic therapy development.","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":"31 7-8","pages":"353-357"},"PeriodicalIF":4.6,"publicationDate":"2024-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.nature.com/articles/s41434-024-00452-2.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140661841","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}
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