Natalia Boukharov, Shipeng Yuan, Wanida Ruangsirluk, Saravanan Ayyadurai, Ashiqur Rahman, Melody Rivera-Hernandez, Shashank Sunkara, Kristin Tonini, Eric Y H Park, Mugdha Deshpande, Rizwana Islam
Fabry disease (FD) is a multisystemic lysosomal storage disorder caused by the loss of α-galactosidase A (α-Gal) function. The current standard of care, enzyme replacement therapies, while effective in reducing kidney pathology when treated early, do not fully ameliorate cardiac issues, neuropathic manifestations, and risk of cerebrovascular events. Adeno-associated virus (AAV)-based gene therapies (AAV-GT) can provide superior efficacy across multiple tissues owing to continuous, endogenous production of the therapeutic enzyme and lower treatment burden. We set out to develop a robust AAV-GT to achieve optimal efficacy with the lowest feasible dose to minimize any safety risks that are associated with high-dose AAV-GTs. In this proof-of-concept study, we evaluated the effectiveness of an rAAV9 vector expressing human GLA transgene under a strong ubiquitous promoter, combined with woodchuck hepatitis virus posttranscriptional regulatory element (rAAV9-hGLA). We tested our GT at three different doses, 5e10 vg/kg, 2.5e11 vg/kg, and 6.25e12 vg/kg in the G3Stg/GLAko Fabry mouse model that has tissue Gb3 substrate levels comparable with patients with FD and develops several early FD pathologies. After intravenous injections of rAAV9-hGLA at 11 weeks of age, we observed dose-dependent increases in α-Gal activity in the key target tissues, reaching as high as 393-fold of WT in the kidneys and 6156-fold in the heart at the highest dose. Complete or near-complete substrate clearance was observed in animals treated with the two higher dose levels tested in all tissues except for the brain. We also found dose-dependent improvements in several pathological biomarkers, as well as prevention of structural and functional organ pathology. Taken together, these results indicate that an AAV-GT under a strong ubiquitous promoter has the potential to address the unmet therapeutic needs in patients with FD at relatively low doses.
{"title":"Developing Gene Therapy for Mitigating Multisystemic Pathology in Fabry Disease: Proof of Concept in an Aggravated Mouse Model.","authors":"Natalia Boukharov, Shipeng Yuan, Wanida Ruangsirluk, Saravanan Ayyadurai, Ashiqur Rahman, Melody Rivera-Hernandez, Shashank Sunkara, Kristin Tonini, Eric Y H Park, Mugdha Deshpande, Rizwana Islam","doi":"10.1089/hum.2023.222","DOIUrl":"10.1089/hum.2023.222","url":null,"abstract":"<p><p>Fabry disease (FD) is a multisystemic lysosomal storage disorder caused by the loss of α-galactosidase A (α-Gal) function. The current standard of care, enzyme replacement therapies, while effective in reducing kidney pathology when treated early, do not fully ameliorate cardiac issues, neuropathic manifestations, and risk of cerebrovascular events. Adeno-associated virus (AAV)-based gene therapies (AAV-GT) can provide superior efficacy across multiple tissues owing to continuous, endogenous production of the therapeutic enzyme and lower treatment burden. We set out to develop a robust AAV-GT to achieve optimal efficacy with the lowest feasible dose to minimize any safety risks that are associated with high-dose AAV-GTs. In this proof-of-concept study, we evaluated the effectiveness of an rAAV9 vector expressing human <i>GLA</i> transgene under a strong ubiquitous promoter, combined with woodchuck hepatitis virus posttranscriptional regulatory element (rAAV9-h<i>GLA</i>). We tested our GT at three different doses, 5e10 vg/kg, 2.5e11 vg/kg, and 6.25e12 vg/kg in the G3Stg/GLAko Fabry mouse model that has tissue Gb3 substrate levels comparable with patients with FD and develops several early FD pathologies. After intravenous injections of rAAV9-h<i>GLA</i> at 11 weeks of age, we observed dose-dependent increases in α-Gal activity in the key target tissues, reaching as high as 393-fold of WT in the kidneys and 6156-fold in the heart at the highest dose. Complete or near-complete substrate clearance was observed in animals treated with the two higher dose levels tested in all tissues except for the brain. We also found dose-dependent improvements in several pathological biomarkers, as well as prevention of structural and functional organ pathology. Taken together, these results indicate that an AAV-GT under a strong ubiquitous promoter has the potential to address the unmet therapeutic needs in patients with FD at relatively low doses.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141544775","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 : 2024-07-09DOI: 10.1089/hum.2024.53426.bfs
Alex Philippidis
{"title":"Boy Dosed with Pfizer's Duchenne Muscular Dystrophy Gene Therapy Dies a Year After Phase II Trial.","authors":"Alex Philippidis","doi":"10.1089/hum.2024.53426.bfs","DOIUrl":"https://doi.org/10.1089/hum.2024.53426.bfs","url":null,"abstract":"","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141563275","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}
Jason C Huang, Ming-Chun Li, I-Chieh Huang, Je-Ming Hu, Wei-Zhi Lin, Yu-Tien Chang
Early diagnosis and intervention are pivotal in reducing colorectal cancer (CRC) incidence and enhancing patient outcomes. In this study, we focused on three genes, AQP8, GUCA2B, and SPIB, which exhibit high coexpression and play crucial roles in suppressing early-stage CRC. Our objective was to identify key miRNAs that can mitigate CRC tumorigenesis and modulate the coexpression network involving these genes. We conducted a comprehensive analysis using large-scale tissue mRNA data from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus to validate the coexpression of AQP8, GUCA2B, and SPIB, and to assess their diagnostic and prognostic significance in CRC. The mRNA-miRNA interactions were examined using MiRNet and the Encyclopedia of RNA Interactomes. Furthermore, using various molecular techniques, we conducted miRNA inhibitor transfection experiments in HCT116 cells to evaluate their effects on cell growth, migration, and gene/protein expression. Our findings revealed that, compared with normal tissues, AQP8, GUCA2B, and SPIB exhibited high coexpression and were downregulated in CRC, particularly during tumorigenesis. OncoMirs, hsa-miR-182-5p, and hsa-miR-27a-3p, were predicted to regulate these genes. MiRNA inhibition experiments in HCT116 cells demonstrated the inhibitory effects of miR-27a-3p and miR-182-5p on GUCA2B mRNA and protein expression. These miRNAs promoted the proliferation of CRC cells, possibly through their involvement in the GUCA2B-GUCY2C axis, which is known to promote tumor growth. While the expressions of AQP8 and SPIB were barely detectable, their regulatory relationship with hsa-miR-182-5p remained inconclusive. Our study confirms that hsa-miR-27a-3p and hsa-miR-182-5p are oncomiRs in CRC. These miRNAs may contribute to GUCY2C dysregulation by downregulating GUCA2B, which encodes uroguanylin. Consequently, hsa-miR-182-5p and hsa-miR-27a-3p show promise as potential targets for early intervention and treatment in the early stages of CRC.
{"title":"Gene Coexpression and miRNA Regulation: A Path to Early Intervention in Colorectal Cancer.","authors":"Jason C Huang, Ming-Chun Li, I-Chieh Huang, Je-Ming Hu, Wei-Zhi Lin, Yu-Tien Chang","doi":"10.1089/hum.2023.207","DOIUrl":"10.1089/hum.2023.207","url":null,"abstract":"<p><p>Early diagnosis and intervention are pivotal in reducing colorectal cancer (CRC) incidence and enhancing patient outcomes. In this study, we focused on three genes, AQP8, GUCA2B, and SPIB, which exhibit high coexpression and play crucial roles in suppressing early-stage CRC. Our objective was to identify key miRNAs that can mitigate CRC tumorigenesis and modulate the coexpression network involving these genes. We conducted a comprehensive analysis using large-scale tissue mRNA data from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus to validate the coexpression of AQP8, GUCA2B, and SPIB, and to assess their diagnostic and prognostic significance in CRC. The mRNA-miRNA interactions were examined using MiRNet and the Encyclopedia of RNA Interactomes. Furthermore, using various molecular techniques, we conducted miRNA inhibitor transfection experiments in HCT116 cells to evaluate their effects on cell growth, migration, and gene/protein expression. Our findings revealed that, compared with normal tissues, AQP8, GUCA2B, and SPIB exhibited high coexpression and were downregulated in CRC, particularly during tumorigenesis. OncoMirs, hsa-miR-182-5p, and hsa-miR-27a-3p, were predicted to regulate these genes. MiRNA inhibition experiments in HCT116 cells demonstrated the inhibitory effects of miR-27a-3p and miR-182-5p on GUCA2B mRNA and protein expression. These miRNAs promoted the proliferation of CRC cells, possibly through their involvement in the GUCA2B-GUCY2C axis, which is known to promote tumor growth. While the expressions of AQP8 and SPIB were barely detectable, their regulatory relationship with hsa-miR-182-5p remained inconclusive. Our study confirms that hsa-miR-27a-3p and hsa-miR-182-5p are oncomiRs in CRC. These miRNAs may contribute to GUCY2C dysregulation by downregulating GUCA2B, which encodes uroguanylin. Consequently, hsa-miR-182-5p and hsa-miR-27a-3p show promise as potential targets for early intervention and treatment in the early stages of CRC.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-07-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141064757","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}
Shun-Qing Liang, Andrew W Navia, Michelle Ramseier, Xuntao Zhou, Michele Martinez, Charles Lee, Chen Zhou, Joae Wu, Jun Xie, Qin Su, Dan Wang, Terence R Flotte, Daniel G Anderson, Alice F Tarantal, Alex K Shalek, Guangping Gao, Wen Xue
Genome editing has the potential to treat genetic diseases in a variety of tissues, including the lung. We have previously developed and validated a dual adeno-associated virus (AAV) CRISPR platform that supports effective editing in the airways of mice. To validate this delivery vehicle in a large animal model, we have shown that intratracheal instillation of CRISPR/Cas9 in AAV5 can edit a housekeeping gene or a disease-related gene in the lungs of young rhesus monkeys. We observed up to 8% editing of angiotensin-converting enzyme 2 (ACE2) in lung lobes after single-dose administration. Single-nuclear RNA sequencing revealed that AAV5 transduces multiple cell types in the caudal lung lobes, including alveolar cells, macrophages, fibroblasts, endothelial cells, and B cells. These results demonstrate that AAV5 is efficient in the delivery of CRISPR/Cas9 in the lung lobes of young rhesus monkeys.
{"title":"AAV5 Delivery of CRISPR/Cas9 Mediates Genome Editing in the Lungs of Young Rhesus Monkeys.","authors":"Shun-Qing Liang, Andrew W Navia, Michelle Ramseier, Xuntao Zhou, Michele Martinez, Charles Lee, Chen Zhou, Joae Wu, Jun Xie, Qin Su, Dan Wang, Terence R Flotte, Daniel G Anderson, Alice F Tarantal, Alex K Shalek, Guangping Gao, Wen Xue","doi":"10.1089/hum.2024.035","DOIUrl":"10.1089/hum.2024.035","url":null,"abstract":"<p><p>Genome editing has the potential to treat genetic diseases in a variety of tissues, including the lung. We have previously developed and validated a dual adeno-associated virus (AAV) CRISPR platform that supports effective editing in the airways of mice. To validate this delivery vehicle in a large animal model, we have shown that intratracheal instillation of CRISPR/Cas9 in AAV5 can edit a housekeeping gene or a disease-related gene in the lungs of young rhesus monkeys. We observed up to 8% editing of <i>angiotensin-converting enzyme 2 (ACE2)</i> in lung lobes after single-dose administration. Single-nuclear RNA sequencing revealed that AAV5 transduces multiple cell types in the caudal lung lobes, including alveolar cells, macrophages, fibroblasts, endothelial cells, and B cells. These results demonstrate that AAV5 is efficient in the delivery of CRISPR/Cas9 in the lung lobes of young rhesus monkeys.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141064818","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}
Julie Vendomèle, Gaëlle Anne Chauveau, Deniz Dalkara, Anne Galy, Sylvain Fisson
After more than two decades of basic research and preclinical studies, adeno-associated virus (AAV)-mediated gene transfer has been tested successfully in clinical trials to treat inherited retinal diseases. Despite the eye's immune-privileged status, some patients display inflammatory events requiring the use of corticoids as an adjunct treatment which led us to question the immune consequences of a subretinal AAV administration. We first characterized anti-transgene immune responses induced in the periphery by injecting increasing doses of AAV8 encoding reporter proteins fused with the HY male antigen into the subretinal space of female C57BL/6 and rd10 mice. Transgene expression was monitored over time with bioluminescence imaging, and T cell immune responses in the spleen were analyzed by IFNγ ELISpot and cytokine multiplex assays. Our data show that AAV8 injections cause pro-inflammatory T cell immune response against the transgene product correlated with the transgene expression level at 2.109 vg and above. In addition, co-injection of immunodominant peptides from the transgene product, along with AAV8, modulates the immune response at all AAV doses tested. Taken together, our data suggest that injection of AAV8 in the subretinal space induces pro-inflammatory peripheral T cell responses to the transgene product that can be modulated by the subretinal-associated immune inhibition mechanism.
经过二十多年的基础研究和临床前研究,AAV 介导的基因转移已成功应用于治疗遗传性视网膜疾病的临床试验中。尽管眼睛具有免疫优势,但一些患者仍会出现炎症反应,需要使用皮质激素作为辅助治疗,这让我们对视网膜下 AAV 给药的免疫后果产生了疑问。我们首先通过向雌性 C57BL/6 和 rd10 小鼠视网膜下间隙注射不断增加剂量的编码与 HY 雄性抗原融合的报告蛋白的 AAV8,确定了在外周诱导的抗转基因免疫反应的特征。转基因的表达随时间变化通过生物发光成像进行监测,脾脏中的 T 细胞免疫反应则通过 IFNγ ELISpot 和细胞因子多重检测进行分析。我们的数据显示,注射 AAV8 会引起针对转基因产物的促炎性 T 细胞免疫反应,这与转基因表达水平在 2.109 vg 及以上相关。此外,在所有测试的 AAV 剂量下,同时注射来自转基因产物的免疫优势肽和 AAV8 可调节免疫反应。总之,我们的数据表明,在视网膜下腔注射 AAV8 会诱发针对转基因产物的促炎性外周 T 细胞反应,这种反应可通过视网膜下相关免疫抑制(SRAII)机制进行调节。
{"title":"Peripheral Cellular Immune Responses Induced by Subretinal Adeno-Associated Virus Gene Transfer Can Be Restrained by the Subretinal-Associated Immune Inhibition Mechanism.","authors":"Julie Vendomèle, Gaëlle Anne Chauveau, Deniz Dalkara, Anne Galy, Sylvain Fisson","doi":"10.1089/hum.2023.191","DOIUrl":"10.1089/hum.2023.191","url":null,"abstract":"<p><p>After more than two decades of basic research and preclinical studies, adeno-associated virus (AAV)-mediated gene transfer has been tested successfully in clinical trials to treat inherited retinal diseases. Despite the eye's immune-privileged status, some patients display inflammatory events requiring the use of corticoids as an adjunct treatment which led us to question the immune consequences of a subretinal AAV administration. We first characterized anti-transgene immune responses induced in the periphery by injecting increasing doses of AAV8 encoding reporter proteins fused with the HY male antigen into the subretinal space of female C57BL/6 and rd10 mice. Transgene expression was monitored over time with bioluminescence imaging, and T cell immune responses in the spleen were analyzed by IFNγ ELISpot and cytokine multiplex assays. Our data show that AAV8 injections cause pro-inflammatory T cell immune response against the transgene product correlated with the transgene expression level at 2.10<sup>9</sup> vg and above. In addition, co-injection of immunodominant peptides from the transgene product, along with AAV8, modulates the immune response at all AAV doses tested. Taken together, our data suggest that injection of AAV8 in the subretinal space induces pro-inflammatory peripheral T cell responses to the transgene product that can be modulated by the subretinal-associated immune inhibition mechanism.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141320817","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 : 2024-07-01Epub Date: 2024-03-18DOI: 10.1089/hum.2023.194
Elizabeth Kropf, David M Markusic, Anna Majowicz, Federico Mingozzi, Klaudia Kuranda
Adeno-associated virus (AAV) vectors represent a novel tool for the delivery of genetic therapeutics and enable the treatment of a wide range of diseases. Success of this new modality is challenged, however, by cases of immune-related toxicities that complicate the clinical management of patients and potentially limit the therapeutic efficacy of AAV gene therapy. While significant progress has been made to manage immune-related liver enzyme elevations following systemic AAV delivery in humans, recent clinical trials utilizing high vector doses have highlighted a new challenge to AAV gene transfer-activation of the complement system. While current in vitro models implicate AAV-specific antibodies in the initiation of the classical complement pathway, evidence from in vivo pre-clinical and clinical studies suggests that the alternative pathway also contributes to complement activation. A convergence of AAV-specific, environmental, and patient-specific factors shaping complement responses likely contributes to differential outcomes seen in clinical trials, from priming of the adaptive immune system to serious adverse events such as hepatotoxicity and thrombotic microangiopathy. Research focused on the interplay of patient-specific and AAV-related factors driving complement activation is needed to understand and identify critical components in the complement cascade to target and devise strategies to mitigate vector-related immune responses.
{"title":"Complement System Response to Adeno-Associated Virus Vector Gene Therapy.","authors":"Elizabeth Kropf, David M Markusic, Anna Majowicz, Federico Mingozzi, Klaudia Kuranda","doi":"10.1089/hum.2023.194","DOIUrl":"10.1089/hum.2023.194","url":null,"abstract":"<p><p>Adeno-associated virus (AAV) vectors represent a novel tool for the delivery of genetic therapeutics and enable the treatment of a wide range of diseases. Success of this new modality is challenged, however, by cases of immune-related toxicities that complicate the clinical management of patients and potentially limit the therapeutic efficacy of AAV gene therapy. While significant progress has been made to manage immune-related liver enzyme elevations following systemic AAV delivery in humans, recent clinical trials utilizing high vector doses have highlighted a new challenge to AAV gene transfer-activation of the complement system. While current <i>in vitro</i> models implicate AAV-specific antibodies in the initiation of the classical complement pathway, evidence from <i>in vivo</i> pre-clinical and clinical studies suggests that the alternative pathway also contributes to complement activation. A convergence of AAV-specific, environmental, and patient-specific factors shaping complement responses likely contributes to differential outcomes seen in clinical trials, from priming of the adaptive immune system to serious adverse events such as hepatotoxicity and thrombotic microangiopathy. Research focused on the interplay of patient-specific and AAV-related factors driving complement activation is needed to understand and identify critical components in the complement cascade to target and devise strategies to mitigate vector-related immune responses.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139512293","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 : 2024-07-01Epub Date: 2024-03-28DOI: 10.1089/hum.2023.188
Mark A Brimble, Christopher L Morton, Stephen M Winston, Isaiah L Reeves, Yunyu Spence, Pei-Hsin Cheng, Junfang Zhou, Amit C Nathwani, Paul G Thomas, Aisha Souquette, Andrew M Davidoff
Liver injury with concomitant loss of therapeutic transgene expression can be a clinical sequela of systemic administration of recombinant adeno-associated virus (rAAV) when used for gene therapy, and a significant barrier to treatment efficacy. Despite this, it has been difficult to replicate this phenotype in preclinical models, thereby limiting the field's ability to systematically investigate underlying biological mechanisms and develop interventions. Prior animal models have focused on capsid and transgene-related immunogenicity, but the impact of concurrently present nontransgene or vector antigens on therapeutic efficacy, such as those derived from contaminating nucleic acids within rAAV preps, has yet to be investigated. In this study, using Ad5-CMV_GFP-immunized immunocompetent BALB/cJ mice, and a coagulation factor VIII expressing rAAV preparation that contains green flourescent protein (GFP) cDNA packaged as P5-associated contaminants, we establish a model to induce transaminitis and observe concomitant therapeutic efficacy reduction after rAAV administration. We observed strong epitope-specific anti-GFP responses in splenic CD8+ T cells when GFP cDNA was delivered as a P5-associated contaminant of rAAV, which coincided and correlated with alanine and aspartate aminotransferase elevations. Furthermore, we report a significant reduction in detectable circulating FVIII protein, as compared with control mice. Lastly, we observed an elevation in the detection of AAV8 capsid-specific T cells when GFP was delivered either as a contaminant or transgene to Ad5-CMV_GFP-immunized mice. We present this model as a potential tool to study the underlying biology of post-AAV hepatotoxicity and demonstrate the potential for T cell responses against proteins produced from AAV encapsidated nontherapeutic nucleic acids, to interfere with efficacious gene transfer.
{"title":"Pre-Existing Immunity to a Nucleic Acid Contaminant-Derived Antigen Mediates Transaminitis and Resultant Diminished Transgene Expression in a Mouse Model of Hepatic Recombinant Adeno-Associated Virus-Mediated Gene Transfer.","authors":"Mark A Brimble, Christopher L Morton, Stephen M Winston, Isaiah L Reeves, Yunyu Spence, Pei-Hsin Cheng, Junfang Zhou, Amit C Nathwani, Paul G Thomas, Aisha Souquette, Andrew M Davidoff","doi":"10.1089/hum.2023.188","DOIUrl":"10.1089/hum.2023.188","url":null,"abstract":"<p><p>Liver injury with concomitant loss of therapeutic transgene expression can be a clinical sequela of systemic administration of recombinant adeno-associated virus (rAAV) when used for gene therapy, and a significant barrier to treatment efficacy. Despite this, it has been difficult to replicate this phenotype in preclinical models, thereby limiting the field's ability to systematically investigate underlying biological mechanisms and develop interventions. Prior animal models have focused on capsid and transgene-related immunogenicity, but the impact of concurrently present nontransgene or vector antigens on therapeutic efficacy, such as those derived from contaminating nucleic acids within rAAV preps, has yet to be investigated. In this study, using Ad5-CMV_GFP-immunized immunocompetent BALB/cJ mice, and a coagulation factor VIII expressing rAAV preparation that contains green flourescent protein (GFP) cDNA packaged as P5-associated contaminants, we establish a model to induce transaminitis and observe concomitant therapeutic efficacy reduction after rAAV administration. We observed strong epitope-specific anti-GFP responses in splenic CD8+ T cells when GFP cDNA was delivered as a P5-associated contaminant of rAAV, which coincided and correlated with alanine and aspartate aminotransferase elevations. Furthermore, we report a significant reduction in detectable circulating FVIII protein, as compared with control mice. Lastly, we observed an elevation in the detection of AAV8 capsid-specific T cells when GFP was delivered either as a contaminant or transgene to Ad5-CMV_GFP-immunized mice. We present this model as a potential tool to study the underlying biology of post-AAV hepatotoxicity and demonstrate the potential for T cell responses against proteins produced from AAV encapsidated nontherapeutic nucleic acids, to interfere with efficacious gene transfer.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139989887","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 : 2024-07-01Epub Date: 2024-04-11DOI: 10.1089/hum.2023.227
Maite Muñoz-Melero, Moanaro Biswas
Adeno-associated virus (AAV) gene therapy is making rapid strides owing to its wide range of therapeutic applications. However, development of serious immune responses to the capsid antigen or the therapeutic transgene product hinders its full clinical impact. Immune suppressive (IS) drug treatments have been used in various clinical trials to prevent the deleterious effects of cytotoxic T cells to the viral vector or transgene, although there is no consensus on the best treatment regimen, dosage, or schedule. Regulatory T cells (Tregs) are crucial for maintaining tolerance against self or nonself antigens. Of importance, Tregs also play an important role in dampening immune responses to AAV gene therapy, including tolerance induction to the transgene product. Approaches to harness the tolerogenic effect of Tregs include the use of selective IS drugs that expand existing Tregs, and skew activated conventional T cells into antigen-specific peripherally induced Tregs. In addition, Tregs can be expanded ex vivo and delivered as cellular therapy. Furthermore, receptor engineering can be used to increase the potency and specificity of Tregs allowing for suppression at lower doses and reducing the risk of disrupting protective immunity. Because immune-mediated toxicities to AAV vectors are a concern in the clinic, strategies that can enhance or preserve Treg function should be considered to improve both the safety and efficacy of AAV gene therapy.
腺相关病毒(AAV)基因疗法因其广泛的治疗应用而突飞猛进。然而,对囊壳抗原或治疗性转基因产品产生严重的免疫反应阻碍了其在临床上的全面应用。免疫抑制(IS)药物治疗已被用于各种临床试验,以防止细胞毒性 T 细胞对病毒载体或转基因产生有害影响,但对于最佳治疗方案、剂量或时间安排还没有达成共识。调节性 T 细胞(Treg)对于维持对自身或非自身抗原的耐受性至关重要。重要的是,调节性 T 细胞在抑制 AAV 基因疗法的免疫反应(包括诱导对转基因产物的耐受)方面也发挥着重要作用。利用Tregs的耐受诱导作用的方法包括使用选择性IS药物来扩增现有的Tregs,并将活化的常规T细胞倾斜为抗原特异性外周诱导Tregs。此外,还可以在体外扩增 Tregs,并将其作为细胞疗法投放。此外,受体工程可用于提高 Tregs 的效力和特异性,从而以较低的剂量进行抑制,并降低破坏保护性免疫的风险。由于 AAV 向量的免疫介导毒性是临床上的一个问题,因此应考虑采用能增强或保护 Treg 功能的策略,以提高 AAV 基因疗法的安全性和有效性。.
{"title":"Role of FoxP3<sup>+</sup> Regulatory T Cells in Modulating Immune Responses to Adeno-Associated Virus Gene Therapy.","authors":"Maite Muñoz-Melero, Moanaro Biswas","doi":"10.1089/hum.2023.227","DOIUrl":"10.1089/hum.2023.227","url":null,"abstract":"<p><p>Adeno-associated virus (AAV) gene therapy is making rapid strides owing to its wide range of therapeutic applications. However, development of serious immune responses to the capsid antigen or the therapeutic transgene product hinders its full clinical impact. Immune suppressive (IS) drug treatments have been used in various clinical trials to prevent the deleterious effects of cytotoxic T cells to the viral vector or transgene, although there is no consensus on the best treatment regimen, dosage, or schedule. Regulatory T cells (Tregs) are crucial for maintaining tolerance against self or nonself antigens. Of importance, Tregs also play an important role in dampening immune responses to AAV gene therapy, including tolerance induction to the transgene product. Approaches to harness the tolerogenic effect of Tregs include the use of selective IS drugs that expand existing Tregs, and skew activated conventional T cells into antigen-specific peripherally induced Tregs. In addition, Tregs can be expanded <i>ex vivo</i> and delivered as cellular therapy. Furthermore, receptor engineering can be used to increase the potency and specificity of Tregs allowing for suppression at lower doses and reducing the risk of disrupting protective immunity. Because immune-mediated toxicities to AAV vectors are a concern in the clinic, strategies that can enhance or preserve Treg function should be considered to improve both the safety and efficacy of AAV gene therapy.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11302314/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140049344","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 : 2024-07-01DOI: 10.1089/hum.2024.38567.bfs
Alex Philippidis
{"title":"Pfizer Weighs Next Steps after DMD Therapy Linked to Boy's Death Fails Phase III Trial.","authors":"Alex Philippidis","doi":"10.1089/hum.2024.38567.bfs","DOIUrl":"10.1089/hum.2024.38567.bfs","url":null,"abstract":"","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141626555","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 : 2024-07-01Epub Date: 2024-02-22DOI: 10.1089/hum.2023.180
Sara Rose Krivoshik, Lindsey Dzielak, April R Masters, Jennifer Hall, Alison J Johnson
Adeno-associated virus (AAV)-based gene therapies have shown promise as novel treatments for rare genetic disorders such as hemophilia A and spinal muscular atrophy. However, cellular immune responses mediated by cytotoxic (CD8+) and helper (CD4+) T cells may target vector-transduced cells as well as healthy immune cells, impacting safety and efficacy. In this study, we describe the optimization and reproducibility of interferon-γ (IFNγ)-based and interleukin-2 (IL-2)-based enzyme-linked immunosorbent spot (ELISpot) assays for measuring T cell responses against AAV peptide antigens. For method optimization, peripheral blood mononuclear cells (PBMCs) were isolated from healthy human donors and stimulated with commercially available major histocompatibility complex (MHC) class I or II-specific peptides as positive controls. Peptide pools were designed from published AAV8 and AAV9 capsid protein sequences and then used to assess the presence of AAV-specific T cell responses. Our results demonstrate a measurable increase in IFNγ and IL-2-producing cells after AAV peptide presentation. Furthermore, there was an observed difference in the magnitude and specificity of response to peptide pools based on AAV serotype and donor. Finally, using individual peptides, we identified a region of the AAV9 capsid protein that can elicit an immunogenic response. This work shows the applicability of ELISpot in assessing anti-AAV immune responses and provides insight into how novel recombinant AAV vectors could be designed to reduce immunogenic potential.
基于腺相关病毒(AAV)的基因疗法有望成为治疗血友病 A 和脊髓性肌肉萎缩症等罕见遗传疾病的新型疗法。然而,由细胞毒性(CD8+)和辅助性(CD4+)T 细胞介导的细胞免疫反应可能会针对载体转导的细胞以及健康的免疫细胞,从而影响安全性和疗效。在这里,我们描述了基于干扰素-γ(IFNγ)和白细胞介素-2(IL-2)的酶联免疫吸附点(ELISpot)测定法的优化和可重复性,该测定法用于测量针对 AAV 多肽抗原的 T 细胞反应。为优化方法,从健康人供体中分离出外周血单核细胞(PBMC),并用市售的主要组织相容性复合体(MHC)I 类或 II 类特异性肽作为阳性对照进行刺激。根据已发表的 AAV8 和 AAV9 包膜蛋白序列设计肽池,然后用于评估 AAV 特异性 T 细胞反应的存在。我们的研究结果表明,AAV 多肽呈现后,IFNγ 和 IL-2 生成细胞明显增加。此外,根据 AAV 血清型和供体的不同,肽池反应的程度和特异性也存在差异。最后,通过使用单个肽,我们确定了 AAV9 帽状蛋白中能引起免疫原性反应的区域。这项工作显示了 ELISpot 在评估抗 AAV 免疫反应方面的适用性,并为如何设计新型重组 AAV 载体以降低免疫原性提供了启示。
{"title":"Development of an Enzyme-Linked Immunosorbent Spot Assay for the Assessment of Adeno-Associated Virus Peptides to Examine Immune Safety.","authors":"Sara Rose Krivoshik, Lindsey Dzielak, April R Masters, Jennifer Hall, Alison J Johnson","doi":"10.1089/hum.2023.180","DOIUrl":"10.1089/hum.2023.180","url":null,"abstract":"<p><p>Adeno-associated virus (AAV)-based gene therapies have shown promise as novel treatments for rare genetic disorders such as hemophilia A and spinal muscular atrophy. However, cellular immune responses mediated by cytotoxic (CD8<sup>+</sup>) and helper (CD4<sup>+</sup>) T cells may target vector-transduced cells as well as healthy immune cells, impacting safety and efficacy. In this study, we describe the optimization and reproducibility of interferon-γ (IFNγ)-based and interleukin-2 (IL-2)-based enzyme-linked immunosorbent spot (ELISpot) assays for measuring T cell responses against AAV peptide antigens. For method optimization, peripheral blood mononuclear cells (PBMCs) were isolated from healthy human donors and stimulated with commercially available major histocompatibility complex (MHC) class I or II-specific peptides as positive controls. Peptide pools were designed from published AAV8 and AAV9 capsid protein sequences and then used to assess the presence of AAV-specific T cell responses. Our results demonstrate a measurable increase in IFNγ and IL-2-producing cells after AAV peptide presentation. Furthermore, there was an observed difference in the magnitude and specificity of response to peptide pools based on AAV serotype and donor. Finally, using individual peptides, we identified a region of the AAV9 capsid protein that can elicit an immunogenic response. This work shows the applicability of ELISpot in assessing anti-AAV immune responses and provides insight into how novel recombinant AAV vectors could be designed to reduce immunogenic potential.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139542280","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}