Pub Date : 2024-07-01Epub Date: 2024-07-05DOI: 10.1089/hum.2024.040
Di Cao, Barry J Byrne, Ype P de Jong, Cox Terhorst, Dongsheng Duan, Roland W Herzog, Sandeep R P Kumar
Adeno-associated virus (AAV) based viral vectors are widely used in human gene therapy and form the basis of approved treatments for several genetic diseases. Immune responses to vector and transgene products, however, substantially complicate these applications in clinical practice. The role of innate immune recognition of AAV vectors was initially unclear, given that inflammatory responses early after vector administration were typically mild in animal models. However, more recent research continues to identify innate immune pathways that are triggered by AAV vectors and that serve to provide activation signals for antigen-presenting cells and initiation of adaptive immune responses. Sensing of the AAV genome by the endosomal DNA receptor toll-like receptor 9 (TLR9) promotes early inflammatory response and interferon expression. Thus, activation of the TLR9>MyD88 pathway in plasmacytoid dendritic cells (pDCs) leads to the conditioning of antigen cross-presenting DCs through type I interferon (IFN-I) and ultimately CD8+ T cell activation. Alternatively, pDCs may also promote CD8+ T cell responses in a TLR9-independent manner by the production of IL-1 cytokines, thereby activating the IL-1R1>MyD88 signaling pathway. AAV can induce cytokine expression in monocyte-derived DCs, which in turn increases antibody formation. Binding of AAV capsid to complement components likely further elevates B cell activation. At high systemic vector doses in humans and in non-human primates, AAV vectors can trigger complement activation, with contributions by classical and alternative pathways, leading to severe toxicities. Finally, evidence for activation of TLR2 by the capsid and of additional innate receptors for nucleic acids has been presented. These observations show that AAV vectors can initiate several and likely redundant innate immune pathways resulting in an exaggerated adaptive immune response.
基于腺相关病毒(AAV)的病毒载体被广泛应用于人类基因疗法,并成为多种遗传疾病获批治疗方法的基础。然而,对载体和转基因产品的免疫反应使这些应用在临床实践中变得非常复杂。AAV 载体的先天性免疫识别作用最初并不明确,因为在动物模型(包括 NHP 研究)中,载体给药后早期的炎症反应通常很轻微。然而,最近的研究不断发现先天性免疫通路由 AAV 载体触发,为抗原递呈细胞提供激活信号,并启动适应性免疫反应。内体 DNA 受体收费样受体 9(TLR9)对 AAV 基因组的感应促进了早期炎症反应和干扰素的表达。因此,浆细胞树突状细胞(pDCs)中 TLR9>MyD88 通路的激活会通过 I 型干扰素(IFN I)调节抗原交叉呈递 DCs,并最终激活 CD8+ T 细胞。另外,pDC 也可能通过产生 IL-1 细胞因子,从而激活 IL-1R1>MyD88 信号通路,以不依赖 TLR9 的方式促进 CD8+ T 细胞应答。AAV 可诱导单核细胞衍生的 DC 中细胞因子的表达,进而增加抗体的形成。AAV 包囊与补体成分结合可能会进一步提高 B 细胞的活化。在人体全身使用高剂量载体的情况下,AAV 载体可引发补体激活,其中包括传统途径和替代途径,从而导致严重中毒。最后,有证据表明,AAV 载体的囊壳会激活 TLR2,并激活核酸的其他先天受体。这些观察结果表明,AAV 载体可以启动多种先天性免疫途径,而且很可能是冗余的,从而导致夸张的适应性免疫反应。
{"title":"Innate Immune Sensing of Adeno-Associated Virus Vectors.","authors":"Di Cao, Barry J Byrne, Ype P de Jong, Cox Terhorst, Dongsheng Duan, Roland W Herzog, Sandeep R P Kumar","doi":"10.1089/hum.2024.040","DOIUrl":"10.1089/hum.2024.040","url":null,"abstract":"<p><p>Adeno-associated virus (AAV) based viral vectors are widely used in human gene therapy and form the basis of approved treatments for several genetic diseases. Immune responses to vector and transgene products, however, substantially complicate these applications in clinical practice. The role of innate immune recognition of AAV vectors was initially unclear, given that inflammatory responses early after vector administration were typically mild in animal models. However, more recent research continues to identify innate immune pathways that are triggered by AAV vectors and that serve to provide activation signals for antigen-presenting cells and initiation of adaptive immune responses. Sensing of the AAV genome by the endosomal DNA receptor toll-like receptor 9 (TLR9) promotes early inflammatory response and interferon expression. Thus, activation of the TLR9>MyD88 pathway in plasmacytoid dendritic cells (pDCs) leads to the conditioning of antigen cross-presenting DCs through type I interferon (IFN-I) and ultimately CD8<sup>+</sup> T cell activation. Alternatively, pDCs may also promote CD8<sup>+</sup> T cell responses in a TLR9-independent manner by the production of IL-1 cytokines, thereby activating the IL-1R1>MyD88 signaling pathway. AAV can induce cytokine expression in monocyte-derived DCs, which in turn increases antibody formation. Binding of AAV capsid to complement components likely further elevates B cell activation. At high systemic vector doses in humans and in non-human primates, AAV vectors can trigger complement activation, with contributions by classical and alternative pathways, leading to severe toxicities. Finally, evidence for activation of TLR2 by the capsid and of additional innate receptors for nucleic acids has been presented. These observations show that AAV vectors can initiate several and likely redundant innate immune pathways resulting in an exaggerated adaptive immune response.</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/PMC11310564/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141418624","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}
Seyed Younes Hosseini, Rahul Mallick, Petri Mäkinen, Seppo Ylä-Herttuala
As the most versatile and precise gene editing technology, prime editing (PE) can establish a durable cure for most human genetic disorders. Several generations of PE have been developed based on an editor machine or prime editing guide RNA (pegRNA) to achieve any kind of genetic correction. However, due to the early stage of development, PE complex elements need to be optimized for more efficient editing. Smart optimization of editor proteins as well as pegRNA has been contemplated by many researchers, but the universal PE machine's current shortcomings remain to be solved. The modification of PE elements, fine-tuning of the host genes, manipulation of epigenetics, and blockage of immune responses could be used to reach more efficient PE. Moreover, the host factors involved in the PE process, such as repair and innate immune system genes, have not been determined, and PE cell context dependency is still poorly understood. Regarding the large size of the PE elements, delivery is a significant challenge and the development of a universal viral or nonviral platform is still far from complete. PE versions with shortened variants of reverse transcriptase are still too large to fit in common viral vectors. Overall, PE faces challenges in optimization for efficiency, high context dependency during the cell cycling, and delivery due to the large size of elements. In addition, immune responses, unpredictability of outcomes, and off-target effects further limit its application, making it essential to address these issues for broader use in nonpersonalized gene editing. Besides, due to the limited number of suitable animal models and computational modeling, the prediction of the PE process remains challenging. In this review, the fundamentals of PE, including generations, potential, optimization, delivery, in vivo barriers, and the future landscape of the technology are discussed.
作为最通用、最精确的基因编辑技术,基因编辑(prime editing,PE)可以持久治愈大多数人类遗传疾病。目前已开发出几代基于编辑机或 pegRNA 的基因编辑技术,可实现各种基因校正。然而,由于处于早期开发阶段,PE 的复杂元件需要优化,以提高编辑效率。许多研究人员都在考虑对编辑蛋白和 pegRNA 进行智能优化,但通用 PE 机器目前的缺陷仍有待解决。可以通过修改 PE 元素、微调宿主基因、操纵表观遗传学和阻断免疫反应来实现更高效的素编辑。此外,参与 PE 过程的宿主因子(如修复和先天免疫系统基因)尚未确定,PE 细胞的环境依赖性仍不甚明了。由于 PE 元件体积庞大,传递是一项重大挑战,通用病毒或非病毒平台的开发工作仍远未完成。带有 RT 缩短变体的 PE 版本仍然太大,无法装入普通病毒载体。新版本应考虑到针对 PE 元件和输送载体的免疫反应。为筛选和验证目的,对主要编辑过程的预测仍有待改进。本综述将讨论 PE 的基本原理,包括世代、潜力、优化、传递、体内障碍以及该技术的未来前景。
{"title":"Insights into Prime Editing Technology: A Deep Dive into Fundamentals, Potentials, and Challenges.","authors":"Seyed Younes Hosseini, Rahul Mallick, Petri Mäkinen, Seppo Ylä-Herttuala","doi":"10.1089/hum.2024.043","DOIUrl":"10.1089/hum.2024.043","url":null,"abstract":"<p><p>As the most versatile and precise gene editing technology, prime editing (PE) can establish a durable cure for most human genetic disorders. Several generations of PE have been developed based on an editor machine or prime editing guide RNA (pegRNA) to achieve any kind of genetic correction. However, due to the early stage of development, PE complex elements need to be optimized for more efficient editing. Smart optimization of editor proteins as well as pegRNA has been contemplated by many researchers, but the universal PE machine's current shortcomings remain to be solved. The modification of PE elements, fine-tuning of the host genes, manipulation of epigenetics, and blockage of immune responses could be used to reach more efficient PE. Moreover, the host factors involved in the PE process, such as repair and innate immune system genes, have not been determined, and PE cell context dependency is still poorly understood. Regarding the large size of the PE elements, delivery is a significant challenge and the development of a universal viral or nonviral platform is still far from complete. PE versions with shortened variants of reverse transcriptase are still too large to fit in common viral vectors. Overall, PE faces challenges in optimization for efficiency, high context dependency during the cell cycling, and delivery due to the large size of elements. In addition, immune responses, unpredictability of outcomes, and off-target effects further limit its application, making it essential to address these issues for broader use in nonpersonalized gene editing. Besides, due to the limited number of suitable animal models and computational modeling, the prediction of the PE process remains challenging. In this review, the fundamentals of PE, including generations, potential, optimization, delivery, <i>in vivo</i> barriers, and the future landscape of the technology are discussed.</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":"141237794","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: 2023-12-06DOI: 10.1089/hum.2023.162
Hildegund C J Ertl
Adeno-associated virus (AAV)-mediated gene therapy has made significant progress in the last few decades. Nevertheless, challenges imposed by the immune system remain. The very high doses of AAV vectors used for some disorders have resulted in serious adverse events (SAEs) or even deaths, demonstrating that AAV vector doses that can safely be injected into patients are limited and for some indications below the therapeutic dose. Currently used immunosuppressive drugs have not prevented the SAEs, indicating that it may be prudent to treat patients with repeated transfer of moderate doses rather than a single injection of high doses of AAV vectors. The former approach has been avoided as AAV vectors elicit neutralizing antibodies that prevent successful reapplication of serologically crossreactive vectors. Immunosuppressive regimens that block B cell responses to AAV vectors or treatments that remove AAV neutralizing antibodies thus need to be developed to allow for a shift from toxic single-dose injections of AAV vectors to repeated treatments with more moderate and safe doses. Preventing or blocking antibody responses would also allow for redosing of patients with declining transgene product expression, or for effective AAV-mediated gene transfer into patients with the pre-existing neutralizing antibodies.
{"title":"Circumventing B Cell Responses to Allow for Redosing of Adeno-Associated Virus Vectors.","authors":"Hildegund C J Ertl","doi":"10.1089/hum.2023.162","DOIUrl":"10.1089/hum.2023.162","url":null,"abstract":"<p><p>Adeno-associated virus (AAV)-mediated gene therapy has made significant progress in the last few decades. Nevertheless, challenges imposed by the immune system remain. The very high doses of AAV vectors used for some disorders have resulted in serious adverse events (SAEs) or even deaths, demonstrating that AAV vector doses that can safely be injected into patients are limited and for some indications below the therapeutic dose. Currently used immunosuppressive drugs have not prevented the SAEs, indicating that it may be prudent to treat patients with repeated transfer of moderate doses rather than a single injection of high doses of AAV vectors. The former approach has been avoided as AAV vectors elicit neutralizing antibodies that prevent successful reapplication of serologically crossreactive vectors. Immunosuppressive regimens that block B cell responses to AAV vectors or treatments that remove AAV neutralizing antibodies thus need to be developed to allow for a shift from toxic single-dose injections of AAV vectors to repeated treatments with more moderate and safe doses. Preventing or blocking antibody responses would also allow for redosing of patients with declining transgene product expression, or for effective AAV-mediated gene transfer into patients with the pre-existing neutralizing antibodies.</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":"49676979","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 : 2024-06-01Epub Date: 2024-04-09DOI: 10.1089/hum.2023.186
Sumaya Abuloha, Shu Niu, Darlene Adirika, Benjamin P Harvey, Mikael Svensson
Cell and gene therapy (CGT) innovations have provided several significant breakthroughs in recent years. However, CGTs often come with a high upfront cost, raising questions about patient access, affordability, and long-term value. This study reviewed cost-effectiveness analysis (CEA) studies that have attempted to assess the long-term value of Food and Drug Administration (FDA)-approved CGTs. Two reviewers independently searched the Tufts Medical Center CEA Registry to identify all studies for FDA-approved CGTs, per January 2023. A data extraction template was used to summarize the evidence in terms of the incremental cost-effectiveness ratio expressed as the cost per quality-adjusted life year (QALY) and essential modeling assumptions, combined with a template to extract the adherence to the Consolidated Health Economic Evaluation Reporting Standards (CHEERS) checklist. The review identified 26 CEA studies for seven CGTs. Around half of the base-case cost-effectiveness results indicated that the cost per QALY was below $100,000-$150,000, often used as a threshold for reasonable cost-effectiveness in the United States. However, the results varied substantially across studies for the same treatment, ranging from being considered very cost-effective to far from cost-effective. Most models were based on data from single-arm trials with relatively short follow-ups, and different long-term extrapolations between studies caused large differences in the modeled cost-effectiveness results. In sum, this review showed that, despite the high upfront costs, many CGTs have cost-effectiveness evidence that can support long-term value. Nonetheless, substantial uncertainty regarding long-term value exists because so much of the modeling results are driven by uncertain extrapolations beyond the clinical trial data.
近年来,细胞和基因疗法创新取得了多项重大突破。然而,细胞和基因疗法往往需要高昂的前期费用,从而引发了有关患者使用、经济承受能力和长期价值的问题。本研究回顾了试图评估经 FDA 批准的细胞和基因疗法长期价值的成本效益分析研究。两名审查员独立检索了塔夫茨医学中心成本效益分析注册表,以确定 2023 年 1 月之前所有关于 FDA 批准的细胞和基因疗法的研究。采用数据提取模板,根据以每质量调整生命年(QALY)成本表示的增量成本效益比和基本建模假设总结证据,并结合模板提取是否符合《卫生经济评估综合报告标准》(CHEERS)检查表。审查确定了七种细胞和基因疗法的 26 项 CEA 研究。大约一半的基础案例成本效益结果表明,每QALY成本低于100,000-150,000美元,这在美国通常被用作合理成本效益的阈值。然而,对于同一治疗方法,不同研究的结果差异很大,有的认为非常具有成本效益,有的则认为远不具有成本效益。大多数模型都是基于随访时间相对较短的单臂试验数据,不同研究之间的长期推断不同,导致模型的成本效益结果差异很大。总之,本综述表明,尽管前期成本较高,但许多细胞和基因疗法的成本效益证据可以支持其长期价值。尽管如此,长期价值仍存在很大的不确定性,因为许多建模结果都是由临床试验数据之外的不确定推断所驱动的。
{"title":"A Review of the Cost-Effectiveness Evidence for FDA-Approved Cell and Gene Therapies.","authors":"Sumaya Abuloha, Shu Niu, Darlene Adirika, Benjamin P Harvey, Mikael Svensson","doi":"10.1089/hum.2023.186","DOIUrl":"10.1089/hum.2023.186","url":null,"abstract":"<p><p>Cell and gene therapy (CGT) innovations have provided several significant breakthroughs in recent years. However, CGTs often come with a high upfront cost, raising questions about patient access, affordability, and long-term value. This study reviewed cost-effectiveness analysis (CEA) studies that have attempted to assess the long-term value of Food and Drug Administration (FDA)-approved CGTs. Two reviewers independently searched the Tufts Medical Center CEA Registry to identify all studies for FDA-approved CGTs, per January 2023. A data extraction template was used to summarize the evidence in terms of the incremental cost-effectiveness ratio expressed as the cost per quality-adjusted life year (QALY) and essential modeling assumptions, combined with a template to extract the adherence to the Consolidated Health Economic Evaluation Reporting Standards (CHEERS) checklist. The review identified 26 CEA studies for seven CGTs. Around half of the base-case cost-effectiveness results indicated that the cost per QALY was below $100,000-$150,000, often used as a threshold for reasonable cost-effectiveness in the United States. However, the results varied substantially across studies for the same treatment, ranging from being considered very cost-effective to far from cost-effective. Most models were based on data from single-arm trials with relatively short follow-ups, and different long-term extrapolations between studies caused large differences in the modeled cost-effectiveness results. In sum, this review showed that, despite the high upfront costs, many CGTs have cost-effectiveness evidence that can support long-term value. Nonetheless, substantial uncertainty regarding long-term value exists because so much of the modeling results are driven by uncertain extrapolations beyond the clinical trial data.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140206781","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 : 2024-06-01Epub Date: 2024-05-23DOI: 10.1089/hum.2023.223
Kumi Mesaki, Haruchika Yamamoto, Stephen Juvet, Jonathan Yeung, Zehong Guan, Akhi Akhter, Yan Yao, Cameron Dickie, Henna Mangat, Aizhou Wang, Gavin W Wilson, Andrea Mariscal, Jim Hu, Alan R Davidson, Benjamin P Kleinstiver, Marcelo Cypel, Mingyao Liu, Shaf Keshavjee
The ongoing advancements in CRISPR-Cas technologies can significantly accelerate the preclinical development of both in vivo and ex vivo organ genome-editing therapeutics. One of the promising applications is to genetically modify donor organs prior to implantation. The implantation of optimized donor organs with long-lasting immunomodulatory capacity holds promise for reducing the need for lifelong potent whole-body immunosuppression in recipients. However, assessing genome-targeting interventions in a clinically relevant manner prior to clinical trials remains a major challenge owing to the limited modalities available. This study introduces a novel platform for testing genome editing in human lungs ex vivo, effectively simulating preimplantation genetic engineering of donor organs. We identified gene regulatory elements whose disruption via Cas nucleases led to the upregulation of the immunomodulatory gene interleukin 10 (IL-10). We combined this approach with adenoviral vector-mediated IL-10 delivery to create favorable kinetics for early (immediate postimplantation) graft immunomodulation. Using ex vivo organ machine perfusion and precision-cut tissue slice technology, we demonstrated the feasibility of evaluating CRISPR genome editing in human lungs. To overcome the assessment limitations in ex vivo perfused human organs, we conducted an in vivo rodent study and demonstrated both early gene induction and sustained editing of the lung. Collectively, our findings lay the groundwork for a first-in-human-organ study to overcome the current translational barriers of genome-targeting therapeutics.
CRISPR-Cas 技术的不断进步可以大大加快体内和体外器官基因组编辑疗法的临床前开发。其中一项前景广阔的应用是在植入前对供体器官进行基因修饰。植入具有长效免疫调节能力的优化供体器官有望减少受体对终生强效全身免疫抑制的需求。然而,由于可用方法有限,在临床试验前以临床相关的方式评估基因组靶向干预仍是一大挑战。本研究引入了一种新型平台,用于在人体肺部进行体外基因组编辑测试,有效模拟了供体器官移植前的基因工程。我们确定了通过 Cas 核酸酶破坏可导致免疫调节基因 IL-10 上调的基因调控元件。我们将这种方法与腺病毒载体(AdV)介导的IL-10递送相结合,为早期(移植后即刻)移植物免疫调节创造了有利的动力学条件。利用体外器官机灌注和精确切割组织切片技术,我们证明了在人类肺部评估 CRISPR 基因组编辑的可行性。为了克服体外灌注人体器官的评估局限性,我们进行了一项体内啮齿动物研究,并证明了肺部的早期基因诱导和持续编辑。总之,我们的研究结果为首次在人体器官中进行研究奠定了基础,从而克服了基因组靶向疗法目前存在的转化障碍。
{"title":"CRISPR-Cas Genome Editing in <i>Ex Vivo</i> Human Lungs to Rewire the Translational Path of Genome-Targeting Therapeutics.","authors":"Kumi Mesaki, Haruchika Yamamoto, Stephen Juvet, Jonathan Yeung, Zehong Guan, Akhi Akhter, Yan Yao, Cameron Dickie, Henna Mangat, Aizhou Wang, Gavin W Wilson, Andrea Mariscal, Jim Hu, Alan R Davidson, Benjamin P Kleinstiver, Marcelo Cypel, Mingyao Liu, Shaf Keshavjee","doi":"10.1089/hum.2023.223","DOIUrl":"10.1089/hum.2023.223","url":null,"abstract":"<p><p>The ongoing advancements in CRISPR-Cas technologies can significantly accelerate the preclinical development of both <i>in vivo</i> and <i>ex vivo</i> organ genome-editing therapeutics. One of the promising applications is to genetically modify donor organs prior to implantation. The implantation of optimized donor organs with long-lasting immunomodulatory capacity holds promise for reducing the need for lifelong potent whole-body immunosuppression in recipients. However, assessing genome-targeting interventions in a clinically relevant manner prior to clinical trials remains a major challenge owing to the limited modalities available. This study introduces a novel platform for testing genome editing in human lungs <i>ex vivo</i>, effectively simulating preimplantation genetic engineering of donor organs. We identified gene regulatory elements whose disruption via Cas nucleases led to the upregulation of the immunomodulatory gene interleukin 10 (<i>IL-10)</i>. We combined this approach with adenoviral vector-mediated <i>IL-10</i> delivery to create favorable kinetics for early (immediate postimplantation) graft immunomodulation. Using <i>ex vivo</i> organ machine perfusion and precision-cut tissue slice technology, we demonstrated the feasibility of evaluating CRISPR genome editing in human lungs. To overcome the assessment limitations in <i>ex vivo</i> perfused human organs, we conducted an <i>in vivo</i> rodent study and demonstrated both early gene induction and sustained editing of the lung. Collectively, our findings lay the groundwork for a first-in-human-organ study to overcome the current translational barriers of genome-targeting therapeutics.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140890555","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 : 2024-06-01Epub Date: 2023-10-04DOI: 10.1089/hum.2023.079
Zi-Wei Yang, Jing-Jing Ji, Yu Jiang, Ya Wu, Jia-Qi Guo, Gen-Shan Ma, Yu-Yu Yao
Objective: Studies have found that high expression of human Kallistatin (HKS) in adipose tissue can improve obesity and its associated comorbidities, but the underlying mechanism of specific regulation is unclear. Methods: An obesity model was built by injecting 8-week-old C57BL/6 mice (n = 6 mice per group) with (Ad.Null and (Ad.HKS adenovirus into epididymal adipose tissue and fed with a high-fat diet (HFD). Insulin resistance-related proteins, AKT and IRS1, were detected in the liver, subcutaneous fat, and skeletal muscle by western blotting after one month of HFD. Epididymal adipose tissue was isolated after 24 h for culture, and exosomes were extracted by differential centrifugation. Enzyme-linked immunosorbent assay detected the expression of HKS protein in serum and exosomes. To examine the role of exosomes in AML12 insulin resistance, we used epididymal adipose tissue-derived exosomes or transfected (Ad.HKS into mature 3T3L1-derived exosomes to interfere with palmitic acid (PA)-induced mouse AML12 insulin resistance model. GW4869 was used to inhibit exosome biogenesis and release. Results: Our results showed that HFD-induced mice with high expression of HKS in epididymal adipose tissue had slower weight gain, lower serum triglycerides, reduced free fatty acids, and improved liver insulin resistance compared with the (Ad.Null group. We also demonstrated that HKS was enriched in epididymal adipose tissue-derived exosomes and released through the exosome pathway. In PA-induced AML12 cells, insulin resistance was alleviated after incubation of the HKS-related exosome; this effect was reversed with GW4869. Conclusion: High expression of HKS in epididymal adipose tissue could lead to its exocrine secretion in the form of exosomes and improve liver insulin resistance by promoting the phosphorylation of AKT. Production of high HKS vesicles might be a possible way to alleviate insulin resistance associated with obesity.
{"title":"Kallistatin Improves High-Fat-Induced Insulin Resistance via Epididymal Adipose Tissue-Derived Exosomes.","authors":"Zi-Wei Yang, Jing-Jing Ji, Yu Jiang, Ya Wu, Jia-Qi Guo, Gen-Shan Ma, Yu-Yu Yao","doi":"10.1089/hum.2023.079","DOIUrl":"10.1089/hum.2023.079","url":null,"abstract":"<p><p><b><i>Objective:</i></b> Studies have found that high expression of human Kallistatin (HKS) in adipose tissue can improve obesity and its associated comorbidities, but the underlying mechanism of specific regulation is unclear. <b><i>Methods:</i></b> An obesity model was built by injecting 8-week-old C57BL/6 mice (<i>n</i> = 6 mice per group) with (Ad.Null and (Ad.HKS adenovirus into epididymal adipose tissue and fed with a high-fat diet (HFD). Insulin resistance-related proteins, AKT and IRS1, were detected in the liver, subcutaneous fat, and skeletal muscle by western blotting after one month of HFD. Epididymal adipose tissue was isolated after 24 h for culture, and exosomes were extracted by differential centrifugation. Enzyme-linked immunosorbent assay detected the expression of HKS protein in serum and exosomes. To examine the role of exosomes in AML12 insulin resistance, we used epididymal adipose tissue-derived exosomes or transfected (Ad.HKS into mature 3T3L1-derived exosomes to interfere with palmitic acid (PA)-induced mouse AML12 insulin resistance model. GW4869 was used to inhibit exosome biogenesis and release. <b><i>Results:</i></b> Our results showed that HFD-induced mice with high expression of HKS in epididymal adipose tissue had slower weight gain, lower serum triglycerides, reduced free fatty acids, and improved liver insulin resistance compared with the (Ad.Null group. We also demonstrated that HKS was enriched in epididymal adipose tissue-derived exosomes and released through the exosome pathway. In PA-induced AML12 cells, insulin resistance was alleviated after incubation of the HKS-related exosome; this effect was reversed with GW4869. <b><i>Conclusion:</i></b> High expression of HKS in epididymal adipose tissue could lead to its exocrine secretion in the form of exosomes and improve liver insulin resistance by promoting the phosphorylation of AKT. Production of high HKS vesicles might be a possible way to alleviate insulin resistance associated with obesity.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9942957","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 : 2024-06-01Epub Date: 2024-06-11DOI: 10.1089/hum.2023.169
Xi Qin, Qikun Yu, Xiang Li, Wei Jiang, Xinchang Shi, Wenxiu Hou, Da Zhang, Zhenzhen Cai, Hua Bi, Wenhong Fan, Youxue Ding, Yichen Yang, Biao Dong, Long Chen, Dehua Huo, Cong Wang, Yong Zhou, Dening Pei, Miao Ye, Chenggang Liang
Currently, adeno-associated virus (AAV) is one of the primary gene delivery vectors in gene therapy, facilitating long-term in vivo gene expression. Despite being imperative, it is incredibly challenging to precisely assess AAV particle distribution according to the sedimentation coefficient and identify impurities related to capsid structures. This study performed the systematic methodological validation of quantifying the AAV empty and full capsid ratio. This includes specificity, accuracy, precision, linearity, and parameter variables involving the sedimentation velocity analytical ultracentrifugation (SV-AUC) method. Specifically, SV-AUC differentiated among the empty, partial, full, and high sedimentation coefficient substance (HSCS) AAV particles while evaluating their sedimentation heterogeneity. The intermediate precision analysis of HE (high percentage of empty capsid) and HF (high percentage of full capsid) samples revealed that the specific species percentage, such as empty or full, was more significant than 50%. Moreover, the relative standard deviation (RSD) could be within 5%. Even for empty or partially less than 15%, the RSD could be within 10%. The accuracy recovery rates of empty capsid were between 103.9% and 108.7% across three different mixtures. When the measured percentage of specific species was more significant than 14%, the recovery rate was between 77.9% and 106.6%. Linearity analysis revealed an excellent linear correlation between the empty, partial, and full in the HE samples. The AAV samples with as low as 7.4 × 1011 cp/mL AAV could be accurately quantified with SV-AUC. The parameter variable analyses revealed that variations in cell alignment significantly affected the overall results. Still, the detection wavelength of 235 nm slightly influenced the empty, partial, and full percentages. Minor detection wavelength changes showed no impact on the sedimentation coefficient of these species. However, the temperature affected the measured sedimentation coefficient. These results validated the SV-AUC method to quantify AAV. This study provides solutions to AAV empty and full capsid ratio quantification challenges and the subsequent basis for calibrating the AAV empty capsid system suitability substance. Because of the AAV structure and potential variability complexity in detection, we jointly calibrated empty capsid system suitability substance with three laboratories to accurately detect the quantitative AAV empty and full capsid ratio. The empty capsid system suitability substance could be used as an external reference to measure the performance of the instrument. The results could be compared with multiple QC (quality control) laboratories based on the AAV vector and calibration accuracy. This is crucial for AUC to be used for QC release and promote gene therapy research worldwide.
{"title":"Methodological Validation of Sedimentation Velocity Analytical Ultracentrifugation Method for Adeno-Associated Virus and Collaborative Calibration of System Suitability Substance.","authors":"Xi Qin, Qikun Yu, Xiang Li, Wei Jiang, Xinchang Shi, Wenxiu Hou, Da Zhang, Zhenzhen Cai, Hua Bi, Wenhong Fan, Youxue Ding, Yichen Yang, Biao Dong, Long Chen, Dehua Huo, Cong Wang, Yong Zhou, Dening Pei, Miao Ye, Chenggang Liang","doi":"10.1089/hum.2023.169","DOIUrl":"10.1089/hum.2023.169","url":null,"abstract":"<p><p>Currently, adeno-associated virus (AAV) is one of the primary gene delivery vectors in gene therapy, facilitating long-term <i>in vivo</i> gene expression. Despite being imperative, it is incredibly challenging to precisely assess AAV particle distribution according to the sedimentation coefficient and identify impurities related to capsid structures. This study performed the systematic methodological validation of quantifying the AAV empty and full capsid ratio. This includes specificity, accuracy, precision, linearity, and parameter variables involving the sedimentation velocity analytical ultracentrifugation (SV-AUC) method. Specifically, SV-AUC differentiated among the empty, partial, full, and high sedimentation coefficient substance (HSCS) AAV particles while evaluating their sedimentation heterogeneity. The intermediate precision analysis of HE (high percentage of empty capsid) and HF (high percentage of full capsid) samples revealed that the specific species percentage, such as empty or full, was more significant than 50%. Moreover, the relative standard deviation (RSD) could be within 5%. Even for empty or partially less than 15%, the RSD could be within 10%. The accuracy recovery rates of empty capsid were between 103.9% and 108.7% across three different mixtures. When the measured percentage of specific species was more significant than 14%, the recovery rate was between 77.9% and 106.6%. Linearity analysis revealed an excellent linear correlation between the empty, partial, and full in the HE samples. The AAV samples with as low as 7.4 × 10<sup>11</sup> cp/mL AAV could be accurately quantified with SV-AUC. The parameter variable analyses revealed that variations in cell alignment significantly affected the overall results. Still, the detection wavelength of 235 nm slightly influenced the empty, partial, and full percentages. Minor detection wavelength changes showed no impact on the sedimentation coefficient of these species. However, the temperature affected the measured sedimentation coefficient. These results validated the SV-AUC method to quantify AAV. This study provides solutions to AAV empty and full capsid ratio quantification challenges and the subsequent basis for calibrating the AAV empty capsid system suitability substance. Because of the AAV structure and potential variability complexity in detection, we jointly calibrated empty capsid system suitability substance with three laboratories to accurately detect the quantitative AAV empty and full capsid ratio. The empty capsid system suitability substance could be used as an external reference to measure the performance of the instrument. The results could be compared with multiple QC (quality control) laboratories based on the AAV vector and calibration accuracy. This is crucial for AUC to be used for QC release and promote gene therapy research worldwide.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140890556","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 : 2024-05-01DOI: 10.1089/hum.2024.28412.bfs
Alex Philippidis
{"title":"Verve Pauses Enrollment in Base Editing Trial after Adverse Events.","authors":"Alex Philippidis","doi":"10.1089/hum.2024.28412.bfs","DOIUrl":"10.1089/hum.2024.28412.bfs","url":null,"abstract":"","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141087657","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 : 2024-05-01Epub Date: 2024-04-19DOI: 10.1089/hum.2023.225
Rebecca Xicluna, Allan Avenel, Céline Vandamme, Marie Devaux, Nicolas Jaulin, Célia Couzinié, Johanne Le Duff, Alicia Charrier, Mickaël Guilbaud, Oumeya Adjali, Gwladys Gernoux
Recombinant adeno-associated virus (rAAV) vectors appear, more than ever, to be efficient viral vectors for in vivo gene transfer as illustrated by the approvals of 7 drugs across Europe and the United States. Nevertheless, preexisting immunity to AAV capsid in humans remains one of the major limits for a successful clinical translation. Whereas a preexisting humoral response to AAV capsid is well documented, the prevalence of preexisting capsid-specific T cell responses still needs to be studied and characterized. In this study, we investigated the prevalence of AAV-specific circulating T cells toward AAV2, 4, 5, 8, 9, and rh10 in a large cohort of healthy donors using the standard IFNγ ELISpot assay. We observed the highest prevalence of preexisting cellular immunity to AAV9 serotype followed by AAV8, AAV4, AAV2, AAVrh10, and AAV5 independently of the donors' serological status. An in-depth analysis of T cell responses toward the 2 most prevalent serotypes 8 and 9 shows that IFNγ secretion is mainly mediated by CD8 T cells for both serotypes. A polyfunctional analysis reveals different cytokine profiles between AAV8 and AAV9. Surprisingly, no IL-2 secretion was mediated by anti-AAV9 immune cells suggesting that these cells may rather be exhausted or terminally differentiated than cytotoxic T cells. Altogether, these results suggest that preexisting immunity to AAV may vary depending on the serotype and support the necessity of using multiparametric monitoring methods to better characterize anticapsid cellular immunity and foresee its impact in rAAV-mediated clinical trials.
{"title":"Prevalence Study of Cellular Capsid-Specific Immune Responses to AAV2, 4, 5, 8, 9, and rh10 in Healthy Donors.","authors":"Rebecca Xicluna, Allan Avenel, Céline Vandamme, Marie Devaux, Nicolas Jaulin, Célia Couzinié, Johanne Le Duff, Alicia Charrier, Mickaël Guilbaud, Oumeya Adjali, Gwladys Gernoux","doi":"10.1089/hum.2023.225","DOIUrl":"10.1089/hum.2023.225","url":null,"abstract":"<p><p>Recombinant adeno-associated virus (rAAV) vectors appear, more than ever, to be efficient viral vectors for <i>in vivo</i> gene transfer as illustrated by the approvals of 7 drugs across Europe and the United States. Nevertheless, preexisting immunity to AAV capsid in humans remains one of the major limits for a successful clinical translation. Whereas a preexisting humoral response to AAV capsid is well documented, the prevalence of preexisting capsid-specific T cell responses still needs to be studied and characterized. In this study, we investigated the prevalence of AAV-specific circulating T cells toward AAV2, 4, 5, 8, 9, and rh10 in a large cohort of healthy donors using the standard IFNγ ELISpot assay. We observed the highest prevalence of preexisting cellular immunity to AAV9 serotype followed by AAV8, AAV4, AAV2, AAVrh10, and AAV5 independently of the donors' serological status. An in-depth analysis of T cell responses toward the 2 most prevalent serotypes 8 and 9 shows that IFNγ secretion is mainly mediated by CD8 T cells for both serotypes. A polyfunctional analysis reveals different cytokine profiles between AAV8 and AAV9. Surprisingly, no IL-2 secretion was mediated by anti-AAV9 immune cells suggesting that these cells may rather be exhausted or terminally differentiated than cytotoxic T cells. Altogether, these results suggest that preexisting immunity to AAV may vary depending on the serotype and support the necessity of using multiparametric monitoring methods to better characterize anticapsid cellular immunity and foresee its impact in rAAV-mediated clinical trials.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":4.2,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140854809","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 : 2024-05-01Epub Date: 2024-04-08DOI: 10.1089/hum.2024.022
Chujiao Lin, Matthew B Greenblatt, Guangping Gao, Jae-Hyuck Shim
Adeno-associated viral (AAV) vectors have emerged as crucial tools in advancing gene therapy for skeletal diseases, offering the potential for sustained expression with low postinfection immunogenicity and pathogenicity. Preclinical studies support both the therapeutic efficacy and safety of these vectors, illustrating the promise of AAV-mediated gene therapy. Emerging technologies and innovations in AAV-mediated gene therapy strategies, such as gene addition, gene replacement, gene silencing, and gene editing, offer new approaches to clinical application. Recently, the increasing preclinical applications of AAV to rare skeletal diseases, such as fibrodysplasia ossificans progressiva (FOP) and osteogenesis imperfecta (OI), and prevalent bone diseases, such as osteoporosis, bone fracture, critical-sized bone defects, and osteoarthritis, have been reported. Despite existing limitations in clinical use, such as high cost and safety, the AAV-mediated gene transfer platform is a promising approach to deliver therapeutic gene(s) to the skeleton to treat skeletal disorders, including those otherwise intractable by other therapeutic approaches. This review provides a comprehensive overview of the therapeutic advancements, challenges, limitations, and solutions within AAV-based gene therapy for prevalent and rare skeletal diseases.
{"title":"Development of AAV-Mediated Gene Therapy Approaches to Treat Skeletal Diseases.","authors":"Chujiao Lin, Matthew B Greenblatt, Guangping Gao, Jae-Hyuck Shim","doi":"10.1089/hum.2024.022","DOIUrl":"10.1089/hum.2024.022","url":null,"abstract":"<p><p>Adeno-associated viral (AAV) vectors have emerged as crucial tools in advancing gene therapy for skeletal diseases, offering the potential for sustained expression with low postinfection immunogenicity and pathogenicity. Preclinical studies support both the therapeutic efficacy and safety of these vectors, illustrating the promise of AAV-mediated gene therapy. Emerging technologies and innovations in AAV-mediated gene therapy strategies, such as gene addition, gene replacement, gene silencing, and gene editing, offer new approaches to clinical application. Recently, the increasing preclinical applications of AAV to rare skeletal diseases, such as fibrodysplasia ossificans progressiva (FOP) and osteogenesis imperfecta (OI), and prevalent bone diseases, such as osteoporosis, bone fracture, critical-sized bone defects, and osteoarthritis, have been reported. Despite existing limitations in clinical use, such as high cost and safety, the AAV-mediated gene transfer platform is a promising approach to deliver therapeutic gene(s) to the skeleton to treat skeletal disorders, including those otherwise intractable by other therapeutic approaches. This review provides a comprehensive overview of the therapeutic advancements, challenges, limitations, and solutions within AAV-based gene therapy for prevalent and rare skeletal diseases.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11302315/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140293395","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}