Katherine J D A Excoffon, Mark D Smith, Lillian Falese, Robert Schulingkamp, Shen Lin, Madhu Mahankali, Poornima K L Narayan, Matthew R Glatfelter, Maria P Limberis, Eric Yuen, Roland Kolbeck
Cystic fibrosis (CF) is a serious genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Approved small molecule therapies benefit the majority of people with CF (pwCF), but unfortunately not all. Gene addition offers a mutation agnostic treatment option for all pwCF. SP-101 is an adeno-associated virus gene therapy vector (AAV2.5T) that has been optimized for efficient human airway cell transduction, and that contains a functional and regulated shortened human CFTR minigene (hCFTRΔR) with a small synthetic promoter/enhancer. To understand SP-101 airway distribution, activity, and the associated immune response, in vivo studies were performed in wild-type and CF ferrets. After single dose inhaled delivery of SP-101, followed by single dose inhaled doxorubicin (an AAV transduction augmenter) or saline, SP-101 vector genomes were detected throughout the respiratory tract. hCFTRΔR mRNA expression was highest in ferrets also receiving doxorubicin and persisted for the duration of the study (13 weeks). Pre-existing mucus in the CF ferrets did not present a barrier to effective transduction. Binding and neutralizing antibodies to the AAV2.5T capsid were observed regardless of doxorubicin exposure. Only a portion of ferrets exhibited a weak T-cell response to AAV2.5T and no T-cell response was seen against hCFTRΔR. These data strongly support the continued development of inhaled SP-101, followed by inhaled doxorubicin, for the treatment of CF.
{"title":"Inhalation of SP-101 Followed by Inhaled Doxorubicin Results in Robust and Durable <i>hCFTRΔR</i> Transgene Expression in the Airways of Wild-Type and Cystic Fibrosis Ferrets.","authors":"Katherine J D A Excoffon, Mark D Smith, Lillian Falese, Robert Schulingkamp, Shen Lin, Madhu Mahankali, Poornima K L Narayan, Matthew R Glatfelter, Maria P Limberis, Eric Yuen, Roland Kolbeck","doi":"10.1089/hum.2024.064","DOIUrl":"10.1089/hum.2024.064","url":null,"abstract":"<p><p>Cystic fibrosis (CF) is a serious genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Approved small molecule therapies benefit the majority of people with CF (pwCF), but unfortunately not all. Gene addition offers a mutation agnostic treatment option for all pwCF. SP-101 is an adeno-associated virus gene therapy vector (AAV2.5T) that has been optimized for efficient human airway cell transduction, and that contains a functional and regulated shortened human CFTR minigene (<i>hCFTRΔR</i>) with a small synthetic promoter/enhancer. To understand SP-101 airway distribution, activity, and the associated immune response, <i>in vivo</i> studies were performed in wild-type and CF ferrets. After single dose inhaled delivery of SP-101, followed by single dose inhaled doxorubicin (an AAV transduction augmenter) or saline, SP-101 vector genomes were detected throughout the respiratory tract. <i>hCFTRΔR</i> mRNA expression was highest in ferrets also receiving doxorubicin and persisted for the duration of the study (13 weeks). Pre-existing mucus in the CF ferrets did not present a barrier to effective transduction. Binding and neutralizing antibodies to the AAV2.5T capsid were observed regardless of doxorubicin exposure. Only a portion of ferrets exhibited a weak T-cell response to AAV2.5T and no T-cell response was seen against hCFTRΔR. These data strongly support the continued development of inhaled SP-101, followed by inhaled doxorubicin, for the treatment of CF.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141999776","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}
Santiago R Castillo, Brandon W Simone, Karl J Clark, Patricia Devaux, Stephen C Ekker
DddA-derived cytosine base editors (DdCBEs) enable the targeted introduction of C•G-to-T•A conversions in mitochondrial DNA (mtDNA). DdCBEs work in pairs, with each arm composed of a transcription activator-like effector (TALE), a split double-stranded DNA deaminase half, and a uracil glycosylase inhibitor. This pioneering technology has helped improve our understanding of cellular processes involving mtDNA and has paved the way for the development of models and therapies for genetic disorders caused by pathogenic mtDNA variants. Nonetheless, given the intrinsic properties of TALE proteins, several target sites in human mtDNA are predicted to remain out of reach to DdCBEs and other TALE-based technologies. Specifically, due to the conventional requirement for a thymine immediately upstream of the TALE target sequences (i.e., the 5'-T constraint), over 150 loci in the human mitochondrial genome are presumed to be inaccessible to DdCBEs. Previous attempts at circumventing this requirement, either by developing monomeric DdCBEs or utilizing DNA-binding domains alternative to TALEs, have resulted in suboptimal specificity profiles with reduced therapeutic potential. Here, aiming to challenge and elucidate the relevance of the 5'-T constraint in the context of DdCBE-mediated mtDNA editing, and to expand the range of motifs that are editable by this technology, we generated DdCBEs containing TALE proteins engineered to recognize all 5' bases. These modified DdCBEs are herein referred to as αDdCBEs. Notably, 5'-T-noncompliant canonical DdCBEs efficiently edited mtDNA at diverse loci. However, they were frequently outperformed by αDdCBEs, which exhibited significant improvements in activity and specificity, regardless of the most 5' bases of their TALE binding sites. Furthermore, we showed that αDdCBEs are compatible with the enhanced DddAtox variants DddA6 and DddA11, and we validated TALE shifting with αDdCBEs as an effective approach to optimize base editing outcomes. Overall, αDdCBEs enable efficient, specific, and unconstrained mitochondrial base editing.
DddA 衍生胞嘧啶碱基编辑器(DdCBEs)能有针对性地将线粒体 DNA(mtDNA)中的 C-G 转换为 T-A。DdCBEs 成对工作,每个臂由一个类似转录激活剂的效应器 (TALE)、半个分裂的双链 DNA 去氨酶和一个尿嘧啶糖基化酶抑制剂组成。这项开创性技术有助于我们更好地了解涉及 mtDNA 的细胞过程,并为开发由致病性 mtDNA 变体引起的遗传疾病的模型和疗法铺平了道路。尽管如此,鉴于 TALE 蛋白的固有特性,人类 mtDNA 中的一些靶位点仍无法被 DdCBE 和其他基于 TALE 的技术所触及。具体来说,由于 TALE 目标序列上游必须有一个胸腺嘧啶的传统要求(即 5'-T 限制),人类线粒体基因组中超过 150 个位点被认为是 DdCBE 无法访问的。以前有人试图通过开发单体 DdCBE 或利用 DNA 结合域替代 TALE 来规避这一要求,但结果都是特异性不理想,治疗潜力降低。为了挑战和阐明 5'-T 约束在 DdCBE 介导的 mtDNA 编辑中的相关性,并扩大该技术可编辑的基团范围,我们生成了含有 TALE 蛋白的 DdCBE,这些 TALE 蛋白经过工程化处理后可识别所有 5' 碱基。这些经过修饰的 DdCBE 在此称为 αDdCBE。值得注意的是,不符合5'-T规范的DdCBE能有效编辑不同位点的mtDNA。然而,αDdCBEs 的表现却常常优于它们,无论它们的 TALE 结合位点的 5' 碱基是多少,αDdCBEs 的活性和特异性都有显著提高。此外,我们还发现 αDdCBE 与增强型 DddAtox 变体 DddA6 和 DddA11 兼容,并验证了 TALE 与 αDdCBE 的转移是优化碱基编辑结果的有效方法。总之,αDdCBEs 可以实现高效、特异和无约束的线粒体碱基编辑。
{"title":"Unconstrained precision mitochondrial genome editing with αDdCBEs.","authors":"Santiago R Castillo, Brandon W Simone, Karl J Clark, Patricia Devaux, Stephen C Ekker","doi":"10.1089/hum.2024.073","DOIUrl":"10.1089/hum.2024.073","url":null,"abstract":"<p><p>DddA-derived cytosine base editors (DdCBEs) enable the targeted introduction of C•G-to-T•A conversions in mitochondrial DNA (mtDNA). DdCBEs work in pairs, with each arm composed of a transcription activator-like effector (TALE), a split double-stranded DNA deaminase half, and a uracil glycosylase inhibitor. This pioneering technology has helped improve our understanding of cellular processes involving mtDNA and has paved the way for the development of models and therapies for genetic disorders caused by pathogenic mtDNA variants. Nonetheless, given the intrinsic properties of TALE proteins, several target sites in human mtDNA are predicted to remain out of reach to DdCBEs and other TALE-based technologies. Specifically, due to the conventional requirement for a thymine immediately upstream of the TALE target sequences (i.e., the 5'-T constraint), over 150 loci in the human mitochondrial genome are presumed to be inaccessible to DdCBEs. Previous attempts at circumventing this requirement, either by developing monomeric DdCBEs or utilizing DNA-binding domains alternative to TALEs, have resulted in suboptimal specificity profiles with reduced therapeutic potential. Here, aiming to challenge and elucidate the relevance of the 5'-T constraint in the context of DdCBE-mediated mtDNA editing, and to expand the range of motifs that are editable by this technology, we generated DdCBEs containing TALE proteins engineered to recognize all 5' bases. These modified DdCBEs are herein referred to as αDdCBEs. Notably, 5'-T-noncompliant canonical DdCBEs efficiently edited mtDNA at diverse loci. However, they were frequently outperformed by αDdCBEs, which exhibited significant improvements in activity and specificity, regardless of the most 5' bases of their TALE binding sites. Furthermore, we showed that αDdCBEs are compatible with the enhanced DddAtox variants DddA6 and DddA11, and we validated TALE shifting with αDdCBEs as an effective approach to optimize base editing outcomes. Overall, αDdCBEs enable efficient, specific, and unconstrained mitochondrial base editing.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-08-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142106920","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}
Katherine Jda Excoffon, Shen Lin, Poornima Kotha Lakshmi Narayan, Sneha Sitaraman, Awal M Jimah, Tyler T Fallon, Melane L James, Matthew R Glatfelter, Maria P Limberis, Mark D Smith, Guia Guffanti, Roland Kolbeck
Cystic fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein. Although CF affects multiple organs, lung disease is the main cause of morbidity and mortality, and gene therapy is expected to provide a mutation-agnostic option for treatment. SP-101 is a recombinant adeno-associated virus (AAV) gene therapy vector carrying a human CFTR minigene, hCFTRΔR, and is being investigated as an inhalation treatment for people with CF. To further understand SP-101 activity, in vitro studies were performed in human airway epithelia (HAE) derived from multiple CF and non-CF donors. SP-101 restored CFTR-mediated chloride conductance, measured via Ussing chamber assay, at a multiplicity of infection (MOI) as low as 5E2 in the presence of doxorubicin, a small molecule known to augment AAV transduction. Functional correction of CF HAE increased with increasing MOI and doxorubicin concentration and correlated with increasing cell-associated vector genomes and hCFTRΔR mRNA expression. Tropism studies using a fluorescent reporter vector and single-cell mRNA sequencing of SP-101-mediated hCFTRΔR mRNA demonstrated broad expression in all cell types after apical transduction, including secretory, ciliated, and basal cells. In summary, SP-101, particularly in combination with doxorubicin, shows promise for a novel CF treatment strategy and strongly supports continued development.
{"title":"SP-101, A Novel Adeno-Associated Virus Gene Therapy for the Treatment of Cystic Fibrosis, Mediates Functional Correction of Primary Human Airway Epithelia From Donors with Cystic Fibrosis.","authors":"Katherine Jda Excoffon, Shen Lin, Poornima Kotha Lakshmi Narayan, Sneha Sitaraman, Awal M Jimah, Tyler T Fallon, Melane L James, Matthew R Glatfelter, Maria P Limberis, Mark D Smith, Guia Guffanti, Roland Kolbeck","doi":"10.1089/hum.2024.063","DOIUrl":"10.1089/hum.2024.063","url":null,"abstract":"<p><p>Cystic fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein. Although CF affects multiple organs, lung disease is the main cause of morbidity and mortality, and gene therapy is expected to provide a mutation-agnostic option for treatment. SP-101 is a recombinant adeno-associated virus (AAV) gene therapy vector carrying a human <i>CFTR</i> minigene, <i>hCFTRΔR</i>, and is being investigated as an inhalation treatment for people with CF. To further understand SP-101 activity, <i>in vitro</i> studies were performed in human airway epithelia (HAE) derived from multiple CF and non-CF donors. SP-101 restored CFTR-mediated chloride conductance, measured via Ussing chamber assay, at a multiplicity of infection (MOI) as low as 5E2 in the presence of doxorubicin, a small molecule known to augment AAV transduction. Functional correction of CF HAE increased with increasing MOI and doxorubicin concentration and correlated with increasing cell-associated vector genomes and <i>hCFTRΔR</i> mRNA expression. Tropism studies using a fluorescent reporter vector and single-cell mRNA sequencing of SP-101-mediated <i>hCFTRΔR</i> mRNA demonstrated broad expression in all cell types after apical transduction, including secretory, ciliated, and basal cells. In summary, SP-101, particularly in combination with doxorubicin, shows promise for a novel CF treatment strategy and strongly supports continued development.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-08-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141999777","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}
Angela E Araujo, Martin Bentler, Xabier Perez Garmendia, Asma Kaleem, Claire Fabian, Michael Morgan, Ulrich T Hacker, Hildegard Büning
All current market-approved gene therapy medical products for in vivo gene therapy of monogenic diseases rely on adeno-associated virus (AAV) vectors. Advances in gene editing technologies and vector engineering have expanded the spectrum of target cells and, thus, diseases that can be addressed. Consequently, AAV vectors are now being explored to modify cells of the hematopoietic system, including hematopoietic stem and progenitor cells (HSPCs), to develop novel strategies to treat monogenic diseases, but also to generate cell- and vaccine-based immunotherapies. However, the cell types that represent important new targets for the AAV vector system are centrally involved in immune responses against the vector and its transgene product as discussed briefly in the first part of this review. In the second part, studies exploring AAV vectors for genetic engineering of HSPCs, T and B lymphocytes, and beyond are presented.
目前市场上批准用于单基因疾病体内基因治疗的所有基因治疗医疗产品都依赖于腺相关病毒(AAV)载体。基因编辑技术和载体工程学的进步扩大了靶细胞的范围,从而也扩大了可治疗疾病的范围。因此,AAV 载体目前正被用于改造造血系统细胞,包括造血干细胞和祖细胞(HSPCs),以开发治疗单基因疾病的新策略,同时也用于产生以细胞和疫苗为基础的免疫疗法。然而,AAV载体系统的重要新靶点细胞类型主要参与了针对载体及其转基因产物的免疫反应,本综述的第一部分对此进行了简要讨论。第二部分将介绍探索 AAV 载体用于 HSPC、T 淋巴细胞和 B 淋巴细胞等基因工程的研究。
{"title":"Adeno-Associated Virus Vectors-a Target of Cellular and Humoral Immunity-are Expanding Their Reach Toward Hematopoietic Stem Cell Modification and Immunotherapies.","authors":"Angela E Araujo, Martin Bentler, Xabier Perez Garmendia, Asma Kaleem, Claire Fabian, Michael Morgan, Ulrich T Hacker, Hildegard Büning","doi":"10.1089/hum.2024.114","DOIUrl":"https://doi.org/10.1089/hum.2024.114","url":null,"abstract":"<p><p>All current market-approved gene therapy medical products for <i>in vivo</i> gene therapy of monogenic diseases rely on adeno-associated virus (AAV) vectors. Advances in gene editing technologies and vector engineering have expanded the spectrum of target cells and, thus, diseases that can be addressed. Consequently, AAV vectors are now being explored to modify cells of the hematopoietic system, including hematopoietic stem and progenitor cells (HSPCs), to develop novel strategies to treat monogenic diseases, but also to generate cell- and vaccine-based immunotherapies. However, the cell types that represent important new targets for the AAV vector system are centrally involved in immune responses against the vector and its transgene product as discussed briefly in the first part of this review. In the second part, studies exploring AAV vectors for genetic engineering of HSPCs, T and B lymphocytes, and beyond are presented.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142080135","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}
Nadine Saber, Mariona Estapé Senti, Raymond M Schiffelers
Lipid nanoparticles (LNPs) are the most clinically advanced drug delivery system for nucleic acid therapeutics, exemplified by the success of the COVID-19 mRNA vaccines. However, their clinical use is currently limited to hepatic diseases and vaccines due to their tendency to accumulate in the liver upon intravenous administration. To fully leverage their potential, it is essential to understand and address their liver tropism, while also developing strategies to enhance delivery to tissues beyond the liver. Ensuring that these therapeutics reach their target cells while avoiding off-target cells is essential for both their efficacy and safety. There are three potential targeting strategies-passive, active, and endogenous-which can be used individually or in combination to target nonhepatic tissues. In this review, we delve into the recent advancements in LNP engineering for delivering nucleic acid beyond the liver.
{"title":"Lipid Nanoparticles for Nucleic Acid Delivery Beyond the Liver.","authors":"Nadine Saber, Mariona Estapé Senti, Raymond M Schiffelers","doi":"10.1089/hum.2024.106","DOIUrl":"10.1089/hum.2024.106","url":null,"abstract":"<p><p>Lipid nanoparticles (LNPs) are the most clinically advanced drug delivery system for nucleic acid therapeutics, exemplified by the success of the COVID-19 mRNA vaccines. However, their clinical use is currently limited to hepatic diseases and vaccines due to their tendency to accumulate in the liver upon intravenous administration. To fully leverage their potential, it is essential to understand and address their liver tropism, while also developing strategies to enhance delivery to tissues beyond the liver. Ensuring that these therapeutics reach their target cells while avoiding off-target cells is essential for both their efficacy and safety. There are three potential targeting strategies-passive, active, and endogenous-which can be used individually or in combination to target nonhepatic tissues. In this review, we delve into the recent advancements in LNP engineering for delivering nucleic acid beyond the liver.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141975598","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}
Teng-Cheong Ha, Michael A Morgan, Adrian J Thrasher, Axel Schambach
Severe combined immunodeficiency (SCID) encompasses rare primary immunodeficiency disorders characterized by deficient T-cell development, which leads to a severely compromised immune system and susceptibility to life-threatening infections. Among SCID subtypes, IL7RA-SCID is caused by mutations in the interleukin 7 receptor alpha chain (IL7RA) and represents a significant subset of patients with limited treatment options. This study investigated the efficacy of a self-inactivating (SIN) alpharetroviral vector (ARV) engineered to deliver a codon-optimized IL7RA cDNA to restore T-cell development in Il7r-knockout mice. We compared the elongation factor 1 alpha short (EFS) promoter and the lymphoid-restricted Lck promoter for their ability to drive IL7RA expression and found that the EFS promoter enabled robust and sustained IL7RA expression that led to the functional rescue of T-lymphopoiesis in vitro and in vivo. Conversely, though effective in vitro, the Lck promoter failed to produce viable T-cell populations in vivo. Our results highlight the potential of using SIN-ARVs as a gene therapy (GT) strategy for treating IL7RA-SCID. Importantly, sustained production of T-lymphocytes was found in both primary and secondary transplant recipient animals with no adverse effects, supporting the safety and feasibility of this approach. Overall, this study provides valuable insights into the development of GT for IL7RA-SCID and underscores the clinical potential of an EFS-driven SIN-ARV to restore IL7RA-deficient immune function.
严重联合免疫缺陷症(SCID)是一种罕见的原发性免疫缺陷疾病,其特点是 T 细胞发育不全,导致免疫系统严重受损,容易受到危及生命的感染。在SCID亚型中,IL7RA-SCID是由白细胞介素7受体α链(IL7RA)突变引起的,是治疗方案有限的重要患者亚型。本研究调查了一种自失活(SIN)α逆转录病毒载体的疗效,该载体被设计为递送经过密码子优化的IL7RA cDNA,以恢复IL7RA基因敲除小鼠的T细胞发育。我们比较了α短伸长因子(EFS)启动子和淋巴限制性Lck启动子驱动IL7RA表达的能力,发现EFS启动子能使IL7RA得到稳健而持续的表达,从而在体外和体内实现T淋巴细胞生成的功能性拯救。相反,Lck 启动子虽然在体外有效,但在体内却不能产生有活力的 T 细胞群。我们的研究结果凸显了使用 SIN-逆转录病毒载体作为基因治疗(GT)策略治疗 IL7RA-SCID 的潜力。重要的是,在初次和二次移植受体动物中都发现了T淋巴细胞的持续生成,且无不良反应,这支持了这种方法的安全性和可行性。总之,这项研究为IL7RA-SCID基因疗法的开发提供了宝贵的见解,并强调了EFS驱动的SIN-alpharetroviral载体恢复IL7RA缺陷免疫功能的临床潜力。
{"title":"Alpharetroviral Vector-Mediated Gene Therapy for IL7RA-Deficient Severe Combined Immunodeficiency.","authors":"Teng-Cheong Ha, Michael A Morgan, Adrian J Thrasher, Axel Schambach","doi":"10.1089/hum.2024.103","DOIUrl":"10.1089/hum.2024.103","url":null,"abstract":"<p><p>Severe combined immunodeficiency (SCID) encompasses rare primary immunodeficiency disorders characterized by deficient T-cell development, which leads to a severely compromised immune system and susceptibility to life-threatening infections. Among SCID subtypes, IL7RA-SCID is caused by mutations in the interleukin 7 receptor alpha chain (IL7RA) and represents a significant subset of patients with limited treatment options. This study investigated the efficacy of a self-inactivating (SIN) alpharetroviral vector (ARV) engineered to deliver a codon-optimized <i>IL7RA</i> cDNA to restore T-cell development in <i>Il7r</i>-knockout mice. We compared the elongation factor 1 alpha short (EFS) promoter and the lymphoid-restricted Lck promoter for their ability to drive IL7RA expression and found that the EFS promoter enabled robust and sustained IL7RA expression that led to the functional rescue of T-lymphopoiesis <i>in vitro</i> and <i>in vivo</i>. Conversely, though effective <i>in vitro</i>, the Lck promoter failed to produce viable T-cell populations <i>in vivo</i>. Our results highlight the potential of using SIN-ARVs as a gene therapy (GT) strategy for treating IL7RA-SCID. Importantly, sustained production of T-lymphocytes was found in both primary and secondary transplant recipient animals with no adverse effects, supporting the safety and feasibility of this approach. Overall, this study provides valuable insights into the development of GT for IL7RA-SCID and underscores the clinical potential of an EFS-driven SIN-ARV to restore IL7RA-deficient immune function.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-08-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141987879","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}
The invention of next-generation CRISPR/Cas gene editing tools, like base and prime editing, for correction of gene variants causing disease, has created hope for in vivo use in patients leading to wider clinical translation. To realize this potential, delivery vehicles that can ferry gene editing tool kits safely and effectively into specific cell populations or tissues are in great demand. In this review, we describe the development of enveloped retrovirus-derived particles as carriers of "ready-to-work" ribonucleoprotein complexes consisting of Cas9-derived editor proteins and single guide RNAs. We present arguments for adapting viruses for cell-targeted protein delivery and describe the status after a decade-long development period, which has already shown effective editing in primary cells, including T cells and hematopoietic stem cells, and in tissues targeted in vivo, including mouse retina, liver, and brain. Emerging evidence has demonstrated that engineered virus-derived nanoparticles can accommodate both base and prime editors and seems to fertilize a sprouting hope that such particles can be further developed and produced in large scale for therapeutic applications.
{"title":"Gene Editing by Ferrying of CRISPR/Cas Ribonucleoprotein Complexes in Enveloped Virus-Derived Particles.","authors":"Jacob Hørlück Janns, Jacob Giehm Mikkelsen","doi":"10.1089/hum.2024.105","DOIUrl":"10.1089/hum.2024.105","url":null,"abstract":"<p><p>The invention of next-generation CRISPR/Cas gene editing tools, like base and prime editing, for correction of gene variants causing disease, has created hope for <i>in vivo</i> use in patients leading to wider clinical translation. To realize this potential, delivery vehicles that can ferry gene editing tool kits safely and effectively into specific cell populations or tissues are in great demand. In this review, we describe the development of enveloped retrovirus-derived particles as carriers of \"ready-to-work\" ribonucleoprotein complexes consisting of Cas9-derived editor proteins and single guide RNAs. We present arguments for adapting viruses for cell-targeted protein delivery and describe the status after a decade-long development period, which has already shown effective editing in primary cells, including T cells and hematopoietic stem cells, and in tissues targeted <i>in vivo</i>, including mouse retina, liver, and brain. Emerging evidence has demonstrated that engineered virus-derived nanoparticles can accommodate both base and prime editors and seems to fertilize a sprouting hope that such particles can be further developed and produced in large scale for therapeutic applications.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-08-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141987880","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}
MicroRNAs (miRNAs) are crucial regulators of gene expression involved in various pathophysiological processes. Their ability to modulate multiple pathways simultaneously and their involvement in numerous diseases make miRNAs attractive tools and targets in therapeutic development. Significant efforts have been made to advance miRNA research in the preclinical stage, attracting considerable investment from biopharmaceutical companies. Consequently, an increasing number of miRNA-based therapies have entered clinical trials for both diagnostic and therapeutic applications across a wide range of diseases. While individual miRNAs can regulate a broad array of mRNA targets, this also complicates the management of adverse effects seen in clinical trials. Several candidates have been discontinued due to toxicity concerns, underscoring the need for comprehensive risk assessments of miRNA therapeutics. Despite no miRNA-based strategies have yet received approval from regulatory agencies, prominent progress in the miRNAs modulation approaches and in the nano-delivery systems have been made in the last decade, leading to the development of novel safe and well-tolerated miRNA drug candidates. In this review, we present recent advances in the development of miRNA therapeutics currently in preclinical or clinical stages for treating both rare genetic disorders and multifactorial common conditions. We also address the challenges related to the safety and targeted delivery of miRNA therapies, as well as the identification of the most effective therapeutic candidates in preclinical and clinical trials.
{"title":"Advances in MicroRNA Therapeutics: from Preclinical to Clinical Studies.","authors":"Simona Brillante, Mariagrazia Volpe, Alessia Indrieri","doi":"10.1089/hum.2024.113","DOIUrl":"https://doi.org/10.1089/hum.2024.113","url":null,"abstract":"<p><p>MicroRNAs (miRNAs) are crucial regulators of gene expression involved in various pathophysiological processes. Their ability to modulate multiple pathways simultaneously and their involvement in numerous diseases make miRNAs attractive tools and targets in therapeutic development. Significant efforts have been made to advance miRNA research in the preclinical stage, attracting considerable investment from biopharmaceutical companies. Consequently, an increasing number of miRNA-based therapies have entered clinical trials for both diagnostic and therapeutic applications across a wide range of diseases. While individual miRNAs can regulate a broad array of mRNA targets, this also complicates the management of adverse effects seen in clinical trials. Several candidates have been discontinued due to toxicity concerns, underscoring the need for comprehensive risk assessments of miRNA therapeutics. Despite no miRNA-based strategies have yet received approval from regulatory agencies, prominent progress in the miRNAs modulation approaches and in the nano-delivery systems have been made in the last decade, leading to the development of novel safe and well-tolerated miRNA drug candidates. In this review, we present recent advances in the development of miRNA therapeutics currently in preclinical or clinical stages for treating both rare genetic disorders and multifactorial common conditions. We also address the challenges related to the safety and targeted delivery of miRNA therapies, as well as the identification of the most effective therapeutic candidates in preclinical and clinical trials.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-08-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141987878","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}
β654-thalassemia is caused by a point mutation in the second intron (IVS-II) of the β-globin gene that activates a cryptic 3' splice site, leading to incorrect RNA splicing. Our previous study demonstrated that when direct deletion of the β654 mutation sequence or the cryptic 3' splice site in the IVS-II occurs, correct splicing of β-globin mRNA can be restored. Herein, we conducted an in-depth analysis to explore a more precise gene-editing method for treating β654-thalassemia. A single-base substitution of the cryptic 3' acceptor splice site was introduced in the genome of a β654-thalassemia mouse model using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9(Cas9)-mediated homology-directed repair (HDR). All of the HDR-edited mice allow the detection of correctly spliced β-globin mRNA. Pathological changes were improved compared with the nonedited β654 mice. This resulted in a more than twofold increase in the survival rate beyond the weaning age of the mice carrying the β654 allele. The therapeutic effects of this gene-editing strategy showed that the typical β-thalassemia phenotype can be improved in a dose-dependent manner when the frequency of HDR is over 20%. Our research provides a unique and effective method for correcting the splicing defect by gene editing the reactive splicing acceptor site in a β654 mouse model.
{"title":"Gene Editing of the Endogenous Cryptic 3' Splice Site Corrects the RNA Splicing Defect in the β<sup>654</sup>-Thalassemia Mouse Model.","authors":"Dan Lu, Xiuli Gong, Xinbing Guo, Qin Cai, Yanwen Chen, Yiwen Zhu, Xiao Sang, Hua Yang, Miao Xu, Yitao Zeng, Dali Li, Fanyi Zeng","doi":"10.1089/hum.2023.202","DOIUrl":"10.1089/hum.2023.202","url":null,"abstract":"<p><p>β<sup>654</sup>-thalassemia is caused by a point mutation in the second intron (IVS-II) of the β-globin gene that activates a cryptic 3' splice site, leading to incorrect RNA splicing. Our previous study demonstrated that when direct deletion of the β<sup>654</sup> mutation sequence or the cryptic 3' splice site in the IVS-II occurs, correct splicing of β-globin mRNA can be restored. Herein, we conducted an in-depth analysis to explore a more precise gene-editing method for treating β<sup>654</sup>-thalassemia. A single-base substitution of the cryptic 3' acceptor splice site was introduced in the genome of a β<sup>654</sup>-thalassemia mouse model using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9(Cas9)-mediated homology-directed repair (HDR). All of the HDR-edited mice allow the detection of correctly spliced β-globin mRNA. Pathological changes were improved compared with the nonedited β<sup>654</sup> mice. This resulted in a more than twofold increase in the survival rate beyond the weaning age of the mice carrying the β<sup>654</sup> allele. The therapeutic effects of this gene-editing strategy showed that the typical β-thalassemia phenotype can be improved in a dose-dependent manner when the frequency of HDR is over 20%. Our research provides a unique and effective method for correcting the splicing defect by gene editing the reactive splicing acceptor site in a β<sup>654</sup> mouse model.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141792336","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}
Jacob A Hoffman, Nathan Denton, Joshua J Sims, Rosemary Meggersee, Zhe Zhang, Kanyin Olagbegi, James M Wilson
Effective use of adeno-associated viruses (AAVs) for clinical gene therapy is limited by their propensity to accumulate in and transduce the liver. This natural liver tropism is associated with severe adverse events at the high doses that can be necessary for achieving therapeutic transgene expression in extrahepatic tissues. To improve the safety and cost of AAV gene therapy, capsid engineering efforts are underway to redirect in vivo AAV biodistribution away from the liver toward disease-relevant peripheral organs such as the heart. Building on previous work, we generated a series of AAV libraries containing variations at three residues (Y446, N470, and W503) of the galactose-binding pocket of the AAV9 VP1 protein. Screening of this library in mice identified the XRH family of variants (Y446X, N470R, and W503H), the strongest of which, HRH, exhibited a 6-fold reduction in liver RNA expression and a 10-fold increase in cardiac RNA expression compared with wild-type AAV9 in the mouse. Screening of our library in a nonhuman primate (NHP) revealed reduced performance of AAV9 and two closely related vectors in the NHP liver compared with the mouse liver. Measurement of the galactose-binding capacity of our library further identified those same three vectors as the only strong galactose binders, suggesting an altered galactose presentation between the mouse and NHP liver. N-glycan profiling of these tissues revealed a 9% decrease in exposed galactose in the NHP liver compared with the mouse liver. In this work, we identified a novel family of AAV variants with desirable biodistribution properties that may be suitable for targeting extrahepatic tissues such as the heart. These data also provide important insights regarding species- and tissue-specific differences in glycan presentation that may have implications for the development and translation of AAV gene therapies.
{"title":"Modulation of AAV9 Galactose Binding Yields Novel Gene Therapy Vectors and Predicts Cross-Species Differences in Glycan Avidity.","authors":"Jacob A Hoffman, Nathan Denton, Joshua J Sims, Rosemary Meggersee, Zhe Zhang, Kanyin Olagbegi, James M Wilson","doi":"10.1089/hum.2024.050","DOIUrl":"10.1089/hum.2024.050","url":null,"abstract":"<p><p>Effective use of adeno-associated viruses (AAVs) for clinical gene therapy is limited by their propensity to accumulate in and transduce the liver. This natural liver tropism is associated with severe adverse events at the high doses that can be necessary for achieving therapeutic transgene expression in extrahepatic tissues. To improve the safety and cost of AAV gene therapy, capsid engineering efforts are underway to redirect <i>in vivo</i> AAV biodistribution away from the liver toward disease-relevant peripheral organs such as the heart. Building on previous work, we generated a series of AAV libraries containing variations at three residues (Y446, N470, and W503) of the galactose-binding pocket of the AAV9 VP1 protein. Screening of this library in mice identified the XRH family of variants (Y446X, N470R, and W503H), the strongest of which, HRH, exhibited a 6-fold reduction in liver RNA expression and a 10-fold increase in cardiac RNA expression compared with wild-type AAV9 in the mouse. Screening of our library in a nonhuman primate (NHP) revealed reduced performance of AAV9 and two closely related vectors in the NHP liver compared with the mouse liver. Measurement of the galactose-binding capacity of our library further identified those same three vectors as the only strong galactose binders, suggesting an altered galactose presentation between the mouse and NHP liver. N-glycan profiling of these tissues revealed a 9% decrease in exposed galactose in the NHP liver compared with the mouse liver. In this work, we identified a novel family of AAV variants with desirable biodistribution properties that may be suitable for targeting extrahepatic tissues such as the heart. These data also provide important insights regarding species- and tissue-specific differences in glycan presentation that may have implications for the development and translation of AAV gene therapies.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141603567","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}