Molecular Therapy-Methods & Clinical Development最新文献_第2页

E2A, VA RNA I, and L4-22k adenoviral helper genes are sufficient for AAV production in HEK293 cells. E2A、VA RNA I和L4-22k腺病毒辅助基因足以在HEK293细胞中产生AAV。

IF 4.6 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-11-13 eCollection Date: 2024-12-12 DOI: 10.1016/j.omtm.2024.101376

Jiten Doshi, Emma Couto, Jillian Staiti, Luk H Vandenberghe, Nerea Zabaleta

{"title":"E2A, VA RNA I, and L4-22k adenoviral helper genes are sufficient for AAV production in HEK293 cells.","authors":"Jiten Doshi, Emma Couto, Jillian Staiti, Luk H Vandenberghe, Nerea Zabaleta","doi":"10.1016/j.omtm.2024.101376","DOIUrl":"10.1016/j.omtm.2024.101376","url":null,"abstract":"The replication-defective adeno-associated virus (AAV) is extensively utilized as a research tool or vector for gene therapy. The production process of AAV remains intricate, expensive, and mechanistically underexplored. With the aim of enhancing AAV manufacturing efficiencies in mammalian cells, we revisited the questions and optimization surrounding the requirement of the various adenoviral helper genes in enabling AAV production. First, we refined the minimal set of adenoviral genes in HEK293 AAV production to E2A, L4-22 K /33 K, and VA RNA I. These findings challenge the previously accepted necessity of adenoviral E4orf6 in AAV production. In addition, we identified L4-22 K genes as crucial helpers for AAV production. Next, a revised minimal adenoviral helper plasmid comprising E2A, L4-22 K, and VA RNA I genes was designed and demonstrated to yield high titer and potent AAV preps in HEK293 transient transfection. Lastly, stable packaging cells harboring inducible E2A and L4-22 K genes were shown to maintain AAV production yields comparable to transient transfection over a culture period of ∼10 weeks. Combined, these findings further our understanding of adenoviral helper function in mammalian AAV production and provide novel plasmid and cell-line reagents with an improved safety profile for potential broad applicability in the research and gene therapy community.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"32 4","pages":"101376"},"PeriodicalIF":4.6,"publicationDate":"2024-11-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11635002/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142820145","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

αFAP-specific nanobodies mediate a highly precise retargeting of modified AAV2 capsids thereby enabling specific transduction of tumor tissues. αFAP特异性纳米抗体可高度精确地再靶向修饰过的AAV2包囊，从而实现对肿瘤组织的特异性转导。

IF 4.6 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-11-12 eCollection Date: 2024-12-12 DOI: 10.1016/j.omtm.2024.101378

Olaniyi Olarewaju, Franziska Held, Pamela Curtis, Cynthia Hess Kenny, Udo Maier, Tadas Panavas, Francois du Plessis

{"title":"αFAP-specific nanobodies mediate a highly precise retargeting of modified AAV2 capsids thereby enabling specific transduction of tumor tissues.","authors":"Olaniyi Olarewaju, Franziska Held, Pamela Curtis, Cynthia Hess Kenny, Udo Maier, Tadas Panavas, Francois du Plessis","doi":"10.1016/j.omtm.2024.101378","DOIUrl":"10.1016/j.omtm.2024.101378","url":null,"abstract":"Due to the refractiveness of tumor tissues to adeno-associated virus (AAV) transduction, AAV vectors are poorly explored for cancer therapy delivery. Here, we aimed to engineer AAVs to target tumors by enabling the specific engagement of fibroblast activation protein (FAP). FAP is a cell surface receptor distinctly upregulated in the reactive tumor stroma, but rarely expressed in healthy tissues. Thus, targeting FAP presents an opportunity to selectively transduce tumor tissues. To achieve this, we modified the capsid surface of AAV2 with an αFAP nanobody to retarget the capsid to engage FAP receptor. Following transduction, we observed a 23- to 80-fold increase in the selective transduction of FAP+ tumor cells in vitro, and greater than 5-fold transduction of FAP+ tumor tissues in vivo. Subsequent optimization of the VP1-nanobody expression cassette further enhanced the transduction efficiency of the modified capsids. Due to the limited αFAP nanobodies repertoires, we broadened the versatility of this high-fidelity platform by screening a naive VHH yeast display library, leading to the identification of several novel αFAP nanobody candidates (KD = 0.1 to >100 nM). Hence, our study offers new opportunity for the application of AAV vectors for highly selective delivery of therapeutics to the tumor stroma.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"32 4","pages":"101378"},"PeriodicalIF":4.6,"publicationDate":"2024-11-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11655695/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142866294","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

CAR-NK cells derived from cord blood originate mainly from CD56^-CD7⁺CD34^-HLA-DR^-Lin^- NK progenitor cells. 来源于脐带血的CAR-NK细胞主要来源于CD56-CD7+CD34-HLA-DR-Lin- NK祖细胞。

IF 4.6 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-11-12 eCollection Date: 2024-12-12 DOI: 10.1016/j.omtm.2024.101374

Tansri Wibowo, Yosuke Kogue, Shunya Ikeda, Moto Yaga, Mana Tachikawa, Makiko Suga, Shuhei Kida, Kumi Shibata, Kazuhito Tsutsumi, Hiraku Murakami, Yasutaka Ueda, Hisashi Kato, Kentaro Fukushima, Jiro Fujita, Tomoaki Ueda, Shinsuke Kusakabe, Akihisa Hino, Michiko Ichii, Chihaya Imai, Daisuke Okuzaki, Atsushi Kumanogoh, Naoki Hosen

{"title":"CAR-NK cells derived from cord blood originate mainly from CD56-CD7+CD34-HLA-DR-Lin- NK progenitor cells.","authors":"Tansri Wibowo, Yosuke Kogue, Shunya Ikeda, Moto Yaga, Mana Tachikawa, Makiko Suga, Shuhei Kida, Kumi Shibata, Kazuhito Tsutsumi, Hiraku Murakami, Yasutaka Ueda, Hisashi Kato, Kentaro Fukushima, Jiro Fujita, Tomoaki Ueda, Shinsuke Kusakabe, Akihisa Hino, Michiko Ichii, Chihaya Imai, Daisuke Okuzaki, Atsushi Kumanogoh, Naoki Hosen","doi":"10.1016/j.omtm.2024.101374","DOIUrl":"10.1016/j.omtm.2024.101374","url":null,"abstract":"Cord blood (CB)-derived chimeric antigen receptor (CAR)-natural killer (NK) cells targeting CD19 have been shown to be effective against B cell malignancies. While human CD56+ NK cells can be expanded in vitro, NK cells can also be differentiated from hematopoietic progenitor cells. It is still unclear whether CAR-NK cells originate from mature NK cells or NK progenitor cells in CB. Here, we determined that CAR-NK cells were predominantly derived from CD56- NK progenitor cells. We first found that substantial numbers of CD19 CAR-NK cells were produced from CD56- CB mononuclear cells after in vitro culture for 2 weeks. Single-cell RNA sequencing analysis of CD56-CD3-CD14-CD19- CB mononuclear cells revealed that these cells could be subdivided into three subpopulations based on the expression of CD34 and human leukocyte antigen (HLA)-DR. NK cells originated primarily from CD34-HLA-DR- cells. In addition, among the CD34-HLA-DR- cells, only CD7+ cells could differentiate into NK cells. These results indicate that CD56-CD7+CD34-HLA-DR- lineage marker (Lin)- cells are the major origin of human CB-derived CAR-NK cells, indicating the importance of developing methods to enhance the quality and quantity of NK cells produced from these NK progenitor cells.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"32 4","pages":"101374"},"PeriodicalIF":4.6,"publicationDate":"2024-11-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11629225/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142807719","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Preclinical efficacy and safety of adeno-associated virus 5 alpha-galactosidase: A gene therapy for Fabry disease. 腺相关病毒 5 α-半乳糖苷酶的临床前疗效和安全性：法布里病的基因疗法。

IF 4.6 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-11-12 eCollection Date: 2024-12-12 DOI: 10.1016/j.omtm.2024.101375

Jolanda M P Liefhebber, Giso Brasser, Elisabeth A Spronck, Roelof Ottenhoff, Lieke Paerels, Maria J Ferraz, Lukas K Schwarz, Nikoleta Efthymiopoulou, Chi-Lin Kuo, Paula S Montenegro-Miranda, Melvin M Evers, Johannes M F G Aerts, Ying Poi Liu

{"title":"Preclinical efficacy and safety of adeno-associated virus 5 alpha-galactosidase: A gene therapy for Fabry disease.","authors":"Jolanda M P Liefhebber, Giso Brasser, Elisabeth A Spronck, Roelof Ottenhoff, Lieke Paerels, Maria J Ferraz, Lukas K Schwarz, Nikoleta Efthymiopoulou, Chi-Lin Kuo, Paula S Montenegro-Miranda, Melvin M Evers, Johannes M F G Aerts, Ying Poi Liu","doi":"10.1016/j.omtm.2024.101375","DOIUrl":"10.1016/j.omtm.2024.101375","url":null,"abstract":"We developed a novel adeno-associated virus 5 gene therapy (AAV5-GLA) expressing human alpha-galactosidase A (GLA) under the control of a novel, small and strong, liver-restricted promoter. We assessed the preclinical potential of AAV5-GLA for treating Fabry disease, an X-linked hereditary metabolic disorder resulting from mutations in the gene encoding GLA that lead to accumulation of the substrates globotriaosylceramide and globotriaosylsphingosine, causing heart, kidney, and central nervous system dysfunction. Effects of intravenously administered AAV5-GLA were evaluated in (1) GLA-knockout mice aged 7-8 weeks (early in disease) and 20 weeks (nociception phenotype manifestation) and (2) cynomolgus macaques during an 8-week period. In both species, AAV5-GLA was observed as safe, generated detectable vector DNA and mRNA levels in liver, and produced stable enzyme activity in liver and plasma. In mice, dose-dependent transgene enzyme activity, cross-correction (substrate reduction) in kidney and heart, and improved nociception lasted over 6 months. Moreover, after delayed administration when animals displayed the nociception phenotype, target organ enzyme activity was present, and accumulated substrates were reduced. Given the strong, durable expression of active GLA with this promoter and favorable profile of adeno-associated virus 5-based gene therapy in humans, AAV5-GLA warrants further investigation in clinical trials for Fabry disease.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"32 4","pages":"101375"},"PeriodicalIF":4.6,"publicationDate":"2024-11-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11646755/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142839256","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Structural characterization and epitope mapping of the AAVX affinity purification ligand. AAVX 亲和纯化配体的结构特征和表位图谱。

IF 4.6 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-11-12 eCollection Date: 2024-12-12 DOI: 10.1016/j.omtm.2024.101377

Mario Mietzsch, Manasi Kamat, Kari Basso, Paul Chipman, Juha T Huiskonen, Robert McKenna

{"title":"Structural characterization and epitope mapping of the AAVX affinity purification ligand.","authors":"Mario Mietzsch, Manasi Kamat, Kari Basso, Paul Chipman, Juha T Huiskonen, Robert McKenna","doi":"10.1016/j.omtm.2024.101377","DOIUrl":"10.1016/j.omtm.2024.101377","url":null,"abstract":"The application of adeno-associated virus (AAV) vectors in human gene therapies requires reproducible and homogeneous preparations for clinical efficacy and safety. For the AAV production process, often scalable affinity chromatography columns are utilized, such as the POROS CaptureSelect AAVX affinity resin, during downstream processing to ensure highly purified AAV vectors. The AAVX ligand is based on a camelid single-domain antibody capturing a wide range of recombinant AAV capsids. Described here is the identification of the AAV8 capsid epitope to AAVX at 2.3 Å resolution using cryo-electron microscopy. The ligand binds near the 5-fold axis of the capsid in a similar manner to the previously characterized AVB affinity ligand but does not conform to the capsid's icosahedral symmetry. The cross-reactivity of AAVX to other AAV capsids is achieved by primarily interacting with the peptide backbone of the AAV capsid's structurally conserved DE and HI loops. These observations will guide AAV capsid engineering efforts to retain the ability of future recombinant capsid designs to be purified using antibody-based affinity ligands.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"32 4","pages":"101377"},"PeriodicalIF":4.6,"publicationDate":"2024-11-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11638594/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142830827","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Exogenous expression of ATP8, a mitochondrial encoded protein, from the nucleus in vivo. 线粒体编码蛋白ATP8在体内的外源表达。

IF 4.6 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-11-06 eCollection Date: 2024-12-12 DOI: 10.1016/j.omtm.2024.101372

David V Begelman, Bhavna Dixit, Carly Truong, Christina D King, Mark A Watson, Birgit Schilling, Martin D Brand, Amutha Boominathan

{"title":"Exogenous expression of ATP8, a mitochondrial encoded protein, from the nucleus in vivo.","authors":"David V Begelman, Bhavna Dixit, Carly Truong, Christina D King, Mark A Watson, Birgit Schilling, Martin D Brand, Amutha Boominathan","doi":"10.1016/j.omtm.2024.101372","DOIUrl":"10.1016/j.omtm.2024.101372","url":null,"abstract":"Replicative errors, inefficient repair, and proximity to sites of reactive oxygen species production make mitochondrial DNA (mtDNA) susceptible to damage with time. We explore in vivo allotopic expression (re-engineering mitochondrial genes and expressing them from the nucleus) as an approach to rescue defects arising from mtDNA mutations. We used a mouse strain C57BL/6J(mtFVB) with a natural polymorphism (m.7778 G>T) in the mitochondrial ATP8 gene that encodes a protein subunit of the ATP synthase. We generated a transgenic mouse with an epitope-tagged recoded mitochondrial-targeted ATP8 gene expressed from the ROSA26 locus in the nucleus and used the C57BL/6J(mtFVB) strain to verify successful incorporation. The allotopically expressed ATP8 protein in transgenic mice was constitutively expressed across all tested tissues, successfully transported into the mitochondria, and incorporated into ATP synthase. The ATP synthase with transgene had similar activity to non-transgenic control, suggesting successful integration and function. Exogenous ATP8 protein had no negative impact on measured mitochondrial function, metabolism, or behavior. Successful allotopic expression of a mitochondrially encoded protein in vivo in a mammal is a step toward utilizing allotopic expression as a gene therapy in humans to repair physiological consequences of mtDNA defects that may accumulate in congenital mitochondrial diseases or with age.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"32 4","pages":"101372"},"PeriodicalIF":4.6,"publicationDate":"2024-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11629202/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142807731","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Comparative assessment of the transduction efficiency and safety associated with the delivery of AAV9-GFP vector via lumbar puncture to cynomolgus macaques with and without anti-AAV9 pre-existing antibodies. AAV9-GFP载体经腰椎穿刺传递给具有和不具有抗aav9抗体的食蟹猴的转导效率和安全性的比较评估。

IF 4.6 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-11-06 eCollection Date: 2024-12-12 DOI: 10.1016/j.omtm.2024.101371

Ghiabe H Guibinga, Janet Do, Binh Chu, Yin Gu, Rie Kikkawa, Xiaoguang Li, Fatih Ozsolak, Timothy MacLachlan

{"title":"Comparative assessment of the transduction efficiency and safety associated with the delivery of AAV9-GFP vector via lumbar puncture to cynomolgus macaques with and without anti-AAV9 pre-existing antibodies.","authors":"Ghiabe H Guibinga, Janet Do, Binh Chu, Yin Gu, Rie Kikkawa, Xiaoguang Li, Fatih Ozsolak, Timothy MacLachlan","doi":"10.1016/j.omtm.2024.101371","DOIUrl":"10.1016/j.omtm.2024.101371","url":null,"abstract":"Administration of AAV-based gene therapies into the intra-cerebrospinal fluid (CSF) compartments via routes such as lumbar puncture (LP) has been implemented as an alternative to intravenous dosing to target the CNS regions. This route enables lower doses, decreases systemic toxicity, and circumvents intravascular pre-existing anti-AAV antibodies. In this study, AAV9-GFP vectors were administered via LP to juvenile cynomolgus macaques with and without pre-existing serum anti-AAV9 antibodies at a 5.0 × 1013 vector genomes per mL (vg/mL) dose and examined for 28 days. CNS and peripheral tissues were surveyed for vector genome, mRNA, and protein expression. Histopathology, clinical pathology, and humoral immune response to the viral capsid and transgene were also assessed. In addition, serum and CSF samples were analyzed to examine 276 proteomic markers curated to evaluate neural injury, organ damage, and inflammatory response. This study reveals no noticeable difference in AAV9-mediated gene transfer in the CNS tissues in the two groups; however, differences were observed for endpoints such as liver enzyme activities, histopathology, and levels of protein markers in the serum and CSF. These findings provide a view into vector transduction efficiency and safety following LP-delivered AAV9 to juvenile cynomolgus macaques with and without pre-existing anti-AAV9 antibodies.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"32 4","pages":"101371"},"PeriodicalIF":4.6,"publicationDate":"2024-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11664412/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142883091","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Generation and maintenance of kidney and kidney cancer organoids from patient-derived material for drug development and precision oncology. 从患者来源的材料中生成和维持肾脏和肾癌类器官，用于药物开发和精确肿瘤学。

IF 4.6 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-11-05 eCollection Date: 2024-12-12 DOI: 10.1016/j.omtm.2024.101368

Jakub Gubala, Valentin Mieville, Daniel Benamran, Jean-Christophe Tille, Massimo Valerio, Patrycja Nowak-Sliwinska

引用次数: 0

Intrathecal or intravenous AAV9-IDUA/RGX-111 at minimal effective dose prevents cardiac, skeletal and neurologic manifestations of murine MPS I. 鞘内或静脉注射最小有效剂量的AAV9-IDUA/RGX-111可预防小鼠MPS I的心脏、骨骼和神经表现。

IF 4.6 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-11-04 eCollection Date: 2024-12-12 DOI: 10.1016/j.omtm.2024.101369

Lalitha R Belur, Avery K Huber, Hillary Mantone, Mason Robertson, Miles C Smith, Andrea D Karlen, Kelley F Kitto, Li Ou, Chester B Whitley, Elizabeth Braunlin, Justin Furcich, Troy C Lund, Davis Seelig, Carolyn A Fairbanks, Nicholas Buss, Kwi Hye Kim, R Scott McIvor

{"title":"Intrathecal or intravenous AAV9-IDUA/RGX-111 at minimal effective dose prevents cardiac, skeletal and neurologic manifestations of murine MPS I.","authors":"Lalitha R Belur, Avery K Huber, Hillary Mantone, Mason Robertson, Miles C Smith, Andrea D Karlen, Kelley F Kitto, Li Ou, Chester B Whitley, Elizabeth Braunlin, Justin Furcich, Troy C Lund, Davis Seelig, Carolyn A Fairbanks, Nicholas Buss, Kwi Hye Kim, R Scott McIvor","doi":"10.1016/j.omtm.2024.101369","DOIUrl":"10.1016/j.omtm.2024.101369","url":null,"abstract":"Mucopolysaccharidosis type I (MPS I) is a rare metabolic disorder caused by deficiency of α-L-iduronidase (IDUA), resulting in glycosaminoglycan (GAG) accumulation and multisystemic disease. Current treatments include hematopoietic stem cell transplantation and enzyme replacement therapy, but these do not address all manifestations of the disease. We infused MPS I mice with an adeno-associated virus 9 (AAV9)-IDUA vector (RGX-111) at doses from 107 to 1010 vector genomes (vg) via intrathecal (IT), intravenous (IV), and intrathecal+intravenous (IT+IV) routes of administration. In mice administered doses ≤109 vg IT or ≤108 vg IV, there was no therapeutic benefit, while in mice administered 109 vg IV, there was a variable increase in IDUA activity with inconclusive neurocognitive and cardiac assessments. However, at the 1010 vg dose, we observed substantial metabolic correction, with restored IDUA levels and normalized tissue GAGs for all treatment groups. Aortic insufficiency was mostly normalized, neurologic deficit was prevented, and microcomputed tomography (micro-CT) analysis showed normalization of skeletal parameters. Histologic analysis showed minimal GAG storage and lysosomal pathology. We thus report a minimal effective dose of 1010 vg (5 × 1011 per kg) RGX-111 for IV and IT routes of administration in MPS I mice, which prevented neurocognitive deficit, cardiac insufficiency, and skeletal manifestations, as a model for genetic therapy of human MPS I.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"32 4","pages":"101369"},"PeriodicalIF":4.6,"publicationDate":"2024-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11646787/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142840384","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Comprehensive analysis of off-target and on-target effects resulting from liver-directed CRISPR-Cas9-mediated gene targeting with AAV vectors. 肝靶向crispr - cas9介导的AAV载体基因靶向的脱靶和靶标效应综合分析

IF 4.6 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-11-04 eCollection Date: 2024-12-12 DOI: 10.1016/j.omtm.2024.101365

Kshitiz Singh, Raffaele Fronza, Hanneke Evens, Marinee K Chuah, Thierry VandenDriessche

{"title":"Comprehensive analysis of off-target and on-target effects resulting from liver-directed CRISPR-Cas9-mediated gene targeting with AAV vectors.","authors":"Kshitiz Singh, Raffaele Fronza, Hanneke Evens, Marinee K Chuah, Thierry VandenDriessche","doi":"10.1016/j.omtm.2024.101365","DOIUrl":"10.1016/j.omtm.2024.101365","url":null,"abstract":"Comprehensive genome-wide studies are needed to assess the consequences of adeno-associated virus (AAV) vector-mediated gene editing. We evaluated CRISPR-Cas-mediated on-target and off-target effects and examined the integration of the AAV vectors employed to deliver the CRISPR-Cas components to neonatal mice livers. The guide RNA (gRNA) was specifically designed to target the factor IX gene (F9). On-target and off-target insertions/deletions were examined by whole-genome sequencing (WGS). Efficient F9-targeting (36.45% ± 18.29%) was apparent, whereas off-target events were rare or below the WGS detection limit since only one single putative insertion was detected out of 118 reads, based on >100 computationally predicted off-target sites. AAV integrations were identified by WGS and shearing extension primer tag selection ligation-mediated PCR (S-EPTS/LM-PCR) and occurred preferentially in CRISPR-Cas9-induced double-strand DNA breaks in the F9 locus. In contrast, AAV integrations outside F9 were not in proximity to any of ∼5,000 putative computationally predicted off-target sites (median distance of 70 kb). Moreover, without relying on such off-target prediction algorithms, analysis of DNA sequences close to AAV integrations outside the F9 locus revealed no homology to the F9-specific gRNA. This study supports the use of S-EPTS/LM-PCR for direct in vivo comprehensive, sensitive, and unbiased off-target analysis.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"32 4","pages":"101365"},"PeriodicalIF":4.6,"publicationDate":"2024-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11626537/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142803323","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0