Molecular Therapy-Methods & Clinical Development最新文献_第10页

Characterization of Residual MicroRNAs in AAV Vector Batches Produced in HEK293 Mammalian Cells and Sf9 Insect Cells 用 HEK293 哺乳动物细胞和 Sf9 昆虫细胞生产的 AAV 向量批次中残留 MicroRNA 的特征

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-07-25 DOI: 10.1016/j.omtm.2024.101305

{"title":"Characterization of Residual MicroRNAs in AAV Vector Batches Produced in HEK293 Mammalian Cells and Sf9 Insect Cells","authors":"","doi":"10.1016/j.omtm.2024.101305","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101305","url":null,"abstract":"With more than 130 clinical trials and eight approved gene therapy products, AAVs stand as one of the most popular vehicles to deliver therapeutic DNA in vivo. One critical quality attribute analyzed in AAV batches is the presence of residual DNA, as it could pose genotoxic risks or induce immune responses. Surprisingly, the presence of small cell-derived RNAs, such as micro-RNAs, has not been previously investigated. In this study, we examined the presence of miRNAs in purified AAV batches produced in mammalian or in insect cells. Our findings revealed that miRNAs were present in all batches, regardless of the production cell line or capsid serotype (2 and 8). Quantitative assays indicated that miRNAs were co-purified with the rAAV particles in a proportion correlated with their abundance in the production cells. The level of residual miRNAs was reduced via an immunoaffinity chromatography purification process including a tangential flow filtration step or by RNase treatment, suggesting that most miRNA contaminants are likely non-encapsidated. In summary, we demonstrate, for the first time, that miRNAs are co-purified with AAV particles. Further investigations are required to determine whether these miRNAs could interfere with the safety or efficacy of AAV-mediated gene therapy.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"5 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2024-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141782918","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

An amplification-free CRISPR/Cas12a assay for titer determination and composition analysis of the rAAV genome 用于 rAAV 基因组滴度测定和成分分析的无扩增 CRISPR/Cas12a 分析法

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-07-22 DOI: 10.1016/j.omtm.2024.101304

{"title":"An amplification-free CRISPR/Cas12a assay for titer determination and composition analysis of the rAAV genome","authors":"","doi":"10.1016/j.omtm.2024.101304","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101304","url":null,"abstract":"The viral genome titer is a crucial indicator for the clinical dosing, manufacturing, and analytical testing of recombinant adeno-associated virus (rAAV) gene therapy products. Although quantitative PCR and digital PCR are the common methods used for quantifying rAAV genome titer, they are limited by inadequate accuracy and robustness. The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a biosensor is being increasingly used in virus detection; however, there is currently no report on its application in the titer determination of gene therapy products. In the present study, an amplification-free CRISPR-Cas12a assay was developed, optimized, and applied for rAAV genome titer determination. The assay demonstrated high precision and accuracy within the detection range of 4 × 109 and 1011 vg/mL. No significant difference was observed between the Cas12a and qPCR assay results (p﹤0.05, t-test). Moreover, Cas12a exhibited similar activity on both single-stranded and double-stranded DNA substrates. Based on this characteristic, the titers of positive-sense and negative-sense strands were determined separately, which revealed a significant difference between their titers for an in-house reference AAV5-IN. This study presents the inaugural report of a Cas12a assay developed for the titer determination and composition analysis of the rAAV genome.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"33 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2024-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141753988","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Atelocollagen supports three-dimensional culture of human induced pluripotent stem cells Atelocollagen 支持人类诱导多能干细胞的三维培养

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-07-20 DOI: 10.1016/j.omtm.2024.101302

{"title":"Atelocollagen supports three-dimensional culture of human induced pluripotent stem cells","authors":"","doi":"10.1016/j.omtm.2024.101302","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101302","url":null,"abstract":"As autologous iPSC therapy requires a custom-made small-lot cell production line, the cell production method differs significantly from the existing processes for producing allogeneic iPSC stocks for clinical use. Specifically, mass culture to produce stock is no longer necessary; instead, a series of operations from iPSC production to induction of differentiation of therapeutic cells must be performed continuously. A 3D culture method using small, closed-cell manufacturing devices is suitable for autologous iPSC therapy. Use of such devices avoids the need to handle many patient-derived specimens in a single clean room; handling of cell cultures in an open system in a cell processing facility increases the risk of infection. In this study, atelocollagen beads were evaluated as a three-dimensional biomaterial to assist 3D culture in the establishment, expansion culture, and induced differentiation of iPSCs. It was found that iPSCs can be handled in a closed-cell device with the same ease as use of 2D culture when Laminin-511 is added to the medium. In conclusion, atelocollagen beads enable 3D culture of iPSCs, and the quality of the obtained cells is at the same level as those derived from 2D culture.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"342 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2024-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141746186","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation 通过糖基化调节可溶性 ACE2 诱饵受体的药代动力学

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-07-19 DOI: 10.1016/j.omtm.2024.101301

{"title":"Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation","authors":"","doi":"10.1016/j.omtm.2024.101301","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101301","url":null,"abstract":"The Spike of SARS-CoV-2 recognizes a transmembrane protease, ACE2, on host cells to initiate infection. Soluble derivatives of ACE2, in which Spike affinity is enhanced and the protein is fused to Fc of an immunoglobulin, are potent decoy receptors that reduce disease in animal models of COVID-19. Mutations were introduced into an ACE2 decoy receptor, including adding custom N-glycosylation sites and a cavity-filling substitution together with Fc modifications, which increased the decoy's catalytic activity and provided small-to-moderate enhancements of pharmacokinetics following intravenous and subcutaneous administration in humanized FcRn mice. Most prominently, sialylation of native glycans increases exposures by orders of magnitude, and the optimized decoy is therapeutically efficacious in a mouse COVID-19 model. Ultimately, an engineered and highly sialylated decoy receptor produced using methods suitable for manufacture of representative drug substance has high exposure with a 5- to 9-day half-life. Finally, peptide epitopes at mutated sites in the decoys generally have low binding to common HLA class II alleles and the predicted immunogenicity risk is low. Overall, glycosylation is a critical molecular attribute of ACE2 decoy receptors and modifications that combine tighter blocking of Spike with enhanced pharmacokinetics elevate this class of molecules as viable drug candidates.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"24 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2024-07-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141746281","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Treating late-onset Tay Sachs disease: Brain delivery with a dual trojan horse protein 治疗晚期 Tay Sachs 病：用双特洛伊木马蛋白输送大脑

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-07-17 DOI: 10.1016/j.omtm.2024.101300

Esther Osher, Yossi Anis, Ruth Singer-Shapiro, Nataly Urshanski, Tamar Unger, Shira Albeck, Oren Bogin, Gary Weisinger, Fortune Kohen, Avi Valevski, Aviva Fattal-Valevski, Liora Sagi, Michal Weitman, Yulia Shenberger, Nadav Sagiv, Ruth Navon, Meir Wilchek, Naftali Stern

{"title":"Treating late-onset Tay Sachs disease: Brain delivery with a dual trojan horse protein","authors":"Esther Osher, Yossi Anis, Ruth Singer-Shapiro, Nataly Urshanski, Tamar Unger, Shira Albeck, Oren Bogin, Gary Weisinger, Fortune Kohen, Avi Valevski, Aviva Fattal-Valevski, Liora Sagi, Michal Weitman, Yulia Shenberger, Nadav Sagiv, Ruth Navon, Meir Wilchek, Naftali Stern","doi":"10.1016/j.omtm.2024.101300","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101300","url":null,"abstract":"Tay-Sachs (TS) disease is a neurodegenerative disease resulting from mutations in the gene encoding the α-subunit (HEXA) of lysosomal β-hexosaminidase A (HexA). We report that (1) recombinant HEXA alone increased HexA activity and decreased GM2 content in human TS glial cells and peripheral mononuclear blood cells; 2) a recombinant chimeric protein composed of HEXA linked to two blood-brain barrier (BBB) entry elements, a transferrin receptor binding sequence and granulocyte-colony stimulating factor, associates with HEXB ; reaches human cultured TS cells lysosomes and mouse brain cells, especially neurons, ; lowers GM2 in cultured human TS cells; lowers whole brain GM2 concentration by approximately 40% within 6 weeks, when injected intravenously (IV) to adult TS-mutant mice mimicking the slow course of late-onset TS; and increases forelimbs grip strength. Hence, a chimeric protein equipped with dual BBB entry elements can transport a large protein such as HEXA to the brain, decrease the accumulation of GM2, and improve muscle strength, thereby providing potential treatment for late-onset TS.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"39 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2024-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141782921","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Toward a large-batch manufacturing process for silicon-stabilized lipid nanoparticles: A highly customizable RNA delivery platform 硅稳定脂质纳米颗粒的大批量制造工艺：高度可定制的 RNA 运送平台

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-07-17 DOI: 10.1016/j.omtm.2024.101299

Suzanne Saffie-Siebert, Nissim Torabi-Pour, Andrew Gibson, Flavia Maria Sutera, Ashkan Dehsorkhi, Paulina Baran-Rachwalska, Skye Quinn

{"title":"Toward a large-batch manufacturing process for silicon-stabilized lipid nanoparticles: A highly customizable RNA delivery platform","authors":"Suzanne Saffie-Siebert, Nissim Torabi-Pour, Andrew Gibson, Flavia Maria Sutera, Ashkan Dehsorkhi, Paulina Baran-Rachwalska, Skye Quinn","doi":"10.1016/j.omtm.2024.101299","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101299","url":null,"abstract":"While lipid nanoparticles (LNPs) are a key enabling technology for RNA-based therapeutics, some outstanding challenges hinder their wider clinical translation and use, particularly in terms of RNA stability and limited shelf life. In response to these limitations, we developed silicon-stabilized hybrid lipid nanoparticles (sshLNPs) as a next-generation nanocarrier with improved physical and temperature stability, as well as the highly advantageous capacity for “post-hoc loading” of RNA. Nevertheless, previously reported sshLNP formulations were produced using lipid thin film hydration, making scale-up impractical. To realize the potential of this emerging delivery platform, a manufacturing process enabling multikilogram batch sizes was required for successful clinical translation and deployment at scale. This was achieved by developing a revised protocol based on solvent injection mixing and incorporating other process adjustments to enable in-flow extrusion of multiliter volumes, while ensuring sshLNPs with the desired characteristics. Optimized procedures for nanoparticle formation, extrusion, and tangential flow filtration (to remove residual organic solvent) currently enable production of 2 kg finished batches. Importantly, sshLNPs produced via the modified large-scale workflow show equivalent physical and functional properties to those derived from the earlier small-scale methods, paving the way for GMP manufacturing protocols to enable vital translational clinical studies.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"40 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2024-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141782920","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Subacute liver injury in two young infants following gene replacement therapy for spinal muscular atrophy 两名婴儿在接受脊髓性肌萎缩症基因替代疗法后出现亚急性肝损伤

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-07-02 DOI: 10.1016/j.omtm.2024.101296

Cassie Ables, Catalina Jaramillo, E. Lynne Wood, Sara Stern, Mouied Alashari, Linda Book, Russell J. Butterfield

{"title":"Subacute liver injury in two young infants following gene replacement therapy for spinal muscular atrophy","authors":"Cassie Ables, Catalina Jaramillo, E. Lynne Wood, Sara Stern, Mouied Alashari, Linda Book, Russell J. Butterfield","doi":"10.1016/j.omtm.2024.101296","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101296","url":null,"abstract":"Spinal muscular atrophy is a neurodegenerative disorder resulting from the irreversible loss of anterior horn cells secondary to homozygous mutations in the survival motor neuron gene . Gene replacement therapy using a recombinant adeno-associated virus 9 vector containing an gene construct, onasemnogene abeparvovec-xioi, was approved by the US Food and Drug Administration in May 2019. Subacute mild elevation of liver function tests following infusion has since been shown to be a common adverse event. Additionally, there have been case reports of liver failure following administration of this therapy and two reported patient deaths. While these adverse events are relatively common, they have not been reported in the youngest treated patients. We present two cases of subacute severe elevation of liver function tests >10–20 times the upper limit of normal, without progression to liver failure, following onasemnogene abeparvovec administration in young infants less than 4 weeks old. Potential mechanisms of injury, management, and implications for future treatment with onasemnogene abeparvovec and other adeno-associated virus vector gene therapies are discussed.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"52 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141586163","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A self-complementary AAV proviral plasmid system to reduce aberrant cross-packaging and ITR promoter activity in AAV vector preparations 减少 AAV 载体制备中异常交叉包装和 ITR 启动子活性的自补体 AAV 后裔质粒系统

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-06-26 DOI: 10.1016/j.omtm.2024.101295

Noah K. Taylor, Matthew J. Guggenbiller, Pranali P. Mistry, Oliver D. King, Scott Q. Harper

{"title":"A self-complementary AAV proviral plasmid system to reduce aberrant cross-packaging and ITR promoter activity in AAV vector preparations","authors":"Noah K. Taylor, Matthew J. Guggenbiller, Pranali P. Mistry, Oliver D. King, Scott Q. Harper","doi":"10.1016/j.omtm.2024.101295","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101295","url":null,"abstract":"Adeno-associated viral vectors (AAVs) are a leading delivery system for gene therapy in animal models and humans. With several Food and Drug Administration-approved AAV gene therapies on the market, issues related to vector manufacturing have become increasingly important. In this study, we focused on potentially toxic DNA contaminants that can arise from AAV proviral plasmids, the raw materials required for manufacturing recombinant AAV in eukaryotic cells. Typical AAV proviral plasmids are circular DNAs containing a therapeutic gene cassette flanked by natural AAV inverted terminal repeat (ITR) sequences, and a plasmid backbone carrying prokaryotic sequences required for plasmid replication and selection in bacteria. While the majority of AAV particles package the intended therapeutic payload, some capsids instead package the bacterial sequences located on the proviral plasmid backbone. Since ITR sequences also have promoter activity, potentially toxic bacterial open reading frames can be produced , thereby representing a safety risk. In this study, we describe a new AAV proviral plasmid for vector manufacturing that (1) significantly decreases cross-packaged bacterial sequences, (2) increases correctly packaged AAV payloads, and (3) blunts ITR-driven transcription of cross-packaged material to avoid expressing potentially toxic bacterial sequences. This system may help improve the safety of AAV vector products.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"45 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2024-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141552022","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

The longitudinal kinetics of AAV5 vector integration profiles and evaluation of clonal expansion in mice 小鼠 AAV5 向量整合的纵向动力学特征和克隆扩增评估

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-06-26 DOI: 10.1016/j.omtm.2024.101294

Ashrafali Mohamed Ismail, Evan Witt, Taren Bouwman, Wyatt Clark, Bridget Yates, Matteo Franco, Sylvia Fong

{"title":"The longitudinal kinetics of AAV5 vector integration profiles and evaluation of clonal expansion in mice","authors":"Ashrafali Mohamed Ismail, Evan Witt, Taren Bouwman, Wyatt Clark, Bridget Yates, Matteo Franco, Sylvia Fong","doi":"10.1016/j.omtm.2024.101294","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101294","url":null,"abstract":"Adeno-associated virus (AAV)-based vectors are used clinically for gene transfer and persist as extrachromosomal episomes. A small fraction of vector genomes integrate into the host genome, but the theoretical risk of tumorigenesis depends on vector regulatory features. A mouse model was used to investigate integration profiles of an AAV serotype 5 (AAV5) vector produced using and HEK293 cells that mimic key features of valoctocogene roxaparvovec (AAV5-hFVIII-SQ), a gene therapy for severe hemophilia A. The majority (95%) of vector genome reads were derived from episomes, and mean (± standard deviation) integration frequency was 2.70 ± 1.26 and 1.79 ± 0.86 integrations per 1,000 cells for - and HEK293-produced vector. Longitudinal integration analysis suggested integrations occur primarily within 1 week, at low frequency, and their abundance was stable over time. Integration profiles were polyclonal and randomly distributed. No major differences in integration profiles were observed for either vector production platform, and no integrations were associated with clonal expansion. Integrations were enriched near transcription start sites of genes highly expressed in the liver ( = 1 × 10) and less enriched for genes of lower expression. We found no evidence of tumorigenesis or fibrosis caused by the vector integrations.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"54 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2024-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141552028","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Probing recombinant AAV capsid integrity and genome release after thermal stress by mass photometry 用质量光度法检测重组 AAV 包囊的完整性和热应力后的基因组释放情况

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-06-24 DOI: 10.1016/j.omtm.2024.101293

Eduard H.T.M. Ebberink, Alisa Ruisinger, Markus Nuebel, Helena Meyer-Berg, Irene R.S. Ferreira, Marco Thomann, Albert J.R. Heck

{"title":"Probing recombinant AAV capsid integrity and genome release after thermal stress by mass photometry","authors":"Eduard H.T.M. Ebberink, Alisa Ruisinger, Markus Nuebel, Helena Meyer-Berg, Irene R.S. Ferreira, Marco Thomann, Albert J.R. Heck","doi":"10.1016/j.omtm.2024.101293","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101293","url":null,"abstract":"Adeno-associated viruses (AAVs) are gaining traction as delivery vehicles for gene therapy although the molecular understanding of AAV-transgene release is still limited. Typically, the process of viral uncoating is investigated () through thermal stress, revealing capsid disintegration at elevated temperatures. To assess the (in)stability of different empty and filled AAV preparations, we used the light-scattering-based interferometric microscopy technique of mass photometry that, on a single-particle basis, determines the molecular weight of AAVs. By introducing a heat-stable DNA plasmid as an internal standard, we quantitatively probed the impact of heat on AAVs. Generally, empty AAVs exhibited greater heat resistance than genome-filled particles. Our data also indicate that upon DNA release, the capsids do not transform into empty AAVs, but seem to aggregate or disintegrate. Strikingly, some AAVs exhibited an intermediate state with disrupted capsids but preserved bound genome, a feature that experimentally only emerged following incubation with a nuclease. Our data demonstrate that the thermal uncoating process is highly AAV specific (i.e., can be influenced by serotype, genome, host system). We argue that nuclease treatment in combination with MP can be used as an additional analytical tool for assessing structural integrity of recombinant and/or clinical AAV vectors.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"49 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2024-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141552023","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0