Article
Virology
Anshuman Das, Madhuvanthi Vijayan, Eric M. Walton, V. Grace Stafford, David N. Fiflis, Aravind Asokan
Summary: This study provides evidence that the double-stranded DNA binding protein NP220, in association with the human silencing hub (HUSH) complex, mediates transcriptional silencing of single-stranded as well as self-comple- mentary rAAV genomes. The AAV capsid appears to play an important role in NP220-mediated silencing of packaged genomes. Modulation of epigenetic pathways could potentially improve rAAV expression.
JOURNAL OF VIROLOGY
(2022)
Article
Biochemistry & Molecular Biology
Melad Farraha, Renuka Rao, Sindhu Igoor, Thi Y. L. Le, Michael A. Barry, Christopher Davey, Cindy Kok, James J. H. Chong, Eddy Kizana
Summary: This study aimed to develop a biological alternative to electronic pacemakers by using gene therapy to convert cardiomyocytes into sinoatrial node-like cells. The results showed that the transduction of hTBX18 gene resulted in morphological, molecular, physiological, and functional changes in the cardiomyocytes, recapitulating the pacemaker phenotype. The use of a clinically relevant vector opens new prospects for the development of biological pacemakers.
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2022)
Article
Multidisciplinary Sciences
Weiheng Su, Maria I. Patricio, Margaret R. Duffy, Jakub M. Krakowiak, Leonard W. Seymour, Ryan Cawood
Summary: The authors describe a self-inhibiting helper plasmid strategy that improves outcomes compared to helper-free approaches.
NATURE COMMUNICATIONS
(2022)
Article
Biotechnology & Applied Microbiology
Zion Lee, Min Lu, Eesha Irfanullah, Morgan Soukup, Daniel Schmidt, Wei-Shou Hu
Summary: This study describes a stable cell line that can be used to quantify the titers of infectious recombinant adeno-associated viruses (rAAVs). It has high sensitivity and eliminates the need for a helper virus, improving assay consistency and allowing high-throughput applications. This cell line can serve as an effective tool for quantifying infectious rAAV titers to advance gene therapy vector biomanufacturing.
HUMAN GENE THERAPY
(2023)
Article
Biotechnology & Applied Microbiology
Ngoc Tam Tran, Emilie Lecomte, Sylvie Saleun, Suk Namkung, Cecile Robin, Kristina Weber, Eric Devine, Veronique Blouin, Oumeya Adjali, Eduard Ayuso, Guangping Gao, Magalie Penaud-Budloo, Phillip W. L. Tai
Summary: Over the past two decades, significant progress has been made in the manufacturing of adeno-associated virus (AAV) vectors to meet the production demands for preclinical and clinical trials. However, the presence of empty AAV capsids and particles containing inaccurate vector genomes remains a concern. Different methods have been utilized to separate empty capsids from full particles, but no single technique can completely eliminate empty or partial capsids. This study reveals that vectors produced by different manufacturing systems exhibit varying degrees of truncated and unresolved species, and purified empty particles actually contain genomes composed of a single truncated and/or unresolved inverted terminal repeat (ITR). These findings provide valuable insights into the efficacy, safety, and quantification of clinical vectors.
HUMAN GENE THERAPY
(2022)
Article
Biotechnology & Applied Microbiology
Riona Hatazawa, Saori Fukuda, Kanako Kumamoto, Fumio Matsushita, Shizuko Nagao, Takayuki Murata, Koki Taniguchi, Taei Matsui, Satoshi Komoto
Summary: In this study, multiple foreign genes were successfully inserted into a single gene segment of rotavirus to determine its efficiency in expressing exogenous genes from its genome. By engineering a truncated NSP1 segment platform, a recombinant rotavirus stably expressing NanoLuc luciferase as a model foreign gene was generated, followed by the demonstration of a replication-competent rotavirus expressing three reporter genes. These results highlight the potential of rotaviruses as expression vectors for multiple foreign genes.
JOURNAL OF GENERAL VIROLOGY
(2021)
Article
Virology
Seyyed Mehdy Elahi, Jennifer Jiang, Nazila Nazemi-Moghaddam, Renald Gilbert
Summary: In this study, researchers developed a method to generate and produce adenovirus vectors (AdVs) without the need for fetal bovine serum (FBS). By using a serum-free medium and a limiting dilution method, they were able to eliminate FBS from the production process and demonstrated that the purified AdV stock was free of replication-competent adenovirus (RCA). The limiting dilution method was also effective in rescuing AdV from highly contaminated stocks.
Article
Chemistry, Medicinal
Salama Ramy, Yuki Ueda, Hiroyuki Nakajima, Miya Hiroi, Yoshiomi Hiroi, Tetsuo Torisu, Susumu Uchiyama
Summary: In this study, a polyionic hydrophilic complex polymer coating was applied to the surfaces of tools used in viral vector quantification analyses. The coating effectively decreased the adsorption of recombinant adeno-associated virus vectors, leading to efficient recovery of vector particles and higher transduction efficiency. This coating has the potential to be widely used in material processing in the gene therapy field.
JOURNAL OF PHARMACEUTICAL SCIENCES
(2022)
Article
Biochemical Research Methods
Aurelien Jacob, Laurie Brun, Paloma Jimenez Gil, Lucie Menard, Mohammed Bouzelha, Frederic Broucque, Aline Roblin, Luk H. Vandenberghe, Oumeya Adjali, Cecile Robin, Achille Francois, Veronique Blouin, Magalie Penaud-Budloo, Eduard Ayuso
Summary: Manufacturing viral vectors for gene delivery is challenging, with a scalable baculovirus-insect cell platform being used for producing rAAV vectors. A faster and safer method based on homologous recombination (HR) has been investigated to avoid technical constraints and safety concerns associated with the Tn7 transposition method. The HR system offers increased stability and avoids undesirable bacterial genes, ensuring the generation of safer rAAV biotherapeutic products.
BIOTECHNOLOGY JOURNAL
(2021)
Article
Biochemistry & Molecular Biology
Kumi Adachi, Taro Tomono, Hironori Okada, Yusuke Shiozawa, Motoko Yamamoto, Yoshitaka Miyagawa, Takashi Okada
Summary: By comparing the properties of rAAV containing DNA plasmids and PCR-amplified transgenes, the study found that rAAV-pAD/L-AD had packaging efficiency comparable to scAAV, but lower transduction efficiency than ss/scAAV. Additionally, rAAV-L-AD reduced plasmid backbone packaging contamination.
Article
Medicine, Research & Experimental
Tam N. T. Nguyen, Sha Sha, Moo Sun Hong, Andrew J. Maloney, Paul W. Barone, Caleb Neufeld, Jacqueline Wolfrum, Stacy L. Springs, Anthony J. Sinskey, Richard D. Braatz
Summary: The study developed a mechanistic model to predict the synthesis process of rAAV viral vectors and validated it based on experimental and literature data. Sensitivity analysis showed that the poor coordination between capsid synthesis and viral DNA replication, as well as the inhibitory function of the Rep protein, may impact rAAV production.
MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT
(2021)
Review
Genetics & Heredity
Sofia Fernandes, Joana Diogo, Ana Sofia Coroadinha
Summary: Adeno-associated viruses (AAV) are widely used in gene therapy due to their non-pathogenicity, ability to transduce both dividing and non-dividing cells, and flexibility in targeting different tissues. The success of AAV gene therapy has been demonstrated by the approval of three products. To meet the requirements of high dosage, safety, and reproducibility in clinical trials, production platforms based on stable mammalian cell lines have been proposed as the best strategy, although the methodologies employed may result in different productivities.
CURRENT GENE THERAPY
(2023)
Review
Biochemical Research Methods
Pranav R. H. Joshi, Alina Venereo-Sanchez, Parminder S. Chahal, Amine A. Kamen
Summary: Significant progress has been made in IC-BEVS for rAAV production, with improvements in Bac-vector stability and reduced baculovirus co-infections, leading to the development of evolved manufacturing strategies.
BIOTECHNOLOGY JOURNAL
(2021)
Article
Biophysics
Sylvie Saleun, Caroline Mas, Aline Le Roy, Magalie Penaud-Budloo, Oumeya Adjali, Veronique Blouin, Christine Ebel
Summary: Recombinant adeno-associated virus virus-derived vectors (rAAVs) are widely used in vivo gene therapies due to their safety profile. However, rAAV production often results in a heterogeneous vector population with undesired empty particles. Analytical ultracentrifugation sedimentation velocity (AUC-SV) is a gold standard technique to measure the relative amounts of each vector subpopulation and components. This letter discusses the principle and practice of using AUC for rAAVs characterization, including the use of interference optics to estimate weight percentages of different capsid populations and the genome size incorporated in rAAV particles.
EUROPEAN BIOPHYSICS JOURNAL WITH BIOPHYSICS LETTERS
(2023)
Article
Biochemical Research Methods
Mette Richner, Nadia Pereira Goncalves, Poul Henning Jensen, Jens Randel Nyengaard, Christian Bjerggaard Vaegter, Asad Jan
Summary: This article introduces a minimally invasive technique called SciNDi for transgene delivery in the extracranial nervous system of adult mice using AAV. Compared to intramuscular delivery, SciNDi achieves widespread transduction in connected neuroanatomical tracts in the sciatic nerve trunk and the lumbar spinal cord.