Article
Virology
Renuka Rao, Melad Farraha, Grant J. Logan, Sindhu Igoor, Cindy Y. Kok, James J. H. Chong, Ian E. Alexander, Eddy Kizana
Summary: This study compared two production methods for cardiotropic rAAVs, finding that rAAV produced by the traditional HEK293 method outperformed vector produced using the baculovirus/Sf9 system in vitro and in vivo. This underscores the importance of thorough assessment of vector performance when using diverse rAAV production methods.
Article
Virology
Jianan Huang, Huanlei Liu, Xiaodong Xu
Summary: The baculovirus expression vector system (BEVS) is widely used for producing recombinant proteins. However, there is a risk of homologous recombination between viral genome and functional baculovirus-derived elements associated with foreign genes, which can lead to defective viruses and decreased recombinant protein yields. Shortening the length of homologous fragments and separating them by point mutations can effectively reduce the unfavorable recombination.
Article
Biotechnology & Applied Microbiology
Phuc Leo H. Vo, Carlotta Ronda, Sanne E. Klompe, Ethan E. Chen, Christopher Acree, Harris H. Wang, Samuel H. Sternberg
Summary: The study introduces a new INTEGRATE system that enables highly accurate and marker-free DNA integration in bacteria at high efficiency. By combining CRISPR arrays and multiple enzymes for multiplexed insertions and deletions, the potential applications of INTEGRATE in complex bacterial communities were demonstrated.
NATURE BIOTECHNOLOGY
(2021)
Article
Biochemical Research Methods
Yue Yu, Cancan Yang, Tong Zhang, Zhenhe Xu, Yuanxing Zhang, Qin Liu
Summary: A rapid and simple YBac system for heterologous protein expression was established in this study. The system utilizes an Ac99KO bacmid as a virus vector and co-transfects it with a donor plasmid into Sf9 cells. The YBac system successfully expressed human thyroid peroxidase (hTPO) and porcine epidemic diarrhea virus-like particles (PEDV VLPs), with the entire process shortened to 10 days and easily prepared components in the laboratory, suggesting great potential for heterologous protein production.
BIOTECHNOLOGY JOURNAL
(2023)
Review
Biotechnology & Applied Microbiology
Mark A. Brimble, Stephen M. Winston, Andrew M. Davidoff
Summary: Recombinant AAV (rAAV) is commonly used delivery vector for clinical gene therapy, but there are several issues to be addressed before its safer and wider implementation. This paper reviews the characteristics, abundances, and consequences of nucleic acid impurities within rAAV, and discusses strategies to improve rAAV purity, aiming to enhance the understanding of these aberrantly packaged DNA species.
Article
Biochemistry & Molecular Biology
Matt W. G. Walker, Sanne E. Klompe, Dennis J. Zhang, Samuel H. Sternberg
Summary: The study reveals the sequence requirements for efficient transposon DNA integration activity mediated by CRISPR-associated transposases (CASTs). The research also identifies a novel cellular factor, integration host factor (IHF), involved in the assembly of the CRISPR-associated transpososome. The findings provide insights for the design of customized payload sequences for genome engineering applications with CAST systems.
NUCLEIC ACIDS RESEARCH
(2023)
Review
Genetics & Heredity
Kateryna Kratzer, Landon J. Getz, Thibaut Peterlini, Jean-Yves Masson, Graham Dellaire
Summary: Gene therapy for genetic diseases has gained increased attention, but faces technical challenges and ethical barriers. Current research is primarily focused on improving gene therapy and gene editing techniques, with less emphasis on the timing and rationale for using such technology.
Article
Multidisciplinary Sciences
Jung-Un Park, Amy Wei-Lun Tsai, Eshan Mehrotra, Michael T. Petassi, Shan-Chi Hsieh, Ailong Ke, Joseph E. Peters, Elizabeth H. Kellogg
Summary: CRISPR-associated transposition systems allow integration of a single DNA cargo directed by guide RNA in one orientation at a fixed distance from a programmable target sequence. Cryo-EM was used to characterize the transposition regulator TnsC, revealing the mechanism behind transposase polarity information transfer. Mechanistic findings include polymerization of ATP-bound TnsC helical filaments, TniQ capping the TnsC filament, and transposase-driven disassembly for element delivery only to unused protospacers. These findings provide a foundation for engineering CRISPR-associated transposition systems for research and therapeutic applications.
Article
Biotechnology & Applied Microbiology
Nikolaus Virgolini, Marco Silvano, Ryan Hagan, Ricardo Correia, Paula M. Alves, Colin Clarke, Antonio Roldao, Ines A. Isidro
Summary: Using single-cell RNA sequencing, this study performed transcriptomics analysis of Sf9 insect cells during the production of rAAV2. The study found an increase in transcriptional heterogeneity during infection, which was linked to the expression of baculovirus genes and rAAV2 transgenes. Additionally, the study highlighted limitations of the dual-baculovirus system in producing rAAV2 particles.
BIOTECHNOLOGY AND BIOENGINEERING
(2023)
Article
Multidisciplinary Sciences
Ramesh Periasamy, Dwani D. Patel, Sanford L. Boye, Shannon E. Boye, Daniel M. Lipinski
Summary: Studies have shown that modifying the rAAV capsid can improve the targeting of retinal VECs. In mouse experiments, the EC5 mutant showed improved retinal vasculature transduction and unexpected cardiac muscle transduction.
Article
Oncology
Benjamin D. Leibowitz, Bonnie V. Dougherty, Joshua S. K. Bell, Joshuah Kapilivsky, Jackson Michuda, Andrew J. Sedgewick, Wesley A. Munson, Tushar A. Chandra, Jonathan R. Dry, Nike Beaubier, Catherine Igartua, Timothy Taxter
Summary: With the introduction of DNA-damaging therapies in cancer treatment, there is a growing need for predictive diagnostics assessing homologous recombination deficiency (HRD) status. The study found that both HRD-DNA and HRD-RNA models have good predictive performance in various types of cancer.
Article
Biochemistry & Molecular Biology
Sonia Albini, Laura Palmieri, Auriane Dubois, Nathalie Bourg, William Lostal, Isabelle Richard
Summary: This study reports a dual AAV vector approach for the expression of a larger dystrophin version and develops a methodology to determine the recombination efficiency as well as the occurrence of unwanted concatemerization events or aberrant expression from the single vectors. The dual AAV approach leads to high transgene reconstitution efficiency and negligible ITR-dependent concatemerization, resulting in remarkable protein restoration in muscles and improvement of muscle pathology. This system is suitable for gene therapy application and has the potential to assess and improve the feasibility of multiple vector approaches for therapeutic translation.
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2023)
Article
Multidisciplinary Sciences
Adriana Gonzalez-Sandoval, Katja Pekrun, Shinnosuke Tsuji, Feijie Zhang, King L. Hung, Howard Y. Chang, Mark A. Kay
Summary: The effectiveness of rAAV vectors varies between species, and a single amino acid change in the vector can alter its epigenomic state and transgene expression levels between species.
NATURE COMMUNICATIONS
(2023)
Article
Engineering, Chemical
Chin-Wei Chang, Liang-Shin Wang, Nam Ngoc Pham, Chih-Che Shen, Mu-Nung Hsu, Nuong Thi Kieu Nguyen, Chia-Yi Yen, Mei-Wei Lin, Jih-Ru Hwu, Yu-Han Chang, Yu-Chen Hu
Summary: Researchers have developed a novel baculovirus vector that efficiently delivers genes into mammalian cells and prolongs transgene expression. This all-in-one vector helps to prevent gene loss during virus production and improves gene delivery efficiency, making it significant for gene therapy applications.
JOURNAL OF THE TAIWAN INSTITUTE OF CHEMICAL ENGINEERS
(2022)
Article
Multidisciplinary Sciences
Miguel M. Alvarez, Josep Biayna, Fran Supek
Summary: Research indicates that TPS3-associated break toxicity is higher in genomic regions with active chromatin, while DSB repair pathway choice and DNA sequence context also play a role in toxicity. The differential toxicity of sgRNA-targeted sites can reduce the power of genetic screens to detect conditional essentiality.
NATURE COMMUNICATIONS
(2022)
Article
Biochemistry & Molecular Biology
Anil Chekuri, Emily M. Logan, Aram J. Krauson, Monica Salani, Sophie Ackerman, Emily G. Kirchner, Jessica M. Bolduc, Xia Wang, Paula Dietrich, Ioannis Dragatsis, Luk H. Vandenberghe, Susan A. Slaugenhaupt, Elisabetta Morini
Summary: Familial dysautonomia (FD) is a neurodegenerative disease caused by a gene mutation, and visual decline leading to blindness is a significant issue. Researchers have generated a mouse model that accurately replicates the splicing defect observed in FD patients and successfully corrected the retinal defect in the mouse model using a splicing modulator compound.
HUMAN MOLECULAR GENETICS
(2022)
Review
Biochemistry & Molecular Biology
S. Zolotukhin, L. H. Vandenberghe
Summary: In vivo therapeutic gene transfer is a novel class of medicines that relies on the safe and effective delivery of genetic material. The adeno-associated virus (AAV) vector has emerged as a preferred gene delivery vehicle, allowing for therapeutic gene expression in various clinical indications. Recent trends in AAV capsid engineering focus on enhancing targeting specificity, safety, and endurance, a significant challenge that is being addressed through techniques such as directed evolution, sequence analysis, and machine learning.
TRENDS IN MOLECULAR MEDICINE
(2022)
Article
Biochemistry & Molecular Biology
Attya Omer-Javed, Gabriele Pedrazzani, Luisa Albano, Sherash Ghaus, Claire Latroche, Maura Manzi, Samuele Ferrari, Martina Fiumara, Aurelien Jacob, Valentina Vavassori, Alessandro Nonis, Daniele Canarutto, Luigi Naldini
Summary: Hematopoietic stem/progenitor cell gene therapy has been successful in treating genetic diseases. Mobilizers provide an opportunity for exogenous cells to efficiently replace depleted bone marrow through competition. This competitive advantage is achieved by rescuing the detrimental effects of mobilization on hematopoietic stem/progenitor cells during ex vivo culture, and can be further enhanced by transient overexpression of engraftment effectors.
Article
Medicine, Research & Experimental
Sophia Grotz, Jessica Schaefer, Kirsten A. Wunderlich, Zdenka Ellederova, Hannah Auch, Andrea Baehr, Petra Runa-Vochozkova, Janet Fadl, Vanessa Arnold, Taras Ardan, Miroslav Veith, Gianluca Santamaria, Georg Dhom, Wolfgang Hitzl, Barbara Kessler, Christian Eckardt, Joshua Klein, Anna Brymova, Joshua Linnert, Mayuko Kurome, Valeri Zakharchenko, Andrea Fischer, Andreas Blutke, Anna Doering, Stepanka Suchankova, Jiri Popelar, Eduardo Rodriguez-Bocanegra, Julia Dlugaiczyk, Hans Straka, Helen May-Simera, Weiwei Wang, Karl-Ludwig Laugwitz, Luk H. Vandenberghe, Eckhard Wolf, Kerstin Nagel-Wolfrum, Tobias Peters, Jan Motlik, M. Dominik Fischer, Uwe Wolfrum, Nikolai Klymiuk
Summary: This study generated the first translational animal model for Usher syndrome type 1, which exhibits characteristics of hearing loss, vestibular dysfunction, and visual impairment similar to human patients. The research also confirmed the therapeutic potential of gene repair and gene therapy for Usher syndrome through in vitro assessments using primary cells from Usher syndrome pigs or patients.
EMBO MOLECULAR MEDICINE
(2022)
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
Multidisciplinary Sciences
Michela Milani, Cesare Canepari, Tongyao Liu, Mauro Biffi, Fabio Russo, Tiziana Plati, Rosalia Curto, Susannah Patarroyo-White, Douglas Drager, Ilaria Visigalli, Chiara Brombin, Paola Albertini, Antonia Follenzi, Eduard Ayuso, Christian Mueller, Andrea Annoni, Luigi Naldini, Alessio Cantore
Summary: Liver gene therapy using lentiviral vectors that integrate into the host cell genome has shown efficient liver gene transfer in mice, dogs, and non-human primates. In this study, we compared engineered coagulation factor VIII transgenes and found that codon-usage optimization and inclusion of an unstructured XTEN peptide significantly increased factor VIII expression in mice and non-human primates. Stable and long-term normal or above-normal factor VIII activity was achieved in hemophilia A mouse models. This research opens up new possibilities for treating hemophilia A using lentiviral gene therapy.
NATURE COMMUNICATIONS
(2022)
Article
Multidisciplinary Sciences
Thibaut Naninck, Nidhal Kahlaoui, Julien Lemaitre, Pauline Maisonnasse, Antoine De Mori, Quentin Pascal, Vanessa Contreras, Romain Marlin, Francis Relouzat, Benoit Delache, Cecile Herate, Yoann Aldon, Marit van Gils, Nerea Zabaleta, Raphael Ho Tsong Fang, Nathalie Bosquet, Rogier W. Sanders, Luk H. Vandenberghe, Catherine Chapon, Roger Le Grand
Summary: Non-human primates (NHPs) are important preclinical models for SARS-CoV-2 infection. This study found that computed X-ray tomography (CT) and [F-18]-FDG positron emission tomography (PET) have significant benefits in monitoring early infection in NHPs, allowing for early detection of inflammation and lung burden.
Article
Cell & Tissue Engineering
Samuele Ferrari, Aurelien Jacob, Daniela Cesana, Marianne Laugel, Stefano Beretta, Angelica Varesi, Giulia Unali, Anastasia Conti, Daniele Canarutto, Luisa Albano, Andrea Calabria, Valentina Vavassori, Carlo Cipriani, Maria Carmina Castiello, Simona Esposito, Chiara Brombin, Federica Cugnata, Oumeya Adjali, Eduard Ayuso, Ivan Merelli, Anna Villa, Raffaella Di Micco, Anna Kajaste-Rudnitski, Eugenio Montini, Magalie Penaud-Budloo, Luigi Naldini
Summary: This study reveals the unexpected load and persistence of AAV genomes and their fragments in gene editing using viral transduction. The accumulation of viral DNA triggers a sustained DNA damage response, leading to frequent integration in cells. Using an integrase-defective lentiviral vector can mitigate the adverse effects and improve gene editing efficiency.
Article
Medicine, Research & Experimental
Ying-Chang Lu, Yi-Hsiu Tsai, Yen-Huei Chan, Chin-Ju Hu, Chun-Ying Huang, Ru Xiao, Chuan-Jen Hsu, Luk H. Vandenberghe, Chen-Chi Wu, Yen-Fu Cheng
Summary: Recessive PJVK mutations cause a deficiency of pejvakin, leading to hereditary hearing impairment. This study used the synthetic Anc80L65 vector to deliver the PJVKtransgene to the inner ear of Pjvk mutant mice, resulting in the restoration of neuron and hair cell density, and partial recovery of sensorineural hearing impairment.
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
Medicine, Research & Experimental
Michael Florea, Fotini Nicolaou, Simon Pacouret, Eric M. Zinn, Julio Sanmiguel, Eva Andres-Mateos, Carmen Unzu, Amy J. Wagers, Luk H. Vandenberghe
Summary: The adeno-associated viral vector (AAV) is a safe and efficient gene therapy platform, but the production efficiency, cost, and scalability remain bottlenecks. Chromatographic methods have the potential to address these challenges but require optimization for each serotype.
MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT
(2023)
Article
Biochemistry & Molecular Biology
Daniele Canarutto, Claudia Asperti, Valentina Vavassori, Simona Porcellini, Elisabetta Rovelli, Marianna Paulis, Samuele Ferrari, Angelica Varesi, Martina Fiumara, Aurelien Jacob, Lucia Sergi Sergi, Ilaria Visigalli, Francesca Ferrua, Luis Ignacio Gonzalez-Granado, Vassilios Lougaris, Andrea Finocchi, Anna Villa, Marina Radrizzani, Luigi Naldini
Summary: This study focused on CD40LG gene editing and developed a PCR assay for detecting large on-target deletions. The results showed that large deletions and donor template integrations were common during gene editing. Counter-selection and enrichment methods can reduce the occurrence of on-target deletions. Optical genome mapping was also used to detect genome-wide rearrangements, but no other recurring events were found.
Meeting Abstract
Biotechnology & Applied Microbiology
Aurelien Jacob, Samuele Ferrari, Daniela Cesana, Marianne Laugel, Stefano Beretta, Angelica Varesi, Daniele Canarutto, Luisa Albano, Anastasia Conti, Ivan Merelli, Raffaella Di Micco, Eugenio Montini, Magalie Penaud-Budloo, Luigi Naldini
