Systemic Elimination ofde novoCapsid Protein Synthesis from Replication-Competent AAV Contamination in the Liver
- 1 May 2011
- journal article
- research article
- Published by Mary Ann Liebert Inc in Human Gene Therapy
- Vol. 22 (5), 625-632
- https://doi.org/10.1089/hum.2011.005
Abstract
The capsid protein synthesis in targeted tissues resulting from residual contaminating replication-competent adeno-associated virus particles (rcAAV) remains a concern for hazardous immune responses that shut down the factor IX expression in the hemophilia B clinical trial. To systematically reduce/eliminate the effects of potential contaminating rcAAV particles, we designed a novel adeno-associated virus (AAV) helper (pH22mir) with a microRNA binding cassette containing multiple copies of liver-specific (hsa-mir-122) and hematopoietic-specific (has-mir-142-3p) sequences to specifically control cap gene expression. In 293 cells, the rep and cap gene from pH22mir functioned similarly to that of conventional helper pH22. The vector yields and compositions from pH22mir and pH22 were indistinguishable. The performance of vector produced in this new system was comparable to that of similar vectors produced by conventional methods. In the human hepatic cell line, the capsid expression was reduced significantly from cap-mir cassette driven by a cytomegalovirus promoter. In the liver, 99.9% of capsid expression could be suppressed and no cap expression could be detected by western blot. In summary, we demonstrated a new concept in reducing de novo capsid synthesis in the targeted tissue. This strategy may not only help AAV vectors in controlling undesirable capsid gene expression, but can also be adopted for lentiviral or adenoviral vector production. Replication-competent adeno-associated viral particles (rcAAV) are an undesirable contaminant of vector preparations that may affect transgene expression or elicit a hazardous immune response. In this study, Yuan and colleagues have designed a recombinant AAV (rAAV) vector production system to tightly control AAV rep and capsid activity from rcAAV particles. According to the authors, this new method does not have any effects on rAAV yield or affect rAAV vector performance and is compatible with all current rAAV production systems.Keywords
This publication has 31 references indexed in Scilit:
- Dystrophin Immunity in Duchenne's Muscular DystrophyNew England Journal of Medicine, 2010
- Sustained transgene expression despite T lymphocyte responses in a clinical trial of rAAV1-AAT gene therapyProceedings of the National Academy of Sciences, 2009
- Naturally occurring singleton residues in AAV capsid impact vector performance and illustrate structural constraintsGene Therapy, 2009
- Large-Scale Adeno-Associated Viral Vector Production Using a Herpesvirus-Based System Enables Manufacturing for Clinical StudiesHuman Gene Therapy, 2009
- Producing Recombinant Adeno-Associated Virus in Foster Cells: Overcoming Production Limitations Using a Baculovirus–Insect Cell Expression StrategyHuman Gene Therapy, 2009
- Cytotoxic-T-Lymphocyte-Mediated Elimination of Target Cells Transduced with Engineered Adeno-Associated Virus Type 2 Vector In VivoJournal of Virology, 2009
- Cellular immune response to cryptic epitopes during therapeutic gene transferProceedings of the National Academy of Sciences, 2009
- Enhanced Factor VIII Heavy Chain for Gene Therapy of Hemophilia AMolecular Therapy, 2009
- Undetectable Transcription of cap in a Clinical AAV Vector: Implications for Preformed Capsid in Immune ResponsesMolecular Therapy, 2009
- Complete Correction of Hemophilia A with Adeno-Associated Viral Vectors Containing a Full-Size Expression CassetteHuman Gene Therapy, 2008