Capsid-expressing DNA in AAV vectors and its elimination by use of an oversize capsid gene for vector production

• Published in: Gene therapy Vol. 18; no. 4; pp. 411 - 417
• Main Authors: Halbert, C L, Metzger, M J, Lam, S-L, Miller, A D
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.04.2011; Nature Publishing Group
• Subjects: 631/250; 631/326/596/2561; Adeno-associated virus; Adenoviruses; Analysis; Anesthesia. Intensive care medicine. Transfusions. Cell therapy and gene therapy; Animal models; Antigens; Applied cell therapy and gene therapy; Biological and medical sciences; Biomedical and Life Sciences; Biomedicine; Biotechnology; Capsid - immunology; Capsid protein; Cell Biology; Cells, Cultured; Clinical trials; Complementation; Deoxyribonucleic acid; Dependovirus - genetics; Dependovirus - immunology; Dependoviruses; DNA; DNA, Viral - immunology; Expression vectors; Fundamental and applied biological sciences. Psychology; Gene Expression; Gene Therapy; Gene Transfer Techniques; Genetic Vectors; Health aspects; Health. Pharmaceutical industry; Human Genetics; Humans; Immune clearance; Immune response; Immune system; Impurities; Industrial applications and implications. Economical aspects; Introns - immunology; Medical sciences; Nanotechnology; Nucleotide sequence; original-article; Packaging; Physiological aspects; Polymerase chain reaction; Transduction, Genetic; Transfusions. Complications. Transfusion reactions. Cell and gene therapy; Virions; United States; AAV vectors; vector immune responses; capsid gene transfer; Immune response; Use; Elimination; Dependovirus; Genetic transfer; Virus; Associated virus; Gene; Parvoviridae; DNA; Production; Capsid; Gene therapy; Vector; Parvovirinae
• ISSN: 0969-7128; 1476-5462
• DOI: 10.1038/gt.2010.167

Adeno-associated virus (AAV) vectors have been shown to mediate persistent transduction in animal models of gene therapy. However, clinical trials with AAV vectors have shown that an immune response to AAV capsid protein can result in clearance of transduced cells. One source of capsid antigen is from the delivered vector virions, but expression of cap DNA impurities in AAV vector preparations might provide an alternative and persistent source of capsid antigen. Here we show that DNA without any AAV sequences can be packaged in AAV virions, and that both cap and rep DNA are packaged into AAV vectors produced by standard methods. Using a sensitive complementation assay, we also observed significant expression of capsid in cultured cells transduced with AAV vectors. In an attempt to solve this problem, we inserted a large intron into the cap gene to generate a capsid expression cassette (captron) that is too large for packaging into AAV virions. Both complementation assays and quantitative reverse-transcription PCR analysis showed that cultured cells infected with AAV vectors made with the captron plasmid expressed no detectable capsid. Elimination of transfer of capsid-expressing DNA may reduce immune responses to AAV vector-transduced cells and promote long-term expression of therapeutic proteins.