Abstract
Adeno associated virus (AAV) is a versatile gene delivery tool, which has been approved as a human gene therapy vector for combating genetic diseases. AAV capsid proteins are the major components that determine the tissue specificity, immunogenicity and in vivo transduction performance of the vector. In this study, the AAV8 capsid glycosylation profile was systemically analyzed by peptide mass fingerprinting utilizing high-resolution mass spectrometry to determine the presence of capsid glycosylation. We identified N-glycosylation on the amino acid N499 of the capsid protein. We characterized the overall sugar profile for vector produced in 293 cells. Multiple N-glycosylated host-cell proteins (HCPs) copurified with AAV8 vectors and were identified by analyzing LC-MS data utilizing a human database and proteome discoverer search engine. The N-glycosylation analysis by MALDI-TOF MS, highlighted the probability of AAV8 interaction with terminal galactosylated N-glycans within the HCPs.
Highlights
Adeno associated virus (AAV) is a dependoparvovirus being used as a gene therapy vector for treating a variety of genetic disorders and acquired diseases
To characterize characterize the sitesite of capsid proteins, we focused on higher-energy C-trap dissociation (HCD)
The identification of glycosylated proteins interacting with AAV8 is interesting given AAV’s known predilection for glycosylated proteins [26,43,44,45] While some of these proteins could be due to issues with purification steps it does beg for further research into the interactions that AAV has with glycans in the cell and their importance in secretion of AAV
Summary
Adeno associated virus (AAV) is a dependoparvovirus being used as a gene therapy vector for treating a variety of genetic disorders and acquired diseases. Low immunogenicity and differential tropism to multiple cell types make AAV a versatile gene delivery system [1]. This non-enveloped virus is approximately 25 nm in diameter and contains a unique linear single-stranded DNA genome [2,3,4]. AAV has a 4.8 kilobase genome flanked by two copies of 145 bp inverted terminal repeats [5]. AAV has two open reading frames (ORFs) consisting of the Rep and Cap genes. The Rep gene encodes four different replicating proteins
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