Adeno-associated virus 2 (AAV2) vector transfer through the blood brain barrier by directed evolution approach

Adeno-associated virus serotype 2 (AAV2) is a relatively safe and efficient gene delivery vehicle. It is a 4.7-kilobase, single-stranded DNA virus that contains two genes, rep and cap. Rep encodes for four proteins necessary for genome replication (Rep78, Rep68, Rep52, and Rep40), and Cap expresses three structural proteins (VP1, VP2, and VP3) necessary for forming the viral capsid. Even though AAV2 is nonpathogenic and shown to be capable of efficient gene delivery in various tissues, there are still problems to overcome. The main purpose of my project is to alter AAV2 using the rabies virus glycoprotein (RVG) peptide to cross the blood brain barrier. The RVG peptide binds to acetylcholine receptors expressed in neuronal cells. Previous research has shown that insertion of peptide sequences could reduce viral production or infectivity (Walters, et al., 2004). Therefore, instead of only inserting the RVG peptide at amino acid 453, we will also use a directed evolution approach to produce mutant AAV2 variants with desired properties.


Figure 1. Two open reading frames of rep and cap.

Peptide insertion into specific capsid locations does not alter antibody neutralization and virus-cell interactions because these properties are distributed throughout the primary sequence of the capsid (Xie, et al., 2002). My project aims to coordinate direct peptide insertion method with nature’s approach to functional diversification. The 453 insertion site has been chosen to maximize acetylcholine receptors’ binding affinities. Looking at the crystal structure of AAV2 reported by Xie et al., we have determined that the 453 insertion site is projected outwards between two loops (Xie, et al., 2002). Therefore, we hope to show insertion at the 453 site will make the RVG peptide more accessible to acetylcholine receptors. Maheshri et al. applied a directed evolution approach to modify AAV2 to evade problems with neutralizing antibodies, tissue transport, and infection of resistant cell types. He was able to create a library of AAV2 variants with different affinities for heparin and different delivery efficiencies in the presence of anti-AAV serum. A library of the capsid mutants was prepared by error-prone PCR, and after several rounds of selection processes, he selected the mutants with different heparin affinities to increase infectivity of non-permissive cells (Maheshri et al., 2006).


Figure 2. Part of three dimensional structure of AAV2 showing the 453 insertion site

For this project, we will first have the entire rep and cap genes with the RVG peptide subjected to mutagenesis and recombination using PCR methods. The mutant DNA will be inserted into a plasmid to create a viral plasmid library with AAV helper-free transfection and HEK 293 cells. High-throughout selection process will be performed to select mutants that have less heparin binding affinities not to interrupt with acetylcholine receptors. After we have a library of AAV2 variants with different properties, we will infect Neuro2a or M17 cells to observe whether replication occurs inside the neuronal cells. We will repeat the process of infecting the cell lines to find out which AAV2 variants has stronger binding affinities for acetylcholine receptors. Also, increasing the concentration of antiserum gradually during neutralization reaction, we can isolate AAV2 variants with improved resistance to antibody neutralization. In the long run, we want isolate the ones that can evade neutralizing antibodies in vivo. This can be tested with an in vivo assay in which the mutants are pre-incubated with antiserum and analyzed for its ability to mediate gene delivery after injection in mouse.

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