Generation of Helper Plasmids Encoding Mutant Adeno-associated Virus Type 2 Capsid Proteins with Increased Resistance against Proteasomal Degradation

Abstract

Objective(s): Adeno-associated virus type 2 (AAV2) vectors are widely used for both experimental and clinical gene therapy. A recent research has shown that the performance of these vectors can be greatly improved by substitution of specific surface-exposed tyrosine residues with phenylalanines. In this study, a fast and simple method is presented to generate AAV2 vector helper plasmids encoding capsid proteins with single, double or triple Y→F mutations.

Materials and methods: A one-step, high-fidelity polymerase chain reaction (PCR) cloning procedure involving the use of two partially overlapping primers to amplify a circular DNA template was applied to produce AAV2 cap genes encoding VP1 mutants with Y→F substitutions in residues 444, 500 or 730. The resulting constructs were used to make the different double and triple mutant by another round of PCR (Y444500F mutant), subcloning (Y444730F and Y500730F mutants) or a combination of both techniques (Y444500730F mutant).

Results: Nucleotide sequence analysis revealed successful introduction of the desired mutations in the AAV2 cap gene and showed the absence of any unintended mutations in the DNA fragments used to assemble the final set of AAV2 vector helper plasmids. The correctness of these plasmids was further confirmed by restriction mapping.

Conclusion: PCR-based, single-step site-directed mutagenesis of circular DNA templates is a highly efficient and cost-effective method to generate AAV2 vector helper plasmids encoding mutant Cap proteins for the production of vector particles with increased gene transfer efficiency.

Keywords: Adeno-associated virus type 2; Capsid protein; Gene therapy; Mutagenesis; Proteasomal degradation; Transduction efficiency; Viral vector.

Figure 1

Schematic diagram of the PCR-based, site-directed mutagenesis procedure used to produce AAV2 vector packaging plasmids encoding capsid proteins with increased resistance against proteasomal degradation. Following synthesis of mutant DNA strands, PCR mixtures are subjected to DpnI digestion to selectively degrade the bacterial input DNA, which differs from the newly synthesized PCR products by being methylated at A residues in the DpnI recognition sequence (i.e. 5’ GATC 3’). Red circle, “old” template DNA; blue pedal, methyl group; green arrow, PCR primer; green circle, “new” PCR product; blue circle, mutant DNA recovered from bacteria

Figure 2

Agarose gel electrophoresis of the pUCBM21.Cap2 amplification products obtained after PCR with primers A001/A002 (lanes 1 and 2), A003/A004 (lanes 3 and 4) or A005/A006 (lanes 5 and 6) in the presence (lanes 1, 3 and 5) or absence (negative controls; lanes 2, 4 and 6) of VELOCITY DNA polymerase. One-tenth of each PCR sample was applied to a 0.8% agarose gel (Tris-acetate-EDTA buffer system) following the removal of proteins, primers and dNTPs by SureClean treatment. Figure shows the PCR products before DpnI treatment. Numbers at the left and right of the gel picture represent sizes in kb. Lane M, GeneRuler DNA Ladder Mix (Fermentas) molecular weight marker

Figure 3

Agarose gel electrophoresis of pUCBM21.Cap2 DNA derivatives after digestion with ApaI, ApaLI, AvaI, BglI or DraI. For comparison, also undigested plasmid DNA was loaded on gel. DNA species indicated by a hashtag and an arrowhead represent supercoiled and denatured plasmid DNA, respectively. Lanes 1 and 2, plasmid pUCBM21.Cap2Y444F; lanes 3 and 4, plasmid pUCBM21.Cap2Y500F; lanes 5 and 6, plasmid pUCBM21.Cap2Y730F; lanes M, GeneRuler DNA Ladder Mix (Fermentas) molecular weight marker

Figure 4

Map of plasmid pUCBM21.Cap2 showing the location of the binding sites for primers M13-R, A007-F, A010-R, A008-F, A011-F and M13-F. Also indicated is the AAV2 sequences (black), relevant restriction enzyme recognition sites, the coding sequences of the structural AAV2 proteins (VP1, VP2 and VP3; green) and of the carboxy-terminal ends of the non-structural AAV2 proteins (Rep78, Rep68, Rep 50 and Rep 40; red), the Escherichia coli bla gene conferring resistance to ampicillin (AmpR; blue) and the pBR322 origin of replication (pBR322 ori; yellow)

Figure 5

Chromatograms of the nucleotide sequences of wild type pUCBM21.Cap2 and of the pUCBM21. Cap2 mutants pUCBM21.Cap2Y444F, pUCBM21.Cap2Y500F and pUCBM21.Cap2Y730F at the sites of mutagenesis. The chromatograms on the left and in the middle panels were obtained using primer A008-F while those on the right panel were acquired with primer A011-F. Green, blue, black and red peaks correspond to A, C, G and T residues, respectively. The underlined nucleotide sequence represents the EcoRI recognition sequence resulting from the mutagenesis of codon 500 in the AAV2 cap gene

Figure 6

Agarose gel electrophoresis of the PCR products obtained after amplification of pUCBM21.Cap2Y500F with primers A001/A002 (lanes 1 and 2) and pUCBM21.Cap2Y444F with primers A003/A004 (lanes 3 and 4) in the presence (lanes 1 and 3) or absence (negative controls; lanes 2 and 4) of VELOCITY DNA polymerase. One-tenth of each PCR sample was applied to a 0.8% agarose gel (Tris-acetate-EDTA buffer system) following the removal of proteins, primers and dNTPs by SureClean treatment and the degradation of the template DNA by DpnI treatment. Numbers on the left and on the right of the gel picture represent sizes in kb. Lane M, GeneRuler DNA Ladder Mix (Fermentas) molecular weight marker

Figure 7

Agarose gel electrophoresis of pUCBM21.Cap2 DNA derivatives after digestion with ApaLI, AvaII, BstXI, DraI or EcoRI. For comparison, also undigested plasmid DNA was loaded on gel. Lanes 1 and 2, plasmid pUCBM21.Cap2Y444500F derived from pUCBM21.Cap2Y444F; lanes 3 and 4, plasmid pUCBM21.Cap2Y444500F derived from pUCBM21.Cap2Y500F; lanes M, GeneRuler DNA Ladder Mix (Fermentas) molecular weight marker. #, supercoiled plasmid DNA

Figure 8

Agarose gel electrophoresis of pUCBM21.Cap2 DNA derivatives after digestion with ApaLI, EcoRI, NcoI, SmiI or XhoI. For comparison, also undigested plasmid DNA was loaded on gel. Lanes 1 and 2, plasmid pUCBM21.Cap2Y444730F; lanes 3 and 4, plasmid pUCBM21.Cap2Y500730F; lanes 5 and 6, plasmid pUCBM21.Cap2Y444500730F; lanes M, GeneRuler DNA Ladder Mix (Fermentas) molecular weight marker. #, supercoiled plasmid DNA