Effect of genome size on AAV vector packaging

Abstract

Adeno-associated virus (AAV) vector genomes have been limited to 5 kilobases (kb) in length because their packaging limit was thought to be similar to the size of the parent AAV genome. Recent reports claim that significantly larger vector genomes can be packaged intact. We examined the packaged vector genomes from plasmid-encoded AAV vectors that ranged from 4.7 to 8.7 kb in length, using AAV types 2, 5, and 8 capsids. Southern blot analysis indicated that packaged AAV vector genomes never exceeded 5.2 kb in length irrespective of the size of the plasmid-encoded vector or the capsid type. This result was confirmed by vector genome probing with strand-specific oligonucleotides. The packaged vector genomes derived from plasmid-encoded vectors exceeding 5 kb were heterogeneous in length and truncated on the 5' end. Despite their truncated genomes, vector preparations produced from plasmid-encoded vectors exceeding 5.2 kb mediated reporter gene expression in vitro at high multiplicity of infection (MOI). The efficiency of expression was substantially lower than that of reporter vectors with genomes <5 kb in length. We propose that transcriptionally functional, intact vector genomes are generated in cells transduced at high MOI from the fragmentary genomes of these larger vectors, probably by recombination.

Figure 1

Electrophoresis of AAV-mCEP290 vectors. (a) Structure of the AAV mouse CEP290 vector. (b) Alkaline agarose electrophoresis and Southern blot analysis of isolated AAV mCEP290 vector genomes. Lanes 1 and 2: 8,343 bp vector plasmid DNA marker, 0.1 and 1 ng, respectively; lanes 3 and 4: 5,067 bp vector plasmid DNA marker, 0.1 and 1 ng, respectively; lanes 5–7: DNA from 1 × 10¹⁰ vector genomes isolated from AAV2 mCEP290, AAV5 mCEP290, and AAV8 mCEP290 vector preparations, respectively. AAV, adeno-associated virus; bp, base pair; CMV, cytomegalovirus; ITR, inverted terminal repeat; kb, kilobase.

Figure 2

Genome integrity of AAV5-mCEP290 vector. (a) Hybridization positions of single-stranded oligonucleotides on the vector genome. (b) Strand-specific dot-blot hybridization of vector genome DNA. Twofold serial dilutions of vector plasmid DNA (top row) and isolated AAV5 mCEP290 vector genome DNA (bottom row). The masses of the vector plasmid DNAs are 50, 25, 12.5, 6.25, 3.1, 1.6, 0.78, and 0.39 ng (wells 1–8, top row). The calculated masses of the isolated vector DNAs are 7, 3.5, 1.7, 0.86, 0.43, 0.21, 0.11, and 0.05 ng (wells 1–8, bottom row). The DNA in the first well on the bottom row was isolated from 2.3 × 10⁹ vector genomes.

Figure 3

Packaging of AAV2 and AAV5 lacZ vectors. (a) Structures of plasmid-encoded vectors ranging from 4.7 to 8.7 kb. All of these vectors contained a 4.4 kb CMV lacZ expression cassette, located adjacent to the ITR (I designation in name) or centrally (C designation in name), and phage lambda DNA was used to increase the total length. (b) Southern blot analysis of isolated vector genome DNA separated on an alkaline agarose gel. 1 × 10¹⁰ vector genomes were analyzed for the AAV5 vectors (upper panel), and 1 × 10⁹ vector genomes were analyzed for the AAV2 vectors (lower panel). Vector plasmid DNA markers in lanes M1: 8.6 kb (1 ng), M2: 5.1 kb (1 ng), M3: 8.6 kb (0.1 ng), and M4 5.1 kb (0.1 ng). The DNA in M2 is an unintended partial digestion. Lanes 1–8 in both panels: genomes isolated from AAV-lacZ-4.7k, AAV-lacZ-I5.2k, AAV-lacZ-I5.7k, AAV-lacZ-I6.7k, AAV-lacZ-I8.7k, AAV-lacZ-C5.7k, AAV-lacZ-C6.7k, and AAV-lacZ-C8.7k vector preparations, respectively. CMV, cytomegalovirus; ITR, inverted terminal repeat; kb, kilobase.

Figure 4

LacZ expression in 293 cells infected with AAV2-lacZ vectors. (a) LacZ expression in 293 cells infected with AAV2-lacZ vectors at MOI 2,000 (left panels) or 10,000 (right panels). (b) Quantification of transduction of the AAV2 lacZ vector set. MOI, multiplicity of infection.

Figure 5

Proposed model for packaging and transduction of AAV2 lacZ vectors. (1) Packaging, (2) uncoating, (3) second-strand DNA synthesis, (4) annealing, and (5) homologous recombination.

Figure 6

Proposed model for fragmentary genome transduction. Right panel: one ITR and intact lacZ cassette contained in DNA fragment <5,050 bp in length. Left panel: intact lacZ cassette and either ITR contained in a DNA fragment >5,050 bp in length. The higher transduction efficiency of vectors with ITR-proximal lacZ expression cassettes may indicate that these vectors package the first 5,050 bp from the 3′ end, which encodes one ITR and an intact lacZ expression, and that this fragmentary genome is capable of mediating gene expression without having to anneal to a second vector. bp, base pair; CMV, cytomegalovirus; ITR, inverted terminal repeat.