Characterization of genome integrity for oversized recombinant AAV vector

Abstract

Application of recombinant adeno-associated virus (rAAV) in gene therapy has been limited by its packaging capacity. Recent studies suggested that rAAV could achieve persistent transgene expression beyond 4.7-kb packaging limit. To clarify the mechanism leading to transgene expression from oversized rAAV vector, we constructed a series of rAAV vectors with genomes ranging from 2.9 to 7.2 kb. A plasmid replication origin and an ampicillin-resistant marker were included in the vector to facilitate the recovery of circularized, post-transduction AAV genome. Southern dot-blot analysis and silver staining confirmed that rAAVs could be produced at varying vector size. However, the vector yields decreased approximately tenfold for oversized vectors as compared to regular ones. Alkaline Southern blot hybridization suggested that the packaged genomes for oversized vectors were truncated. In the cells transduced by the above vectors, circularized rAAV monomers could be rescued at 24 hours after infection. Few recovered AAV genomes were >5 kb regardless of the initial vector size. In mice receiving the above vectors, larger circularized rAAV genomes could be recovered for oversized vectors at day 21 after vector administration. Our studies suggested that the partially packaged rAAV sequences may complement each other to restore full expression cassette.

Figure 1

Illustration of AAV vector genomes. The stuffer fragments were partial sequences of factor VIII gene. The vectors were named according to their rAAV genome size. The DNA fragment used for the probe for Southern blot hybridization was identified, which corresponds to the sequences in the ampicillin-resistant gene. AAV, adeno-associated virus; amp^R, an ampicillin-resistant gene, β-lactamase; ITR, inverted terminal repeat sequence; ori, a plasmid replication region of pUC18; ↓, AleI site; *, BamHI site; Δ, SmaI site.

Figure 2

Oversized AAV vectors are inefficient in packaging. rAAV vectors were packaged using AAV5 capsids using triple plasmid transfection method as described in the Materials and Methods section. The resulting rAAV vectors were purified by two rounds of cesium chloride–gradient ultracentrifugation. (a) rAAV vectors in each gradient fraction were quantified by use of Southern dot-blotting. Each dot represented 2 µl of purified vectors from each gradient fraction. The membrane was hybridized with a probe to a region that is identical in all vectors. (b) Detection of capsid proteins by silver staining. A volume of 1 × 10¹⁰ viral particles for each vector were used for the silver staining. rAAV, recombinant adeno-associated virus; Std, the standard for quantification of the purified viral genomes; VP1, VP2, VP3, are AAV capsid proteins.

Figure 3

Analysis of the rAAV genome integrity. The size of the single-stranded DNAs packaged in rAAV capsids were analyzed using alkaline agarose gel electrophoresis. After extensive DNaseI treatment, the packaged DNAs from different vectors were resolved on 1% alkaline agarose gel. The membrane was hybridized with a probe specific to the ampicillin-resistant gene. rAAV, recombinant adeno-associated virus.

Figure 4

Rescued rAAV genomes from HeLa cells. HeLa cells were transduced with similar amounts of rAAVs at a multiplicity of infection of 10,000. After 24-hour infection, low-molecular-weight DNAs were extracted and transformed into competent cells. The ampicillin-resistant colonies growing on Luria–Bertani plates were counted for each vector. The experiment was repeated twice. rAAV, recombinant adeno-associated virus.

Figure 5

Summary of the deleted regions in the rescued rAAV genomes for DB7.2. All plasmids rescued from HeLa cells and mouse muscle tissues were analyzed by restriction enzyme digestion and DNA sequencing. The percentage was calculated as dividing the total number of rescued plasmids by the number of classified plasmids. The semi-filled box illustrates missing region from recovered rAAV genome. amp^R, an ampicillin-resistant gene, β-lactamase; ITR, inverted terminal repeat sequence; ori, a plasmid replication region of pUC18; rAAV, recombinant adeno-associated virus.