Packaging capacity of adeno-associated virus serotypes: impact of larger genomes on infectivity and postentry steps

Abstract

The limited packaging capacity of adeno-associated virus (AAV) precludes the design of vectors for the treatment of diseases associated with larger genes. Autonomous parvoviruses, such as minute virus of mice and B19, while identical in size (25 nm), are known to package larger genomes of 5.1 and 5.6 kb, respectively, compared to AAV genomes of 4.7 kb. One primary difference is the fact that wild-type (wt) AAV utilizes three capsid subunits instead of two to form the virion shell. In this study, we have characterized the packaging capacity of AAV serotypes 1 through 5 with and without the Vp2 subunit. Using reporter transgene cassettes that range in size from 4.4 to 6.0 kb, we determined that serotypes 1 through 5 with and without Vp2 could successfully package, replicate in, and transduce cells. Dot blot analysis established that packaging efficiency was similar for all vector cassettes and that the integrity of encapsidated genomes was intact regardless of size. Although physical characterization determined that virion structures were indistinguishable from wt, transduction experiments determined that all serotype vectors carrying larger genomes (5.3 kb and higher) transduced cells less efficiently (within a log) than AAV encapsidating wt size genomes. This result was not unique to reporter genes and was observed for CFTR vector cassettes ranging in size from 5.1 to 5.9 kb. No apparent advantage in packaging efficiency was observed when Vp2 was present or absent from the virion. Further analysis determined that a postentry step was responsible for the block in infection and specific treatment of cells upon infection with proteasome inhibitors increased transduction of AAV encapsidating larger DNA templates to wt levels, suggesting a preferential degradation of virions encapsidating larger-than-wt genomes. This study illustrates that AAV is capable of packaging and protecting recombinant genomes as large as 6.0 kb but the larger genome-containing virions are preferentially degraded by the proteasome and that this block can be overcome by the addition of proteasome inhibitors.

FIG. 1.

Vector sequence for the pAVCNst packaging cassettes (10). Each cassette is composed of serotype 2 ITRs with a CMV immediate-early (CMVie) promoter driving CAT gene expression. A stuffer region was designated for cloning in DNA sequences to expand the packagable genome. Letters on the left indicate different vectors. The sizes of the stuffer DNA along with total vector genome size are shown in the second and third columns, respectively.

FIG. 2.

Southern blot of vector DNA isolated directly from AAV virions and run on an alkaline agarose gel. The marker lanes contain DNA fragments produced from pAVCNst K cassette that are 4 kb and 5 kb in size. The DNase control lane represents the pAVCNst K cassette digested with DNase I to show that it was functional. Each set of bracketed lanes represents genomes isolated from AAV encapsidating each pAVCNst vector. The numbers above each bracketed set signify the size of the vectors. The first two lanes in each bracketed set are wt capsid, and the second set of two lanes is DNA isolated from the ACA (Vp2-less) mutants. DNase was added to the lysates of the second and fourth lanes in each bracketed set (marked with asterisks). AAV DNA was labeled with a probe specific for the CAT gene. The single-stranded vector DNAs appear as broad, blurry bands that are characteristic for AAV genomic DNA isolated from AAV capsids.

FIG. 3.

(Top) Southern blot analysis of vector DNA isolated from infected 293 cells in the presence of transfected adenovirus helper (XX680) and AAV2 helper (pXR2). AAV DNA was isolated from infected 293 cells by Hirt extraction and run on a 0.7% agarose gel. AAV DNA was labeled with a probe specific for the CAT gene. The marker lanes contain DNA fragments produced from the pAVCNst K cassette that are 4 kb and 5 kb in size. Replicated dimer genomes for the 5.3- and 6-kb genomes can be detected with extended exposure times. (Bottom) Southern blot of vector DNA isolated from 293 cells infected with CsCl-purified AAV1 and 3 E, K, and L vectors in the presence of transfected adenovirus helper (XX680) and AAV2 helper (pXR2). The sizes of the E (4,675 nucleotides [nts]), K (5,302 nts), and L (6,019 nts) vector genomes are depicted at the top of the Southern blot. The correctly sized genomes and replication products are present within the cells. It is evident that the larger vectors are not as efficient in delivery as the AAV encapsidating genomes of near wt size.

FIG. 4.

Transmission electron micrographs of AAV2 E, K, and L preparations. Peak fractions of the FPLC heparin column-purified AAV2 encapsidating the E, K, and L genomes were stained in 2% uranyl acetate and imaged by transmission electron microscopy. The small arrowhead shows a genome containing rAAV2. The large arrowhead shows empty rAAV2 particles. The size and structure of each AAV vector is consistent. However, more empty particles are evident in the K and L AAV2 vector preparations. nts, nucleotides.

FIG. 5.

Functional assay for the CAT transgene. HeLa cells (6 × 10⁵) were infected with 500 viral genomes/cell with an adenovirus MOI of 5. Cell homogenate was collected from the HeLa cells at 24 h postinfection. CAT assays were carried out on 30 μl of protein homogenate from the infected HeLa cells. Transduction was normalized to the protein concentration for each sample. CAT activity is represented on the vertical axes in counts per minute (cpm) of radioactivity, and the horizontal axes indicate the sizes of the AAV vectors. (Top panels) Graphical representation of CAT transduction with wt capsid AAV2 and AAV4. (Middle panels) Graphical representation of CAT transduction for the Vp2-less (ACA) AAV2 and AAV4. (Bottom panels) CAT transduction profiles of CsCl-purified AAV1 E (4,675 nucleotides), K (5,302 nucleotides), and L (6,019 nucleotides) and heparin column-purified AAV2 E, K, and L. CAT activity was measured to be three- to sevenfold lower for the K and L vectors than for the E and F vectors for serotypes 1 through 5.

FIG. 6.

CAT transduction profile of proteasome inhibitor (LLnL)-treated HeLa cells. HeLa cells (6 × 10⁵) were infected with 500 viral genomes/cell of AAV1 E, K, and L vectors with an adenovirus MOI of 5 and 40 μM LLnL. Cell homogenate was collected from the HeLa cells at 24 h postinfection. CAT assays were carried out on 30 μl of protein homogenate from the infected HeLa cells. Transduction was normalized to the protein concentration for each sample. The gray bars represent the untreated HeLa cells, and the black bars represent HeLa cells treated with 40 μM LLnL. LLnL is shown to increase CAT activity preferentially for the K and L vectors compared to the E vector.

FIG. 7.

(Left) Southern blot analysis of CFTR vector DNA isolated from infected 293 cells with CsCl-purified AAV2-CFTR (5.1 kb), AAV2-CMV-CFTR (5.6 kb), and AAV2-CMV(I)-CFTR (5.9 kb) virus in the presence of transfected adenovirus helper (XX680) and AAV2 helper (pXR2). CFTR vector DNA was isolated from infected 293 cells by Hirt extraction and run on a 0.7% agarose gel. CFTR vector DNA was labeled with a probe specific for the CFTR gene. Replicated monomer and dimer products can be seen for each vector at the correct size based on the 4.7- and 5.7-kb markers. The arrows present in the AAV2-CMV-CFTR and AAV2-CMV(I)-CFTR lanes represent the locations of the replicated dimer products that are present on longer exposures. (Right) Reverse transcription-PCR of poly(A) RNA isolated from AAV2-CFTR-, AAV2-CMV-CFTR-, and AAV2-CMV(I)-CFTR-infected HeLa cells. HeLa cells (5 × 10⁵) were infected with each virus at approximately 2,000 viral genomes/cell. The poly(A) RNA was then isolated from the infected HeLa cells at 48 h postinfection as described in Materials and Methods. Primers specific for the virally delivered CFTR gene were used to detect and amplify 450 bp of the CFTR transcripts. Each CFTR vector is capable of transducing the cell line of interest.

FIG. 8.

Predicted model based on AAV2 trafficking. (I) The first step in AAV2 infection is binding to its primary receptor heparan sulfate proteoglycan and to a secondary receptor. (II) AAV becomes endocytosed via clathrin-coated pits and is brought into the cell in an early endosome. (III) The early endosome then matures into a late endosome as the pH begins to drop to around 5. A pH-dependent conformational change occurs that is thought to expose the N terminus of Vp1, providing the phospholipase activity aiding in endosome escape. (IV) At this point in the pathway, AAV either fails to escape the late endosome, where it later becomes degraded by the lysosome, or escapes into the cytoplasm perinuclearly, where it becomes ubiquitinated. (V) The ubiquitinated virions are then recognized by cytoplasmic proteasomes on their way to the nucleus where they are degraded, but those that avoid interaction with the proteasomes reach the nucleus for genome delivery.