Inverted terminal repeat sequences are important for intermolecular recombination and circularization of adeno-associated virus genomes

Abstract

The relatively small package capacity (less than 5 kb) of adeno-associated virus (AAV) vectors has been effectively doubled with the development of dual-vector heterodimerization approaches. However, the efficiency of such dual-vector systems is limited not only by the extent to which intermolecular recombination occurs between two independent vector genomes, but also by the directional bias required for successful transgene reconstitution following concatemerization. In the present study, we sought to evaluate the mechanisms by which inverted terminal repeat (ITR) sequences mediate intermolecular recombination of AAV genomes, with the goal of engineering more efficient vectors for dual-vector trans-splicing approaches. To this end, we generated a novel AAV hybrid-ITR vector characterized by an AAV-2 and an AAV-5 ITR at opposite ends of the viral genome. This hybrid genome was efficiently packaged into either AAV-2 or AAV-5 capsids to generate infectious virions. Hybrid AV2:5 ITR viruses had a significantly lower capacity to form circular intermediates in infected cells than homologous AV2:2 and AV5:5 ITR vectors despite their similar capacity to express an encoded enhanced green fluorescent protein (EGFP) transgene. To examine whether the divergent ITR sequences contained within hybrid AV2:5 ITR vectors could direct intermolecular recombination in a tail-to-head fashion, we generated two hybrid ITR trans-splicing vectors (AV5:2LacZdonor and AV2:5LacZacceptor). Each delivered one exon of a beta-galactosidase minigene flanked by donor or acceptor splice sequences. These hybrid trans-splicing vectors were compared to homologous AV5:5 and AV2:2 trans-splicing vector sets for their ability to reconstitute beta-galactosidase gene expression. Results from this comparison demonstrated that hybrid ITR dual-vector sets had a significantly enhanced trans-splicing efficiency (6- to 10-fold, depending on the capsid serotype) compared to homologous ITR vectors. Molecular studies of viral genome structures suggest that hybrid ITR vectors provide more efficient directional recombination due to an increased abundance of linear-form genomes. These studies provide direct evidence for the importance of ITR sequences in directing intermolecular and intramolecular homologous recombination of AAV genomes. The use of hybrid ITR AAV vector genomes provides new strategies to manipulate viral genome conversion products and to direct intermolecular recombination events required for efficient dual-AAV vector reconstitution of the transgene.

FIG. 1.

Characterization of circular AV5:5/5 vector genomes. To evaluate the ability of the AV5:5/5 virus to form circular intermediates, we used an EGFP shuttle vector to rescue replication-competent plasmids from bacteria following transformation with Hirt DNA from AV5:5/5eGFP-infected HeLa cells (500 DNase-resistant particles/cell). (A) Schematic diagram of the AV5:5eGFP viral genome. (B) The predominant form of rescued circular rAAV-5 intermediates is represented as a double-D circular monomer. The positions of the BamHI sites used for restriction mapping and the primers (EL1080 and EL1088) used for sequencing of the double-D ITR junction are shown. (C) Restriction enzyme and Southern blotting analyses of rescued AV5:5eGFP circular intermediates are displayed. Plasmids from 19 randomly chosen colonies were analyzed by BamHI digestion, followed by Southern blotting with ³²P-labeled AAV-5 ITR and EGFP probes. The major bands of BamHI digestion from an AV5:5eGFP double-D circular monomer are expected to migrate as a ≈200-bp double-D ITR fragment, a 620-bp cytomegalovirus promoter fragment, a 760-bp EGFP cDNA fragment, and a ≈3-kb band containing the simian virus 40 polyadenylation site and the ampicillin resistance and ori sequences. Isolated clones incurring deletions (Δ) within the ITR junction are marked above the gel. (D) The sequence of ITR junctions from AV5:5eGFP circular intermediates is shown in panel C. The EL1081 (5′-CAAGTGGGCAGTTTACCGTA-3′) and EL1088 (5′-CATTAATGCAGCTGGCACGA-3′) primers were used for sequencing the AAV-5 ITR junction as positioned in panel B. The sequence results from individual rescued circular intermediates are aligned against a predicted double-D AAV-5 head-to-tail ITR junction. BamHI sites in the sequence are marked with a box. Known deletions confirmed by sequencing are marked with dashed lines, and regions that could not be sequenced due to secondary structure are marked with solid lines. Sequences from all other clones not shown were identical to p1.

FIG. 2.

Dual rAAV vector reconstitution of a β-galactosidase minigene through heterodimerization with the AV2:2 or AV5:5 vector. (A) Schematic representation of the rAAV-5-based lacZ trans-splicing vectors used to assess heterodimerization. Only heterodimerization of these two vectors in a tail-to-head orientation is capable of reconstituting a functional lacZ gene product. 5′-LacZ, the first half of the lacZ genome; 3′-LacZ, the second half of the lacZ genome; SD, splice donor; SA, splice acceptor; RSV, promoter/enhancer; SV40, polyadenylation sequences; Amp, ampicillin resistance gene; Ori, bacterial replication origin. (B) Ferret fetal fibroblasts were coinfected with the AV5:5/5LacZdonor or AV5:5/5LacZacceptor virus at a multiplicity of infection of 2,500 DNase-resistant particles/cell. Cells were then functionally evaluated for β-galactosidase expression by histochemical X-Gal staining at 7 days postinfection. (C) The efficiency of heterodimerization for AV2:2 and AV5:5 vector genomes were compared in HeLa cells with dual-vector trans-splicing reconstitution of β-galactosidase expression. All viruses were packaged into AAV-5 capsids. HeLa cells were infected at a multiplicity of infection of 2,500 DNase-resistant particles/cell with each virus alone or in combination, as indicated. β-Galactosidase activity was quantified at 3 days postinfection. Data represent the mean (± standard error of the mean) of four independent infections.

FIG. 3.

Hybrid ITR viral genomes produce infectious virus with efficiency similar to that of genomes with homogenous ITR structure. (A) Homology alignment of AAV-2 and AAV-5 ITRs generated by Higgins' arithmetic with DNASIS software. Identical nucleotides in both sequences are indicated by shading. The Rep protein-binding motif is underlined, the terminal resolution site motif (trs) is marked with a box, and the site of cleavage in each is indicated with vertical arrows. (B) Schematic structure of the hybrid ITR proviral plasmid pAV2:5eGFP and AV2:5eGFP viral genome. The hybrid ITR vector was packaged into either an AAV-2 or AAV-5 capsid to assemble the AV2:5/2eGFP and AV2:5/5eGFP infectious viruses, respectively. Similar vectors with uniform AAV-2 and AAV-5 ITRs on either end of the genome were used to generate AV2:2/2eGFP and AV5:5/5eGFP viruses, respectively (not diagrammed). (C) Alkaline Southern blot analysis was used to evaluate viral genome integrity from purified AV2:5/2eGFP, AV2:2/2eGFP, and AV5:5eGFP viruses. A uniform band from each vector was visualized with a ³²P-labeled EGFP probe in this Southern blot and confirmed the packaging of intact viral genomes for each virus type. (D) Slot blot analysis evaluating DNA from purified viruses with different probes against the AAV-2 ITR, AAV-5 ITR, the EGFP transgene, or stuffer sequences in the backbone of proviral plasmids. pCat is a control plasmid with no sequence homology to any of the probes. pAV2:5eGFP is a control proviral plasmid used to compare hybridization signals for all probes used. Results demonstrate equivalent packaging of both AAV-2 and AAV-5 ITRs in the hybrid vectors and nearly equivalent packaging of stuffer sequences (≈10%) in all vectors. (E) Slot blot analysis was used to evaluate the efficiency of plus- and minus-strand viral genome packaging. Oligonucleotide probes against the sense and antisense strands of EGFP cDNA were used for this analysis. The minus strand of the viral genome was detected with a 44-mer probe, 5′-TCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAG-3′, and the plus viral strand was detected with a complementary probe, 5′-CTTGTAGTTGCCGTCGTCCTTGAAGAAGATGGTGCGCTCCTGGA-3′. Oligonucleotide probes were end labeled with ³²P by using T4 polynucleotide kinase and [γ-³²P]ATP; 4 × 10⁸ and 2 × 10⁹ DNase-resistant particles of AV2:5/2eGFP, AV2:2/2eGFP, and AV5:5/5eGFP were used for the slot blot hybridization assay. The results demonstrated equivalent ratios of plus- and minus-strand viral genomes for all vectors analyzed. (F) The infectious titer of hybrid ITR viruses (AV2:5/2eGFP and AV2:5/5eGFP) was compared to that achieved with native AV2:2/2eGFP and AV5:5/5eGFP viruses following infection in HeLa cells. HeLa cells were infected with 500 DNase-resistant particles/cell of AV2:2/2eGFP, AV5:5/5eGFP, AV2:5/2eGFP, or AV2:5/5eGFP, and EGFP fluorescent photomicrographs were taken at 48 h postinfection. Hybrid ITR viruses demonstrate levels of transduction similar to that of the native vectors for each serotype. Note that transduction with the AAV-5 capsid virus is approximately four- to fivefold lower than that achieved with AAV-2 capsid vectors in HeLa cells.

FIG. 4.

Hybrid AV2:5 ITR viral genomes have reduced capacity to form circular intermediates. HeLa cells were infected at a multiplicity of infection of 500 DNase-resistant particles/cell with AV2:2/2eGFP, AV5:5/2eGFP, or AV2:5/2eGFP virus. The low-molecular-weight Hirt DNA was extracted at 1 day postinfection, and 1/10th of the DNA was used to transform E. coli Sure cells. (A) Total rescued ampicillin-resistant CFU and head-to-tail circular intermediates with double-D ITR junctions are indicated. Results indicate the mean (± standard error of the mean) for triplicate assays for each experimental point. (B) The predicted structure of a head-to-tail circularized hybrid ITR genome is given with restriction enzyme sites and sequence primers used for molecular characterization. (C) Restriction enzyme analysis of 24 circular intermediates rescued following AV2:5/2eGFP infection. Isolated plasmids p12, p14, p17, p18, and p21 (marked by arrows) were the only five clones that demonstrated an intact 800-bp EGFP SacII fragment and a ≈200-bp ITR SphI fragment that matched the predicted double-D circular intermediate structure seen with the AV2:2 and AV5:5 genomes. These clones were further analyzed by sequencing of the ITR junction with primers EL1080 and EL1081. (D) Sequence results of the five clones containing a double-D ITR junction. The upper line depicts consistent sequences found in all clones and includes an intact D′ sequence from the AAV-5 and AAV-2 ITRs. The boldface partial sequence is the area flanking the ITRs where the SphI site (boxed) resides. Below is the remaining sequence between the two D sequences for each of the individual clones, as marked. The sequence homology to AAV-5 ITR is indicated in black, and homology to AAV-2 ITR is indicated in red. The various palindromic segments of the ITR are indicated by D, A′ B, B′, C, C′, and A, according to the standard nomenclature. The D′ and A segments of the AAV-2 ITR and the A′ and D segments of AAV-5 are marked with underlining. Segments with deletions are marked by an asterisk.

FIG. 5.

Southern blot analysis of Hirt DNA extracted from AV2:5/2eGFP- and AV2:2/2eGFP-infected HeLa cells. HeLa cells were infected with AV2:2/2eGFP and AV2:5/2eGFP at a multiplicity of infection of 500 DNase-resistant particles/cell, and Hirt DNAs were extracted at different time points postinfection. One-quarter of the Hirt DNA from each sample was resolved on a 1.2% agarose gel, and Southern blotting of viral DNA was performed with a ³²P-labeled probe against the cytomegalovirus promoter. (A) Schematic representation of the predicted StuI digestion products from possible double-stranded transduction intermediates. H-H, head-to-head; T-T, tail-to-tail; H-T, head-to-tail. (B) Hirt DNA samplesat 2 days postinfection were digested with StuI and analyzed by Southern blotting with a cytomegalovirus probe. The open arrows point to a 0.94-kb band end fragment diagnostic for the existence of linear viral genomes. Solid arrows point to bands diagnostic for monomer supercoiled circular intermediates in the uncut sample (2.8 kb) and linear full-length genomes in the StuI-digested sample (4.7 kb). The migration of single-stranded viral DNA (ssDNA) is also marked. U, undigested; C, StuI digested. (C) Undigested Hirt DNAs extracted from AV2:2/2eGFP- and AV2:5/2eGFP-infected HeLa cells at 6 h, 1 day, 2 days, 3 days, and 5 days postinfection were analyzed by Southern blotting. Solid arrows point to a 2.8-kb band previously characterized as supercoiled circular monomers (32), which is visible only AV2:2/2eGFP-infected cells.

FIG. 6.

Comparison of dual-vector heterodimerization between hybrid and homologous ITR vectors. Dual-vector reconstitution of a lacZ minigene was used to evaluate the extent of tail-to-head intermolecular recombination with hybrid and homologous ITR vectors. (A) Schematic representation of the hybrid ITR trans-splicing viral genomes, AV5:2LacZdonor and AV2:5LacZacceptor, which were used to generate AAV-2 serotype viruses AV5:2/2LacZdonor and AV2:5/2LacZacceptor, respectively. The AV2:2/2LacZdonor and AV2:2/2LacZacceptor viruses (not shown) had similar structures but contained AAV-2 ITRs on both ends of the genome. (B to D) HeLa cells were infected at a multiplicity of infection of 2,500 DNase-resistant particles/cell with each virus alone or in combination, as indicated. β-Galactosidase activity was quantified at 3 days postinfection. This analysis was performed with (B) AAV-2-encapsidated viruses and (C) AAV-5-encapsidated viruses. (D) Mixing experiments with combinations of hybrid and homologous ITR donor and acceptor vectors were also performed to assess sequence specificity in ITR recombination. Vector combinations are shown schematically on the graph, with open and solid arrowheads indicating AAV-2 and AAV-5 ITRs, respectively. Data represent the mean (± standard error of the mean) of seven independent infections.

FIG. 7.

Hybrid ITR vectors facilitate directional heterodimerization more efficiently than homologous ITR vectors. Hybrid ITR vectors have been shown to more efficiently reconstitute dual-vector trans-spliced transgenes than homologous ITR vectors. Based on molecular analysis of circular and linear intermediates from these two vector types, working hypotheses regarding intermolecular and intramolecular recombination events can be derived for (A) homologous ITR vectors and (B) hybrid ITR vectors. (A) Predicted transduction intermediates and possible intermolecular heterodimers are shown for homologous ITR vectors. With this vector type, 25% of linear dimers are capable of reconstituting a dually encoded minigene (marked by an arrow). Circular dimers are derived either from recombination between circular monomers or by circularization of linear dimers; 50% of circular dimers generated by either of these two processes would be expected to reconstitute a functional minigene. (B) In contrast, hybrid ITR vectors inefficiently form circular monomers, and 50% of linear heterodimers would be expected to reconstitute a functional minigene. However, self-circularization of these two forms of linear heterodimers is also expected to occur via ITR-mediated homologous recombination. If such events occur, all the resulting circular heterodimers will be competent to functionally trans-splice, and the probability of reconstituting the functional minigene will be even higher. This working model helps to explain how hybrid ITR vectors may be capable of greatly enhancing dual-vector trans-splicing approaches by altering the equilibrium of various viral genome intermediates within the cell.