Hybrid adeno-associated virus bearing nonhomologous inverted terminal repeats enhances dual-vector reconstruction of minigenes in vivo.

We have previously demonstrated that hybrid adeno-associated viral (AAV) vectors bearing nonhomologous inverted terminal repeats (ITRs) enhance directional intermolecular recombination and the efficiency of dual-AAV vector trans-splicing in cultured cells. Using hybrid-ITR vectors carrying two exons of a lacZ minigene, we demonstrate that this dual-vector approach also mediates higher levels (3- to 6-fold) of gene reconstitution in mouse skeletal muscle, liver, and heart. Inhibition of the proteasome by systemic administration of Doxil (Food and Drug Administration-approved lipid-formulated doxorubicin) further enhanced dual-vector trans-splicing 6- to 12-fold in two mouse strains. Hence, using hybrid-ITR AAV vectors in combination with proteasome modulation enhanced dual-vector delivery of a transgene approximately 36-fold over the current dual-vector trans-splicing approaches. These data provide in vivo evidence that ITR sequence-dependent homologous recombination, rather than nonhomologous end joining, is the predominant mechanism for AAV genome heterodimerization. Hence, enhanced directional recombination provided by hybrid-ITR vectors may be a useful in vivo strategy for improving dual-vector delivery of transgenes larger than the AAV packaging limit.

Unique biologic properties of recombinant AAV1 transduction in polarized human airway epithelia.

The choice of adeno-associated virus serotypes for clinical applications is influenced by the animal model and model system used to evaluate various serotypes. In the present study, we sought to compare the biologic properties of rAAV2/1, rAAV2/2, and rAAV2/5 transduction in polarized human airway epithelia using viruses purified by a newly developed common column chromatography method. Results demonstrated that apical transduction of human airway epithelia with rAAV2/1 was 100-fold more efficient than rAAV2/2 and rAAV2/5. This transduction profile in human airway epithelia (rAAV2/1 >> rAAV2/2 = rAAV2/5) was significantly different from that seen following nasal administration of these vectors to mouse lung (rAAV2/5 > rAAV2/1 >> rAAV2/2), emphasizing differences in transduction of these serotypes between these two species. In stark contrast to rAAV2/2 and rAAV2/5, rAAV2/1 transduced both the apical and basolateral membrane of human airway epithelia with similar efficiency. However, the overall level of transduction across serotypes did not correlate with vector internalization. We hypothesized that differences in post-entry processing of these serotypes might influence the efficiency of apical transduction. To this end, we tested the effectiveness of proteasome inhibitors to augment nuclear translocation and gene expression from the three serotypes. Augmentation of rAAV2/1 apical transduction of human polarized airway epithelia was 10-fold lower than that for rAAV2/2 and rAAV2/5. Cellular fractionation studies demonstrated that proteasome inhibitors more significantly enhanced rAAV2/2 and rAAV2/5 translocation to the nucleus than rAAV2/1. These results demonstrate that AAV1 transduction biology in human airway epithelia differs from that of AAV2 and AAV5 by virtue of altered ubiquitin/proteasome sensitivities that influence nuclear translocation.

Spliceosome-mediated RNA trans-splicing with recombinant adeno-associated virus partially restores cystic fibrosis transmembrane conductance regulator function to polarized human cystic fibrosis airway epithelial cells.

We previously reported that spliceosome-mediated RNA trans-splicing (SMaRT), using recombinant adenoviral vectors expressing pre-trans-splicing molecules (PTMs), could partially restore cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel activity to polarized human DeltaF508 CF airway epithelia. Although these studies proved that SMaRT could correct CFTR mRNA defects, recombinant adenoviral infection from the basolateral surface was required because of inefficient infection from the apical membrane. Hence, applications of SMaRT technology for CF gene therapy require further testing with alternative, more clinically viable, vector systems. Furthermore, because recombinant adeno-associated virus (rAAV) vectors have packing limitations with respect to the size of the CFTR transgene insert, SMaRT correction of CFTR has the added attraction of a smaller transgene cassette. In the present study, we investigated whether rAAV vectors could effectively rescue CFTR chloride conductance in polarized human CF airway epithelial cells, using a SMaRT approach. AAV vectors were generated to carry a PTM engineered to bind intron 9 of CFTR pre-mRNA and then trans-splice the normal sequence for human CFTR exons 10-24 into the endogenous pre-mRNA. Human CF polarized airway epithelia were infected from the apical membrane with rAAV2 or rAAV5 CFTR-PTM vectors in the presence of proteasome-modulating agents (doxorubicin and N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal) to enhance transduction. Epithelia were then evaluated for cAMP-sensitive short-circuit currents 2 weeks postinfection. Levels of CFTR correction seen with rAAV2 (1.07 +/- 0.24 microA) and rAAV5 (0.90 +/- 0.20 microA) CFTR-PTM vectors were similar, representing conductance equivalent to 14.2 and 13.6% of that observed in non-CF human polarized epithelia, respectively. RT-PCR analysis demonstrated the existence of wild-type CFTR transcript in CFTR-PTM-corrected epithelia, whereas only DeltaF508 mRNA was detected in polarized cells infected with control rAAV LacZ-PTM vectors. These results provide evidence that rAAV vectors are capable of using SMaRT to correct CFTR function after apical infection of human CF airway epithelia. The ability of CFTR-PTM-mediated correction to maintain endogenous CFTR regulation of the transgene product may further improve the efficacy of gene therapy for CF.

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

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.

Targeted correction of single-base-pair mutations with adeno-associated virus vectors under nonselective conditions.

Recombinant adeno-associated virus (rAAV) vectors possess the unique ability to introduce genetic alterations at sites of homology in genomic DNA through a mechanism thought to predominantly involve homologous recombination. We have investigated the efficiency of this approach using a mutant enhanced green fluorescent protein (eGFP) fluorescence recovery assay that facilitates detection of gene correction events in living cells under nonselective conditions. Our data demonstrate that rAAV infection can correct a mutant eGFP transgene at an efficiency of 0.1% in 293 cells, as determined by fluorescence-activated cell-sorting analysis. Gene repair was also confirmed using clonal expansion of GFP-positive cells and sequencing of the eGFP transgene. These results support previous findings demonstrating the efficacy of rAAV for gene targeting. In an effort to improve gene-targeting efficiencies, we evaluated several agents known to increase rAAV transduction (i.e., expression of an expressed gene), including genotoxic stress and proteasome inhibitors, but observed no correlation between the level of gene repair and rAAV transduction. Interestingly, however, our results demonstrated that enrichment of G(1)/S-phase cells in the target population through the addition of thymidine moderately (approximately 2-fold) increased gene correction compared to cells in other cell cycle phases, including G(0)/G1, G(1), and G(2)/M. These results suggest that the S phase of the cell cycle may more efficiently facilitate gene repair by rAAV. Transgenic mice expressing the mutant GFP were used to evaluate rAAV targeting efficiencies in primary fetal fibroblast and tibialis muscles. However, targeting efficiencies in primary mouse fetal fibroblasts were significantly lower (approximately 0.006%) than in 293 cells, and no correction was seen in tibialis muscles following rAAV infection. To evaluate the molecular structures of rAAV genomes that might be responsible for gene repair, single-cell injection studies were performed with purified viral DNA in a mutant eGFP target cell line. However, the failure of direct cytoplasm- or nucleus-injected rAAV DNA to facilitate gene repair suggests that some aspect of intracellular viral processing may be required to prime recombinant viral genomes for gene repair events.

Distinct classes of proteasome-modulating agents cooperatively augment recombinant adeno-associated virus type 2 and type 5-mediated transduction from the apical surfaces of human airway epithelia.

Tripeptidyl aldehyde proteasome inhibitors have been shown to effectively increase viral capsid ubiquitination and transduction of recombinant adeno-associated virus type 2 (rAAV-2) and rAAV-5 serotypes. In the present study we have characterized a second class of proteasome-modulating agents (anthracycline derivatives) for their ability to induce rAAV transduction. The anthracycline derivatives doxorubicin and aclarubicin were chosen for analysis because they have been shown to interact with the proteasome through a mechanism distinct from that of tripeptidyl aldehydes. Our studies demonstrated that doxorubicin and aclarubicin also significantly augmented rAAV transduction in airway cell lines, polarized human airway epithelia, and mouse lungs. Both tripeptidyl aldehyde and anthracycline proteasome-modulating agents similarly augmented nuclear accumulation of rAAV in A549 and IB3 airway cell lines. However, these two cell types demonstrated cell specificity in the ability of N-acetyl-L-leucyl-L-leucyl-L-norleucine (LLnL) or doxorubicin to augment rAAV transduction. Interestingly, the combined administration of LLnL and doxorubicin resulted in substantially increased transduction (>2,000-fold) following apical infection of human polarized epithelia with either rAAV-2 or rAAV-5. In summary, the cell type specificity of LLnL and doxorubicin to induce rAAV transduction, together with the ability of these compounds to synergistically enhance rAAV transduction in polarized airway epithelial induction, suggests that these two classes of compounds likely modulate different proteasome functions that affect rAAV transduction. Findings from this study provide new insights into how modulation of proteasome function can be effectively used to augment rAAV transduction in airway epithelia for gene therapy of cystic fibrosis.

Immunization with live HPV-16-transformed mouse cells expressing the herpes simplex thymidine kinase and either GM-CSF or IL-2.

From mouse (C57BL/6) HPV-16 transformed cells denoted MK16/1/IIIABC (MK16) a cellular thymidine kinase deficient (cTK-) cell line was isolated. These cTK- cells were transduced by bicistronic recombinant adeno-associated viruses (rAAV) carrying the herpes simplex virus thymidine kinase gene and the gene for either the mouse granulocyte-macrophage colony stimulating factor (GM-CSF) or mouse interleukin-2 (IL-2). Transduced cells were highly sensitive to minute amounts of ganciclovir (GCV) and synthesized moderate amounts of the respective cytokines. A number of cell clones were tested for the cytokine production. The two best producer cell lines, the GM-CSF-producing cells denoted B9 and the IL-2-producing cells denoted 181, were selected for further experiments. Neither B9 nor 181 cells were tumorigenic in syngeneic animals. As inducers of antitumour immunity against challenge with MK16 cells, B9 cells proved superior to the 181 cells. GCV treatment did not markedly influence the level of immunity induced.

Second-strand genome conversion of adeno-associated virus type 2 (AAV-2) and AAV-5 is not rate limiting following apical infection of polarized human airway epithelia.

Recombinant adeno-associated virus type 5 (rAAV-5) is known to efficiently transduce airway epithelia via apical infection. In contrast, rAAV-2 has been shown to be inherently ineffective at transducing airway epithelia from the apical surface. However, tripeptide proteasome inhibitors (such as LLnL) can dramatically enhance rAAV-2 transduction from the apical surface of human polarized airway epithelia by modulating the intracellular trafficking and processing of the virus. To further investigate potential differences between rAAV-2 and rAAV-5 that might explain their altered ability to transduce airway epithelia from the apical membrane, we examined the functional involvement of the ubiquitin/proteasome pathway and rate-limiting aspects of second-strand synthesis for these two rAAV serotypes. To this end, we conducted studies to compare the extent to which LLnL alters transduction efficiencies with both rAAV-2 and rAAV-2/5 by using luciferase and enhanced green fluorescent protein (EGFP) reporter vectors. Our results demonstrate that the coadministration of LLnL at the time of viral infection significantly enhanced transduction of both rAAV-2/5 and rAAV-2 from the apical surface of airway epithelia. Although rAAV-2/5 was slightly more effective at transducing epithelia from the apical membrane, rAAV-2 transduction was superior to that of rAAV-2/5 in the presence of proteasome inhibitors. Interestingly, the basolateral membrane entry pathways for both serotypes were not significantly affected by the addition of LLnL, which suggests that apical and basolateral infectious pathways possess distinctive intracellular processing pathways for both rAAV-2 and rAAV-5. Studies comparing the transduction of short self-complementary (scAAV) to full-length conventional AAV EGFP vectors suggested that second-strand synthesis of rAAV genomes was not rate limiting for either serotype or altered by proteasome inhibitors following apical infection of polarized airway epithelia. These findings suggest that both rAAV-2 and rAAV-5 share similar intracellular viral processing barriers that involve the ubiquitin/proteasome system, but do not appear to involve second-strand synthesis.

Ubiquitination of both adeno-associated virus type 2 and 5 capsid proteins affects the transduction efficiency of recombinant vectors.

In the presence of complementing adeno-associated virus type 2 (AAV-2) Rep proteins, AAV-2 genomes can be pseudotyped with the AAV-5 capsid to assemble infectious virions. Using this pseudotyping strategy, the involvement of the ubiquitin-proteasome system in AAV-5 and AAV-2 capsid-mediated infections was compared. A recombinant AAV-2 (rAAV-2) proviral luciferase construct was packaged into both AAV-2 and AAV-5 capsid particles, and transduction efficiencies in a number of cell lines were compared. Using luciferase expression as the end point, we demonstrated that coadministration of the viruses with proteasome inhibitors not only increased the transduction efficiency of rAAV-2, as previously reported, but also augmented rAAV-5-mediated gene transfer. Increased transgene expression was independent of viral genome stability, since there was no significant difference in the amounts of internalized viral DNA in the presence or absence of proteasome inhibitors. Western blot assays of immunoprecipitated viral capsid proteins from infected HeLa cell lysates and in vitro reconstitution experiments revealed evidence for ubiquitin conjugation of both AAV-2 and AAV-5 capsids. Interestingly, heat-denatured virus particles were preferential substrates for in vitro ubiquitination, suggesting that endosomal processing of the viral capsid proteins is a prelude to ubiquitination. Furthermore, ubiquitination may be a signal for processing of the capsid at the time of virion disassembly. These studies suggest that the previously reported influences of the ubiquitin-proteasome system on rAAV-2 transduction are also active for rAAV-5 and provide a clearer mechanistic framework for understanding the functional significance of ubiquitination.

Tumour-inhibitory and antimetastatic effects of IL-2 in mice carrying MHC class I- tumours of HPV16 origin.

Oncogenic, moderately immunogenic, MHC class I- and class II-, B7-, MK16/1/III ABC (MK16) cells were previously established by co-transfection of HPV16 E6/E7 and activated H-ras oncogene DNA into C57BL/6 kidney cells. Subcutaneous transplantation of these cells produced progressively growing local neoplasms which metastasized spontaneously to lungs and lymph nodes. The MK16 cells were implanted into syngeneic mice and used to examine whether the tumour lacking the signal molecules required for the induction of and sensitivity to T cell immunity is susceptible to local IL-2 treatment and IL-2 gene therapy. Peritumoural administration of human rIL-2 or murine IL-2 gene-modified MK16 tumour vaccine inhibited growth of subcutaneous MK16 tumour transplants and reduced the number of their lung metastases. Spleen cells from MK16 tumour-immunized mice were not cytolytic when allowed to react with the MK16 target cells, although they efficiently lysed the MHC class I+ malignant TC1 cells, obtained from C57BL/6 lung cell cultures after transfection with the same plasmids as those used for the derivation of the MK16 cells. However, when the MK16 cells were cultivated in vitro in the presence of IFNgamma, they acquired, together with the expression of MHC class I molecules, the sensitivity to the cytolytic effect of spleen cells from the MK16 tumour-immunized mice. These results indicate that experimental tumours which are MHC class I- and mimick in this respect a high proportion of human HPV16-associated carcinomas are suitable for IL-2 treatment.