Assessment of optimal transduction of primary human skin keratinocytes by viral vectors

BACKGROUND: Genetically modified keratinocytes generate transplantable self-renewing epithelia suitable for delivery of therapeutic polypeptides. However, the variety of viral vectors and experimental conditions currently used make fragmented or contradictory the information on the transduction efficiency of the human primary keratinocytes. To compare the suitability of the most currently used viral vectors for efficient gene transfer to human keratinocytes, we have performed a comparative study using a panel of recombinant constructs. METHODS: For each vector, the transduction efficiency and the persistence of the transgene expression were quantified by fluorescence microscopy and flow cytometry analysis of the infected cells. RESULTS: We show that: (1) canine and human adenoviral vectors achieve a highly efficient but transient transduction of both primary and immortalized keratinocytes; (2) the adenovirus-associated virus (AAV) vectors transduce immortalized keratinocytes, albeit with a short-lived gene expression (<4 days), but fail to infect primary keratinocytes; and (3) under appropriate conditions, the oncoretroviral and lentiviral vectors can permanently transduce up to 100% of primary keratinocytes, but the highly clonogenic keratinocytes are more efficiently targeted by lentiviral vectors. CONCLUSIONS: Therefore, AAV vectors are unsuitable to transduce primary keratinocytes, while human and canine adenoviral vectors appears to be appropriate to achieve short-term delivery of therapeutic products. Recombinant retroviruses provide sustained expression of the transgene, but the lentiviral vectors are the most suitable for ex vivo gene therapy because of their ability to transduce clonogenic primary keratinocytes.     

Transduction by adeno-associated virus vectors in the rabbit airway: efficiency, persistence, and readministration.

The ability of recombinant adeno-associated virus (AAV) vectors to integrate into the host genome and to transduce nondividing cells makes them attractive as vehicles for gene delivery. In this study, we assessed the ability of several AAV vectors to transduce airway cells in rabbits by measuring marker gene expression. AAV vectors that transferred either a beta-galactosidase (beta-gal) or a human placental alkaline phosphatase (AP) gene were delivered to one lobe of the rabbit lung by use of a balloon catheter placed under fluoroscopic guidance. We observed vector-encoded beta-gal or AP staining almost exclusively in the epithelial and smooth muscle cells in the bronchus at the region of balloon placement. The overall efficiency of transduction in the balloon-treated bronchial epithelium was low but reached 20% in some areas. The majority of the staining was in ciliated cells but was also observed in basal cells and airway smooth muscle cells. We observed an 80-fold decrease in marker-positive epithelial cells during the 60-day period after vector infusion, whereas the number of marker-positive smooth muscle cells stayed constant. Although treatment with the topoisomerase inhibitor etoposide dramatically enhanced AAV transduction in primary airway epithelial cells in culture, treatment of rabbits did not improve transduction rates in the airway. Vector readministration failed to produce additional transduction events, which correlated with the appearance of neutralizing antibodies. These results indicate that both readministration and immune modulation will be required in the use of AAV vectors for gene therapy to the airway epithelium.     

Adeno-associated virus (AAV) serotypes 2, 4 and 5 display similar transduction profiles and penetrate solid tumor tissue in models of human glioma

BACKGROUND: Adeno-associated viral (AAV) vectors are potent delivery vehicles for gene transfer strategies directed at the central nervous system (CNS), muscle and liver. However, comparatively few studies have described AAV-mediated gene transfer to tumor tissues. We have previously demonstrated that while AAV2 and Adenoviral (Ad) 5 vectors have similar broad host ranges in tumor-derived cell lines, AAV2 was able to penetrate human glioblastoma biopsy spheroids and xenografts more efficiently than Ad 5 vectors. These results suggested that AAV vectors could be suitable for therapeutic gene delivery to solid tumor tissue. In the present work, the transduction efficacy of AAV serotypes 4 and 5 were compared to AAV2, both in vitro and in intracranial GBM xenografts derived from patient biopsies implanted into nude rats. METHODS: AAV vector serotypes 2, 4, and 5 containing either the green fluorescent protein (GFP) or the bacterial beta-galactosidase (lacZ) reporter gene were added to five different human glioma cell lines, to multicellular spheroids generated from glioblastoma patient biopsies, and to spheroids xenografted intracranially in nude rats. Transduction efficiency was assessed by fluorescence imaging, histochemistry, immunohistochemistry and flow cytometry. RESULTS: While all three AAV serotypes were able to transduce the glioma cell lines when added individually or when they were administered in concert, AAV2 transduced the glioma cells most effectively compared to AAV4 or AAV5. Upon infecting glioblastoma spheroids in vitro, all three AAV serotypes efficiently transduced cells located at the surface as well as within deeper layers of the spheroids. In addition, similarly to what was observed for AAV2 16, both AAV4 and AAV5 were able to transduce human glioblastoma xenografts implanted intracranially. CONCLUSIONS: In addition to the widely used AAV2 serotype, AAV4 and AAV5 serotypes may also be used to transduce biologically diverse glioma cell lines. They also penetrate and transduce solid human tumor tissue derived from patient biopsies. Therefore, the data presented here provide a proof of principle for developing AAV4 and AAV5 as treatment vehicles for human malignant gliomas.     

Adeno-Associated Virus Type 6 (AAV6) Vectors Mediate Efficient Transduction of Airway Epithelial Cells in Mouse Lungs Compared to That of AAV2 Vectors

Although vectors derived from adeno-associated virus type 2 (AAV2) promote gene transfer and expression in many somatic tissues, studies with animal models and cultured cells show that the apical surface of airway epithelia is resistant to transduction by AAV2 vectors. Approaches to increase transduction rates include increasing the amount of vector and perturbing the integrity of the epithelia. In this study, we explored the use of vectors based on AAV6 to increase transduction rates in airways. AAV vectors were made using combinations of rep, cap, and packaged genomes from AAV2 or AAV6. The packaged genomes encoded human placental alkaline phosphatase and contained terminal repeat sequences from AAV2 or AAV6. We found that transduction efficiency was primarily dependent on the source of Cap protein, defined here as the vector pseudotype. The AAV6 and AAV2 pseudotype vectors exhibited different tropisms in tissue-cultured cells, and cell transduction by AAV6 vectors was not inhibited by heparin, nor did they compete for entry in a transduction assay, indicating that AAV6 and AAV2 capsid bind different receptors. In vivo analysis of vectors showed that AAV2 pseudotype vectors gave high transduction rates in alveolar cells but much lower rates in the airway epithelium. In contrast, the AAV6 pseudotype vectors exhibited much more efficient transduction of epithelial cells in large and small airways, showing up to 80% transduction in some airways. These results, combined with our previous results showing lower immunogenicity of AAV6 than of AAV2 vectors, indicate that AAV6 vectors may provide significant advantages over AAV2 for gene therapy of lung diseases like cystic fibrosis.     

Repeat transduction in the mouse lung by using adeno-associated virus vectors with different serotypes

Vectors derived from adeno-associated virus type 2 (AAV2) promote gene transfer and expression in the lung; however, we have found that while gene expression can persist for at least 8 months in mice, it was reduced dramatically in rabbits over a period of 2 months. The efficiency and persistence of AAV2-mediated gene expression in the human lung have yet to be determined, but it seems likely that readministration will be necessary over the lifetime of an individual. Unfortunately, we have found that transduction by a second administration of an AAV2 vector is blocked, presumably due to neutralizing antibodies generated in response to the primary vector exposure. Here, we have explored the use of AAV2 vectors pseudotyped with capsid proteins from AAV serotypes 2, 3, and 6 for readministration in the mouse lung. We found that an AAV6 vector transduced airway epithelial and alveolar cells in the lung at rates that were at least as high as those of AAV2 pseudotype vectors, while transduction rates mediated by AAV3 were much lower. AAV6 pseudotype vector transduction was unaffected by prior administration of an AAV2 or AAV3 vector, and transduction by an AAV2 pseudotype vector was unaffected by prior AAV6 vector administration, showing that cross-reactive neutralizing antibodies against AAV2 and AAV6 are not generated in mice. Interestingly, while prior administration of an AAV2 vector completely blocked transduction by a second AAV2 pseudotype vector, prior administration of an AAV6 vector only partially inhibited transduction by a second administration of an AAV6 pseudotype vector. Analysis of sera obtained from mice and humans showed that AAV6 is less immunogenic than AAV2, which helps explain this finding. These results support the development of AAV6 vectors for lung gene therapy both alone and in combination with AAV2 vectors.     

Specific adeno-associated virus serotypes facilitate efficient gene transfer into human and non-human primate mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) show great promise for ex vivo gene and cell-mediated therapies. The immunophenotype and in vitro differentiation capacity of primary baboon MSCs was demonstrated to be near-identical to that observed in human MSCs. To optimize gene transfer efficiency, we compared the efficiency of serotypes 1, 2, 3, 4, 5, 6, and 8 of adeno-associated virus (AAV) vectors for their ability to mediate transduction of human and baboon MSCs. AAV serotype 2 vectors were the most efficient in transducing MSCs from humans and baboons. As a reference, human Ad293 cells were transduced with these seven AAV serotypes, and were found to have the highest transduction levels followed by baboon MSCs, and then human MSCs. The order of increasing transduction efficiency for the serotypes tested was similar for human and baboon MSCs, but was different for human Ad293 cells. The transduction efficiency of MSCs isolated from different individuals was comparable within the same species. We also demonstrated that baboon MSCs transduced with AAV serotype 2 vectors retain their potential to differentiate into adipocytes in vitro, and can incorporate into injured muscle tissue of NODSCID mice in vivo. We detected beta-galactosidase reporter gene expression in host muscle tissue for up to 9 weeks in this study, indicating engraftment of transduced baboon MSCs and sustained transgene expression in vivo.     

Optimizing the transduction efficiency of capsid-modified AAV6 serotype vectors in primary human hematopoietic stem cells in vitro and in a xenograft mouse model in vivo

Background aimsAlthough recombinant adeno-associated virus serotype 2 (AAV2) vectors have gained attention because of their safety and efficacy in numerous phase I/II clinical trials, their transduction efficiency in hematopoietic stem cells (HSCs) has been reported to be low. Only a few additional AAV serotype vectors have been evaluated, and comparative analyses of their transduction efficiency in HSCs from different species have not been performed.MethodsWe evaluated the transduction efficiency of all available AAV serotype vectors (AAV1 through AAV10) in primary mouse, cynomolgus monkey and human HSCs. The transduction efficiency of the optimized AAV vectors was also evaluated in human HSCs in a murine xenograft model in vivo.ResultsWe observed that although there are only six amino acid differences between AAV1 and AAV6, AAV1, but not AAV6, transduced mouse HSCs well, whereas AAV6, but not AAV1, transduced human HSCs well. None of the 10 serotypes transduced cynomolgus monkey HSCs in vitro. We also evaluated the transduction efficiency of AAV6 vectors containing mutations in surface-exposed tyrosine residues. We observed that tyrosine (Y) to phenylalanine (F) point mutations in residues 445, 705 and 731 led to a significant increase in transgene expression in human HSCs in vitro and in a mouse xenograft model in vivo.ConclusionsThese studies suggest that the tyrosine-mutant AAV6 serotype vectors are the most promising vectors for transducing human HSCs and that it is possible to increase further the transduction efficiency of these vectors for their potential use in HSC-based gene therapy in humans.     

Targeted adeno-associated virus vector transduction of nonpermissive cells mediated by a bispecific F(ab'gamma)2 antibody

We have developed a system for the targeted delivery of adeno-associated virus (AAV) vectors. Targeting is achieved via a bispecific F(ab')2 antibody that mediates a novel interaction between the AAV vector and a specific cell surface receptor expressed on human megakaryocytes. Targeted AAV vectors were able to transduce megakaryocyte cell lines, DAMI and MO7e, which were nonpermissive for normal AAV infection, 70-fold above background and at levels equivalent to permissive K562 cells. Transduction was shown to occur through the specific interaction of the AAV vector-bispecific F(ab')2 complex and cell-associated targeting receptor. Importantly, targeting appeared both selective and restrictive as the endogenous tropism of the AAV vector was significantly reduced. Binding and internalization through the alternative receptor did not alter subsequent steps (escape from endosomes, migration to nucleus, or uncoating) required to successfully transduce target cells. These results demonstrate that AAV vectors can be targeted to a specific cell population and that transduction can be achieved by circumventing the normal virus receptor.     

Improved short-term engraftment of lentivirally versus gammaretrovirally transduced allogeneic canine repopulating cells

Gammaretroviral vectors require cell division for efficient transduction. Thus, extended cell culture times are necessary for efficient transduction with gammaretroviral vectors, which in turn can lead to stem cell loss and impaired engraftment. Lentiviral vectors transduce nondividing cells and are therefore able to transduce stem cells in short transduction protocols. Here, we compared the short-term engraftment of lentivirally and gammaretrovirally transduced canine allogeneic DLA-matched littermate cells. A reduced conditioning regimen of 400 cGy total body irradiation was used in preparation for clinical studies. Two dogs received a graft of gammaretrovirally transduced CD34-selected cells. CD34(+) cells were prestimulated for 30 h and then exposed twice to concentrated RD114 pseudotype vector. Three dogs received lentivirally transduced CD34-selected cells. Cells were transduced overnight with concentrated VSV-G pseudotype lentiviral vector. The animals in the lentiviral group showed a significantly faster granulocyte recovery. VNTR analysis 40-50 days after transplantation revealed higher donor chimerism for the lentiviral group compared to the retroviral group. These data suggest that short lentiviral transduction protocols may be superior to extended gammaretroviral transduction protocols with respect to engraftment potential of transduced CD34(+) hematopoietic repopulating cells.     

Adeno-associated virus type 2-mediated transfer of ecotropic retrovirus receptor cDNA allows ecotropic retroviral transduction of established and primary human cells.

The cellular receptors that mediate binding and internalization of retroviruses have recently been identified. The concentration and accessibility of these receptors are critical determinants in accomplishing successful gene transfer with retrovirus-based vectors. Murine retroviruses containing ecotropic glycoproteins do not infect human cells since human cells do not express the receptor that binds the ecotropic glycoproteins. To enable human cells to become permissive for ecotropic retrovirus-mediated gene transfer, we have developed a recombinant adeno-associated virus type 2 (AAV) vector containing ecotropic retroviral receptor (ecoR) cDNA under the control of the Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter (vRSVp-ecoR). Established human cell lines, such as HeLa and KB, known to be nonpermissive for murine ecotropic retroviruses, became permissive for infection by a retroviral vector containing a bacterial gene for resistance to neomycin (RV-Neo(r)), with a transduction efficiency of up to 47%, following transduction with vRSVp-ecoR, as determined by the development of colonies that were resistant to the drug G418, a neomycin analog. No G418-resistant colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. Southern and Northern blot analyses revealed stable integration and long-term expression, respectively, of the transduced murine ecoR gene in clonal isolates of HeLa and KB cells. Similarly, ecotropic retrovirus-mediated Neo(r) transduction of primary human CD34+ hematopoietic progenitor cells from normal bone marrow was also documented, but only following infection with vRSVp-ecoR. The retroviral transduction efficiency was approximately 7% without prestimulation and approximately 14% with prestimulation of CD34+ cells with cytokines, as determined by hematopoietic clonogenic assays. No G418-resistant progenitor cell colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. These results suggest that sequential transduction of primary human cells with two different viral vectors may overcome limitations encountered with a single vector. Thus, the combined use of AAV- and retrovirus-based vectors may have important clinical implications for ex vivo and in vivo human gene therapy.     

Hybrid Adeno-Associated Viral Vectors Utilizing Transposase-Mediated Somatic Integration for Stable Transgene Expression in Human Cells

Recombinant adeno-associated viral (AAV) vectors have been shown to be one of the most promising vectors for therapeutic gene delivery because they can induce efficient and long-term transduction in non-dividing cells with negligible side-effects. However, as AAV vectors mostly remain episomal, vector genomes and transgene expression are lost in dividing cells. Therefore, to stably transduce cells, we developed a novel AAV/transposase hybrid-vector. To facilitate SB-mediated transposition from the rAAV genome, we established a system in which one AAV vector contains the transposon with the gene of interest and the second vector delivers the hyperactive Sleeping Beauty (SB) transposase SB100X. Human cells were infected with the AAV-transposon vector and the transposase was provided in trans either by transient and stable plasmid transfection or by AAV vector transduction. We found that groups which received the hyperactive transposase SB100X showed significantly increased colony forming numbers indicating enhanced integration efficiencies. Furthermore, we found that transgene copy numbers in transduced cells were dose-dependent and that predominantly SB transposase-mediated transposition contributed to stabilization of the transgene. Based on a plasmid rescue strategy and a linear-amplification mediated PCR (LAM-PCR) protocol we analysed the SB100X-mediated integration profile after transposition from the AAV vector. A total of 1840 integration events were identified which revealed a close to random integration profile. In summary, we show for the first time that AAV vectors can serve as template for SB transposase mediated somatic integration. We developed the first prototype of this hybrid-vector system which with further improvements may be explored for treatment of diseases which originate from rapidly dividing cells.     

Herpes simplex virus type 1/adeno-associated virus rep(+) hybrid amplicon vector improves the stability of transgene expression in human cells by site-specific integration

Herpes simplex virus type 1 (HSV-1) amplicon vectors are promising gene delivery tools, but their utility in gene therapy has been impeded to some extent by their inability to achieve stable transgene expression. In this study, we examined the possibility of improving transduction stability in cultured human cells via site-specific genomic integration mediated by adeno-associated virus (AAV) Rep and inverted terminal repeats (ITRs). A rep(-) HSV/AAV hybrid amplicon vector was made by inserting a transgene cassette flanked with AAV ITRs into an HSV-1 amplicon backbone, and a rep(+) HSV/AAV hybrid amplicon was made by inserting rep68/78 outside the rep(-) vector 3' AAV ITR sequence. Both vectors also had a pair of loxP sites flanking the ITRs. The resulting hybrid amplicon vectors were successfully packaged and compared to a standard amplicon vector for stable transduction frequency (STF) in human 293 and Gli36 cell lines and primary myoblasts. The rep(+), but not the rep(-), hybrid vector improved STF in all three types of cells; 84% of Gli36 and 40% of 293 stable clones transduced by the rep(+) hybrid vector integrated the transgene into the AAVS1 site. Due to the difficulty in expanding primary myoblasts, we did not assess site-specific integration in these cells. A strategy to attempt further improvement of STF by "deconcatenating" the hybrid amplicon DNA via Cre-loxP recombination was tested, but it did not increase STF. These data demonstrate that introducing the integrating elements of AAV into HSV-1 amplicon vectors can significantly improve their ability to achieve stable gene transduction by conferring the AAV-like capability of site-specific genomic integration in dividing cells.     

Factors Influencing Adeno-Associated Virus-Mediated Gene Transfer to Human Cystic Fibrosis Airway Epithelial Cells: Comparison with Adenovirus Vectors

Adeno-associated virus (AAV) vectors appear promising for use in gene therapy in cystic fibrosis (CF) patients, yet many features of AAV-mediated gene transfer to airway epithelial cells are not well understood. We compared the transduction efficiencies of AAV vectors and adenovirus (Ad) vectors in immortalized cell lines from CF patients and in nasal epithelial primary cultures from normal humans and CF patients. Similar dose-dependent relationships between the vector multiplicities of infection and the efficiencies of lacZ gene transfer were observed. However, levels of transduction for both Ad and recombinant AAV (rAAV) were significantly lower in the airway epithelial cell than in the control cell lines HeLa and HEK 293. Transduction efficiencies differed among cultured epithelial cell types, with poorly differentiated cells transducing more efficiently than well-differentiated cells. A time-dependent increase in gene expression was observed after infection for both vectors. For Ad, but not for AAV, this increase was dependent on prolonged incubation of cells with the vector. Furthermore, for rAAV (but not for rAd), the delay in maximal transduction could be abrogated by wild-type Ad helper infection. Thus, although helper virus is not required for maximal transduction, it increases the kinetics by which this is achieved. Expression of Ad E4 open reading frame 6 or addition of either hydroxyurea or camptothecin resulted in increased AAV transduction, as previously demonstrated for nonairway cells (albeit to lower final levels), suggesting that second-strand synthesis may not be the sole cause of inefficient transduction. Finally, the efficiency of AAV-mediated ex vivo gene transfer to lung cells was similar to that previously described for Ad vectors in that transduction was limited to regions of epithelial injury and preferentially targeted basal-like cells. These studies address the primary factors influencing rAAV infection of human airway cells and should impact successful gene delivery in CF patients.     

Design and packaging of adeno-associated virus gene targeting vectors

Adeno-associated virus (AAV) vectors can transduce cells by several mechanisms, including (i) gene addition by chromosomal integration or episomal transgene expression or (ii) gene targeting by modification of homologous chromosomal sequences. The latter process can be used to correct a variety of mutations in chromosomal genes with high fidelity and specificity. In this study, we used retroviral vectors to introduce mutant alkaline phosphatase reporter genes into normal human cells and subsequently corrected these mutations with AAV gene targeting vectors. We find that increasing the length of homology between the AAV vector and the target locus improves gene correction rates, as does positioning the mutation to be corrected in the center of the AAV vector genome. AAV-mediated gene targeting increases with time and multiplicity of infection, similar to AAV-mediated gene addition. However, in contrast to gene addition, genotoxic stress did not affect gene targeting rates, suggesting that different cellular factors are involved. In the course of these studies, we found that (i) vector genomes less than half of wild-type size could be packaged as monomers or dimers and (ii) packaged dimers consist of inverted repeats with covalently closed hairpins at either end. These studies should prove helpful in designing AAV gene targeting vectors for basic research or gene therapy.     

Adeno-associated virus vector integration junctions.

Vectors derived from adeno-associated virus (AAV) have the potential to stably transduce mammalian cells by integrating into host chromosomes. Despite active research on the use of AAV vectors for gene therapy, the structure of integrated vector proviruses has not previously been analyzed at the DNA sequence level. Studies on the integration of wild-type AAV have identified a common site-specific integration locus on human chromosome 19; however, most AAV vectors do not appear to integrate at this locus. To improve our understanding of AAV vector integration, we analyzed the DNA sequences of several integrated vector proviruses. HeLa cells were transduced with an AAV shuttle vector, and integrated proviruses containing flanking human DNA were recovered as bacterial plasmids for further analysis. We found that AAV vectors integrated as single-copy proviruses at random chromosomal locations and that the flanking HeLa DNA at integration sites was not homologous to AAV or the site-specific integration locus of wild-type AAV. Recombination junctions were scattered throughout the vector terminal repeats with no apparent site specificity. None of the integrated vectors were fully intact. Vector proviruses with nearly intact terminal repeats were excised and amplified after infection with wild-type AAV and adenovirus. Our results suggest that AAV vectors integrate by nonhomologous recombination after partial degradation of entering vector genomes. These findings have important implications for the mechanism of AAV vector integration and the use of these vectors in human gene therapy.     

Integrating Adenovirus–Adeno-Associated Virus Hybrid Vectors Devoid of All Viral Genes

Recently, we demonstrated that inverted repeat sequences inserted into first-generation adenovirus (Ad) vector genomes mediate precise genomic rearrangements resulting in vector genomes devoid of all viral genes that are efficiently packaged into functional Ad capsids. As a specific application of this finding, we generated adenovirus-adeno-associated virus (AAV) hybrid vectors, first-generation Ad vectors containing AAV inverted terminal repeat sequences (ITRs) flanking a reporter gene cassette inserted into the E1 region. We hypothesized that the AAV ITRs present within the hybrid vector genome could mediate the formation of rearranged vector genomes (DeltaAd.AAV) and stimulate transgene integration. We demonstrate here that DeltaAd.AAV vectors are efficiently generated as by-products of first-generation adenovirus-AAV vector amplification. DeltaAd.AAV genomes contain only the transgene flanked by AAV ITRs, Ad packaging signals, and Ad ITRs. DeltaAd.AAV vectors can be produced at a high titer and purity. In vitro transduction properties of these deleted hybrid vectors were evaluated in direct comparison with first-generation Ad and recombinant AAV vectors (rAAVs). The DeltaAd.AAV hybrid vector stably transduced cultured cells with efficiencies comparable to rAAV. Since cells transduced with DeltaAd.AAV did not express cytotoxic viral proteins, hybrid viruses could be applied at very high multiplicities of infection to increase transduction rates. Southern analysis and pulsed-field gel electrophoresis suggested that DeltaAd.AAV integrated randomly as head-to-tail tandems into the host cell genome. The presence of two intact AAV ITRs was crucial for the production of hybrid vectors and for transgene integration. DeltaAd.AAV vectors, which are straightforward in their production, represent a promising tool for stable gene transfer in vitro and in vivo.     

Efficient mouse airway transduction following recombination between AAV vectors carrying parts of a larger gene

The small packaging capacity of adeno-associated virus (AAV) vectors limits the utility of this promising vector system for transfer of large genes. We explored the possibility that larger genes could be reconstituted following homologous recombination between AAV vectors carrying overlapping gene fragments. An alkaline phosphatase (AP) gene was split between two such AAV vectors (rec vectors) and packaged using AAV2 or AAV6 capsid proteins. Rec vectors having either capsid protein recombined to express AP in cultured cells at about 1-2% of the rate observed for an intact vector. Surprisingly, the AAV6 rec vectors transduced lung cells in mice almost as efficiently as did an intact vector, with 10% of airway epithelial cells, the target for treatment of cystic fibrosis (CF), being positive. Thus AAV rec vectors may be useful for diseases such as CF that require transfer of large genes.     

Gene delivery to airway epithelial cells in vivo: a direct comparison of apical and basolateral transduction strategies using pseudotyped lentivirus vectors

Lentivirus vectors are being investigated as gene delivery vehicles for cystic fibrosis airway gene therapy. Vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped vectors transduce airway epithelia via receptors that are located predominantly on the basolateral surface of the airway epithelium. Effective transduction with VSV-G-pseudotyped vectors requires the use of a pre-treatment that disrupts epithelial tight junctions, allowing access to these basolateral receptors. In contrast, it has been reported that apically targeted lentiviral vectors allow efficient gene transfer in the absence of any pre-treatment. In a direct comparison of transduction by a VSV-G-pseudotyped vector, in combination with a pre-treatment with lysophosphatidylcholine (LPC), and the same vector pseudotyped with the apically targeted baculovirus GP64 envelope (without any pre-treatment), the GP64 vector was found to be significantly less efficient. However, when a pre-treatment with LPC was used the level of transduction with the GP64-pseudotyped lentiviral vector was not significantly different to that resulting from the VSV-G-pseudotyped vector. The cell types transduced with each vector were essentially the same, with the majority of cells transduced being respiratory (ciliated cells). However, unlike the VSV-G-pseudotyped vector, which results in persisting gene expression, transduction with the GP64-pseudotyped vector resulted in gene expression that declined to undetectable levels over six months, whether or not an LPC pre-treatment was used.