Production and Characterization of Improved Adenovirus Vectors with the E1, E2b, and E3 Genes Deleted

Adenovirus (Ad)-based vectors have great potential for use in the gene therapy of multiple diseases, both genetic and nongenetic. While capable of transducing both dividing and quiescent cells efficiently, Ad vectors have been limited by a number of problems. Most Ad vectors are engineered such that a transgene replaces the Ad E1a, E1b, and E3 genes; subsequently the replication-defective vector can be propagated only in human 293 cells that supply the deleted E1 gene functions in trans. Unfortunately, the use of high titers of E1-deleted vectors has been repeatedly demonstrated to result in low-level expression of viral genes still resident in the vector. In addition, the generation of replication-competent Ad (RCA) by recombination events with the E1 sequences residing in 293 cells further limits the usefulness of E1-deleted Ad vectors. We addressed these problems by isolating new Ad vectors deleted for the E1, E3, and the E2b gene functions. The new vectors can be readily grown to high titers and have several improvements, including an increased carrying capacity and a theoretically decreased risk for generating RCA. We have also demonstrated that the further block to Ad vector replication afforded by the deletion of both the E1 and E2b genes significantly diminished Ad late gene expression in comparison to a conventional E1-deleted vector, without destabilization of the modified vector genome. The results suggested that these modified vectors may be very useful both for in vitro and in vivo gene therapy applications.     

Generation of Adenovirus Vectors Devoid of All Viral Genes by Recombination between Inverted Repeats

Direct or inverse repeated sequences are important functional features of prokaryotic and eukaryotic genomes. Considering the unique mechanism, involving single-stranded genomic intermediates, by which adenovirus (Ad) replicates its genome, we investigated whether repetitive homologous sequences inserted into E1-deleted adenoviral vectors would affect replication of viral DNA. In these studies we found that inverted repeats (IRs) inserted into the E1 region could mediate predictable genomic rearrangements, resulting in vector genomes devoid of all viral genes. These genomes (termed DeltaAd.IR) contained only the transgene cassette flanked on both sides by precisely duplicated IRs, Ad packaging signals, and Ad inverted terminal repeat sequences. Generation of DeltaAd.IR genomes could also be achieved by coinfecting two viruses, each providing one inverse homology element. The formation of DeltaAd.IR genomes required Ad DNA replication and appeared to involve recombination between the homologous inverted sequences. The formation of DeltaAd. IR genomes did not depend on the sequence within or adjacent to the inverted repeat elements. The small DeltaAd.IR vector genomes were efficiently packaged into functional Ad particles. All functions for DeltaAd.IR replication and packaging were provided by the full-length genome amplified in the same cell. DeltaAd.IR vectors were produced at a yield of approximately 10(4) particles per cell, which could be separated from virions with full-length genomes based on their lighter buoyant density. DeltaAd.IR vectors infected cultured cells with the same efficiency as first-generation vectors; however, transgene expression was only transient due to the instability of deleted genomes within transduced cells. The finding that IRs present within Ad vector genomes can mediate precise genetic rearrangements has important implications for the development of new vectors for gene therapy approaches.     

Genome size and structure determine efficiency of postinternalization steps and gene transfer of capsid-modified adenovirus vectors in a cell-type-specific manner

Adenovirus serotype 5 (Ad5) vectors containing Ad B-group fibers have become increasingly popular as gene transfer vectors because they efficiently transduce human cell types that are relatively refractory to Ad5 infection. So far, most B-group fiber-containing vectors have been first-generation vectors, deleted of E1 and/or E3 genes. Transduction with these vectors, however, results in viral gene expression and is associated with cytotoxicity and immune responses against transduced cells. To circumvent these problems, we developed fiber-chimeric Ad vectors devoid of all viral genes that were produced either by the homologous recombination of first-generation vectors or by using the Cre/lox-based helper virus system. In this study we compared early steps of infection between first-generation (35-kb genome) and Ad vectors devoid of all viral genes with genome sizes of 28 kb and 12.6 kb. All vectors possessed an Ad35-derived fiber knob domain, which uses CD46 as a primary attachment receptor. Using immortalized human hematopoietic cell lines and primary human CD34-positive hematopoietic cells, we found that the Ad genome size did not affect the efficiency of virus attachment to and internalization into cells. Furthermore, independently of the genome length and structure, all vectors migrated to the nucleus through late endosomal and lysosomal cellular compartments. However, the vector containing the short 12.6-kb genome was unable to efficiently escape from endosomes and deliver its DNA into the nucleus. Moreover, compared to other vectors, these Ad particles were less stable and had an abnormal capsid protein composition, including a lack of capsid-stabilizing protein IX. Our data indicate that the size and structure of the packaged viral genomes can affect the integrity of Ad particles, which in turn results in lower infectivity of Ad vectors.     

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.     

Molecular characterization of replication-competent variants of adenovirus vectors and genome modifications to prevent their occurrence.

Adenovirus (Ad) vectors for gene therapy are made replication defective by deletion of E1 region genes. For isolation, propagation, and large-scale production of such vectors, E1 functions are supplied in trans from a stable cell line. Virtually all Ad vectors used for clinical studies are produced in the 293 cell, a human embryonic kidney cell line expressing E1 functions from an integrated segment of the left end of the Ad type 5 (Ad5) genome. Replication-competent vector variants that have regained E1 sequences have been observed within populations of Ad vectors grown on 293 cells. These replication-competent variants presumably result from recombination between vector and 293 cell Ad5 sequences. We have developed Ad2-based vectors and have characterized at the molecular level examples of replication-competent variants. All such variants analyzed are Ad2-Ad5 chimeras in which the 293 cell Ad5 E1 sequences have become incorporated into the viral genome by legitimate recombination events. A map of Ad5 sequences within the 293 cell genome developed in parallel is consistent with the proposed recombination events. To provide a convenient vector production system that circumvents the generation of replication-competent variants, we have modified the Ad2 vector backbone by deleting or rearranging the protein IX coding region normally present downstream from the E1 region such that the frequency of recombination between vector and 293 cell Ad5 sequences is greatly reduced. Twelve serial passages of an Ad2 vector lacking the protein IX gene were carried out without generating replication-competent variants. In the course of producing and testing more than 30 large-scale preparations of vectors lacking the protein IX gene or having a rearranged protein IX gene, only three examples of replication-competent variants were observed. Use of these genome modifications allows use of conventional 293 cells for production of large-scale preparations of Ad-based vectors lacking replication-competent variants.     

A new type of adenovirus vector that utilizes homologous recombination to achieve tumor-specific replication

We have developed a new class of adenovirus vectors that selectively replicate in tumor cells. The vector design is based on our recent observation that a variety of human tumor cell lines support DNA replication of adenovirus vectors with deletions of the E1A and E1B genes, whereas primary human cells or mouse liver cells in vivo do not. On the basis of this tumor-selective replication, we developed an adenovirus system that utilizes homologous recombination between inverted repeats to mediate precise rearrangements within the viral genome resulting in replication-dependent activation of transgene expression in tumors (Ad.IR vectors). Here, we used this system to achieve tumor-specific expression of adenoviral wild-type E1A in order to enhance viral DNA replication and spread within tumor metastases. In vitro DNA replication and cytotoxicity studies demonstrated that the mechanism of E1A-enhanced replication of Ad.IR-E1A vectors is efficiently and specifically activated in tumor cells, but not in nontransformed human cells. Systemic application of the Ad.IR-E1A vector into animals with liver metastases achieved transgene expression exclusively in tumors. The number of transgene-expressing tumor cells within metastases increased over time, indicating viral spread. Furthermore, the Ad.IR-E1A vector demonstrated antitumor efficacy in subcutaneous and metastatic models. These new Ad.IR-E1A vectors combine elements that allow for tumor-specific transgene expression, efficient viral replication, and spread in liver metastases after systemic vector application.     

Development and assessment of human adenovirus type 11 as a gene transfer vector

Adenovirus vectors based on human serotype 5 (Ad5) have successfully been used as gene transfer vectors in many gene therapy-based approaches to treat disease. Despite their widespread application, many potential therapeutic applications are limited by the widespread prevalence of vector-neutralizing antibodies within the human population and the inability of Ad5-based vectors to transduce important therapeutic target cell types. In an attempt to circumvent these problems, we have developed Ad vectors based on human Ad serotype 11 (Ad11), since the prevalence of neutralizing antibodies to Ad11 in humans is low. E1-deleted Ad11 vector genomes were generated by homologous recombination in 293 cells expressing the Ad11-E1B55K protein or by recombination in Escherichia coli. E1-deleted Ad11 genomes did not display transforming activity in rodent cells. Transduction of primary human CD34+ hematopoietic progenitor cells and immature dendritic cells was more efficient with Ad11 vectors than with Ad5 vectors. Thirty minutes after intravenous injection into mice that express one of the Ad11 receptors (CD46), we found, in a pattern and at a level comparable to what is found in humans, Ad11 vector genomes in all analyzed organs, with the highest amounts in liver, lung, kidney, and spleen. Neither Ad11 genomes nor Ad11 vector-mediated transgene expression were, however, detected at 72 h postinfusion. A large number of Ad11 particles were also found to be associated with circulating blood cells. We also discovered differences in in vitro transduction efficiencies and in vivo biodistributions between Ad11 vectors and chimeric Ad5 vectors possessing Ad11 fibers, indicating that Ad11 capsid proteins other than fibers influence viral infectivity and tropism. Overall, our study provides a basis for the application of Ad11 vectors for in vitro and in vivo gene transfer and for gaining an understanding of the factors that determine Ad tropism.     

Replication-deficient human adenovirus type 35 vectors for gene transfer and vaccination: efficient human cell infection and bypass of preexisting adenovirus immunity

Replication-deficient human adenovirus type 5 (Ad5) can be produced to high titers in complementing cell lines, such as PER.C6, and is widely used as a vaccine and gene therapy vector. However, preexisting immunity against Ad5 hampers consistency of gene transfer, immunological responses, and vector-mediated toxicities. We report the identification of human Ad35 as a virus with low global prevalence and the generation of an Ad35 vector plasmid system for easy insertion of heterologous genes. In addition, we have identified the minimal sequence of the Ad35-E1B region (molecular weight, 55,000 [55K]), pivotal for complementation of fully E1-lacking Ad35 vector on PER.C6 cells. After stable insertion of the 55K sequence into PER.C6 cells a cell line was obtained (PER.C6/55K) that efficiently transcomplements both Ad5 and Ad35 vectors. We further demonstrate that transduction with Ad35 is not hampered by preexisting Ad5 immunity and that Ad35 efficiently infects dendritic cells, smooth muscle cells, and synoviocytes, in contrast to Ad5.     

Replication properties of human adenovirus in vivo and in cultures of primary cells from different animal species

Oncolytic adenoviruses have emerged as a promising approach for the treatment of tumors resistant to other treatment modalities. However, preclinical safety studies are hampered by the lack of a permissive nonhuman host. Screening of a panel of primary cell cultures from seven different animal species revealed that porcine cells support productive replication of human adenovirus type 5 (Ad5) nearly as efficiently as human A549 cells, while release of infectious virus by cells from other animal species tested was diminished by several orders of magnitude. Restriction of productive Ad5 replication in rodent and rabbit cells seems to act primarily at a postentry step. Replication efficiency of adenoviral vectors harboring different E1 deletions or mutations in porcine cells was similar to that in A549 cells. Side-by-side comparison of the viral load kinetics in blood of swine and mice injected with Ad5 or a replication-deficient adenoviral vector failed to provide clear evidence for virus replication in mice. In contrast, evidence suggests that adenovirus replication occurs in swine, since adenoviral late gene expression produced a 13.5-fold increase in viral load in an individual swine from day 3 to day 7 and 100-fold increase in viral DNA levels in the Ad5-infected swine compared to the animal receiving a replication-deficient adenovirus. Lung histology of Ad5-infected swine revealed a severe interstitial pneumonia. Although the results in swine are based on a small number of animals and need to be confirmed, our data strongly suggest that infection of swine with human adenovirus or oncolytic adenoviral vectors is a more appropriate animal model to study adenoviral pathogenicity or pharmacodynamic and toxicity profiles of adenoviral vectors than infection of mice.     

Development and characterization of a binary gene expression system based on bacteriophage T7 components in adenovirus vectors

To explore the utility of the bacteriophage T7 binary system in adenovirus (Ad) vectors we constructed three Ad5-based vectors containing the T7 RNA polymerase (T7pol) gene in either early region 1 (E1) or E3. The recombinant Ad vectors were either deficient (AdT7pol1, AdT7pol2) or competent (AdT7pol3) for replication in human cells other than Ad5 transformed (293) cells. To test the ability of the T7 polymerase produced by these vectors to drive gene expression, a reporter vector was constructed with an E1 substitution comprising the bacterial β-galactosidase (βGal) (lacZ) gene under the control of the T7 gene 10 promoter (T7pro) and linked to the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) (AdBHG10T7βGal). Coinfections were performed with the various AdT7pol vectors and the reporter vector, and expression was analysed in three different human cell lines: 293, A549 and MRC-5. Depending on the AdT7pol vector used, different levels of expression were obtained from the reporter gene. In 293 cells, expression was detected following infection at very low multiplicities of infection (moi) with all of the T7pol vectors when coinfected with the reporter vector AdBHG10T7βGal. In A549 and MRC-5 cells very little expression was detected using AdT7pol1 or pol2 and efficient expression was only obtained when relatively high moi values of the replication-competent vector were used in the coinfections. We also constructed a single vector containing both elements of the T7 system (T7pol in E3 and T7 promoter driving expression of the chloramphenicol acetyl transferase (cat) gene in E1). This vector proved difficult to rescue but was stable once isolated. Finally, experiments performed to evaluate the `leakiness' of the Ad-T7 system detected very little expression from the T7pro in the absence of T7 polymerase suggesting this system may be useful for the cloning and expression of genes encoding cytotoxic proteins.     

Transfer of the full-length dystrophin-coding sequence into muscle cells by a dual high-capacity hybrid viral vector with site-specific integration ability

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene, making it a potential target for gene therapy. There is, however, a scarcity of vectors that can accommodate the 14-kb DMD cDNA and permanently genetically correct muscle tissue in vivo or proliferating myogenic progenitors in vitro for use in autologous transplantation. Here, a dual high-capacity adenovirus-adeno-associated virus (hcAd/AAV) vector with two full-length human dystrophin-coding sequences flanked by AAV integration-enhancing elements is presented. These vectors are generated from input linear monomeric DNA molecules consisting of the Ad origin of replication and packaging signal followed by the recently identified AAV DNA integration efficiency element (p5IEE), the transgene(s) of interest, and the AAV inverted terminal repeat (ITR). After infection of producer cells with a helper Ad vector, the Ad DNA replication machinery, in concert with the AAV ITR-dependent dimerization, leads to the assembly of vector genomes with a tail-to-tail configuration that are efficiently amplified and packaged into Ad capsids. These dual hcAd/AAV hybrid vectors were used to express the dystrophin-coding sequence in rat cardiomyocytes in vitro and to restore dystrophin synthesis in the muscle tissues of mdx mice in vivo. Introduction into human cells of chimeric genomes, which contain a structure reminiscent of AAV proviral DNA, resulted in AAV Rep-dependent targeted DNA integration into the AAVS1 locus on chromosome 19. Dual hcAd/AAV hybrid vectors may thus be particularly useful to develop safe treatment modalities for diseases such as DMD that rely on efficient transfer and stable expression of large genes.     

Induction of endogenous genes following infection of human endothelial cells with an E1(-) E4(+) adenovirus gene transfer vector

Recombinant adenovirus (Ad) gene transfer vectors are effective at transferring exogenous genes to a variety of cells and tissue types both in vitro and in vivo. However, in the process of gene transfer, the Ad vectors induce the expression of target cell genes, some of which may modify the function of the target cell and/or alter the local milieu. To develop a broader understanding of Ad vector-mediated induction of endogenous gene expression, genes induced by first-generation E1(-) E4(+) Ad vectors in primary human umbilical vein endothelial cells were identified by cDNA subtraction cloning. The identified cDNAs included signaling molecules (lymphoid blast crisis [LBC], guanine nucleotide binding protein alpha type S [Galpha-S], and mitogen kinase [MEK5]), calcium-regulated/cytoskeletal proteins (calpactin p11 and p36 subunits, vinculin, and spinocerebellar ataxia [SCA1]), growth factors (insulin-like growth factor binding protein 4 and transforming growth factor beta2), glyceraldehyde-6-phosphate dehydrogenase, an expressed sequence tag, and a novel cDNA showing homology to a LIM domain sequence. Two- to sevenfold induction of the endogenous gene expression was observed at 24 h postinfection, and induction continued up to 72 h, although the timing of gene expression varied among the identified genes. In contrast to that observed in endothelial cells, the Ad vector-mediated induction of gene expression was not found following Ad vector infection of primary human dermal fibroblasts or human alveolar macrophages. Empty Ad capsids did not induce endogenous gene expression in endothelial cells. Interestingly, additional deletion of the E4 gene obviated the upregulation of genes in endothelial cells by the E1(-) E3(-) Ad vector, suggesting that genes carried by the E4 region play a central role in modifying target cell gene expression. These findings are consistent with the notion that efficient transfer of exogenous genes to endothelial cells by first-generation Ad vectors comes with the price that these vectors also induce the expression of a variety of cellular genes.     

A gene transfer vector-cell line system for complete functional complementation of adenovirus early regions E1 and E4.

The improvements to adenovirus necessary for an optimal gene transfer vector include the removal of virus gene expression in transduced cells, increased transgene capacity, complete replication incompetence, and elimination of replication-competent virus that can be produced during the growth of first-generation adenovirus vectors. To achieve these aims, we have developed a vector-cell line system for complete functional complementation of both adenovirus early region 1 (E1) and E4. A library of cell lines that efficiently complement both E1 and E4 was constructed by transforming 293 cells with an inducible E4-ORF6 expression cassette. These 293-ORF6 cell lines were used to construct and propagate viruses with E1 and E4 deleted. While the construction and propagation of AdRSV beta gal.11 (an E1-/E4- vector engineered to contain a deletion of the entire E4 coding region) were possible in 293-ORF6 cells, the yield of purified virus was depressed approximately 30-fold compared with that of E1- vectors. The debilitation in AdRSV beta gal.11 vector growth was found to correlate with reduced fiber protein and mRNA accumulation. AdCFTR.11A, a modified E1-/E4- vector with a spacer sequence placed between late region 5 and the right inverted terminal repeat, efficiently expressed fiber and grew with the same kinetic profile and virus yield as did E1- vectors. Moreover, purified AdCFTR.11A yields were equivalent to E1- vector levels. Since no overlapping sequences exist in the E4 regions of E1-/E4- vectors and 293-ORF6 cell lines, replication-competent virus cannot be generated by homologous recombination. In addition, these second-generation E1-/E4- vectors have increased transgene capacity and have been rendered virus replication incompetent outside of the new complementing cell lines.     

Expression of the chloramphenicol acetyl transferase gene in human cells under the control of early adenovirus subgroup C promoters: effect of E1A gene products from other subgroups on gene expression

A hierarchy of dominance has been observed in HeLa cells co-infected with two serotypes of adenovirus belonging to different subgroups. DNA replication and late protein synthesis of one serotype are inhibited by those of the other. The degree of inhibitory effect has the following decreasing order: adenovirus type 3 (Ad3) and Ad7 (subgroup B), Ad9 (D), Ad4 (E), Ad12 (A), Ad2 and Ad5 (C) [Delsert and D'Halluin, Virus Res. 1 (1984) 365-380]. HeLa cells were first transfected with recombinant plasmids carrying Ad5 E2A or E3 promoters fused to the chloramphenicol acetyl transferase gene (cat), and then infected with human Ad belonging to different subgroups. All the serotypes tested were found to be able to stimulate both E2A and E3 promoters. When HeLa cells were co-transfected with either of the previous plasmids, plus a second plasmid carrying the Ad3 E1A region, the same stimulatory effect was observed. However, an inhibitory effect on Ad5 E2A and E3 promoters seemed to occur when both Ad2 E1A (subgroup C) and Ad3 E1A (subgroup B) genes were present together. To determine which one of the early products was responsible for the observed repression effect, and to assign the target on the genome of subgroup C Ad, a plasmid was constructed in which the sequences at the 5' end of the Ad2 E1A region were fused to the structural sequences of the cat gene. In HeLa cells transfected with this plasmid, CAT activity was significantly increased after co-transfection with a plasmid carrying the Ad2 E1A region, but decreased with a plasmid carrying the Ad3 E1A region.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anti-tumor immunotherapy despite immunity to adenovirus using a novel adenoviral vector Ad5 [E1-, E2b-]-CEA

Adenovirus serotype 5 (Ad5) has been widely used in clinical trials because it expresses inserted transgenes robustly and augments the innate immune response. Strategies to improve Ad5 vectors that can circumvent Ad5 immunity have become a critical issue, especially for use as a cancer immunotherapeutic in which repeated immunization is required. In this study, we constructed a novel Ad5 vector with unique deletions of the viral DNA polymerase and the pre-terminal protein region (Ad5 [E1-, E2b-]). This vector contains the carcinoembryonic antigen (CEA) gene insert and is designed to induce cell-mediated immunity (CMI) against the tumor-associated target. The CEA immunogenicity and in vivo anti-tumor effects of repeated immunizations with Ad5 [E1-, E2b-]-CEA compared with those observed with current generation Ad5 [E1-]-CEA were tested in Ad5 pre-immunized mice. We report that Ad5-immune mice immunized multiple times with Ad5 [E1-, E2b-]-CEA induced CEA-specific CMI responses that were significantly increased over those detected in Ad5-immune mice immunized multiple times with a current generation Ad5 [E1-]-CEA. Ad5 immune mice bearing CEA-expressing tumors that were treated with Ad5 [E1-, E2b-]-CEA had increased anti-tumor response as compared with Ad5 [E1-]-CEA treated mice. These results demonstrate that Ad5 [E1-, E2b-]-CEA can induce CMI immune responses which result in tumor growth inhibition despite the presence of pre-existing Ad5 immunity. Multiple re-immunizations using the same vector platform are now possible with the novel Ad5 [E1-, E2b-] platform.     

Increased in vitro and in vivo gene transfer by adenovirus vectors containing chimeric fiber proteins.

Alteration of the natural tropism of adenovirus (Ad) will permit gene transfer into specific cell types and thereby greatly broaden the scope of target diseases that can be treated by using Ad. We have constructed two Ad vectors which contain modifications to the Ad fiber coat protein that redirect virus binding to either alpha(v) integrin [AdZ.F(RGD)] or heparan sulfate [AdZ.F(pK7)] cellular receptors. These vectors were constructed by a novel method involving E4 rescue of an E4-deficient Ad with a transfer vector containing both the E4 region and the modified fiber gene. AdZ.F(RGD) increased gene delivery to endothelial and smooth muscle cells expressing alpha(v) integrins. Likewise, AdZ.F(pK7) increased transduction 5- to 500-fold in multiple cell types lacking high levels of Ad fiber receptor, including macrophage, endothelial, smooth muscle, fibroblast, and T cells. In addition, AdZ.F(pK7) significantly increased gene transfer in vivo to vascular smooth muscle cells of the porcine iliac artery following balloon angioplasty. These vectors may therefore be useful in gene therapy for vascular restenosis or for targeting endothelial cells in tumors. Although binding to the fiber receptor still occurs with these vectors, they demonstrate the feasibility of tissue-specific receptor targeting in cells which express low levels of Ad fiber receptor.     

A helper-dependent system for adenovirus vector production helps define a lower limit for efficient DNA packaging.

Adenoviruses (Ads) are intermediate-sized mammalian DNA viruses with a double-stranded linear genome of 36 kb. The icosohedral virion has been shown to accommodate up to 105% of the wild-type genome length, and genomes larger than this size are either unpackageable or extremely unstable, frequently undergoing DNA rearrangement. Here we show that the Ad virion also has a lower packaging limit of approximately 75% of the wild-type genome length. We have constructed a series of vectors with sizes ranging from 15.1 to 33.6 kb and used these to show that in our Cre/loxP helper-dependent system (R. J. Parks, L. Chen, M. Anton, U. Sankar, M. A. Rudnicki, and F. L. Graham, Proc. Natl. Acad. Sci. USA 93:13565-13570, 1996), vectors with genomes greater than or equal to 27.7 kb are packaged with equal efficiencies, whereas vectors with smaller genomes are inefficiently packaged. A 15.1-kb vector, approximately half the size of the wild-type adenovirus genome, was packaged with an efficiency intermediate between that of the small (21.3- to 25.7-kb) and large (27.7- to 33.5-kb) vectors. Analysis of vector DNA after amplification in helper virus-infected cells showed that vectors below 75% of the Ad genome had undergone DNA rearrangements, whereas larger vectors were unaltered. The 15.1-kb vector was recovered primarily as a mix of head-to-tail and tail-to-tail covalent dimers, with a size of 30 kb. We conclude that the Ad virion can efficiently accommodate viral DNA of greater than 75% of the viral genome but that smaller viral genomes tend to undergo rearrangement, resulting in a final size of greater than approximately 27 kb before they can be efficiently packaged. Knowledge of the lower limit to Ad DNA packaging should allow for the design of better and more stable vectors.     

Efficient generation and amplification of high-capacity adeno-associated virus/adenovirus hybrid vectors

Effective gene therapy is dependent on safe gene delivery vehicles that can achieve efficient transduction and sustained transgene expression. We are developing a hybrid viral vector system that combines in a single particle the large cloning capacity and efficient cell cycle-independent nuclear gene delivery of adenovirus (Ad) vectors with the long-term transgene expression and lack of viral genes of adeno-associated virus (AAV) vectors. The strategy being pursued relies on coupling the AAV DNA replication mechanism to the Ad encapsidation process through packaging of AAV-dependent replicative intermediates provided with Ad packaging elements into Ad capsids. The generation of these high-capacity AAV/Ad hybrid vectors takes place in Ad early region 1 (E1)-expressing cells and requires an Ad vector with E1 deleted to complement in trans both AAV helper functions and Ad structural proteins. The dependence on a replicating helper Ad vector leads to the contamination of AAV/Ad hybrid vector preparations with a large excess of helper Ad particles. This renders the further propagation and ultimate use of these gene delivery vehicles very difficult. Here, we show that Cre/loxP-mediated genetic selection against the packaging of helper Ad DNA can reduce helper Ad vector contamination by 99.98% without compromising hybrid vector rescue. This allowed amplification of high-capacity AAV/Ad hybrid vectors to high titers in a single round of propagation.