Last-generation adenovirus vectors, also called helper-dependent or gutless adenovirus, are very attractive for gene therapy because the associated in vivo immune response is highly reduced compared to first- and second-generation adenovirus vectors, while maintaining high transduction efficiency and tropism. Nowadays, gutless adenovirus is administered in different organs, such as the liver, muscle or the central nervous system achieving high-level and long-term transgene expression in rodents and primates. However, as devoid of all viral coding regions, gutless vectors require viral proteins supplied in trans by a helper virus. To remove contamination by a helper virus from the final preparation, different systems based on the excision of the helper-packaging signal have been generated. Among them, Cre-loxP system is mostly used, although contamination levels still are 0.1–1% too high to be used in clinical trials. Recently developed strategies to avoid/reduce helper contamination were reviewed.
Gene therapy for most genetic diseases requires expression of the therapeutic protein for the whole life of the patient. In order to be efficient for the treatment of genetic disorders, a gene therapy vector has to meet several conditions: (i) safety, which can be better achieved with a nonintegrative vector, as it avoids the risk for insertional mutagenesis; (ii) ability to be easily and inexpensively produced at a large-scale in the laboratory; (iii) stability in target cells, which is favored with low-immunogenic vectors; and (iv) high-capacity allowing the possibility of introducing full-length DNA sequences for most genes, long full-length cDNAs, endogenous promoters or additional regulatory sequences such as enhancers or insulators, which can provide a tightly regulated expression of the therapeutic gene, similar to physiologic conditions. Adenoviruses and especially gutless adenoviruses seem to accomplish most of these conditions as they are not integrative, they have a capacity of 36 kb, they can be easily produced at high titers in the laboratory and can be delivered to a large mass of cells, and, contrary to the first-generation adenovirus in which the expression of wild-type adenoviral genes stimulates the immune system, gutless adenoviruses show long-term stability in many tissues. As seen in Table 1, adenovirus is one of the first elections in clinical trials, being the most used vector in the last 5 years (1999–2004) (visit www.wiley.co.uk/genmed/clinical for more information). Nowadays, adenovirus vectors are applied to treat cancer, monogenic disorders, vascular diseases and others complications.
Basically, wild-type adenovirus is associated with mild diseases like conjunctivitis, pharyngitis and, in a high percentage, with colds or acute respiratory diseases but not to tumoral pathways or other viral alterations.1 In the early 1950s, adenovirus was used for the first time as a preventive vaccine for respiratory diseases. These studies supposed a great advance since they allowed the knowledge of aspects related to associated immune responses and adverse secondary effects.
As gene transfer vector, the adenovirus has a high transfection efficiency, both in quiescent and in dividing cells, it does not integrate; allows easy capsid modification in order to retarget its tropism to different tissues; and high titers (up to 1013 particles/ml) are routinely obtained. In addition, it is noteworthy that in the last 10 years, new scale-up adenovirus production systems have been developed, facilitating its use in human clinical trials.
Well-characterized human serotypes Ad2 and Ad5 from group C are the classic adenoviruses used as vectors. In order to produce safe and nonreplicative vectors, first-generation adenoviral vectors containing the whole viral genome with the exception of the E1 region were developed.2 To propagate first-generation adenoviruses, several E1-expressing cell lines have been generated: 293,3 911,4 N52.E65 and PER.C6.6
Although E1-deleted vectors cannot replicate in vivo, residual expression from adenoviral genes triggers a cytotoxic T lymphocyte (CTL) immune response towards infected cells,7 which finally leads to the elimination of transduced cells and, therefore, to the lost of therapeutic gene expression.
To avoid this problem, second-generation adenoviral vectors combining deletion of different early regions (E1±E3 and E2/E4) were generated (Figure 1). Deletion of these regions permits to accommodate up to 14 kb and hence increase the vector cloning capacity.8, 9 However, second-generation Ad vectors still do not avoid in vivo-associated immunogenicity and toxicity due to residual gene expression from remaining viral genes.
Third-generation vectors, called gutless or gutted Ad, devoid of all coding viral regions were recently generated. They are also called helper-dependent adenoviruses because of the need of a helper adenovirus that carries all coding regions, and high-capacity adenoviruses because they can accommodate up to 36 kb of DNA. Briefly, the gutless adenovirus only keeps the 5′ and 3′ inverted terminal repeats (ITRs) and the packaging signal (Ψ) from the wild-type adenovirus.
Vector capsids package efficiently only 75–105% of the whole adenovirus genome.10, 11, 12, 13, 14 As therapeutic expression cassettes usually do not reach up to 36 kb, there is a need to use stuffer DNA in order to complete the genome size for encapsidation. Initially, it was believed that stuffer DNA's unique role was the participation in the packaging of the vector genome. Thus, the first DNA stuffer used was from lambda phage, yeast, bacterial and non-human DNA.14 However, first injections of gutless vectors carrying lambda DNA elicited CTL immune response because peptides from stuffer were presented in the cell membrane, and it became evident that DNA stuffer played an important role in the stabilization of viral DNA into the cell.15, 16 The first choice was then DNA from mammalian and human introns since they seemed to favor maintenance of the gutless genome into the cell for long periods of time.15 For example, intronic sequences from the HPRT gene containing matrix attachment regions (MAR) elements have been used as stuffer DNA showing increased DNA stability and no induction of a CTL response. Sequences from other human loci have also been used with similar or better results.17
However, not all intronic sequences are appropriate DNA stuffer and, thus, proper candidates should avoid the following sequences (follow next characteristics) (i) coding regions, (ii) repetitive sequences (like alu sequences), (iii) hot spots for recombination, (iv) regions that can interfere with the expression of the transgene, and (v) toxic or immunogenic regions. On the contrary, MAR that seem to stabilize the adenovirus genome into the nucleus and permit long periods of gene expression are recommended.17
Production of gutless adenovirus vectors
Since gutless vectors are devoid of all viral genes, proteins needed for its genome replication, packaging and capsid formation must be supplied in trans. This is achieved by coinfection of the gutless with a helper adenovirus. However, since both helper and gutless vectors have the same viral capsid, separation must be addressed before purification. Thus far, strategies have been based on reducing the packaging efficiency of the helper genome compared to the gutless genome, either by mutating its packaging signal,18, 19, 20 by the different size of its genome13 (genomes bigger or smaller than the optimal do not package efficiently), or by specific elimination of its packaging signal during viral production.21
Initial strategies consisted in the use of helper-dependent adenoviruses carrying a defective packaging signal.13 These vectors had extensive regions of deleted viral genome, but they still have some coding regions. Cotransfection of a wild-type adenovirus as a helper with this gutless permitted to propagate both adenoviruses. After several passages, the gutless adenovirus was purified by CsCl2 density gradient. However, high levels of helper virus and multiple recombinations between both vectors were detected. This, together with important complications in large-scale production and purification, led to the development of new strategies.
An important advance was the specific removal of the packaging signal by Cre recombinase. Parks and collaborators21 developed a production system where the helper adenovirus had a packaging signal flanked by two loxP sites, and amplification was performed in Cre recombinase-expressing cell lines (293Cre). When the helper adenovirus entered the cell, its packaging signal was excised preventing the inclusion of its genome into the viral particle, but retaining all coding regions for the viral proteins needed to produce the gutless vectors (Figure 2). Using this system, average helper contamination ranged over 0.1–10% compared to gutless vectors. These levels of helper contamination seem to be due to the limited efficiency of packaging signal excision associated to low recombinase activity or with low endogenous levels of recombinases. This is caused either by adenovirus-mediated host cell shut off or by cytotoxicity of high levels of Cre recombinase.22, 23 Although the final level of helper contamination is low, further reduction is desirable to minimize any potential toxicity associated with the helper virus, especially when high doses are required.24
Other recombinases such as FLP have also been used to remove the packaging signal. FLP is a yeast site-specific recombinase, which catalyzes recombination between frt sites.25 Philip Ng and collaborators26 created new FLP-expressing cell lines (293FLP and 293CreFLP) and a helper adenovirus with a packaging signal flanked by frt sites. Cre and FLP seem to have equivalent efficiency in the production of gutless adenovirus with a final helper contamination of 0.1–1%. A recent and elegant improvement in the Cre-loxP system has been the reversion of the packaging signal of the helper virus in order to avoid generation of a replication competent adenovirus (RCA) by recombination with the gutless vector, which has reduced the helper contamination to levels down to 0.02–0.1%.18 However, Cre and FLP are bidirectional recombinases that permit excision of the packaging signal and also its re-entrance, favoring contamination by the helper adenovirus. Improving the excision efficiency of Cre and FLP in cell lines also improves the opposite reaction. Thus, the use of unidirectional recombinases, such as ΦC31, is an attractive alternative to reduce contamination levels with the helper adenovirus. ΦC31 is a unidirectional recombinase, which recognizes homologous sequences called attB/attP and shows an excision efficiency similar to that of Cre in 293 cells.27 When ΦC31 excises attB/attP-flanked sequences, it creates new sequences called attR/attL preventing the opposite reaction. Thus, the helper adenovirus carrying an attB/attP-flanked packaging signal opens the door to a new gutless production system (authors' unpublished results).
Another approach to reduce helper Ad contamination is based on size-restricted protein IX-deleted helper. pIX is a hexon-associated protein essential for packaging full-length viral genomes. A 293 pIX-expressing cell line permitting the growth of a full-length pIX-defective helper was generated.28 The use of gutless vectors with genomes smaller than 36 kb gives a selective advantage in the pIX system. Plaque-forming unit assay showed a reduction by 1000 times of helper contamination compared to the classical Cre-loxP system. However, southern blot analysis revealed helper contamination levels of 0.2% (similar to Cre-loxP), meaning that the majority of helper adenoviruses was packaged into the capsid. Combining both Cre and pIX size-restricted systems allowed to reduce the number of helper adenovirus particles, but defective helper virions still remain in the final preparation.
To address the helper contamination issue, other strategies are based on using nonadenovirus vectors as helpers, like herpes simplex virus-129 and baculovirus.30 However, low production efficiency29 and 2% of RCA generation30 make their use not feasible in large-scale production.
As important as the design of a helper-free gutless adenovirus system is its production in large or industrial scale. Large-scale production of gutless adenovirus is complex and less efficient since it requires successive time-consuming gutless:helper adenovirus coinfections. Unfortunately, this process increases the risk of reorganizations in the vector genome by homologous recombination with viral E1 sequences present in permissive 293 cells, which can finally lead to the generation of RCAs. To avoid this, several laboratories have developed permissive cells, such as N52.E65 and PERC6,6 that do not contain viral sequences prone to recombine. However, to achieve large quantities of high quality helper-free gutless vectors needed for clinical assays, it is fundamental to develop and improve the methodology in the amplification and purification steps as well as in vector quality. Thus, development of several cell lines able to grow in suspension, like human 293S,31 PER.C6,32 and 293Cre suspension cell lines18 facilitates large-scale amplification in bioreactors, although purification methods like non-ultracentrifuge-based methods and elimination of helper contamination still need to be improved.
Gutless adenovirus and immune response
Systemic delivery of first-generation adenoviral vectors is known to induce a strong host's immune response, resulting in the rapid elimination of vector-transduced cells and the generation of neutralizing antibodies against the transgene products and the adenovirus capsid. Both nonspecific innate and adaptive immune responses are involved when first- and second-generation adenoviral vectors are administered (see Schagen et al33 for an extensive review). Thus, the innate immune response is rapidly developed after virus entry by induction of inflammatory gene expression and further recruitment of macrophages, neutrophil and natural killer cells, leading to an 80–90% of first-generation vector removal from the liver in 24 h.34 Basically, innate immunity is triggered by the adenovirus particle, is Ad-dose dependent and does not require viral gene expression.35, 36, 37
In a second step, adaptive cellular and humoral immune responses are developed about 4–7 days after delivery. At this time, a second peak of cytokine and chemokine gene expression and inflammation occurs leading to lymphocytic infiltrates and to the induction of adenovirus-specific CTL.35 Initially, cellular immune response is activated when antigen-presenting cells (APCs) uptake adenovirus particles, process the particles into small oligopeptides and present them through the major histocompatibility complex (MHC) class-I molecules at the cell surface. Further binding of CD8+ T cells to the MHC class-I/peptide complex induces formation of Ad-or transgene-product-specific CTLs. Therefore, the novo synthesis does not seem to be required to initiate the process.33 However, for late inflammation, the expression of viral genes still encoded within Ad vectors plays a significant role. In immunocompetent hosts, this response limits the duration of transgene expression and results in adenovirus vector clearance within a few weeks of administration.7, 34
On the other hand, adaptive humoral immune response is initiated by the binding of adenovirus particles to the surface immunoglobulin of B cells.33 After internalization and virus processing, the adenovirus-derived epitopes are presented at the surface of the B cell by MHC-II molecules. Exposure of these cells to cytokines from activated CD4+-Th2 helper cells will result in differentiated plasma cells secreting antibodies towards the adenoviral capsid.38 High titers of antibodies against capsid proteins, either pre-existing because of previous exposure to natural virus or generated as a result of vector administration, may inhibit subsequent dosing with the same vector.
Different strategies to circumvent innate and adaptive immune responses have been developed. However, most of them present secondary complications and/or their use in human patients is questionable. These strategies include macrophage depletion,39, 40 use of immunosuppressive agents (cyclosporin A, cyclophosphamide, dexamethasone, FK506, Interleukin-12 and deoxypergualin),41, 42, 43, 44, 45, 46 use of antibodies to deplete CTLs,47, 48 blockade of costimulatory interactions between APCs, T and B cells,49, 50, 51, 52 intrathymic administration of adenovirus,53 oral tolerization,54 use of vectors derived from non-crossreacting serotypes,55, 56 use of adenoviruses from other species57, 58 and coating vectors with inert chemicals like polyethylene glycol (PEG).59, 60, 61
Diverse in vivo studies in mice suggested that, in the absence of an immune response, first-generation adenoviral vector DNA is maintained as a stable episome in the host cell.41, 62, 63 Last-generation helper-dependent or gutless adenovirus vectors display reduced long-term toxicity and prolonged transgene expression compared to first-generation vectors after administration to peripheral organs of immunologically naïve animals.56, 64, 65, 66, 67, 68
Lack of coding viral genes may account for reduced adaptive cellular immune response after systemic delivery of gutless vectors. Initially, gutless vectors are capable of transducing dendritic cells and stimulating Ad-specific T-cell responses, independent of viral gene transcription.69 However, the expression of viral genes is required for T cells to exert their effector functions in the liver,70 which possibly explains the vector persistence and improved transgene expression following transduction with gutless vectors compared to results with first-generation Ad vectors.
As expected, systemic delivery of gutless vectors still induces adaptive humoral response against the vector capsid as for first-generation Ad vectors. Indeed, the development of Ad-specific antibodies does not contribute to the elimination of Ad-transduced cells and therefore does not affect the persistence of transgene expression. However, Ad-specific antibodies will bind the readministered Ad vector and thereby prevent cell entry and promote opsonization by macrophages.
Humoral response can also be developed towards circulating antigen induced by gutless adenoviral transfer.70 Vectors that mediate transgene expression in APCs trigger antibody formation because they increase the probability of neoantigen presentation by APCs,71 and, hence, careful selection of tissue-specific promoters may significantly improve adenovirus-associated toxicity profiles and diminish or abolish APC transduction and transgene expression.72 In addition, systemic administration of gutless vectors in a clinical setting might be inefficient because of the presence of circulating neutralizing antibodies against the same or crossreactive serotype as a consequence of a natural infection or as a result of previous vector administration. A large proportion of the human population has significant levels of antibodies against adenoviruses;73 thus, the capacity to overcome a pre-existing immunity is a fundamental problem. Different successful strategies to circumvent pre-existing immunity have been applied, such as the use of alternative gutless serotypes15 and the use of a non-human gutless adenovirus.58 Thus, administration of gutless CAV-2 vectors allows the stable, high-level expression in neurons throughout the rat central nervous system (CNS).20 It is noteworthy that even in the presence of an active peripheral immunization with an adenovirus that completely eliminates expression from first-generation vectors within 60 days, gutless vectors are able to maintain a long-term transgene expression in the brain74, 75 due to reduced inflammation both in intensity and in duration in the brains of the preimmunized animals.
As innate immune responses are dependent on the viral capsid or particle, innate responses stimulated by gutless vectors are similar to those stimulated by first-generation adenovirus vectors. Thus, dose-dependent acute inflammation was reported by Brunetti-Pierri and colleagues76 in non-human primates following the administration of high-dose gutless vectors. However, innate response, as these recently reported, may be reduced by PEG modification, probably due to lower vector uptake by Kupffer cells in vivo.77, 78
Surprisingly, gutless vectors have also been proved to be very efficient in vaccination, due to their longer duration of expression, their lower antiviral reactivity and their higher levels of transgene protein in dendritic cells compared to the same amount of first-generation Ad vectors.79
In vivo administration of gutless adenovirus
Gutless adenoviruses have been administered in vivo to different tissues in rodents, dogs and also to non-human primates. Owing to the high efficiency of adenoviruses in targeting hepatocytes, most of the toxicity studies were carried out in the liver, following intravenous administration of the vector.68 Gutless vectors have been shown to express non-immunogenic transgenes during the whole life of the mouse, while expression driven by first-generation adenovirus lasted at most for 3 months.80, 81, 82
Therapeutic genes carried by gutless vectors have been administered to the liver of mouse models for different diseases, such as hemophilia A and B,83, 84 obesity,66 familial hypercholesterolemia,81, 85, 86, 87 ornithine transcarbamylase deficiency,88 diabetes89 and chronic viral hepatitis.90, 91 Therapeutic levels of most proteins have been documented for a long-term duration. In some cases the antibody response against a nonendogenous transgene led to a significant decrease in the circulating levels of the therapeutic protein. However, when the transgene expression was driven by a liver-specific promoter, the immune response was lower, efficacy of the therapeutic protein increased and secondary effects derived from systemic circulation of the therapeutic protein were undetectable.90, 92 The encouraging results obtained in rodents had promoted the preclinical studies in larger animal models. This is the case for hemophilia A and B dogs,72, 83 which resulted in minimal acute liver toxicity and transient expression of the transgene allowing for partial or complete correction of hemophilia. As in rodents, the use of liver-specific promoters avoided the development of neutralizing antibodies against the therapeutic protein, although the expression was lower than with a viral ubiquitous promoter.72
Long-term expression of gutless-driven transgene was also documented in baboons for more than 1 year; however, a decrease in the levels of the protein was observed over time.56 Toxicity in non-human primates was assessed using different doses of gutless adenovirus and the results obtained showed that administration of high doses of adenovirus (1013 viral particles/kg) induced a strong activation of the innate inflammatory response that caused the death of the animal.76
In order to circumvent the decrease in transgene expression overtime, readministration with gutless vectors has been attempted in mice using different adenovirus serotypes. While readministration with different serotypes is possible using first-generation adenovirus,55, 56, 93, 94, 95 the level of transgene expression is lower after the second administration, probably due to crossreacting CTLs between the different serotype proteins.96 However, as gutless vectors do not contain viral genes, readministration of a different serotype leads to the same levels of the therapeutic protein as in naïve animals without showing liver toxicity.15, 81 Liver toxicity was also absent after repeated administration of the same serotype of gutless;67 however, it resulted in a drop of 30- to 100-fold in transgene expression compared to naïve animals.15
Efficiency of regulated promoters was also evaluated using gutless vectors as they offer the possibility of incorporating all the genetic components of an inducible system plus other additional sequences such as insulators or silencers that have been shown to avoid leakiness of the uninduced promoter.97, 98 When these promoters were driven by first-generation adenoviruses, only one induction was possible; a significant hepatotoxicity was developed when the animals were treated with the inducer, followed by a rapid loss of vector DNA.99 This was not the case with gutless vectors, where expression was reinduced three and four times over a period of 2 months. Similar results were obtained with different inducible systems such as tetracycline, tamoxifen or mifepristone.97, 98, 99
Stability of gutless vectors was evaluated during hepatocyte cell cycle. Surprisingly, after two-third partial hepatectomy, gutless vector genomes were reduced by 50%, while first-generation Ad vector copies were reduced by 71%. More importantly, episomal plasmid DNA-injected mice showed a reduction of 99% in DNA copy number. The decrease in vector copy number/cell correlated with transgene expression. Several hypotheses have been proposed to explain the persistence of episomal gutless vectors in dividing hepatocytes, like centromeric function of the Ad genome or nuclear retention activity of the Ad terminal protein, but conclusive results have not been obtained to date.100
Adenoviruses target the liver very efficiently; however, they are less effective in other cell types lacking the CAR or adenovirus receptor. Retargeting of gutless vectors has been attempted by the incorporation of polylysine or RGD in the H–I loop of the adenoviral fiber protein, which can be obtained by adding fiber-modified helper viruses in the last amplification step of the gutless vector production. Polylysine has been shown to effectively transduce mature muscle cells in vitro and in vivo,101 while RGD increased the efficiency of transducing ovarian carcinoma cell lines, primary vascular smooth muscle cells and primary human endothelial cells.102
Duchenne muscular dystrophy (DMD), an X-linked lethal disorder that affects 1 in 3500 males, is caused by genetic mutations in the dystrophin gene. The cDNA for full-length dystrophin is approximately of 14 kb, far above the size of most gene transfer viral vectors. Thus, the highcapacity of gutless adenoviruses has opened the possibility to treat DMD animal models103 not only with one full-length dystrophin cDNA but also with two copies of the therapeutic gene.104, 105 In these studies, neonate skeletal muscles of mdx mice injected with gutless adenovirus expressed dystrophin for the duration of the experiment, up to 1 year. At this point, 52% of the muscle fibers showed transgene expression and functional correction of muscle contractility, and improved histopathology was reported, correlating with the level of dystrophin expression. However, a four-fold decline in the vector DNA copy number was observed from 10 days to 1 year after vector injection, and the treated mice developed a significant humoral response. No transduced muscle fiber loss was reported, indicating that the decline in DNA copies could be due to instability of the vector DNA into the host cell.104 However, loss of DNA copy number/cell has no consequences in dystrophin expression or muscle function in experiments performed in mice because dystrophin is stable for 26 weeks. When skeletal muscle fibers were transduced before birth (in utero at embryonic day 16), expression of the reporter protein was stable at least for 5 months despite the development of antibodies against the transgene and the adenoviral capsid.106 However, in this case, the authors reported no loss of DNA copy number. Lower levels of toxicity compared to first-generation eneration Ad that resulted in higher survival rates of animals transduced in utero were also documented.
Mdx muscle goes through cycles of necrosis followed by regeneration, which could account for the higher efficiency of infection of muscle fibers from young and old mdx mice by adenovirus vectors compared to control animals as reported by the group of JS Chamberlain.107 On the contrary, the high turnover of muscle cells in the mdx mice can decrease the stability of nonintegrative vectors like adenovirus. In 1-year old mdx mice, transduction of 25–30% of skeletal muscle cross-sectional area with gutless vectors carrying the dystrophin cDNA under the regulation of a muscle-specific promoter lead to 40% correction of their high susceptibility of contraction-induced injury 1 month after treatment.107 However, longer time points were not evaluated, but mild immune cell infiltration was reported using mouse dystrophin cDNA.
Administration of recombinant protein to the CNS requires the breakdown of the blood–brain barrier, a procedure that should not be used for repetitive readmnistrations. Thus, permanent transduction of neurons offers the possibility of stable treatment of diseases affecting the CNS. Gene transfer to the CNS has been explored using different vector systems. Despite the immunoprotection of the CNS, results with first-generation adenovirus show a decrease in the expression of the transgene 2 months after transduction, correlating with the disappearance of the adenoviral DNA.108 Acute loss of transduced cells and chronic inflammation could account for this decline, mainly at high doses.109 On the contrary, gutless adenovirus expression can be detected in the CNS 1 year after striatal injections.20, 110 Moreover and contrary to what was reported for other tissues, preimmunization with the same serotype of adenovirus does not alter gutless stability in the CNS, and does not significantly reduce the initial infection of the CNS by gutless vectors, although a transient acute brain inflammation is shown.75 Gutless vectors have also been used to successfully express therapeutic genes in the retina either by transplanting cells previously transduced with the virus or by direct injection of the vector.110, 111
This is a fascinating moment for gutless adenovirus vectors: their use permits long-term expression in vivo with reduced and transient cellular immune response in animal models for human diseases and, moreover, the increasing number of groups working in this field favors technological advances to escape preimmune response by developing non-human gutless adenovirus20 or to increase stability by generating integrative gutless vectors,116, 117 or vectors with replication capacity.118 However, their use in clinical assays is questionable since helper contamination levels are still high, and large-scale production in bioreactors is not yet fully developed. Therefore, future studies will be needed in these areas to finally bring gutless adenovirus to the clinic.
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We would like to acknowledge Dr Mercè Monfar for critically reading the manuscript. Our work is supported by MCYT-SAF2003-03256, Marató TV3-2002-031632 and Instituto de Salud Carlos III (C03/08). AB has a contract from the Ramon y Cajal Program (Ministerio Educación y Ciencia, Spain), and RA is a recipient of an FI-Generalitat fellowship.
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Alba, R., Bosch, A. & Chillon, M. Gutless adenovirus: last-generation adenovirus for gene therapy. Gene Ther 12, S18–S27 (2005). https://doi.org/10.1038/sj.gt.3302612
- helper-dependent vectors
- in vivo gene therapy
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