Evolution of Adenoviruses as Gene Therapy Vectors
Authors: Jayanta Roy-Chowdhury, Marshall S. Horwitz
Journal: Molecular Therapy (2002)

The concept of using adenoviruses as vectors for expressing foreign genes originated from a serendipitous observation of an experiment of nature. During the 1960s, human adenovirus types 4 and 7 were being propagated in primary monkey kidney cell cultures for formulation as vaccines to prevent acute respiratory disease (ARD) in military recruits. After the discovery of the simian virus 40 (SV40), the adenovirus vaccines were found to be contaminated with this new virus [1Heubner R.J. Chanock R.M. Rubin B.A. Casey M.J. Induction by adenovirus type 7 of tumor in hamster having the antigenic characteristics of SV40 virus.Proc. Natl. Acad. Sci. USA. 1964; 52: 1333-1349Crossref Scopus (35) Google Scholar, 2Rowe W.P. Baum S.G. Studies of adenovirus SV40 hybrid viruses. II. Defectiveness of the hybrid particles.J. Exp. Med. 1965; 122: 955-966Crossref Scopus (14) Google Scholar]. The SV40 virions were successfully eliminated by treating the vaccine lots with antibodies against SV40 viral (V) antigens. However, with the subsequent discovery of the SV40 T antigen, it was observed that tissue cultures infected with the adenovirus vaccine strains generated the T antigen, despite the absence of live SV40 virions. This perplexing phenomenon was explained by the finding that the adenoviral genomes contained insertions of SV40 genomic DNA containing the coding sequences for the T antigen. Subsequent studies revealed that the SV40 T antigen served an obligatory helper function for the proliferation of human adenoviruses in monkey cells. The initial adeno-SV40 hybrids were defective because of the insertion of the SV40 sequences within regions of the adenoviral genome that were critical for the growth of the adenovirus. These hybrid viruses could be propagated only by co-infection of the tissue culture cells by intact (non-hybrid) adenoviruses and as such were the first helper-dependent adenoviruses. Later, non-defective adenovirus type 2/SV40 hybrids were isolated that could be cultured without the assistance of helper adenoviruses [3Lewis Jr., A.M. Levin M.J. Wiese W.H. Crumpacker C.S. Henry P.H. A nondefective (competent) adenovirus-SV40 hybrid isolated from the AD.2-SV40 hybrid population.Proc. Natl. Acad. Sci. USA. 1969; 63: 1128-1135Crossref Scopus (55) Google Scholar]. Interestingly, this was possible because the foreign sequences were integrated into early transcription region 3 (E3), which contains a cassette of genes involved in host immunoregulation [reviewed in 4Horwitz M.S. Adenovirus immunoregulatory genes and their cellular targets.Virology. 2001; 279: 1-8Crossref PubMed Scopus (78) Google Scholar], but is not essential for the growth of adenoviruses in cultured cells. The discovery that foreign DNA sequences could integrate and be expressed in the adenoviral genome led to studies in which DNA segments were deliberately introduced into the virus for transfer and expression in mammalian cells. Rational use of adenoviruses as gene transfer agents benefited greatly from previous research on elucidating their structure, strategies for replication, transcription maps, and processing of macromolecules. For example, mRNA splicing, the existence of introns, and capped polyadenylated mRNAs were discovered first during studies of adenovirus replication, and novel immunoregulatory proteins were shown to be coded by its genome [reviewed in 5Shenk, T.2001. Adenoviridae. In Fields' VirologyD. M. Knipe and P. M. Howley, Eds., pp. 2265–2228. Lippincott-Williams & Wilkins, Philadelphia.Google Scholar]. Adenovirus DNA was the first eukaryotic viral DNA to be totally replicated in vitro. Using cell-free systems, these studies further elucidated protein priming of new DNA strands [reviewed in 6Horwitz, M. S.1990. Adenoviridae and their replication. In Fields' VirologyB. Fieldset al., Eds., pp. 1679–1721. Raven Press, New York.Google Scholar]. Adenovirus type-12 was the first human virus shown to cause tumors in animals, which led to a long quest for human cancer viruses. Although subsequent searches did not reveal any epidemiological or molecular genetic link of adenoviruses to human tumors [7Mackey J.K. Rigden P.M. Green M. Do highly oncogenic group A human adenoviruses cause human cancer? Analysis of human tumors for adenovirus 12 transforming DNA sequences.Proc. Natl. Acad. Sci. USA. 1976; 73: 4657-4661Crossref Scopus (23) Google Scholar], the ability of these viruses to transform cultured cells and to produce tumors in animals established them as important experimental tools for probing the mechanisms of oncogenesis. The use of adenoviruses to deliver foreign DNAs to cells has at least four general applications, and there are hundreds of reports describing such experiments in recent years. The first goal is to correct a congenital absence or mutation in an essential protein such as an enzyme, transcription factor, or structural protein. The second goal is to deliver genes to kill or control cell growth of cancer cells. This is being pursued by engineering adenoviruses to contain genes such as p53, which is commonly mutated in many tumor cells, but whose wild-type protein monitors and induces apoptosis in cells in which DNA repair is incomplete [8Fujiwara T. Introduction of chemosensitivity in human lung cancer cells in vivo by adenovirus-mediated transfer of the wild-type p53 gene.Cancer Res. 1994; 54: 2287-2291PubMed Google Scholar]. Alternatively, adenoviruses such as ONYX 015 (dl1520), with deletions in the E1B anti-apoptotic genes, are being studied for their potential to induce cell death selectively in tumor cells [9Dix B.R. Edwards S.J. Braithwaite A.W. Does the antitumor adenovirus ONYX-015/dl1520 selectively target cells defective in the p53 pathway?.J. Virol. 2001; 75: 5443-5447Crossref PubMed Scopus (68) Google Scholar, 10Bischoff J.R. An adenovirus mutant that replicates selectively in p53-deficient humor tumor cells.Science. 1996; 274: 373-376Crossref PubMed Scopus (1579) Google Scholar]. Adenoviruses coding for cell-specific cytokines are being used to improve blood flow by endothelial-based mechanisms in peripheral arterial disease [11Rajagopalan S. Shah M. Luciano A. Crystal R. Nabel E.G. Adenovirus-mediated gene transfer of VEGF(121) improves lower-extremity endothelial function and flow reserve.Circulation. 2001; 104: 753-755Crossref PubMed Scopus (121) Google Scholar]. In contrast, prevention of arterial wall overgrowth after angioplasty has also been a target of adenovirus vectors [12Lamfers M.L. In vivo suppression of restenosis in balloon-injured rat carotid artery by adenovirus-mediated gene transfer of the cell surface-directed plasmin inhibitor ATF.BPTI.Gene Ther. 2001; 8: 534-541Crossref PubMed Scopus (35) Google Scholar]. The third goal is to use adenoviruses to deliver genes encoding epitopes from other infectious agents for the purpose of immunization [13Morin J.E. Recombinant adenovirus induces antibody response to hepatitis B virus surface antigen in hamsters.Proc. Natl. Acad. Sci. USA. 1987; 84: 4626-4630Crossref Scopus (98) Google Scholar], and the fourth goal is to achieve high levels of expression of proteins, especially in tissue cultured cells that are difficult to transfect by other means. In all of these examples, the adenovirus DNA remains episomal, with the advantage of not disrupting any cell genes and the disadvantage of not integrating for permanent expression of the delivered foreign gene. Adenovirus DNA can be integrated into chromosome 19 by adding the capacity to make the adeno-associated virus (AAV) rep protein [14Recchia A. Site-specific integration mediated by a hybrid adenovirus/adeno-associated virus vector.Proc. Natl. Acad. Sci. USA. 1999; 96: 2615-2620Crossref PubMed Scopus (160) Google Scholar]. Alternatively, a loxP-flanked Epstein-Barr virus (EBV) replicon has been inserted into an adenovirus vector. Expression of Cre recombinase from a second adenovirus has been shown to release the transcription unit that could replicate coordinately during the cell cycle [15Leblois H. Stable transduction of actively dividing cells via a novel adenoviral/episomal vector.Mol. Ther. 2000; 1: 314-322Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar]. Other advantages of the adenovirus system for gene therapy are that it can target cells that are not replicating and can be grown to high titers. Two important early milestones in the generation of recombinant adenoviruses were achieved by Frank Graham of McMasters University in Canada and his collaborators. The first was the stable transfection of human embryonic kidney cells with sheared fragments of adenoviral DNA. The transformed cells, named "293" cells, express the E1A and E1B adenoviral proteins, which include transcription factors required for expression of other viral genes [16Graham F.L. Smiley J. Russell W.C. Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5.J. Gen. Virol. 1977; 36: 59-72Crossref PubMed Scopus (3507) Google Scholar]. Although the 293 cells were made to answer questions about adenoviral transformation, they have been used routinely as convenient host cells for providing transactivating transcription factors to complement replication-defective adenovirus vectors and to package recombinant adenoviral vectors deleted of E1 genes. Although DNA preparations from adenovirions contain only linear DNA with inverted terminal repeats and proteins covalently linked to each 5' end, Graham and colleagues found that a small portion of the viral DNA in adenovirus-infected cells was joined covalently at the ends to form closed circles [17Graham F.L. Covalently closed circles of human adenovirus DNA are infectious.EMBO J. 1984; 3: 2917-2922PubMed Google Scholar]. To learn more about the structure and possible function of these circular forms, the adenoviral DNA genome was cloned in two segments in plasmids. When cotransfected into 293 cells, the two plasmids recombined to generate infectious adenovirus. Although the covalently closed circular forms of viral DNA are probably not important in the life cycle of adenoviruses, the principles learned from these experiments led to the development of an efficient and flexible system for construction of adenoviral vectors entirely from plasmids [18Bett A.J. Haddara W. Prevec L. Graham F.L. An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3.Proc. Natl. Acad. Sci. USA. 1994; 91: 8802-8806Crossref PubMed Scopus (689) Google Scholar]. Before the development of this system, generation of recombinant adenoviruses required both the virus and a bacterial plasmid containing the gene of interest flanked by viral DNA sequences for homologous recombination into either the E1 or E3 regions. Insertions of a foreign gene in place of the E1 region resulted in a replication-compromised adenovirus vector that required 293 cells for growth of stock viruses. The resulting virus could enter many different types of cells, but was generally defective for replication. Alternatively, cloning a foreign gene in place of the deleted E3 region produced a replication-competent adenovirus deleted of some of its immunoregulatory genes. Cloning foreign genes into E1 and E3 deleted "first-generation" vectors accommodated foreign DNAs up to 8.3 kb. Despite the absence of the E1 region containing transactivation factors, recombinant adenoviruses may exhibit some expression of the immunogenic viral coat proteins, encoded by the late genes. The accumulated knowledge of adenoviral biology garnered over the past five decades has been used to prevent this "leakiness" of expression by the recombinant vector. The subsequent generations of adenovirus vectors were modified by replacing wild-type genes encoding functions necessary for viral DNA replication with temperature-sensitive mutations in these essential genes or by complementing deletion mutants by expressing these replication proteins in trans in modified helper cell lines [19Amalfitano A. Production and characterization of improved adenovirus vectors with the E1, E2b, E3 genes deleted.J. Virol. 1998; 72: 926-933PubMed Google Scholar]. Alternatively, selective deletions in the open reading frames of the E4 region also made the resulting viruses more defective [20Raper S.E. Selective gene transfer into the liver of non-human primates with E1-deleted, E2A-defective, or E1-E4 deleted recombinant adenoviruses.Hum. Gene Ther. 1998; 9: 671-679Crossref PubMed Scopus (45) Google Scholar]. Subsequently, Ko Mitani and Thomas Caskey designed new helper-dependent adenoviruses and, with the help of Frank Graham, generated vectors deleted of all the regions coding for viral proteins [21Mitani K. Graham F.L. Caskey C.T. Kochanek S. Rescue, propagation, and partial purification of a helper virus-dependent adenovirus vector.Proc. Natl. Acad. Sci. USA. 1995; 92: 3854-3858Crossref PubMed Scopus (222) Google Scholar]. These viruses, often referred to as "gutless" viruses, contained only the necessary cis-acting elements, including the two origins of DNA replication at the physical ends of the viral genome and the packaging sequences within the first 500 base pairs of the left-hand end. Generation of these vectors requires the use of a complementing helper adenovirus, which can be separated from the recombinant virus by virtue of their difference in DNA content. In the Graham laboratory, nearly complete elimination of contaminating helper virus was achieved by rendering its packaging sequence removable by using the Cre-mediated excision system [22Parks R.J. A helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signal.Proc. Natl. Acad. Sci. USA. 1996; 93: 13565-13570Crossref PubMed Scopus (606) Google Scholar]. However, the expectation that the helper-dependent viruses would be non-immunogenic was not fulfilled. Although the helper-dependent vectors may demonstrate prolonged transgene expression after a single injection due to a reduction in the CTL response, antibody responses to the capsid proteins are still present and reduce the effectiveness of subsequent adenovirus infection (see below). It also has been shown that the immune response to adenoviral vectors does not require de novo viral mRNA or protein synthesis [23Kafri T. Cellular immune response to adenoviral vector infected cells does not require de novo viral gene expression: implications for gene therapy.Proc. Natl. Acad. Sci. USA. 1998; 95: 11377-11382Crossref PubMed Scopus (224) Google Scholar]. For example, replication-incompetent adenoviruses, which were further treated with psoralen and ultraviolet light to completely inactivate transcription, retained immunogenicity through the production of antibodies to the input capsids, as well as through MHC class I and chemokinedependent mechanisms [24Borgland S.L. Bowen G.P. Wong N.C. Libermann T.A. Muruve D.A. Adenovirus vector-induced expression of the C-X-C chemokine IP-10 is mediated through capsid-dependent activation of NF-kB.J. Virol. 2000; 74: 3941-3947Crossref PubMed Scopus (132) Google Scholar]. Because a major hurdle in exploiting adenoviral vectors is the immunogenicity of the viral proteins, numerous attempts have been made to understand the immune response elicited by adenoviruses and to apply this knowledge to abrogate the host response both to the vector and the inserted transgene. In fact, the recognition that there were 51 separate human adenoviruses was possible because of the unique neutralizing antibodies produced against hexons, penton base, and fiber proteins of the virus capsid. Neutralizing antibodies and cytotoxic T lymphocyte (CTL) responses to adenoviral antigens are generated after injection of these vectors. The neutralizing antibodies prevent transgene expression on subsequent administration of vectors of the same serotype. However, vectors made of different serotypes would not be neutralized by the humoral immune response to the first vector. The strategy of sequential administration of adenovirus vectors of multiple serotypes, each carrying the same transgene, was introduced by investigators at the Wyeth laboratory led by Paul Hung. This approach has been used to boost the immune response when using adenoviruses as vaccine vectors [13Morin J.E. Recombinant adenovirus induces antibody response to hepatitis B virus surface antigen in hamsters.Proc. Natl. Acad. Sci. USA. 1987; 84: 4626-4630Crossref Scopus (98) Google Scholar, 25Hse, K. H. L., et al.1991. Immunogenicity and protective efficacy of adenovirus vectored respiratory syncytial virus vaccine. In Vaccines 91R. M. Chanock, H. S. Ginsberg, F. Brown, and R. A. Lerner, Eds., p. 293. Cold Spring Harbor Laboratory Press, New York.Google Scholar]. CTL responses against adenoviral antigens result in host immune attack against cells infected by the recombinant virus and the elimination of such transgene-expressing cells. Recombinant adenoviruses generated in 293 cells also have low-level contamination of wild-type adenoviruses, a result of stable recombination of the vector sequences with the E1 sequences present in the 293 cell genome. Synthesis of viral proteins by the wild-type adenovirus would, of course, lead to a marked immune response. This problem of preventing the production of low levels of wild-type adenoviruses was solved by constructing new cells, which contain a more limited E1 region that prevents the reconstitution of the wild-type viral genome [26Fallaux F.J. New helper cells and matched early region 1-deleted adenovirus vectors prevent generation of replication-competent adenoviruses.Hum. Gene Ther. 1998; 9: 1909-1917Crossref PubMed Scopus (395) Google Scholar]. However, it seems that prolongation of transgene expression or repeated administration of the adenovirus vector by abrogation of the immune response requires more than the elimination of the small amount of replication-competent adenoviruses from the preparations. Approaches toward inhibiting the immune response to recombinant adenoviruses by methods specifically directed toward the adenoviruses or more generally immunosuppressive strategies have been explored in rodents. For example, the host can be tolerized to adenoviral antigens. In rodents, both the humoral and cell-mediated immune response to adenoviral antigens could be inhibited markedly by administration of the vector during the neonatal period [27Takahashi M. Long term correction of bilirubin-UDP-glucuronosyltransferase deficiency in Gunn rats by administration of a recombinant adenovirus during the neonatal period.J. Biol. Chem. 1996; 271: 1-7Google Scholar], injection of adenoviral proteins into the host thymus [28Ilan Y.P. Induction of central tolerance by intrathymic inoculation of adenoviral antigens into the host thymus permits long-term gene therapy in Gunn rats.J. Clin. Invest. 1996; 98: 2640-2647Crossref PubMed Scopus (98) Google Scholar], or repeated oral administration of the adenoviral proteins [29Ilan Y. Oral tolerization to adenoviral antigens permits long-term gene expression using recombinant adenoviral vectors.J. Clin. Invest. 1997; 99: 1098-1106Crossref PubMed Scopus (122) Google Scholar]. Oral tolerization to adenoviral proteins is mediated by immunomodulatory cells, which suppress immune responses after both the initial and subsequent exposure to the viral antigens. However, the safety and efficacy of these measures in humans have not been verified. Second, a recombinant adenovirus could be designed to co-express a target transgene along with a gene expressing an immunomodulatory protein. For example, adenoviral vectors, expressing the adenoviral-E3 immunoregulatory genes and human UGT1A1 or factor IX, exhibited a blunted immune response, and vector expression was prolonged [30Ilan Y. Insertion of the adenoviral E3 region into a recombinant viral vector prevents antiviral humoral and cellular immune responses and permits long term gene expression.Proc. Natl. Acad. Sci. USA. 1997; 94: 2587-2592Crossref PubMed Scopus (172) Google Scholar, 31Poller W. Stabilization of transgene expression by incorporation of E3 region genes into an adenoviral factor IX vector and by transient anti-CD4 treatment of the host.Gene Ther. 1996; 3: 521-530PubMed Google Scholar]. Injection of CTLA4-Ig, a soluble inhibitor of the B7-CD28 costimulatory pathway, prolonged the transgene expression from an adenovirus vector, but did not allow transgene expression upon readministration of the vector [32Kay M.A. In vivo hepatic gene therapy: complete albeit transient correction of factor IX deficiency in hemophilia B dogs.Proc. Natl. Acad. Sci. USA. 1994; 91: 2353-2357Crossref PubMed Scopus (251) Google Scholar]. However, coexpression of CTLA4-Ig with a transgene by a single adenovirus vector resulted in the abrogation of both CTL and the primary or secondary antibody responses to adenoviral antigens, permitting transgene expression following repeated administration of the vector [33Reddy B. The effect of CD28/B7 blockade on alloreactive T and B cells after liver cell transplantation.Transplantation. 2001; 71: 801-811Crossref PubMed Scopus (21) Google Scholar]. Because a high plasma level of CTLA4-Ig is expected to inhibit any immune response, there will be a window of time after the administration of this vector during which the host may be expected to be unable to mount a de novo immune response to any infectious agent. Local transgene expression has been shown in human tissues by injection of adenovirus vectors into tumors or by topical application to the airway epithelium by introduction of the vector into the trachea as an aerosol. However, the tissue distribution of these vectors and the efficiency of recombinant adenoviruses in expressing transgenes in human tissues after systemic administration remain uncertain. In rodents, mice, and rabbits, following intravenous injection, the great majority of recombinant adenoviral particles are rapidly concentrated in the liver, where there is an exuberant expression of the transgene. However, it is difficult to obtain adenovirus transgene expression in the liver of primates, such as baboons or humans [34Nunes F.A. Furth E.E. Wilson J.M. Raper S.E. Gene transfer into the History.qxd 3/14/02 3:01 PM Page 344 liver of nonhuman primates with E1-deleted recombinant adenoviral vectors: safety of readministration.Hum. Gene Ther. 1999; 10: 2515-2526Crossref PubMed Scopus (118) Google Scholar, 35Raper S.E. A pilot study of in vivo liver-directed gene transfer with an adenoviral vector in partial ornithine transcarbamylase deficiency.Hum. Gene Ther. 2002; 13: 163-175Crossref PubMed Scopus (314) Google Scholar]. For example, an adenovirus vector, infused into the right branch of the hepatic artery in baboons, expressed transgene in the liver only at doses that were near or at toxic levels, and there was widespread dissemination of the vector DNA. In experiments conducted by James Wilson and collaborators at the University of Pennsylvania, low or undetectable levels of hepatocyte transgene expression were observed in ornithine transcarbamylase (OTC)-deficient human patients after injection of a high dose of a recombinant adenovirus carrying the human OTC gene [35Raper S.E. A pilot study of in vivo liver-directed gene transfer with an adenoviral vector in partial ornithine transcarbamylase deficiency.Hum. Gene Ther. 2002; 13: 163-175Crossref PubMed Scopus (314) Google Scholar]. Dose escalation, with the aim of enhancing transgene expression, resulted in the death of an 18-year-old patient, presumably from the uncontrolled release of vasoactive chemokines and cytokines without achieving the goal of sufficient OTC expression in the liver [36Somia N. Verma I.M. Gene therapy: trials and tribulations.Nat. Rev. Genet. 2000; 1: 91-99Crossref PubMed Scopus (579) Google Scholar]. It has been shown that adenoviruses can induce the release of chemokines even without transcription of any viral genes, and that it occurs primarily at high doses of the virus. Apart from the ethical and procedural issues evoked by this tragic event, the data on transgene expression raise serious questions regarding the efficiency of adenoviral vectors in transferring genes to human tissues after introduction into the circulation. In 1997 the coxsackie B cell surface receptor was discovered by Jeffrey Bergelson and Robert Finberg and, in collaboration with our laboratory (M.S.H.), the receptor was proven to mediate adenoviral infection [37Bergelson J.M. Isolation of a common receptor for coxsackie B viruses and adenoviruses 2 and 5.Science. 1997; 275: 1320-1323Crossref PubMed Scopus (2625) Google Scholar, 38Bergelson J.M. The murine CAR homolog is a receptor for coxsackie B viruses and adenoviruses.J. Virol. 1998; 72: 415-419Crossref PubMed Google Scholar]. The tissue distribution of this receptor, named CAR (coxsackie adenovirus receptor), provided a partial explanation for the differences in organ specificity of transgene expression between humans and various experimental animals, such as rodents. Further studies on CAR demonstrated that it mediates the binding and internalization of five of the six adenovirus subgroups. CAR is a component of the tight junction that forms a barrier to the passage of solutes between epithelial cells, but does not appear on the apical surface of respiratory epithelium [39Cohen C.J. The coxsackievirus and adenovirus receptor is a transmembrane component of the tight junction.Proc. Natl. Acad. Sci. USA. 2001; 98: 15191-15196Crossref PubMed Scopus (554) Google Scholar]. The knob at the tip of the fiber of the adenoviral penton binds to CAR, which is abundant in mouse liver, but is expressed at low levels in human liver [37Bergelson J.M. Isolation of a common receptor for coxsackie B viruses and adenoviruses 2 and 5.Science. 1997; 275: 1320-1323Crossref PubMed Scopus (2625) Google Scholar, 38Bergelson J.M. The murine CAR homolog is a receptor for coxsackie B viruses and adenoviruses.J. Virol. 1998; 72: 415-419Crossref PubMed Google Scholar]. Absence of CAR from the apical surface of respiratory epithelium may be one of the reasons that the early experiments performed by Ronald Crystal and collaborators were not efficient in delivering adenoviruses carrying the cystic fibrosis (CF) gene to the lungs of patients with CF [40Crystal R.G. Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis.Nat. Genet. 1994; 8: 42-51Crossref PubMed Scopus (791) Google Scholar]. In addition to the fiber–CAR interaction, the adenoviral penton base interacts with αv-integrins, which act as co-receptors and assist in viral endocytosis into clathrincoated vesicles [41Nemerow G.R. Cell receptors involved in adenovirus entry.Virology. 2000; 274: 1-4Crossref PubMed Scopus (161) Google Scholar]. Removal of the interaction with both CAR and integrin–αv is needed for reducing the native tropism of adenovirus vectors. Several investigators are engineering adenovirus fibers to alter their tropism [42Einfeld D.A. Reducing the native tropism of adenovirus vectors requires removal of both CAR and integrin interactions.J. Virol. 2001; 75: 11284-11291Crossref PubMed Scopus (176) Google Scholar]. Other ways to alter tissue expression of adenovirus transgenes include the introduction of tissue-specific promoters controlling the transgene. For example, tissue-specific promoters were incorporated into the adenovirus genome, resulting in cell-specific expression of transgenes [43Friedman J.M. Babiss L.E. Clayton D.F. Darnell Jr., J.E. Cellular promoters incorporated into the adenovirus genome: cell specificity of albumin and immunoglobulin expression.Mol. Cell. Biol. 1986; 6: 3791-3797Crossref PubMed Scopus (57) Google Scholar]. This strategy is currently being exploited in several clinically relevant expression systems, including delivery to prostate tumors [44Shi C.X. Hitt M. Ng P. Graham F.L. Superior tissue-specific expression from tyrosinase and prostate-specific antigen promoters/enhancers in helperdependent compared with first-generation adenoviral vectors.Hum. Gene Ther. 2002; 13: 211-224Crossref Scopus (38) Google Scholar]. The immune response to adenoviral proteins and targeted tissue delivery remain major hurdles to the successful clinical application of adenovirus vectors. Abolishing the expression of viral proteins from the vector DNA is not sufficient to prevent the immune response to proteins in the input virus. Tolerization of the host to adenoviral proteins or engineering adenovirus vectors that co-express immunomodulatory genes can prolong the expression of transgenes carried by recombinant adenoviruses and permit repeated administration. However, the safety of such procedures in human subjects has not been tested. Efficient gene targeting to specific human tissues using systemically administered adenoviral vectors has not yet been achieved. The high gene transfer efficiency, particularly to the liver, that makes adenovirus vectors popular in animal research will not be realized in humans without significant alterations of adenovirus tropism. At the present state of technology, the therapeutic usefulness of adenovirus vectors seems to be most promising in vaccine development and targeted transgene expression for applications that require limited periods of gene expression. Examples of the latter include injection into tumors for the treatment of malignancies or local application to vascular endothelial surfaces for stimulating regrowth of vessels or prevention of restenosis after angioplasty. In each of these examples, the delivered transgene would be tailored for the desired biologic effects. However, the ingenuity of the many laboratories dedicated to improving adenovirus vectors is leading to further developments, including vectors with sufficient space to accommodate genes to promote integration, altered tropism, and modulation of the host immune response. One novel application by Michel Perricaudet, another pioneer in the use and design of adenovirus vectors, used the EBV replicon for sustained expression from a replicating episomal transcriptional unit [15Leblois H. Stable transduction of actively dividing cells via a novel adenoviral/episomal vector.Mol. Ther. 2000; 1: 314-322Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar]. Thus, there is considerable optimism that adenovirus vectors will achieve an important place in human gene therapy.