A multitasking oncolytic adenovirus vector
Authors: Karoly Toth, Konstantin Doronin, Ann E Tollefson, William S.M Wold
Journal: Molecular Therapy (2003)

Human adenovirus vectors hold great promise as “drugs” to treat cancer. They are relatively easy to grow and manipulate, are very well understood, infect dividing and non-dividing cells from a variety of tissues, and do not cause serious disease in adults. Thousands of papers have been published by many academic and industrial groups on the potential use of adenovirus vectors in cancer gene therapy. These vectors fall into two general categories: replication-defective and replication-competent (oncolytic) [1Green N.K. Seymour L.W. Adenoviral vectors systemic delivery and tumor targeting.Cancer Gene Ther. 2002; 9: 1036-1042Crossref PubMed Scopus (80) Google Scholar]. In this issue of Molecular Therapy, Bauzon et al. [2Bauzon, M., Castro, D., Karr, M., Hawkins, L. K., and Hermiston, T. W. (2003). Regulated, multi-gene expression from a replicating adenovirus using native viral promoters. Mol. Ther., this issueGoogle Scholar] describe a potential step forward in the design of replication-competent adenovirus vectors. Nearly all replication-defective vectors have the E1A and E1B transcription units deleted and replaced with an expression cassette that drives expression of a cancer-fighting gene by an exogenous promoter. Since the E1A proteins are required for adenovirus replication, these vectors do not replicate. However, the therapeutic gene can be expressed efficiently and can cause death in infected cells. Replication-defective vectors act on cells that become infected when the vector is administered—usually just a fraction of the cells in the tumor mass. Some therapeutic genes expressed from non-replicating vectors give rise to a “bystander effect” by which they induce death of uninfected cells. Repeated administration of the vector increases efficacy. Replication-competent adenovirus vectors destroy infected cells through the natural process of adenovirus replication. Following release from the originally infected cell, these vectors infect other cells and are expected to destroy the tumor via repeated cycles of infection and vector release. Even though replication-competent vectors could theoretically spread to all the cells within a tumor, it seems unlikely that they would do so; tumors have an elaborate architecture and often contain necrotic areas. This may explain the observation that in clinical trials, oncolytic adenovirus vectors are rarely effective as a single modality [3Reid T. Warren R. Kirn D. Intravascular adenoviral agents in cancer patients lessons from clinical trials.Cancer Gene Ther. 2002; 9: 979-986Crossref PubMed Scopus (211) Google Scholar]. It may be possible to improve replication-competent vectors by engineering them to express secreted, therapeutic proteins. The choice of anti-cancer gene is a more delicate matter in the case of oncolytic vectors than in the case of replication-deficient vectors. Secreted cytotoxic proteins may kill susceptible tumor cells surrounding an infected cell, thus creating a barrier for vector spread. A molecule that affects some other aspect of the tumor survival, such as the tumor vasculature or the immune response against the tumor, may be more efficacious. In the best of all worlds, the secreted protein would be effective against tumors that have metastasized. Also, it should have minimal effect on non-cancerous tissue. A variety of potential anti-cancer genes have been expressed from oncolytic adenovirus vectors [4Hermiston T.W. Kuhn I. Armed therapeutic viruses strategies and challenges to arming oncolytic viruses with therapeutic genes.Cancer Gene Ther. 2002; 9: 1022-1035Crossref PubMed Scopus (126) Google Scholar]. A number of research groups have constructed replicating adenoviruses in which a foreign gene is inserted in place of some or all of the E3 transcription unit. None of the E3 genes is essential for adenovirus replication in cultured cells. This was shown early on by the isolation of adenovirus-SV40 recombinants in which the large T antigen of the SV40 virus was found to be incorporated in place of the deleted E3 region of adenovirus [5Kelly Jr., T.J. Lewis Jr., A.M. Use of non-defective adenovirus-simian virus 40 hybrids for mapping the simian virus 40 genome.J. Virol. 1973; 12: 643-652PubMed Google Scholar]. E3 proteins function to protect infected cells against immune killer cells [6Horwitz M. Adenovirus immunoregulatory genes and their cellular targets.Virology. 2001; 279: 1-8Crossref PubMed Scopus (77) Google Scholar, 7Mahr J.A. Gooding L.R. Immune evasion by adenoviruses.Immunol. Rev. 1999; 168: 121-130Crossref PubMed Scopus (121) Google Scholar, 8Wold W.S.M. Doronin K. Toth K. Kuppuswamy M. Lichtenstein D.L. Tollefson A.E. Immune responses to adenoviruses viral evasion mechanisms and their implications for the clinic.Curr. Opin. Immunol. 1999; 11: 380-386Crossref PubMed Scopus (162) Google Scholar], and that function is not relevant in cell culture. The E3 genes are not essential for replication in vivo, at least in the lungs of cotton rats, although they do reduce the inflammatory response [9Ginsberg H.S. Lundholm-Beauchamp U. Horswood R.L. Pernis B. Wold W.S.M. Chanock R.M. Prince G.A. Role of early region 3 (E3) in pathogenesis of adenovirus disease.Proc. Natl. Acad. Sci. USA. 1989; 86: 3823-3827Crossref PubMed Scopus (235) Google Scholar]. The E3 genes are expressed at early stages of infection from a single promoter (the E3 promoter) as a set of overlapping mRNAs derived via alternative splicing and alternative polyadenylation of a single pre-mRNA (Fig. 1) [10Bhat B.M. Wold W.S.M. Genetic analysis of mRNA synthesis in adenovirus region E3 at different stages of productive infection by RNA-processing mutants.J. Virol. 1986; 60: 54-63PubMed Google Scholar, 11Tollefson A.E. Scaria A. Saha S.K. Wold W.S.M. The 11,600-MW protein encoded by region E3 of adenovirus is expressed early but is greatly amplified at late stages of infection.J. Virol. 1992; 66: 3633-3642Crossref PubMed Scopus (77) Google Scholar]. The genes in the “major late” transcription unit, which is expressed following initiation of viral DNA replication, are also expressed via alternative splicing and polyadenylation from a single promoter, the “major late” promoter. Recently, Terry Hermiston, Lynda Hawkins, and colleagues have taken advantage of the intricate splicing pattern of the E3 region, replacing a given ORF with a cDNA of interest, namely that for Tumor Necrosis Factor α (TNFα). The genes replaced were the E3-6.7K and E3- gp19K genes coded in the E3A region [12Hawkins L.K. Johnson L. Bauzon M. Nye J.A. Castro D. Kitzes G.A. Young M.D. Holt J.K. Trown P. Hermiston T.W. Gene delivery from the E3 region of replicating human adenovirus evaluation of the 6.7K/gp19K region.Gene Ther. 2001; 8: 1123-1131Crossref PubMed Scopus (82) Google Scholar], the E3 RIDα, RIDβ, and E3-14.7K genes from the E3B region [13Hawkins L.K. Hermiston T. Gene delivery from the E3 region of replicating human adenovirus evaluation of the E3B region.Gene Ther. 2001; 8: 1142-1148Crossref PubMed Scopus (80) Google Scholar], and the Adenovirus Death Protein (adp) (formerly named E3-11.6K) gene from the E3A region [14Hawkins L.K. Hermiston T.W. Gene delivery from the E3 region of replicating human adenovirus evaluation of the ADP region.Gene Ther. 2001; 8: 1132-1141Crossref PubMed Scopus (63) Google Scholar]. The mRNAs for the E3-6.7K, E3-gp19K, RIDα, RIDβ and E3-14.7K proteins are expressed early in infection, and these proteins accumulate in cells as infection proceeds. ADP is expressed in very low amounts from the E3 promoter at early times and in high abundance from the major late promoter at very late times of infection [11Tollefson A.E. Scaria A. Saha S.K. Wold W.S.M. The 11,600-MW protein encoded by region E3 of adenovirus is expressed early but is greatly amplified at late stages of infection.J. Virol. 1992; 66: 3633-3642Crossref PubMed Scopus (77) Google Scholar, 15Tollefson A.E. Scaria A. Hermiston T.W. Ryerse J.S. Wold L.J. Wold W.S.M. The adenovirus death protein (E3-11.6K) is required at very late stages of infection for efficient cell lysis and release of adenovirus from infected cells.J. Virol. 1996; 70: 2296-2306Crossref PubMed Google Scholar]. Interestingly, when these E3 genes were replaced with the cDNA for TNFα, TNFα was expressed in a manner similar to the native E3 genes, and the remaining E3 proteins were expressed with similar kinetics to those in the wild-type adenovirus. This observation could not have been taken for granted, as earlier research showed that even minor modifications in one E3 gene can alter the expression pattern of other E3 genes [16Brady H.A. Scaria A. Wold W.S.M. Map of cis-acting sequences that determine alternative pre-mRNA processing in the E3 complex transcription unit of adenovirus.J. Virol. 1992; 66: 5914-5923PubMed Google Scholar, 17Scaria A. Wold W.S.M. Fine-mapping of sequences that suppress splicing in the E3 complex transcription unit of adenovirus.Virology. 1994; 205: 406-416Crossref PubMed Scopus (11) Google Scholar]. Based on these results, Bauzon et al. [2Bauzon, M., Castro, D., Karr, M., Hawkins, L. K., and Hermiston, T. W. (2003). Regulated, multi-gene expression from a replicating adenovirus using native viral promoters. Mol. Ther., this issueGoogle Scholar] have further developed the E3 gene replacement strategy in that they constructed a single virus in which the adp gene has been replaced by the cDNA for TNFα and the E3-6.7K plus E3-gp19K genes have been replaced by the cDNA for MCP-3, a pro-inflammatory chemokine. As was the case with the singly substituted viruses, the timing and level of expression of MCP-3 and TNFα were similar to that of the E3 genes that were replaced, i.e. TNFα was highly expressed as is the case with ADP, and MCP-3 was made in lesser levels as is the case with E3-6.7K and E3-gp19K. This ability to synthesize different amounts of proteins should be a desirable feature of a vector. The quantities of the therapeutic proteins expressed from the vector exceeded what would be expected from a non-replicating vector, which is a very important advantage. Valuable packaging capacity is not wasted on foreign promoters, and, more importantly, the expression of the genes of interest is tied to virus replication. If the replication of the vector itself is limited to cancerous cells then expression of MCP-3 and TNFα should be confined to tumor cells. It will be very interesting to see if MCP-3 and TNFα expressed from the virus enhance the oncolytic activity. Of all the E3 proteins, ADP is the only one that is expressed predominantly at late rather then early stages of infection (Fig. 1). The function of ADP is to facilitate the lysis of the infected cell and thereby the egress of progeny virus [15Tollefson A.E. Scaria A. Hermiston T.W. Ryerse J.S. Wold L.J. Wold W.S.M. The adenovirus death protein (E3-11.6K) is required at very late stages of infection for efficient cell lysis and release of adenovirus from infected cells.J. Virol. 1996; 70: 2296-2306Crossref PubMed Google Scholar, 18Tollefson A.E. Ryerse J.S. Scaria A. Hermiston T.W. Wold W.S.M. The E3-11.6kDa Adenovirus Death Protein (ADP) is required for efficient cell death characterization of cells infected with adp mutants.Virology. 1996; 220: 152-162Crossref PubMed Scopus (167) Google Scholar]. It was demonstrated that the overexpression of ADP from adenovirus vectors increases the lytic properties of the vector [19Doronin K. Toth K. Kuppuswamy M. Ward P. Tollefson A.E. Wold W.S.M. Tumor-specific, replication-competent adenovirus vectors overexpressing the Adenovirus Death Protein.J. Virol. 2000; 74: 6147-6155Crossref PubMed Scopus (183) Google Scholar, 20Doronin K. Toth K. Kuppuswamy M. Krajcsi P. Tollefson A.E. Wold W.S.M. Overexpression of the ADP (E3-11.6K) protein increases cell lysis and spread of adenovirus.Virology. 2003; 305: 378-387Crossref PubMed Scopus (93) Google Scholar]. Thus, although replacement of adp is probably the most suitable choice to demonstrate the preservation of timing of expression and to produce high levels of therapeutic proteins, it may have a deleterious effect on the anti-cancer efficacy of the vector in terms of its ability to spread within tumors. In addition to adp, it may be beneficial in some cases to retain other E3 genes in the vector; e.g., retention of the RID genes could prevent apoptosis of the infected cell by immune killer cells and thereby prolong expression of the therapeutic genes [6Horwitz M. Adenovirus immunoregulatory genes and their cellular targets.Virology. 2001; 279: 1-8Crossref PubMed Scopus (77) Google Scholar, 7Mahr J.A. Gooding L.R. Immune evasion by adenoviruses.Immunol. Rev. 1999; 168: 121-130Crossref PubMed Scopus (121) Google Scholar, 8Wold W.S.M. Doronin K. Toth K. Kuppuswamy M. Lichtenstein D.L. Tollefson A.E. Immune responses to adenoviruses viral evasion mechanisms and their implications for the clinic.Curr. Opin. Immunol. 1999; 11: 380-386Crossref PubMed Scopus (162) Google Scholar]. A vector in which all E3 genes are replaced with therapeutic genes can be envisioned [2Bauzon, M., Castro, D., Karr, M., Hawkins, L. K., and Hermiston, T. W. (2003). Regulated, multi-gene expression from a replicating adenovirus using native viral promoters. Mol. Ther., this issueGoogle Scholar]. The expression pattern of the curative proteins could be similar to that of the wild-type E3 proteins. This would give the vector designer some control over the amount of individual proteins expressed. This new approach could be an advance in combating cancer.