Oncolytic Adenovirus: Getting There Is Half the Battle
Authors: P. Joel Ross, Robin J. Parks
Journal: Molecular Therapy (2003)

In recent years, there has been intense research into retar- geting Adenovirus (Ad) for applications in gene therapy. The goal is to ablate the natural tropism of the virus, while concomitantly targeting it to specific cell types [1Wickham, T. J. (2003). Ligand-directed targeting of genes to the site of disease. Nat. Med. 9: 135_9.Google Scholar]. However, Bernt et al. report in this issue of Molecular Therapy that retar- geting may be insufficient to rectify the significant problem of viral loss after systemic administration [2Bernt, K. M., Ni, S., Li, Z.-Y., Shayakhmetov, D. M., and Lieber, A. (2003). The effect of sequestration by non-target tissues on anti-tumor efficacy of systemically applied, conditionally replicating adenovirus vectors. Mol. Ther. This Issue.Google Scholar]. They found that other factors, such as virus stability in blood and translocation through the hepatic sinusoids, might also pres- ent barriers that contribute to poor transduction of target tis- sue. They conclude that there exist hurdles other than the limitation of viral tropism that must be overcome before sys- temically delivered Ad can efficiently target diseased tissue.The study by Bernt et al. explores the efficacy of tumor- targeted, conditionally replicating Ad (CRAd). CRAd have great potential as cancer therapeutics. These viruses have been designed to target tumor cells specifically for lytic infection, and have been used to treat metastatic cancer [3Dubensky, T. W. Jr. (2002). (Re-)Engineering tumor cell-selective replicating aden- oviruses: a step in the right direction toward systemic therapy for metastatic disease. Cancer Cell 1: 307_9.Google Scholar]. However, a major limitation of CRAd has been poor tar- get-cell transduction following systemic administration [4Green, N. K., and Seymour, L. W. (2002). Adenoviral vectors: systemic delivery and tumor targeting. Cancer Gene Ther. 9: 1036_42.Google Scholar]. Ad serotype 5, the most commonly used platform for such applications, is able to transduce many cell types and tissues efficiently. This can be problematic, as sequestration of CRAd by nontarget tissue limits their therapeutic potential. CRAd replication is targeted to tumor cells by mutation, deletion, and/or regulated expression of viral genes that are required for Ad replication [5Haviv, Y. S., and Curiel, D. T (2003). Engineering regulatory elements for conditionally-replicative adenoviruses. Curr. Gene Ther. 3: 357_85.Google Scholar]. Successful replication in nor- mal cells requires circumvention of the cell-cycle block imposed by Rb and the apoptotic response initiated by p53. Mutant viruses that are unable to overcome these hurdles do not replicate in normal cells. However, tumor cells often have disabled Rb and p53 pathways; this allows selective replication of mutant Ad in tumor cells, a phenomenon referred to as oncolysis [5Haviv, Y. S., and Curiel, D. T (2003). Engineering regulatory elements for conditionally-replicative adenoviruses. Curr. Gene Ther. 3: 357_85.Google Scholar].Two viral genes, E1A and E1B (which are responsible for neutralizing Rb and p53, respectively), are deleted from the CRAd used by Bernt et al. and the replication of the resulting virus is tumor selective [6Steinwaerder, D. S., Carlson, C. A., Otto, D. L., Li, S., Ni, Z.-Y., and Lieber, A. (2001). Tumor-specific gene expression in hepatic metastases by a replication-activated ade- novirus vector. Nat. Med. 7: 240_3.Google Scholar]. Additionally, these viruses con- tain a transgene that is activated only upon Ad replication. Replication-coupled recombination between inverted repeats (IR) flanking the transgene places it downstream of a strong promoter, resulting in transcriptional activation. Bernt et al. used viruses encoding either a lacZ reporter con- struct or, for studies of oncolysis, a vector encoding E1A. E1A is able to transactivate the expression of other viral genes; therefore, low level, replication-dependent expression of E1A led to greatly enhanced Ad.IR replication. The combi- nation of tumor-specific replication and replication-depend- ent expression of a transgene makes this an appealing sys- tem for CRAd development.Preliminary in vitro studies by Bernt et al. suggested, per- haps not surprisingly, that increasing the number of CRAd that infect a given tumor cell results in greater oncolysis. These data implied that a similar effect might be observed in vivo. However, due to the widespread expression of the cel- lular receptors used by Ad5 (the Coxsackie-Adenovirus receptor and specific aV integrin heterodimers), nontarget tissue may sequester Ad.IR, effectively reducing the amount of virus available for the infection of tumor cells. This led the authors to question whether targeting of Ad.IR to tumor cells could lead to enhanced oncolysis in vivo. In order to “retarget” Ad5, they replaced the Ad5 fiber with that of Ad35. The chimeric virus, Ad5/35, had a reduced capacity for the transduction of normal mouse tissue, while its abili- ty to transduce human tumor cells remained intact [7Shayakhmetov, D. M., Papayannopoulou, T., Stamatoyannopoulos, S. J., and Lieber, A. (2000). Efficient gene transfer into human CD34(+) cells by a retargeted aden- ovirus vector. J. Virol. 74: 2567_83.Google Scholar]. The oncolytic efficacy of “targeted” (Ad5/35) and “untargeted” (Ad5) Ad.IR was compared in an in vitro model that consist- ed of human tumor cells (which are readily transduced by both Ad5 and Ad5/35) surrounded by mouse hepatocytes (which are transduced only by Ad5). Using this model, oncolysis was nearly three fold greater after infection with Ad5/35.IR. The vectors were also compared in a similar model in vivo (mouse liver metastases derived from human tumor cells). Unexpectedly, the authors observed no signifi- cant difference between treatments, despite the fact that Ad5.IR infected both metastases and regular mouse tissues, whereas Ad5/35 transduction was restricted to tumor cells. These surprising data indicated that other factors, aside from sequestration by nontarget tissue, contribute to Ad loss after systemic administration.Paradoxically, targeting did not seem to increase the amount of CRAd available for transduction of tumor cells, as both viruses exhibited similar oncolysis. Other tissues did not take up Ad5/35, in contrast to the results with Ad5, sug- gesting that much of the virus remained in the serum. However, both the amount of CRAd in the blood and the kinetics of viral clearance were similar for Ad5.IR and Ad5/35.IR. So, where was the Ad5/35 going? The authors found that entrapment within the liver sinusoids might present a major impediment to virus transduction of target tissue, as both viruses accumulated there soon after delivery. Transduction of liver metastases requires successful trafficking of the virus through the fenestrae of the endothelial lin- ing of the sinusoids and subsequent diffusion of the Ad to the tumor cells. Bernt et al. suggest that variable rates of either transendothelial transfer or diffusion of the virus may be to blame for the disappointing results observed using Ad5/35 in vivo. They also suggest that stability of the virus in blood may be a limiting factor. Regardless, future research must elucidate the precise trafficking route of Ad after sys- temic administration, what specific factors contribute to Ad loss, and how these factors can be overcome.How should Ad be delivered for the treatment of disease? Studies have shown that bypassing the liver can significant- ly enhance transduction of other target tissues, such as the vessels and capillaries of the lung, intestinal wall, and renal glomeruli [8Ye, X., Jerebtsova, M., and Ray, P. E. (2000). Liver bypass significantly increases the transduction efficiency of recombinant adenoviral vectors in the lung, intestine, and kidney. Hum. Gene Ther. 11: 621 _7Google Scholar]. However, even when the portal vein and hepatic artery were clamped for 30 minutes at the time of viral administration, significant transgene expression was detected in the liver. The bone marrow and spleen also con- tain sinusoids, and may therefore represent additional tis- sues in which the vectors can be trapped, even if the liver can be avoided. Finally, sequestration in the liver is prob- lematic not only for systemic administration of Ad. A large proportion of Ad injected directly into a tumor ends up in the liver [9Bramson, J. L., Hitt, M., Gauldie, J., and Graham, F. L. (1997). Pre-existing immunity to adenovirus does not prevent tumor regression following intratumoral adminstra- tion of a vector expressing IL-12 but inhibits viral dissemination. Gene Ther. 4: 1069_76.Google Scholar]. Thus, even Ad delivered locally may be lost due to sequestration in the sinusoids. The choice of method for vector administration must be made on a case-by-case (or disease-by-disease) basis, as each method has both advan- tages and disadvantages. However, the data discussed above provide some additional food for thought.The results obtained by Bernt et al. have important impli- cations, not only for the improvement of CRAd, but also for the development of Ad vectors for gene therapy. Successful gene therapy depends upon adequate expression of a therapeutic transgene in the appropriate tissues [1Wickham, T. J. (2003). Ligand-directed targeting of genes to the site of disease. Nat. Med. 9: 135_9.Google Scholar]. However, even targeted Ad is unable to transduce the appropriate tis- sues if it is trapped in the hepatic sinusoids. Gene therapy researchers tend to believe that transductional targeting is the key to successful delivery of therapeutic transgenes. As long as nontarget tissue is avoided, adequate amounts of vector will be available for the transduction of tissues and cells of interest. However, the results obtained by Bernt et al. show that there are other factors contributing to vector loss after systemic administration. These factors, heretofore underappreciated by vectorologists, must be clearly identi- fied and overcome if Ad vectors are to attain their full poten- tial in the clinic. In recent years, there has been intense research into retar- geting Adenovirus (Ad) for applications in gene therapy. The goal is to ablate the natural tropism of the virus, while concomitantly targeting it to specific cell types [1Wickham, T. J. (2003). Ligand-directed targeting of genes to the site of disease. Nat. Med. 9: 135_9.Google Scholar]. However, Bernt et al. report in this issue of Molecular Therapy that retar- geting may be insufficient to rectify the significant problem of viral loss after systemic administration [2Bernt, K. M., Ni, S., Li, Z.-Y., Shayakhmetov, D. M., and Lieber, A. (2003). The effect of sequestration by non-target tissues on anti-tumor efficacy of systemically applied, conditionally replicating adenovirus vectors. Mol. Ther. This Issue.Google Scholar]. They found that other factors, such as virus stability in blood and translocation through the hepatic sinusoids, might also pres- ent barriers that contribute to poor transduction of target tis- sue. They conclude that there exist hurdles other than the limitation of viral tropism that must be overcome before sys- temically delivered Ad can efficiently target diseased tissue. The study by Bernt et al. explores the efficacy of tumor- targeted, conditionally replicating Ad (CRAd). CRAd have great potential as cancer therapeutics. These viruses have been designed to target tumor cells specifically for lytic infection, and have been used to treat metastatic cancer [3Dubensky, T. W. Jr. (2002). (Re-)Engineering tumor cell-selective replicating aden- oviruses: a step in the right direction toward systemic therapy for metastatic disease. Cancer Cell 1: 307_9.Google Scholar]. However, a major limitation of CRAd has been poor tar- get-cell transduction following systemic administration [4Green, N. K., and Seymour, L. W. (2002). Adenoviral vectors: systemic delivery and tumor targeting. Cancer Gene Ther. 9: 1036_42.Google Scholar]. Ad serotype 5, the most commonly used platform for such applications, is able to transduce many cell types and tissues efficiently. This can be problematic, as sequestration of CRAd by nontarget tissue limits their therapeutic potential. CRAd replication is targeted to tumor cells by mutation, deletion, and/or regulated expression of viral genes that are required for Ad replication [5Haviv, Y. S., and Curiel, D. T (2003). Engineering regulatory elements for conditionally-replicative adenoviruses. Curr. Gene Ther. 3: 357_85.Google Scholar]. Successful replication in nor- mal cells requires circumvention of the cell-cycle block imposed by Rb and the apoptotic response initiated by p53. Mutant viruses that are unable to overcome these hurdles do not replicate in normal cells. However, tumor cells often have disabled Rb and p53 pathways; this allows selective replication of mutant Ad in tumor cells, a phenomenon referred to as oncolysis [5Haviv, Y. S., and Curiel, D. T (2003). Engineering regulatory elements for conditionally-replicative adenoviruses. Curr. Gene Ther. 3: 357_85.Google Scholar]. Two viral genes, E1A and E1B (which are responsible for neutralizing Rb and p53, respectively), are deleted from the CRAd used by Bernt et al. and the replication of the resulting virus is tumor selective [6Steinwaerder, D. S., Carlson, C. A., Otto, D. L., Li, S., Ni, Z.-Y., and Lieber, A. (2001). Tumor-specific gene expression in hepatic metastases by a replication-activated ade- novirus vector. Nat. Med. 7: 240_3.Google Scholar]. Additionally, these viruses con- tain a transgene that is activated only upon Ad replication. Replication-coupled recombination between inverted repeats (IR) flanking the transgene places it downstream of a strong promoter, resulting in transcriptional activation. Bernt et al. used viruses encoding either a lacZ reporter con- struct or, for studies of oncolysis, a vector encoding E1A. E1A is able to transactivate the expression of other viral genes; therefore, low level, replication-dependent expression of E1A led to greatly enhanced Ad.IR replication. The combi- nation of tumor-specific replication and replication-depend- ent expression of a transgene makes this an appealing sys- tem for CRAd development. Preliminary in vitro studies by Bernt et al. suggested, per- haps not surprisingly, that increasing the number of CRAd that infect a given tumor cell results in greater oncolysis. These data implied that a similar effect might be observed in vivo. However, due to the widespread expression of the cel- lular receptors used by Ad5 (the Coxsackie-Adenovirus receptor and specific aV integrin heterodimers), nontarget tissue may sequester Ad.IR, effectively reducing the amount of virus available for the infection of tumor cells. This led the authors to question whether targeting of Ad.IR to tumor cells could lead to enhanced oncolysis in vivo. In order to “retarget” Ad5, they replaced the Ad5 fiber with that of Ad35. The chimeric virus, Ad5/35, had a reduced capacity for the transduction of normal mouse tissue, while its abili- ty to transduce human tumor cells remained intact [7Shayakhmetov, D. M., Papayannopoulou, T., Stamatoyannopoulos, S. J., and Lieber, A. (2000). Efficient gene transfer into human CD34(+) cells by a retargeted aden- ovirus vector. J. Virol. 74: 2567_83.Google Scholar]. The oncolytic efficacy of “targeted” (Ad5/35) and “untargeted” (Ad5) Ad.IR was compared in an in vitro model that consist- ed of human tumor cells (which are readily transduced by both Ad5 and Ad5/35) surrounded by mouse hepatocytes (which are transduced only by Ad5). Using this model, oncolysis was nearly three fold greater after infection with Ad5/35.IR. The vectors were also compared in a similar model in vivo (mouse liver metastases derived from human tumor cells). Unexpectedly, the authors observed no signifi- cant difference between treatments, despite the fact that Ad5.IR infected both metastases and regular mouse tissues, whereas Ad5/35 transduction was restricted to tumor cells. These surprising data indicated that other factors, aside from sequestration by nontarget tissue, contribute to Ad loss after systemic administration. Paradoxically, targeting did not seem to increase the amount of CRAd available for transduction of tumor cells, as both viruses exhibited similar oncolysis. Other tissues did not take up Ad5/35, in contrast to the results with Ad5, sug- gesting that much of the virus remained in the serum. However, both the amount of CRAd in the blood and the kinetics of viral clearance were similar for Ad5.IR and Ad5/35.IR. So, where was the Ad5/35 going? The authors found that entrapment within the liver sinusoids might present a major impediment to virus transduction of target tissue, as both viruses accumulated there soon after delivery. Transduction of liver metastases requires successful trafficking of the virus through the fenestrae of the endothelial lin- ing of the sinusoids and subsequent diffusion of the Ad to the tumor cells. Bernt et al. suggest that variable rates of either transendothelial transfer or diffusion of the virus may be to blame for the disappointing results observed using Ad5/35 in vivo. They also suggest that stability of the virus in blood may be a limiting factor. Regardless, future research must elucidate the precise trafficking route of Ad after sys- temic administration, what specific factors contribute to Ad loss, and how these factors can be overcome. How should Ad be delivered for the treatment of disease? Studies have shown that bypassing the liver can significant- ly enhance transduction of other target tissues, such as the vessels and capillaries of the lung, intestinal wall, and renal glomeruli [8Ye, X., Jerebtsova, M., and Ray, P. E. (2000). Liver bypass significantly increases the transduction efficiency of recombinant adenoviral vectors in the lung, intestine, and kidney. Hum. Gene Ther. 11: 621 _7Google Scholar]. However, even when the portal vein and hepatic artery were clamped for 30 minutes at the time of viral administration, significant transgene expression was detected in the liver. The bone marrow and spleen also con- tain sinusoids, and may therefore represent additional tis- sues in which the vectors can be trapped, even if the liver can be avoided. Finally, sequestration in the liver is prob- lematic not only for systemic administration of Ad. A large proportion of Ad injected directly into a tumor ends up in the liver [9Bramson, J. L., Hitt, M., Gauldie, J., and Graham, F. L. (1997). Pre-existing immunity to adenovirus does not prevent tumor regression following intratumoral adminstra- tion of a vector expressing IL-12 but inhibits viral dissemination. Gene Ther. 4: 1069_76.Google Scholar]. Thus, even Ad delivered locally may be lost due to sequestration in the sinusoids. The choice of method for vector administration must be made on a case-by-case (or disease-by-disease) basis, as each method has both advan- tages and disadvantages. However, the data discussed above provide some additional food for thought. The results obtained by Bernt et al. have important impli- cations, not only for the improvement of CRAd, but also for the development of Ad vectors for gene therapy. Successful gene therapy depends upon adequate expression of a therapeutic transgene in the appropriate tissues [1Wickham, T. J. (2003). Ligand-directed targeting of genes to the site of disease. Nat. Med. 9: 135_9.Google Scholar]. However, even targeted Ad is unable to transduce the appropriate tis- sues if it is trapped in the hepatic sinusoids. Gene therapy researchers tend to believe that transductional targeting is the key to successful delivery of therapeutic transgenes. As long as nontarget tissue is avoided, adequate amounts of vector will be available for the transduction of tissues and cells of interest. However, the results obtained by Bernt et al. show that there are other factors contributing to vector loss after systemic administration. These factors, heretofore underappreciated by vectorologists, must be clearly identi- fied and overcome if Ad vectors are to attain their full poten- tial in the clinic.