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Summary:

We are attempting to convert adenovirus into a drug to treat human cancer. We have developed several genetically engineered adenoviruses, termed "vectors", that have specialized features (Fig. 1). These vectors are designed to replicate specifically in solid tumors in cancer patients, to destroy the tumors, and to have minimal side effects. In particular, the vectors are designed such that they cannot harm normal non-cancerous tissues in the body.

Background on Adenoviruses

Adenoviruses are ubiquitous viruses that cause relatively mild upper respiratory tract infections in young children and induce life-long immunity. The virus particle consists of an outer protein shell surrounding a DNA chromosome that contains 34 genes. When adenovirus infects cells, the virus particle penetrates the cell and the viral genes are expressed as protein molecules that carry out various functions that usurp the cell and convert it into a factory for virus replication (reproduction).

fig1 Figure 1 is a schematic representation of the viral genome (the chromosome). The horizontal bar depicts the genome. The arrows indicate "transcription units", groups of genes from which messenger RNA and protein molecules are synthesized. The E1A region encodes the E1A proteins, which function to prepare the cell for virus replication and to induce genes in the E1B, E2, E3, and E4 transcription units. Adenovirus DNA begins to replicate, then genes from the "major late" transcription unit are expressed. Major late genes encode protein components of the virus particle. Beginning at one day postinfection, virus particles assemble in the cell. The cell then breaks apart (lyses) and releases progeny viruses that can infect other cells. One infectious virus particle yields about 10,000 progeny viruses. The adenovirus protein named ADP mediates the process of cell lysis.

Rationale Behind the VirRx Vectors

One of our vectors is named KD3, and it represents our platform technology. KD3 has three key features, namely a mutation in the "E1A" gene, overexpression of an adenovirus protein named "ADP", and deletion of a group of genes named "E3" (Fig. 1). KD1 is similar to KD3.

The E1A protein (which is coded by the E1A gene) has two major functions in adenovirus infections. One function is to turn on expression of all other adenovirus genes so that adenoviral proteins are made and virions can form. The other function is to deregulate the cell cycle of quiescent cells so that viral gene expression and DNA replication can occur efficiently. Regarding the latter function, take for example a lung epithelial cell that becomes infected. This type of cell does not synthesize DNA, and it cannot support the replication of the adenovirus DNA genome (the chromosome). The "cell deregulation" portion of the E1A protein forces the cell to make the enzymes required to synthesize DNA. Also, the cell becomes dedifferentiated, assuming a state that is conducive to adenoviral DNA replication. After this occurs, viral genes are expressed well and the viral genome can replicate.

KD3 has a mutation in the E1A gene that abolishes the ability of the E1A protein to force cells to make DNA. (Specifically, the E1A protein can no longer release the E2F transcription facter from the pRB cell cycle suppressor, nor can it inactivate the p300/CBP transcriptional co-activator which blocks cell cycle progression). As such, KD3 cannot replicate in lung epithelia or other normal cells of the body. Cancer cells, on the other hand, have a deregulated cell cycle (they are not quiescent) and they are able to synthesize DNA. Inasmuch as the cell deregulation function of E1A is not required in cancer cells, KD3 can replicate in cancer cells. The mutation in E1A is a safety feature of KD3.

The second key feature of KD3 is that it is engineered to overexpress ADP (Fig. 1). As mentioned, ADP mediates cell lysis and release of adenovirus from cells. With KD3, cells are lysed more efficiently than with natural adenoviruses, and as a result, KD3 spreads very efficiently from cell-to-cell. In a tumor, KD3 is expected to spread very efficiently from cell-to-cell, thereby abolishing the tumor. Overexpression of ADP is an efficacy feature of KD3.

The third key feature of KD3 is that it lacks genes in the E3 transcription unit (Fig. 1). The E3 proteins function to protect adenovirus-infected cells from attack by killer cells of the immune system. This protection is partial, and eventually the immune system overcomes the infection. With KD3, there is no protection from the immune system. As such, there is a reduced possibility of a disseminated KD3 infection in the patient. Lack of E3 genes is a safety feature of KD3

We have shown that KD3 replicates within and destroys many different types of human cancer cell lines growing in the laboratory in plastic dishes. As expected, it does not replicate in normal non-cancerous cells under the same conditions.

fig2KD3 also suppresses the growth of human cancer cells as tumors in mice. (In these studies, mice lacking an immune system are used, otherwise the human tumors would be rejected). In the typical experiment shown in Fig. 2, human prostate cancer cells (a cell line named LNCaP) were injected at two sites under the skin of 16 immunodeficient mice, and 32 tumors were allowed to form. The mice were then divided into two groups. With the control group, buffered saline was injected directly into the tumors. With the experimental group, 2 x 108 infectious particles of KD3 were injected into the tumors. The growth of the tumors at different days following injection was measured. The results are presented in Fig. 2 as the fold-increase in tumor size versus time following injection.

With the control group, the tumors increased in size by 13-fold over 50 days. With the KD3 group, the tumors did not grow after 68 days. One-third of the tumors totally regressed. The mice did not appear to suffer side effects. Similar results were obtained in a parallel experiment in which KD3 was injected into the blood stream rather than the tumor (data not shown). In other experiments in which KD3 was injected into human lung or colon tumors growing in mice, KD3 suppressed the growth of the tumors. Altogether, our results indicate that KD3 is an efficacious and safe agent against cancer in this animal model.

KD3 should be effective against many different types of tumors, and it contains two safety features, the E1A mutation and the E3 deletion. We have developed additional KD-based vectors that have a third safety feature and that targets the vectors to tumors of a particular type. In these vectors, we have removed a small section of the KD1 or KD3 chromosome, termed a "promoter", which is required for expression of genes in the E4 transcription unit (Fig. 1, bottom). The E4 promoter has been replaced with one of several "promoters" that control genes that are only expressed in cancer cells (Fig. 1). For example, one vector has a promoter that is specific to lung cells. With this vector, the E4 proteins are expressed only in lung cells. Since the E4 proteins are required for adenovirus to replicate, the vector will only replicate in lung cancer cells. Since the vector has the E1A mutation, it will only replicate in cancerous cells of the lung.

Future Research

We will continue with pre-clinical studies to characterize our vectors. In the near future, we plan to begin Phase I trials for human cancer.

Recent Relevant Publications

Tollefson, A.E., Scaria, A., Hermiston, T.W., Ryerse, J.S., Wold, L.J., and Wold, W.S.M. (1996). 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. 70:2296 -2306.

Tollefson, A.E., Ryerse, J.S., Scaria, A., Hermiston, T.W., and Wold, W.S.M. (1996). The E3-11.6KDa Adenovirus Death Protein (ADP) is required for efficient cell death: characterization of cells infected with adp mutants. Virology 220:152-162.

Doronin, K., Toth, K., Kuppuswamy, M., Ward, P., Tollefson, A.E., and Wold, W.S.M. (2000). Tumor specific, replication-competent adenovirus vectors overexpressing the Adenovirus Death Protein. J. Virol. 74:6147-6155.

Doronin, K., Kuppuswamy, K., Toth, K., Tollefson, A.E., Krajcsi, P., Krougliak, V., and Wold, W.S.M. (2001). Tissue-specific, tumor-selective, replication-competent adenovirus vector for cancer gene therapy. J. Virol. 75:3314-3324.

Tollefson, A.E., Toth, K., Doronin, K., Kuppuswamy, M., Doronina, O.A., Lichtenstein, D.L., Hermiston, T.W., Smith, C.A., and Wold, W.S.M. (2001). Inhibition of trail-induced apoptosis and forced internalization of trail receptor 1 by adenovirus proteins. J. Virol. 75:8875-8887.

Habib, N.A., Mitry, R., Seth, P., Kuppuswamy, M., Doronin, K., Toth, K., Krajcsi, P., Tollefson, A.E., and Wold, W.S.M. (2002). Adenovirus replication-competent vectors (KD1, KD3) complement the cytotoxicity and transgene expression from replication-defective vectors (Ad-GFP, Ad-Luc). Cancer Gene Ther. 9:651-654.

Toth, K., Kuppuswamy, M., Doronin, K., Doronin, O.A., Lichtenstein, D.L., Tollefson, A.E., and Wold, W.S.M. (2002). Construction and characterization of E1-minus replication-defective adenovirus vectors that express E3 proteins from the E1 region. Virology 301:99-108.

Lichtenstein, D.L., Krajcsi, P., Esteban, D.J., Tollefson, A.E., and Wold, W.S.M. (2002). Adenovirus RIDß subunit contains a tyrosine residue that is critical for RID-mediated receptor internalization and inhibition of Fas- and TRAIL-induced apoptosis. J. Virol. 76:11329-11342.

Doronin, K., Toth, K., Kuppuswamy, M., Krajcsi, P., Tollefson, A.E., and Wold, W. S. M. (2003). Overexpression of the ADP (E3-11.6K) protein increases cell lysis and spread of adenovirus. Virology 305:378-387.

Toth, K., Tarakanova, V., Doronin, K., Ward, P., Kuppuswamy, M., Locke, J. L., Dawson, J.E., Kim, H. J., and Wold, W.S.M. (2003). Radiation increases the activity of oncolytic adenovirus cancer gene therapy vectors that overexpress the ADP (E3-11.6K) protein. Cancer Gene Therapy 10:193-200.

Toth, K., Doronin, K., Tollefson, A.E., and Wold, W.S.M. (2003). A multitasking oncolytic adenovirus vector. Molec. Ther. 7: 435-437.

Ying, B., and Wold, W.S.M. (2003). Adenovirus ADP protein (E3-11.6K), which is required for efficient cell lysis and virus release, interacts with human MAD2B. Virology 313:224-234.

Tollefson, A.E., Scaria, A., Ying, B., and Wold, W.S.M. (2003). Mutations within the ADP (E3-11.6K) protein alter processing and localization of ADP and the kinetics of cell lysis of adenovirus-infected cells. J. Virol. 77:7764-7778.

Tarakonova, V., and Wold, W.S.M. (2003). Transforming Growth Factor Beta 1 receptor II is downregulated by E1A in adenovirus-infected cells. J. Virol. 77:9324-9336.

Zanardi, T.A., Yei, S., Lichtenstein, D.L., Tollofson, A.E., and Wold, W.S.M. (2003). Distinct domains in the adenovirus E3 RID alpha protein are required for deregulation of Fas and the epidermal growth factor receptor. J. Virol. 77:11685-11696.

Lichtenstein, D.L., Toth, K., Doronin, K., Tollefson, A.E., and Wold, W.S.M. (2003) Functions and mechanisms of action of the adenovirus E3 proteins. International Reviews of Immunology, 23:75-111.

Toth, K., Djeha, H., Ying, B., Tollefson, A.E., Kuppuswamy, M., Doronin, K., Krajcsi, P., Lipinski, K., Wrighton, C.J., and Wold, W.S.M. (2004) An oncolytic adenovirus vector combining enhanced cell-to-cell spreading, mediated by the ADP cytolytic protein, with selective replication in cancer cells with deregulated Wnt-signaling. Cancer Research 64, 3638-3644.

Lichtenstein, D.L., Doronin, K., Toth, K., Kuppuswamy, M., Wold, W.S.M., and Tollefson, A.E. (2004) Adenovirus E3-6.7K protein is required in conjunction with the E3-RID protein complex for the internalization and degradation of TRAIL Receptor 2. Journal of Virology 78:12297-12307.

Lichtenstein, D.L. and Wold, W.S.M. (2004) Experimental infections of humans with wild-type adenoviruses and with replication-competent adenovirus vectors: replication, safety, and transmission. Cancer Gene Therapy 11:819-829.

Toth, K., Doronin, K., Kuppuswamy, M., Ward, P., Tollefson, A.E., and Wold, W.S.M. (2005) Adenovirus immunoregulatory E3 proteins prolong transplants of human cells in immunocompetent mice. Virus Research 108:149-159.

Toth, K., Spencer, J.F., Tollefson, A.E., Kuppuswamy, M., Doronin, K., Lichtenstein, D.L., La Regina, M.C., Prince, G.A., and Wold, W.S.M. (2005) Cotton rat tumor model for the evaluation of oncolytic adenoviruses. Human Gene Therapy 16:139-146.
Kuppuswamy, M., Spencer, J.F., Doronin, K., Tollefson, A.E., Wold, W.S.M., and Toth, K. (2005) Oncolytic adenovirus vector that overproduces the cytolytic ADP protein and replicates selectively in tumor cells due to regulation of E4 gene expression by the hTERT promoter. Gene Therapy 12:1608-1617.

Kanj, S.S., Dandashi, N., El-Hed, A., Harik, H., Maalouf, M., Kozhaya, L., Mousallem, T., Tollefson, A.E., Wold, William, W.S.M., Chalfant, C.E., and Dbaibo, G.S. (2006) Ceramide regulates SR protein phosphorylation duringadenoviral infection. Virology 345:280-289.

Tollefson, A.E., and Wold W.S.M., Editors, (2006) Adenovirus Methods and Protocols, 2nd edition, Humana Press, in press.

Thomas, M.A., Spencer, J.F., and Wold, William S.M. (2006) The use of the Syrian hamster as an animal model for oncolytic adenovirus vectors. In “Adenovirus Methods and Protocols, 2nd Edition” (eds. W.S.M. Wold and A.E. Tollefson), The Humana Press, Inc., Totowa, New Jersey, (Methods in Molecular Biology), in press.

Tollefson, A.E. and Wold, W.S.M. (2006) Preparation and titration of CsCl-banded adenovirus stock. In “Adenovirus Methods and Protocols, 2nd Edition” (eds. W.S.M. Wold and A.E. Tollefson), The Humana Press, Inc., Totowa, New Jersey, (Methods in Molecular Biology), in press.

Toth, K., Spencer, J.F., and Wold, W.S.M. (2006) Immunocompetent, semi-permissive cotton rat tumor model for the evaluation of oncolytic adenoviruses. In “Adenovirus Methods and Protocols, 2nd Edition” (eds. W.S.M. Wold and A.E. Tollefson), The Humana Press, Inc., Totowa, New Jersey, (Methods in Molecular Biology), in press.

Thomas, M.A., Lichtenstein D.L., and Wold, W.S.M. (2006) A real-time PCR method to rapidly assay adenovirus stocks. In “Adenovirus Methods and Protocols, 2nd Edition” (eds. W.S.M. Wold and A.E. Tollefson), The Humana Press, Inc., Totowa, New Jersey, (Methods in Molecular Biology), in press.

Thomas, M.A., Spencer, J.F., La Regina, M.C., Tollefson, A.E., Toth, K., and Wold, W.S.M.. (2006) The Syrian hamster as a permissive immunocompetent animal model for the study of oncolytic adenovirus vectors, Cancer Research 66:1-7.