Gene Therapy and Oncolytic Viruses

Gene Replacement

Cancers develop through loss of function of key regulatory genes known as tumor suppressors as well as through activation of proto-oncogenes. Loss of p14, p16, p53, or PTEN occurs at high frequencies in most tumor types. Loss of Adenomatous Polyposis Coli ( APC ) occurs in the majority of colorectal cancers, and loss of RB is frequent in small-cell lung cancers, among many other examples. Mutations or deletions inactivate products of these genes, or their expression is suppressed by hypermethylation. Clonal evolution of tumors depends on loss of these functions: replacement of functional versions of these genes should therefore reverse this process. Indeed, when tumor suppressors are reexpressed in tumor cells by gene delivery, tumor cells die or growth is arrested. This has been shown for APC, p16, p21, p27, RB, p14ARF, p53, PTEN, APC, and BRCA1, BRCA2, among others.

In contrast, normal cells receiving additional copies of these genes appear to be unaffected, at least in the cases that have been tested so far. This has been documented most clearly for p53: delivery of this tumor suppressor to normal bronchial epithelial cells had no effect on cell growth, suggesting a therapeutic window of more than 2 orders of magnitude. This may be because tumor cells have additional defects that make them more sensitive to the effects of reexpressing p53. For example, almost all tumor cells have a defect in the RB checkpoint and therefore are less able to undergo growth arrest. Normal cells, in contrast, undergo G ₁ arrest when p53 is active, and this can protect them from apoptosis. In addition, the negative regulator of p53, Mdm2, is itself a p53 effector. In tumor cells lacking p53, mdm2 levels are often low. In normal cells, Mdm2 constantly degrades p53 and maintains low p53 activity. Therefore, p53 expressed ectopically in normal cells is degraded, whereas in cancer cells it is relatively stable. In addition, p53 promotes a bystander effect on uninfected tumor cells, possibly through antiangiogenesis, secretion of soluble proapoptotic proteins, and immune upregulation. In agreement with these theoretical considerations, clinical safety and some evidence for anti-tumor activity of an adenoviral vector expresses wild-type p53 under the control of a Rous-sarcoma-virus promoter (Gendicine) have been demonstrated and led to approval of this agent in China for use in head and neck cancer. Fever and flu-like symptoms were the main adverse events; no severe side effects occurred. Long-lasting responses with this agent were observed in studies in patients with head and neck cancer. Very high response rates, including 64% complete responses, were observed following intratumoral injection of Gendicine in combination with radiation therapy. However, a similar virus that delivers wild-type p53 under the control of a constitutively active CMV promoter (AdCMV-p53; Advexin) failed to complete a Phase III clinical trial successfully in the United States, and development of this agent stopped in 2008. This example illustrates the fact that poor delivery systems and inefficient access to tumor cells have limited the potential of gene-replacement therapy for treating cancer, despite compelling theoretical arguments for its development.

Gene Knockout

Thymidine Kinase

The first GDEPT system described was the thymidine kinase gene of the herpes simplex virus (HSV-TK) in combination with the prodrug nucleoside analog ganciclovir. The enzyme thymidine kinase (TK) is naturally present in bacteria, viruses, and mammals, where it is involved in the salvage pathway of nucleotide biosynthesis. Thus, high TK activity is found in proliferating cells such as cancer cells. TK also converts ganciclovir to ganciclovir monophosphate, which is subsequently converted by cellular kinases into the toxic ganciclovir triphosphate nucleotide. The HSV-TK is three orders of magnitude more efficient than any human kinase catalyzing this first activation step. Hence, several gene therapy approaches combining HSV-TK and ganciclovir have been developed.

For instance, retroviral vectors were used to deliver HSV-TK in a brain tumor model. Because retroviruses integrate only in proliferating cells, gene delivery and expression would be tumor selective in the context of normal, nonproliferating brain cells. A further advantage of this approach was thought to result from the “bystander effect,” the killing of uninfected neighboring cells that occurs when HSV-TK–expressing cells are exposed to ganciclovir, which can be observed in vitro and in vivo. To increase transduction of target cells with retroviruses, virus-producing cells (VPCs) were used to inoculate target tumors instead of virus suspension. Preclinical studies in a rat glioma model demonstrated that this delivery of a retrovirus expressing HSV-TK resulted in high transduction levels and frequent tumor regressions following ganciclovir administration. Initial clinical studies were promising and demonstrated in responses of small glioblastomas following VPC injection. However, when standard therapy (surgical resection and radiotherapy) was compared to standard therapy plus injection of retrovirus-producing cells in a Phase III trial, no differences in progression-free and overall survival were observed between the two groups. Smaller, preoperative studies suggested that lack of transduction of tumor cells is the dominant reason for the failure of this approach. Despite the disappointing results of this trial, it represents an early and innovative effort to use gene therapy to kill cancer cells selectively.

Cytosine Deaminase

In contrast to thymidine kinase, the enzyme cytosine deaminase (CD) is not present in mammalian cells, but in several bacteria and fungi. It catalyzes the amidine hydrolysis of cytosine to uracil and ammonia, and several cytosine analogs such as halogenated cytosines are substrates as well. One such substrate is the prodrug 5′-fluorocytosine (5′-FC) which is activated by CD to 5′-fluorouracil (5′-FU). 5′-FU kills cells mainly by inhibition of thymidilate synthetase and by incorporation into DNA. The safety and efficacy of a CD and 5′-FC prodrug therapy are currently being assessed in Phase I/II trials in which 5′-FC and a recombinant Bifidobacterium longum (a live bacterium normally found in the digestive tract) that has been modified to produce CD are given orally to patients with various solid tumors.