Connexin 43-Enhanced Suicide Gene Therapy Using Herpesviral Vectors

Abstract

Tumor cell transduction with the herpes simplex virus (HSV) thymidine kinase (tk) gene and treatment with ganciclovir (GCV) is a widely studied cancer gene therapy. Connexin (Cx)-dependent gap junctions between cells facilitate the intercellular spread of TK-activated GCV, thereby creating a bystander effect that improves tumor cell killing. However, tumor cells often have reduced connexin expression, thus thwarting bystander killing and the effectiveness of TK/GCV gene therapy. To improve the effectiveness of this therapy, we compared an HSV vector (TOCX) expressing Cx43 in addition to TK with an isogenic tk vector (TOZ.1) for their abilities to induce bystander killing of Cx-positive U-87 MG human glioblastoma cells and Cx-negative L929 fibrosarcoma cells in vitro and in vivo. The results showed that low-multiplicity infection of U-87 MG cells with TOCX only minimally increased GCV-mediated cell death compared with infection by TOZ.1, consistent with the endogenous level of Cx in these cells. In contrast, bystander killing of L929 cells was markedly enhanced by vector-mediated expression of Cx. In vivo experiments in which U-87 MG cells were preinfected at low multiplicity and injected into the flanks of nude mice showed complete cures of all animals in the TOCX group following GCV treatment, whereas untreated animals uniformly formed fatal tumors. TOCX injection into U-87 MG intradermal and intracranial tumors resulted in prolonged survival of the host animals in a GCV-dependent manner. Together, these results suggest that the combination of TK and Cx may be beneficial for the treatment of human glioblastoma.

HSV vector cytotoxicity is inversely correlated with effective TK/GCV suicide gene therapy of rat gliosarcoma

“…For example, we have recently demonstrated in animal models that radiosurgery combined with vector-directed production of tumor necrosis factor alpha can improve the efficacy of HSV-TK/GCV therapy for glioblastoma 34 and that expression of connexin 43 from an HSV-TK vector improves the GCV bystander effect in tumors with reduced intercellular communication through gap junctions. 29 These findings, together with our present results, suggest that multi-modal therapies combining conventional treatments with the use of highly defective vectors capable of delivering multiple therapeutic gene products may provide unparalleled new opportunities for effective clinical treatment of recurrent glioma.…”

Development and application of replication-incompetent HSV-1-based vectors

“…12 Furthermore, rat connexin 43 gene has been successfully used to improve the HSV-1 TK/GCV killing of glioma cells by increasing the TK/GCV proximal bystander effect. 50 Recently, an HSV-1-derived replication-defective vector (T0-IFI16) has been developed, which has been shown to efficiently transduce an interferon-inducible gene (IFI16), into primary human umbilical vein endothelial cells (HUVEC), which are usually poorly transfectable. 51 We have also been able to infect HUVEC cells with similar HSV-1-based vectors expressing antiangiogenic fusion proteins endostatin::angiostatin and endostatin::kringle 5.…”

Connexin 26 enhances the bystander effect in HSVtk/GCV gene therapy for human bladder cancer by adenovirus/PLL/DNA gene delivery

“…Similarly, combination therapy of HSVtk and Cx 43 compared with an isogenic HSVtk vector significantly induced the bystander killing effect in Cx-negative L929 fibrosarcoma cells in vitro and Cx-positive U-87 MG human glioblastoma cells in vivo. 48 The recombinant adenovirus systems can efficiently deliver genes both in vitro and in vivo but require a helper cell line and homologous recombination to generate a recombinant adenovirus. As a result, it may take several months for these labor-intensive and time-consuming techniques to generate the recombinant adenovirus vector.…”