VSV-G pseudotyped, MuLV-based, semi-replication-competent retrovirus for cancer treatment

Abstract

Low levels of gene delivery in vivo using replication-defective retroviral vectors have severely limited their application for clinical protocols. To overcome this problem, we describe here a semi-replication-competent retrovirus (s-RCR) in which the gag-pol and envelope (VSV-G, vesicular stomatitis virus G protein) genes were split into two vectors. This system offers potential advantages over both replicationdefective vectors, in terms of efficiency of in vivo spread through a tumor, and all-in-one replication-competent vectors in terms of the payload of therapeutic genes that can be carried. We achieved a viral titer of s-RCR viruses approximately 70-fold higher than VSV-G pseudotyped, replicationdefective vectors. In addition, s-RCR vectors induced tumor killing by the cytotoxicity of VSV-G during viral spread. Inclusion of the herpes simplex virus thymidine kinase (HSVtk30) gene into vectors significantly improved tumor killing activity followed by ganciclovir (GCV) treatment in vitro under conditions of low-level viral replication. However, at high levels of viral spread, VSV-G-mediated cytotoxicity predominated. Xenografts of human fibrosarcoma HT1080 cells, preinfected by semi-replicative green fluorescent protein vectors (semi-GFP), were completely non-tumorigenic in nude mice. Implantation of cells preinfected by semireplicative TK30 vectors (semi-TK30) mixed with parental HT1080 cells at a ratio of 1:1 efficiently prevented tumor growth in mice treated by GCV. Direct intratumoral injection of HT1080 tumors growing in nude mice, or B16 murine melanoma in immunocompetent mice, with semi-TK30 viruses significantly prolonged survival. Injection of autologous cells (B16) producing semi-TK30 vector into B16 tumors prolonged survival only in mice treated with GCV but not with phosphate-buffered saline (PBS). In contrast, when xenogeneic cells (293T) producing semi-TK30 vectors were injected into B16 tumors, an optimal survival advantage was obtained in mice treated with PBS rather than GCV. These data indicate that complex interactions exist between direct cytotoxicity of VSV-G and HSVtk expression when placed in the context of additional immune parameters, which combine to determine the efficacy of the therapy. Taken together, our data suggest that s-RCR vectors have some potential advantages for development to deliver genes into tumors for cancer treatment but that a combination of factors will impact on the decision as to whether the s-RCR strategy is worth developing to full clinical trials.

Identification of parameters required for efficient lentiviral vector transduction and engraftment of human cord blood CD34+ NOD/SCID-repopulating cells

“…Longer virus exposure periods could adversely affect the cells in two ways even though the transduction efficiency remains quite similar. One is that cell viability decreases (Figure 3C and 3D), which could be explained by VSV-G envelope toxicity to the transduced cells 30, 31. The other is a decrease in the GFP + CD34 + CD38 − population, which might relate to the cell cycle changes discussed above.…”

Tumor Anti-angiogenic Gene Therapy with Microencapsulated Recombinant CHO Cells

“…These methods are primarily the in vivo direct injection of the viral vectors. The viral vectors that have been used include adenovirus, 31 adeno-associated virus 29,43 and retrovirus, 41 and each method has its own advantages and limitations. Microencapsulation of recombinant cells represents a novel alternative non-viral approach to gene therapy in which therapeutic protein is sustainedly and long-term delivered by microencapsulated recombinant cells.…”

Interference of CD40L-Mediated Tumor Immunotherapy by Oncolytic Vesicular Stomatitis Virus

“…2f ). We also constructed several additional VSVs, expressing transgenes that we believed would enhance adaptive T cell responses to tumor-associated antigens, based on our previous experience with the B16ova model, including heat shock protein-70 (hsp70) (Daniels et al, 2004;Qiao et al, 2006) and CCL21 (Thanarajasingam et al, 2007). However, similar to our experience with VSV-CD40L, neither VSV-hsp70 nor VSV-CCL21 generated significantly improved therapy compared with VSV-GFP when given two, six, or even nine times directly into B16ova tumors (data not shown).…”