A novel system for mitigation of ectopic transgene expression induced by adenoviral vectors

Abstract

Adenoviral vectors possess a number of attributes which render them useful gene delivery vehicles for systemic gene therapy. In particular, the in vivo transduction efficiencies achievable with these agents are greater than with currently available alternative vector systems. 1 However, measures to control specifically the distribution of delivered transgene expression must be superimposed on the basic vector for optimal applicability. Various approaches to this problem have been proposed, including imparting both transductional and transcriptional targeting properties to Ad vectors. 2-7 While some measure of success has been achieved with these approaches, ectopic transgene expression in non-target organs remains a potential problem, even when vectors that have been modified so as not to recognize the native Ad receptor, CAR, are used. Thus, additional, complementary, strategies may be superimposed upon these systems to afford an even greater measure of control.To complement strategies which primarily aim to ensure that transgene expression is sufficiently high in target tissues, we have focused on a strategy to deliberately switch transgene expression off at a site where expression might otherwise be disadvantageous. With respect to Ad vector administration, the majority of vector localization and subsequent transgene expression (using nonselective promoters) is seen in the liver, and as a consequence, hepatotoxicity resulting from ectopic transgene expression is a major concern. In the context of cancer gene therapy using suicide genes, for example, this toxicity has resulted in dose limiting morbidity and mortality in animal models. [8][9][10] This problem is clearly an important issue, in addition to concerns about expression of residual viral genes. Thus, an approach which switches transgene delivery off in the liver may improve the safety of suicide gene therapy.Based on these considerations, we here explore a novel 'liver off' system. The approach uses the cre recombinase (Cre)/LoxP system, whereby the Cre enzyme catalyzes recombination at specific DNA sequences (LoxP sites). This technique has previously been used in the generation of transgenic knock-out mice and as an 'on' switch in an Ad vector context by excising a DNA stuffer sequence inserted between promoters and transgenes. [11][12][13][14][15][16][17][18] The utility of this system to effectively switch off the expression of an Ad-delivered transgene has not been reported. Thus, we hypothesized that a system could be developed whereby the transgene expression resulting from Ad vectors containing LoxP flanked expression cassettes could be effectively switched off by administration of a second vector delivering the Cre gene.As initial proof of principle we developed a strategy that utilizes the Cre/LoxP system to switch off luciferase reporter gene expression, and evaluated efficacy both in vitro and in vivo. We show here that this approach can indeed inactivate transgene expression and importantly, the approach has efficacy in the liver in vivo....

Electroporation-Enhanced Nonviral Gene Transfer for the Prevention or Treatment of Immunological, Endocrine and Neoplastic Diseases

“…Also, a gene therapy for deficiencies in intracellular enzymes or structural proteins requires functional correction at the cell level, thus targeting the largest number of cells is a major factor determining therapeutic success. While recombinant viral vectors have been widely used to introduce new genes into the liver, their usefulness may be mitigated by side effects, potential safety concerns, and the immunologic reaction to viral components, often precluding redosing [Favre, D. et al, 2001;Nathwani, A.C. et al, 2002;Reynolds, P.N. et al, 2001;Thoma, C. et al, 2000].…”