An improved helper-dependent adenoviral vector allows persistent gene expression after intramuscular delivery and overcomes preexisting immunity to adenovirus

Abstract

Helper-dependent adenoviral vectors deleted of all viral coding sequences have shown an excellent gene expression profile in a variety of animal models, as well as a reduced toxicity after systemic delivery. What is still unclear is whether long-term expression and therapeutic dosages of these vectors can be obtained also in the presence of a preexisting immunity to adenovirus, a condition found in a high proportion of the adult human population. In this study we performed intramuscular delivery of helper-dependent vectors carrying mouse erythropoietin as a marker transgene. We found that low doses of helper-dependent adenoviral vectors can direct long-lasting gene expression in the muscles of fully immunocompetent mice. The best performance-i.e., 100% of treated animals showing sustained expression after 4 months-was achieved with the latest generation helper-dependent backbones, which replicate and package at high efficiency during vector propagation. Moreover, efficient and prolonged transgene expression after intramuscular injection was observed with limited vector load also in animals previously immunized against the same adenovirus serotype. These data suggest that human gene therapy by intramuscular delivery of helper-dependent adenoviral vectors is feasible. R eplication-deficient adenoviral (Ad) vectors deleted of one or more early genes (first-and second-generation Ad vectors) are among the most efficient vehicles for in vivo gene delivery, but their utilization for therapeutic purposes is limited by the transient nature of transgene expression and the systemic toxicity, which are both due to inflammatory and immune responses triggered by the residual expression of viral proteins (1-5). The development of Ad vectors deleted of all viral coding sequences offers the prospect of a safer and more efficient way to deliver genes (6). Production of fully deleted Ad vectors, also called helper-dependent (HD) Ad vectors, is possible thanks to the supply in trans of the viral proteins required for replication and packaging by a helper first-generation virus (7).Previous studies by several laboratories have shown improved performances of HD vs. first-generation vectors after intravenous (i.v.) injection as to efficacy of transduction, longevity of transgene expression, and systemic toxicity (8-11). These studies strongly suggested that latest generation adenoviruses may be useful for human applications. However, delivery of Ad vectors (including HD vectors) to human recipients may be rendered particularly difficult when a preexisting immunity against an adenovirus serotype identical to, or cross-reactive with, the one used for gene transfer has previously developed as a consequence of a natural infection. This interfering effect is expected to be particularly pronounced in the case of i.v. delivery, because transducing vector particles will be exposed to circulating neutralizing antibodies before reaching the main target organ, the liver. It is therefore important to evaluate performance, therapeutic efficacy, an...

Inflammation and adaptive immune responses to adenoviral vectors injected into the brain: peculiarities, mechanisms, and consequences

“…Similar mechanisms occur in muscle, where injection of HC-Adv in preimmunized animals also leads to a reduction, but not elimination, of transgene expression. 64 Cross-presentation of adenoviral capsid antigens has been demonstrated, 65,66 and supports this hypothesis. Furthermore, activation of CTLs against capsid protein antigens has been reported to occur upon injection of inactivated adenoviral vectors 67 in some experiments, although in others, neutralizing antibodies or an immune response capable of eliminating brain transgene expression was not detected upon injection of inactivated adenovirus.…”

Dangerous liaisons: the role of “danger” signals in the immune response to gene therapy

“…The removal of viral coding sequences has led to a reduction in immune responsiveness and even long-term transgene expression, with some "gutless" vectors demonstrating maintained therapeutic output for up to 2 years after treatment. [101][102][103] Similar gains have been made by using tissue-specific promoters to drive transgene expression. [104][105][106] Carrying out these modifications has led not only to a decrease in viral and transgene expression in APCs, but also to a reduction in danger signals supplied with the delivery vehicle, including potentially immunostimulatory sequences present in viral promoters.…”

Gene therapy progress and prospects: therapeutic angiogenesis for limb and myocardial ischemia

“…This technology may improve transgene expression despite the presence of antiadenoviral neutralizing antibodies. 7 Production of fully deleted vectors is made possible by a helper virus that provides viral proteins required for replication and packaging. A study of fully deleted adenoviral vectors, also known as gutless or helperdependent, carrying a marker transgene, erythropoietin, demonstrated that intramuscular delivery resulted in efficient and prolonged expression in both immunocomTherapeutic angiogenesis for limb and myocardial ischemia TA Khan et al petent mice and those immunized against the adenovirus serotype.…”

“…A study of fully deleted adenoviral vectors, also known as gutless or helperdependent, carrying a marker transgene, erythropoietin, demonstrated that intramuscular delivery resulted in efficient and prolonged expression in both immunocomTherapeutic angiogenesis for limb and myocardial ischemia TA Khan et al petent mice and those immunized against the adenovirus serotype. 7 The results of the study are clinically important considering the significant proportion of the population with pre-existing immunity to adenovirus. In addition, recombinant adeno-associated viruses (AAV) are potential vectors for therapeutic angiogenesis.…”