Pharmacologic gene regulation is a key technology, necessary to achieve safe, long-term gene transfer. The approaches described in the scientific literature all share in common the creation of artificial transcription factors by fusing a DNA-binding domain, a drug-binding domain and a transcription activation domain. These transcription factors activate the transgene expression upon binding of the pharmacologic agent (antibiotics of the tetracycline family, insect hormone, progesterone antagonist, or immunosuppressor drug) to the drug-binding domain. The major limitations to the use of these systems for human gene and cell therapies are the toxicity of the inducer molecule and the immunogenicity of the chimeric transcription factor. Thus, the gene regulation systems should operate with clinically approved drugs with safety records that do not conflict with the therapeutic gene expression regimen. This work focuses on the characterization of the immunogenicity of a tetracycline-activated transcription factor commonly used in preclinical gene therapy, rtTA2-M2, and its impact on reporter gene expression. We demonstrate that intramuscular injection of plasmid or adenoviral vectors encoding rtTA-M2 in outbred primates generates a cellular and humoral immune response to this transcription factor. The immune response to rtTA2-M2 blunts the duration of the expression the rtTA2-M2-controlled transgene in primates, presumably by destruction of the cells that coexpress rtTA2-M2 and the reporter or therapeutic gene. This immune response may result directly from the vectors used in this study, which prompts the development of new gene transfer vectors enabling safe and efficient pharmacologic gene regulation in clinic.
Neutralizing antibody is associated with the prevention and clearance of influenza virus infection. Microneutralization (MN) and hemagglutination inhibition (HI) assays are currently used to evaluate neutralizing antibody responses against human and avian influenza viruses, including H5N1. The MN assay is somewhat labor intensive, while HI is a surrogate for neutralization. Moreover, use of replication competent viruses in these assays requires biosafety level 3 (BSL-3) containment. Therefore, a neutralization assay that does not require BSL-3 facilities would be advantageous. Toward this goal, we generated a panel of pseudotypes expressing influenza hemagglutinin (HA) and neuraminidase (NA) and developed a pseudotype-based neutralization (PN) assay. Here we demonstrate that HA/NA pseudotypes mimic release and entry of influenza virus and that the PN assay exhibits good specificity and reveals quantitative difference in neutralizing antibody titers against different H5N1 clades and subclades. Using immune ferret sera, we demonstrated excellent correlation between the PN, MN, and HI assays. Thus, we conclude that the PN assay is a sensitive and quantifiable method to measure neutralizing antibodies against diverse clades and subclades of H5N1 influenza virus.
The ongoing epizootic of highly pathogenic avian H5N1 influenza and its direct transmissibility and high pathogenicity in humans has led to renewed interest in the development of influenza vaccines with enhanced immunogenicity. Influenza vaccines are currently under development against influenza strains that are potentially pandemic threats, such as H5N1, as well as against the current seasonal influenza strains for use in populations susceptible to severe influenza disease. Influenza vaccines may be generally divided into two types: seasonal vaccines for use in a population that is largely primed to subtypes of the circulating influenza A strains and pandemic influenza vaccines that are designed to protect against influenza A viruses of a hemagglutinin (HA) subtype, to which the vast majority of the population is immunologically naive. Pandemic influenza vaccines can be further subdivided into prepandemic vaccines produced for use prior to or just after the declaration of a pandemic, and pandemic influenza vaccines that would be produced and used only after a pandemic is declared. Prepandemic influenza vaccines are formulated using HA and neuraminidase, which are likely to be antigenically similar to the influenza virus subtype deemed to pose the most probable pandemic threat. Enhanced vaccine immunogenicity is desirable for pandemic influenza vaccines and for seasonal vaccines used in target populations, such as the elderly, in which vaccine responses against the circulating influenza subtypes may be weak. Various methods to enhance the immunogenicity of influenza vaccines are under evaluation. Along with dose escalation and alternative delivery routes, strategies for improving the immunogenicity of influenza vaccines have focused on the use of immunologic adjuvants. An adjuvanted seasonal influenza vaccine, Fluad, has been licensed in some countries in Europe since 1997 for the elderly population, and a number of clinical trials have been completed or are in progress evaluating the use of adjuvants with pandemic and seasonal influenza vaccines. This review will focus on the use of emulsion-based adjuvants for enhancing the immunogenicity of pandemic influenza vaccines and of seasonal influenza vaccines in target populations.
A novel assembly of two structurally related 14-membered ring macrocyclic hepatitis C virus protease inhibitors is presented. Key to their successful construction was an ultimate ring-closing metathesis step on the respective highly functionalized dienyl-ureas. In the case of IDX316, this procedure significantly outperformed the original macrocyclizations in terms of reaction conditions, impurity profile, product isolation, and basic efficiency metrics. Simple nonchromatographic purification methods achieved sub-10-ppm ruthenium content in the isolated product. Overall yields to IDX316 from all five starting materials ranged from 11 to 40%, and the estimated process mass intensity was improved by a factor of 50 relative to the original unscalable discovery-based routes. Application of similar methodology in the case of IDX320 and first scale-up to halfkilogram batch sizes was demonstrated.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.