Despite more than two decades of research and development on nucleic acid vaccines, there is still no commercial product for human use. Taking advantage of the recent innovations in systemic delivery of short interfering RNA (siRNA) using lipid nanoparticles (LNPs), we developed a self-amplifying RNA vaccine. Here we show that nonviral delivery of a 9-kb self-amplifying RNA encapsulated within an LNP substantially increased immunogenicity compared with delivery of unformulated RNA. This unique vaccine technology was found to elicit broad, potent, and protective immune responses, that were comparable to a viral delivery technology, but without the inherent limitations of viral vectors. Given the many positive attributes of nucleic acid vaccines, our results suggest that a comprehensive evaluation of nonviral technologies to deliver self-amplifying RNA vaccines is warranted.vaccine platform | SAM vaccine | respiratory syncytial virus | HIV
Vaginal instillation of small-interfering RNA (siRNA) using liposomes has led to silencing of endogenous genes in the genital tract and protected against challenge from infectious disease. Although siRNA lipoplexes are easily formulated, several of the most effective transfection agents available commercially may be toxic to the mucosal epithelia and none are able to provide controlled or sustained release. Here, we demonstrate an alternate approach, using nanoparticles composed entirely of FDA-approved materials. To render these materials effective for gene silencing we developed novel approaches to load them with high amounts of siRNA. A single dose of siRNA-loaded nanoparticles to the mouse female reproductive tract caused efficient and sustained gene silencing. Knockdown of gene expression was observed proximal (in the vaginal lumen) and distal (in the uterine horns) to the site of topical delivery. In addition, nanoparticles penetrated deep into the epithelial tissue. This is the first report demonstrating that biodegradable polymer nanoparticles are effective delivery vehicles for siRNA in the vaginal mucosa.
Rapid, multiplexed, sensitive and specific molecular detection is of great demand in gene profiling, drug screening, clinical diagnostics and environmental analysis. One of the major challenges in multiplexed analysis is to identify each specific reaction with a distinct label or 'code'. Two encoding strategies are currently used: positional encoding, in which every potential reaction is preassigned a particular position on a solid-phase support such as a DNA microarray, and reaction encoding, where every possible reaction is uniquely tagged with a code that is most often optical or particle based. The micrometer size, polydispersity, complex fabrication process and nonbiocompatibility of current codes limit their usability. Here we demonstrate the synthesis of dendrimer-like DNA-based, fluorescence-intensity-coded nanobarcodes, which contain a built-in code and a probe for molecular recognition. Their application to multiplexed detection of the DNA of several pathogens is first shown using fluorescence microscopy and dot blotting, and further demonstrated using flow cytometry that resulted in detection that was sensitive (attomole) and rapid.
Drug delivery to mucosal epithelia is severely limited by the mucus gel, which is a physical diffusion barrier as well as an enzymatic barrier in some sites. Loading of drug into polymer particles can protect drugs from degradation and enhance their stability. To improve efficacy of nanoparticulate drug carriers, it has been speculated that polymers such as poly(ethylene)glycol (PEG) incorporated on the particle surface will enhance transport in mucus. In the present study, we demonstrate the direct influence of PEG on surface properties of poly(lactic-co-glycolic)acid (PLGA) nanoparticles (d = 170 ± 57 nm). PEG of various molecular weights (MW = 2, 5, 10 kDa) were incorporated at a range of densities from 5 -100% on the particle surface. Our results indicate PEG addition improves dispersion, neutralize charge, and enhance particle diffusion in cervical mucus in a manner strongly dependent on polymer MW and density. Diffusion of PEGylated particles was 3 -10× higher than unmodified PLGA particles. These findings improve the understanding of, and confirm a possible direction for, the rational design of effective carriers for mucosal drug/vaccine delivery.
Self-amplifying messenger RNA (mRNA) of positive-strand RNA viruses are effective vectors for in situ expression of vaccine antigens and have potential as a new vaccine technology platform well suited for global health applications. The SAM vaccine platform is based on a synthetic, self-amplifying mRNA delivered by a nonviral delivery system. The safety and immunogenicity of an HIV SAM vaccine encoding a clade C envelope glycoprotein formulated with a cationic nanoemulsion (CNE) delivery system was evaluated in rhesus macaques. The HIV SAM vaccine induced potent cellular immune responses that were greater in magnitude than those induced by self-amplifying mRNA packaged in a viral replicon particle (VRP) or by a recombinant HIV envelope protein formulated with MF59 adjuvant, anti-envelope binding (including anti-V1V2), and neutralizing antibody responses that exceeded those induced by the VRP vaccine. These studies provide the first evidence in nonhuman primates that HIV vaccination with a relatively low dose (50 µg) of formulated self-amplifying mRNA is safe and immunogenic.
The rate of molecular transport through the mucus gel can be an important determinant of efficacy for therapeutic agents delivered by oral, intranasal, intravaginal/rectal, and intraocular routes. Transport through mucus can be described by mathematical models based on principles of physical chemistry and known characteristics of the mucus gel, its constituents, and of the drug itself. In this paper, we review mathematical models of molecular diffusion in mucus, as well as the techniques commonly used to measure diffusion of solutes in the mucus gel, mucus gel mimics, and mucosal epithelia.
Intravaginal (ivag) delivery, which is a proven way to confer local protection against STD’s contracted via the reproductive tract, is complicated by the mucus gel barrier, the hormone cycle, and the harsh mucosal environment, which leads to low residence-time for administered agents. Polymer delivery vehicles may be useful in overcoming these barriers for delivery of agents. We explored the fate of nanoparticles (NP) made from poly(lactide-co-glycolide) (PLGA) in the mouse reproductive tract after ivag delivery. The nanoparticles were modified to display avidin (Avid-NP) or 2kDa PEG (PEG-NP) on their surface. Vaginal retention fractions for both muco-adhesive Avid-NP and stealthy PEG-NP were 5x higher than unmodified PLGA particles (NP). The amount of particles associated with mucus differed across formulations (Avid-NP>NP>PEG-NP). PEG-NP was found at higher concentration in the tissue than Avid-NP and NP up to 6 hr after delivery, and particles were found within epithelial cells and the underlying submucosal stromal and fibroblast cells of the vaginal tissue. Our results demonstrate that surface properties of nanoparticles can impact their fates following ivag delivery and that PEG-modified nanoparticles are potentially useful in ivag delivery.
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