The Skin You Are In: Design-of-Experiments Optimization of Lipid Nanoparticle Self-Amplifying RNA Formulations in Human Skin Explants.

Messenger RNA (mRNA) is a promising tool for biotherapeutics, and self-amplifying mRNA (saRNA) is particularly advantageous, because it results in abundant protein expression and production is easily scalable. While mRNA therapeutics have been shown to be highly effective in small animals, the outcomes do not scale linearly when these formulations are translated to dose-escalation studies in humans. Here, we utilize a design of experiments (DoE) approach to optimize the formulation of saRNA lipid nanoparticles in human skin explants. We first observed that luciferase expression from saRNA peaked after 11 days in human skin. Using DoE inputs of complexing lipid identity, lipid nanoparticle dose, lipid concentration, particle concentration, and ratio of zwitterionic to cationic lipids, we optimized the saRNA-induced luciferase expression in skin explants. Lipid identity and lipid concentration were found to be significant parameters in the DoE model, and the optimized formulation resulted in ∼7-fold increase in luciferase expression, relative to initial 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) formulation. Using flow cytometry, we observed that optimized formulations delivered the saRNA to ∼2% of the resident cells in the human skin explants. Although immune cells comprise only 7% of the total population of cells in skin, immune cells were found to express ∼50% of the RNA. This study demonstrates the powerful combination of using a DoE approach paired with clinically relevant human skin explants to optimize nucleic acid formulations. We expect that this system will be useful for optimizing both formulation and molecular designs of clinically translational nucleic acid vaccines and therapeutics.

Long-lived tissue resident HIV-1 specific memory CD8+ T cells are generated by skin immunization with live virus vectored microneedle arrays.

The generation of tissue resident memory (TRM) cells at the body surfaces to provide a front line defence against invading pathogens represents an important goal in vaccine development for a wide variety of pathogens. It has been widely assumed that local vaccine delivery to the mucosae is necessary to achieve that aim. Here we characterise a novel micro-needle array (MA) delivery system fabricated to deliver a live recombinant human adenovirus type 5 vaccine vector (AdHu5) encoding HIV-1 gag. We demonstrate rapid dissolution kinetics of the microneedles in skin. Moreover, a consequence of MA vaccine cargo release was the generation of long-lived antigen-specific CD8+ T cells that accumulate in mucosal tissues, including the female genital and respiratory tract. The memory CD8+ T cell population maintained in the peripheral mucosal tissues was attributable to a MA delivered AdHu5 vaccine instructing CD8+ T cell expression of CXCR3+, CD103+, CD49a+, CD69+, CD127+ homing, retention and survival markers. Furthermore, memory CD8+ T cells generated by MA immunization significantly expanded upon locally administered antigenic challenge and showed a predominant poly-functional profile producing high levels of IFNγ and Granzyme B. These data demonstrate that skin vaccine delivery using microneedle technology induces mobilization of long lived, poly-functional CD8+ T cells to peripheral tissues, phenotypically displaying hallmarks of residency and yields new insights into how to design and deliver effective vaccine candidates with properties to exert local immunosurveillance at the mucosal surfaces.

Microneedle-mediated delivery of viral vectored vaccines.

INTRODUCTION: Microneedle array platforms are a promising technology for vaccine delivery, due to their ease of administration with no sharp waste generated, small size, possibility of targeted delivery to the specified skin depth and efficacious delivery of different vaccine formulations, including viral vectors. Areas covered: Attributes and challenges of the most promising viral vector candidates that have advanced to the clinic and that have been leveraged for skin delivery by microneedles; The importance of understanding the immunobiology of antigen-presenting cells in the skin, in particular dendritic cells, in order to generate further improved skin vaccination strategies; recent studies where viral vectors expressing various antigens have been coupled with microneedle technology to examine their potential for improved vaccination. Expert opinion: Simple, economic and efficacious vaccine delivery methods are needed to improve health outcomes and manage possible outbreaks of new emerging viruses. Understanding what innate/inflammatory signals are required to induce both immediate and long-term responses remains a major hurdle in the development of the effective vaccines. One approach to meet these needs is microneedle-mediated viral vector vaccination. In order for this technology to fulfil this potential the industry must invest significantly to further develop its design, production, biosafety, delivery and large-scale manufacturing.