Laser Poration for Transdermal Allergen Immunotherapy

For almost a century, allergen immunotherapy has been used as the only antigen-specific immunomodulatory therapy for allergic diseases. Allergen extracts or recombinant allergens are administered either by subcutaneous injection (SCIT) or, more recently, via the sublingual route (SLIT). SCIT requires weekly injections over months or even years and can potentially cause severe side effects. In contrast, SLIT appears to be a safer, but less efficient alternative that requires higher doses and even more frequent (daily) applications because antigen uptake via the mucosa is limited. Divided into two areas - the epidermis and the dermis - the skin is considered a promising target for immunotherapy because it is rich in antigen presenting cells. Both layers are populated by different subsets of skin derived dendritic cells, which differ in surface markers and their immunological properties. Langerhans cells in the epidermis are commonly believed to represent the first line of defence against pathogens. However, recent studies suggest a more regulatory role for Langerhans cells and that instead dermal dendritic cells residing in the dermis are crucial for the development of cutaneous immune reactions. In the proposed project, we intend to precisely target allergen-encoding plasmid DNA or recombinant allergens either to the epidermis or to the dermis of mice. This will be achieved by using a proprietary laser device developed by Pantec Biosolutions, which enables skin ablation in 5-10 micrometer steps, thereby creating controlled aqueous micropores allowing for high diffusion rates over prolonged periods of time (48h). Vaccine formulations applied to such pore arrays are taken up at similar levels compared to subcutaneous injections. For increased patient compliance and long term exposure of the pore arrays with the respective DNA-based or recombinant vaccine, dermal patches incorporating the vaccine in a gel formulation will be developed. This technology enables more efficient immunostimulation, and therefore reduces the necessary application frequency compared to SLIT, while retaining high patient compliance associated with a painless method that can be applied by the patient at home. Detailed characterization of the immune responses elicited with the various vaccines targeting the two different skin compartments will be performed. The relevance of Langerhans cells for the induction of immune responses will be investigated using transgenic mice. Finally, after establishing the optimal parameters for laser settings and vaccine formulations, we will investigate the applicability of this novel vaccine delivery platform for specific immunotherapy in our well established mouse model of type I allergy.

Specific immunotherapy by application of allergen to skin micropores generated via far infrared laser beams was found to be equally effective compared to standard intervention performed by subcutaneous injections in a mouse model of allergic asthma. As the common method for therapy of allergic diseases is based on up to 100 injections of allergen (extracts) over a period of 3 to 5 years and frequently causes local or systemic side effects, this approach suffers from poor patient compliance. Furthermore, the subcutaneous fat tissue is not an immunologically competent target. Novel needle-free delivery methods, which increase efficacy and patient compliance, are under intense investigation. We employed a laser device originally developed for delivery of small molecules such as drugs or hormones to create aqueous micropores with variable depth and density on small areas of mouse skin and subsequently applied grass pollen allergen in solution to these pores. Within two days, healing of the skin areas was completed. As the skin is rich in immunocompetent cells and effectively drained by regional lymph nodes, transcutaneous immunization was found to induce powerful immune responses. When allergen was applied via skin micropores to mice, which had been previously sensitized to allergen, the therapy was found to alleviate allergic symptoms at least equally effective compared to standard subcutaneous injections. Moreover, unwanted side effects induced by the therapy itself, which accompanied the conventional subcutaneous therapy, were diminished in the transcutaneously treated animals. These findings are in line with results from other studies demonstrating efficacy of specific immunotherapy via the skin in mouse models and allergic patients. However, these studies used methods, which are not as reproducible but simultaneously highly versatile as laser microporation, to circumvent the outermost cornified skin layers for enhanced uptake. Data derived from the project establish laser-facilitated transcutaneous immunotherapy as a safe, pain-free and efficacious method for treatment of allergic diseases, which avoids the use of needle injections. This approach could also become relevant for cancer immunotherapy and vaccination against infectious diseases in the future. The immunological mechanisms induced by immunization via laser-generated micropores and the roles of different skin resident immune cell types involved will be closely investigated in a follow-up project.