The objective of this proposal is to develop an effective and safe epicutaneous immunotherapy (EPIT) for peanut allergy. Among various allergen-specific immunotherapies, EPIT is the safest but least effective therapy. It is extremely challenging to deliver allergens into the epidermis via intact skin, despite that the non-vascularized tissue imposes little anaphylactic risk. To enhance epidermic delivery of allergens without provoking any significant skin irritation, we used ablative fractional laser to generate an array of self- renewable microchannels in the epidermis, onto which an antigen powder-based microarray patch was applied resulting in ~80% delivery efficiency in ~1 hr via both human and pig skins, concurrent with little skin irritation. We also identified that a combination of CpG and 1,25-dihydroxyvitamin D3 (VD3) could robustly augment TGF-? and IL-10 production at the site of allergen delivery and vigorously enhance T regulatory (Treg) cell responses systemically. Only 3 doses of EPIT over two weeks sufficiently inhibited allergen- specific IgE responses in mice, which was unattainable by 8 times of conventional subcutaneous immunotherapy (SCIT) over three weeks. To further tailor this technology for clinical uses, we propose to deliver powdered allergens directly by Powder-Laden, Dissolvable MicroNeedle Arrays (PLD-MNAs), which no longer needs laser to perforate the skin. The MNA will be made of highly biocompatible and dissolvable hyaluronic acid (HA) with a cave in the basal portion of each MN. The cave will be about 25~30 m in depth, similar to the depth of mouse epidermis and can be filled with allergen powder directly. The MNA will be dissolved in 15~20 min after skin insertion, exposing the powder in the epidermis that continuously and actively attracts antigen presenting cells (APCs). We will fabricate PLD-MNAs by loading a mixture of CpG, VD3, and peanut allergens (PNA) into caved MNAs. The resultant PLD-MNAs will be applied into the skin of sensitized C3H/HeJ mice, followed by evaluation of delivery efficiency, skin irritation, innate immunity, any PNA leakage into blood as well as humoral and Th1/Treg cell responses against PNA. Skin irritation and any trace amount of PNA leaking into blood will be also assessed in sensitized newborn pigs to confirm safety of the therapy. Aim 2 will seek in a murine peanut allergy model whether 3 doses of the PLD-MNAs are superior to conventional 8 doses of conventional SCIT in treatment of peanut allergy. The PLD-MNA, if successful, would confer safer and faster treatments of peanut allergy and can be readily extended to other IgE-mediated allergies.