ABSTRACT The importance of communications between nerve cells and immune cells in skin has been increasingly recognized, as are the contributions of sensory neurons and the neuropeptides they produce to skin inflammation and disease. Topical therapeutics that target neuro-immune interactions are needed to curtail aberrant skin inflammation in diseases such as psoriasis and atopic dermatitis and to alleviate the disease-associated pathology, pain, and itch. Our ongoing research aims to develop effective topical options to complement systemic immune-targeting biologics, providing targeted relief and improving patient outcomes. The goals of this application are to develop recombinant, cell-penetrating botulinum proteases that inhibit the release of neuropeptides and cytokines in neurons and immune cells, respectively, to examine the in vivo penetration of topically applied proteases into skin with mild barrier perturbation, and to test the utility of the engineered proteases in reducing skin inflammation. Botulinum neurotoxins (BoNTs), such as Botox®, have been widely used in clinics as medicines and cosmetics. BoNT specifically targets motor neurons and deliver its light chain (LC) protease to the cytosol, where it proteolytically cleaves SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors) proteins and blocks neurotransmitter release. Prior studies also suggest that BoNTs are able to suppress neuropeptide secretion, such as substance P (SP) and calcitonin gene-related peptide (CGRP), in the skin via disrupting SNARE-mediated vesicle fusion and lead to improvement of inflammation in psoriasis. However, the therapeutic potential of conventional BoNTs in psoriasis is inherently restricted because BoNTs preferentially target motor neurons over sensory neurons and they are ineffective on immune cells. To overcome these limitations, we have engineered recombinant cell-penetrating LC proteases of BoNT that can autonomously enter cells and selectively cleave SNAREs in sensory neurons and/or immune cells to regulate neuropeptide secretion and immunological effects. Here, we propose to further develop and characterize cell-penetrating LC proteases and examine how they inhibit vesicle fusion and secretion in cultured neurons and immune cells (Aim 1). We will also characterize the activity of the engineered LC variants in mouse skin with topical delivery and perform preclinical studies to examine how topically applied LCs affect skin inflammatory response using mouse models of psoriasis (Aim 2). Completion of this work will result in a new and widely-applicable strategy to regulate inflammation in skin that can be further developed as topical therapeutic candidates for the treatment of many skin diseases.