Topically-delivered Mutation-specific Gene Targeting for Epidermolytic Ichthyosis

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DESCRIPTION (provided by applicant): AuraSense Therapeutics (AST), along with Northwestern University, is applying a recently developed technology using oligonucleotide-functionalized nanoparticles (called spherical nucleic acids or SNAs) to control protein expression. These easily synthesized nanoparticles can have either DNA or RNA shells, and are less than 50 nm in hydrodynamic diameter. SNA constructs are a powerful new way of regulating gene expression. These constructs consist of a dense spherical array of highly oriented oligonucleotides, templated on a nanoparticle, and are remarkable in their ability to readily enter cells, resistance to nuclease degradation, low toxicity and minimal immunostimulation. Critically, SNAs are also capable of penetrating biological barriers such as the stratum corneum and blood brain barrier, increasing the number of organs that can be treated using gene regulation. AST and Northwestern will design SNAs against the KRT10 R156H (hotspot) mutation for the treatment of epidermolytic ichthyosis (EI), a life-altering orphan skin disease that is a prototype for dominant negative keratin gene disorders. These SNAs will be evaluated in vitro and in a reconstituted human epidermal model of the disease. In Phase I we will choose the siRNA SNAs that knock down selectively the mutant KRT10 by recognizing specifically the R156H site and will test the functional impact of topically-applied mutation-specific knockdown in 3-D organotypic raft cultures generated from patient KCs. Aim 1: Optimize the specific knockdown of the hotspot KRT10 R156H/c.467G>A mutation in immortalized KCs isolated from patients with EI using KRT10 SNAs. Initially, candidate SNAs, and controls (scrambled SNAs or vehicle) will be added in 24-well plates to a prototype c.467G>A EI immortalized cell line or control normal immortalized KCs, and knockdown of the mutant and wild type genes will be screened by allele-specific RT- qPCR at 72 h. The goal is to observe greater 70% knockdown and less than 20% knockdown of the mutant and wild-type K10 gene, respectively, as measured with RT-PCR. Aim 2: Suppress the K10 hot-spot mutation in reconstituted human epidermal equivalents. The two SNAs that perform best from Aim 1 will be tested in cultured reconstituted human epidermal equivalents (3-D raft cultures) from normal and EI patient keratinocytes. Our goal is to observe greater 70% knockdown of the mutant gene and less than 20% knockdown of the wild-type K10 gene as measured with RT-PCR, and to reverse the defect clinically and histologically in the reconstituted 3-D human epidermal model. Relationship to Phase II. In a Phase II research and development effort, we will validate this approach in humanized grafted mice for their ability to control EI. We will also demonstrate SNA safety in Phase II, allowing us to enter into a commercial partnership upon completion of Phase II of the program and have sufficient data for filing an IND.