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SANTA CRUZ, CA, United States

Kanada M.,Stanford University | Bachmann M.H.,Stanford University | Hardy J.W.,Stanford University | Frimannson D.O.,Stanford University | And 9 more authors.
Proceedings of the National Academy of Sciences of the United States of America

Extracellular vesicles (EVs), specifically exosomes and microvesicles (MVs), are presumed to play key roles in cell-cell communication via transfer of biomolecules between cells. The biogenesis of these two types of EVs differs as they originate from either the endosomal (exosomes) or plasma (MVs) membranes. To elucidate the primary means through which EVs mediate intercellular communication, we characterized their ability to encapsulate and deliver different types of macromolecules from transiently transfected cells. Both EV types encapsulated reporter proteins and mRNA but only MVs transferred the reporter function to recipient cells. De novo reporter protein expression in recipient cells resulted only from plasmid DNA (pDNA) after delivery via MVs. Reporter mRNA was delivered to recipient cells by both EV types, but was rapidly degraded without being translated. MVs also mediated delivery of functional pDNA encoding Cre recombinase in vivo to tissues in transgenic Crelox reporter mice. Within the parameters of this study, MVs delivered functional pDNA, but not RNA, whereas exosomes from the same source did not deliver functional nucleic acids. These results have significant implications for understanding the role of EVs in cellular communication and for development of EVs as delivery tools. Moreover, studies using EVs from transiently transfected cells may be confounded by a predominance of pDNA transfer. © 2015, National Academy of Sciences. All rights reserved. Source

Transderm, Inc. | Date: 2013-02-04

The present invention provides for transdermal delivery devices having microneedle arrays, as well as methods for their manufacture and use. In one embodiment, a transdermal delivery device is provided. The transdermal delivery device includes a polymer layer which has microneedles projecting from one of its surfaces. The microneedles are compositionally homogenous with the polymer base layer.

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 340.86K | Year: 2010

DESCRIPTION (provided by applicant): Although RNA interference offers high potential as a novel therapeutic approach for treating skin disorders, delivery concerns have hampered their progression to the clinic. The most efficient and effective way to deliver functionally-active siRNA is through intradermal injection. We have shown that the high pressure generated by intradermal injection of large volumes is sufficient to deliver nucleic acid to skin (similar to what is observed in high pressure tail vein injection that delivers siRNA to the liver). Indeed, the intradermal injection of large volumes of our lead clinical siRNA inhibitor TD101 to foot lesions led to marked improvement in pachyonychia congenita symptoms in a recently completed Phase 1b clinical trial. The intense pain associated with this mode of administration (oral pain medication and regional nerve blocks were required to mitigate the pain) appears to prevent future use of this method. The use of intradermal injection bypasses the stratum corneum barrier and also provides a hydrodynamic pressure that appears to facilitate siRNA uptake by keratinocytes. We, and others, have developed patient-friendly technologies that allow siRNA to be delivered across the stratum corneum barrier, including a topical formulation (GeneCream) as well as hollow dissolvable microneedle arrays that penetrate into the epidermis and release their siRNA cargo. Unfortunately, without the pressure associated with injection of high volumes, little functional siRNA enters the cell. Our long-term goal is to identify patient-friendly technologies that will allow effective and efficient siRNA delivery into the appropriate skin compartment and also facilitate uptake by keratinocytes and incorporation into the RNA induced silencing complex (RISC). In this Phase I proposal, we develop a human epidermal equivalent system (i.e. regenerated human skin), in which pre-existing gene expression (CD44, mutant keratin 6a and/or reporter) can be monitored. This model system will be used to evaluate and optimize Traversa's PTD-DRBD siRNA delivery technology (facilitates siRNA uptake by cells) as well as other commercially-available delivery uptake technologies including Invivofectamine, Accell, etc. The most promising of these will be used in combination with our stratum corneum delivery technologies (GeneCream, microneedle arrays and/or iontophoresis). Skin equivalents will be prepared from transduced (with EGFP reporter) or untreated normal primary human keratinocytes as well as keratinocytes derived from pachyonychia congenita patient biopsies. In Phase 2, the best technologies identified in Phase 1 will be tested in mouse models including immunocompromised mice harboring human skin grafts. The most effective combination(s) of these technologies will then be subjected to toxicology and pharmacokinetics studies in preparation for evaluation in the clinic. PUBLIC HEALTH RELEVANCE: Despite the exciting discoveries of the underlying genes and mutations responsible for a large number of skin disorders, few if any novel clinical treatments have emerged. The purpose of this proposal is to exploit RNA interference to selectively inhibit expression of disease-relevant genes. In Phase 1, we develop human skin equivalents (organotypic skin model) in which gene expression can be readily monitored for successful delivery of functional siRNA and test siRNA technologies that facilitate siRNA uptake by skin keratinocytes in combination with existing technologies that allow delivery across the stratum corneum skin barrier. In Phase 2, we will test the technology developed in Phase 1 in animal models. The best combination(s) of technologies will be further evaluated under GLP conditions in animal toxicology and pharmacokinetic assays in preparation for human trials.

Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 224.66K | Year: 2016

ABSTRACT The rare genetic disorder pachyonychia congenita PC results from dominant mutations in the inducible keratins K including K a K b K and K While disabling painful plantar keratoderma is commonly accepted as the primary symptom affecting patient quality of life hyperhidrosis appears to contribute to blister formation and pain Supporting the involvement of hyperhidrosis to PC pain several teams worldwide have shown that multiple intradermal injections of botulinum toxin BTX known to effectively treat hyperhidrosis substantially reduce pain and blistering in PC patients similar results were observed with the related genodermatosis epidermolysis bullosa simplex Unfortunately the number of dermal injections associated with the current treatment protocol makes this procedure costly and difficult on the patient regional nerve blocks or general anesthesia are used We propose to use TransDermandapos s proprietary Flex PAD delivery system to administer BTX to the skin in a patient friendly manner with little or no pain and with no requirement for anesthetic In Phase we aim to demonstrate that BTX can be effectively and efficiently loaded on Flex PADs and that the resulting drug product has sufficient stability to be evaluated in mouse models and for future clinical trial use The ability of the Flex PADs to delivery BTX will be evaluated in head to head studies with intradermal injection of BTX and scored for its ability to block pilocarpine induced sweating in mouse paws In Phase we refine and streamline manufacture and loading of the Flex PADs with BTX and perform IND enabling stability and toxicity studies in mice and minipig models in preparation for human studies !NARRATIVE Much progress has been made over the past two decades in identifying the underlying genes and mutations responsible for a large number of genodematoses with over disorders identified from mutations in intermediate filament genes alone Despite these discoveries few clinical treatments have emerged that modulate these molecular targets In the skin disorder pachyonychia congenita PC the causative genes for PC are expressed in sweat glands and the structure of the sweat gland is grossly altered and malformed Preliminary human studies suggest that botulinum toxin is effective in reducing hyperhidrosis and pain in PC Although injected botulinum toxin reduced PC symptoms administration is cumbersome and costly with regional nerve blocks or general anesthesia required This project seeks to exploit the ability of TransDermandapos s Flex PAD delivery platform to deliver large charged proteins such as botulinum toxin in a patient friendly i e little or no pain fashion Patient friendly delivery of botulinum toxin by Flex PADs has potential applicability to benefit not only PC patients but also other patients with hyperhidrosis and associated pain including psoriasis such as epidermolysis bullosa simplex

A method for keratin hyperproliferation disorders such as corns, calluses, or keratosis pilaris (KP) by administering to a subject experiencing the disorder a therapeutically effective amount of an RNA sequence which inhibits expression of a gene encoding for a keratin selected from the group consisting of K6a, K6b, K16, K17, and combinations thereof.

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