Transcutaneous delivery of non-viral interleukin-10 gene therapy for the topical treatment of psoriasis and other inflammatory skin disorders

Abstract

Methods and compositions for treating an inflammatory skin disorder or a symptom thereof in a subject in need, via the transdermal delivery of a nucleic acid expression construct encoding Interleukin- 10 (IL- 10) that is expressed in the affects cells.

Description

[0001] TRANSCUTANEOUS DELIVERY OF NON- VIRAL INTERLEUKIN- 10 GENE THERAPY FOR THE TOPICAL TREATMENT OF PSORIASIS AND OTHER INFLAMMATORY SKIN DISORDERS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 713,195, filed October 29, 2024. The entire specification and figures of the above-referenced application are hereby incorporated, in their entirety by reference.

[0004] SEQUENCE LISTINGS

[0005] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on October 29, 2025, is named 90245.01321-Sequence-Listing. xml and is 18,896 bytes in size.

[0006] TECHNICAL FIELD

[0007] This invention relates to methods of transdermally delivering an expression construct encoding an endogenous interluking- 10 (IL- 10) to treat immune-mediated topical skin disorders, and in particular psoriasis in a subject in need thereof.

[0008] BACKGROUND

[0009] Inflammatory skin diseases such as psoriasis are a common ailment affecting millions worldwide. Based on U.S. 2020 census data, 7.55 million U.S. adults have been diagnosed with psoriasis. Psoriasis is a chronic immune-mediated topical skin disorder characterized by local inflammation, epidermal hyperplasia, and leukocyte infiltration. Skin lesions caused by psoriasis are characterized by T-cell and neutrophil infiltration into the dermis and epidermis with hyperproliferation and aberrant keratinocyte differentiation. The inflammatory process seen in psoriasis skin lesions includes overexpression of proinflammatory cytokines (e.g., TNF-oc, interferon (IFN)-y) thought to be important in the pathogenesis of the disease.

[0010] Mechanisms underlying skin inflammation characteristic of human psoriasis are not well understood, leading to the development and utilization of several rodent models of over the years. Of these, topical imiquimod (IMQ) has perhaps gained the most traction as creating a clinically- relevant model of human psoriatic skin. First described in 2009 by Van Der Fits et al., IMQ is widely accepted as creating a skin condition in rodents that mirrors human psoriasis in many respects. Amongst its effects, IMQ is a strong ligand for epidermal Toll-like receptors 7 / 8 causing activation of macrophages, monocytes, and dendritic cells. These immune changes are induced by topical IMQ application onto shaved rodent skin, inducing significant psoriasis-like inflammatory effects. Like human psoriasis, the IMQ-induced immune response in skin is characterized by the production of interferons (IFN), pro-inflammatory cytokines and chemokines.

[0011] Currently, treatment approaches for human psoriasis aim to alleviate symptoms and prevent disease progression. These approaches encompass topical therapies (corticosteroids, vitamin D), phototherapy (narrowband UV-B, broadband UV-B, and PUVA), oral systemic medications (methotrexate, apremilast, acitretin, and cyclosporine), and inhibitors of tumor necrosis factor-alpha (TNF-a), interleukin- 17 (IL- 17), or IL-23. Despite advances, there are still several persistent challenges that undermine successful psoriasis treatment.

[0012] Among these challenges, adverse effects, treatment resistance, long-term safety concerns, variability in treatment response among individuals, and high costs associated with some therapies limit their widespread use and efficacy. A major challenge, beyond adverse effects, is inadequate response to treatment, leading to high-rates of treatment failure. This highlights the need for better therapeutic strategies for psoriasis.

[0013] To address this challenge, Applicant describe herein the therapeutic potential of transcutaneously delivered plasmid DNA encoding the anti-inflammatory cytokine IL-10 (pDNA- rlLlO). Initially described as a T-helper 2 (32)-derived cytokine, IL-10 has potent antiinflammatory properties that play a central role in preventing inflammation-induced damage to the host and maintaining normal tissue homeostasis.

[0014] SUMMARY OF THE INVENTION

[0015] As noted above, psoriasis is a chronic immune-mediated skin disorder characterized by intense local inflammation, epidermal hyperplasia, and leukocyte infiltration. Current treatment approaches for psoriasis aim to alleviate symptoms and prevent disease progression, including systemically administered drugs with whole body side effects. Despite some advances in psoriasis treatment, success has been quite limited. To begin to address this challenge, Applicants evaluated whether transcutaneous delivery of an endogenous anti-inflammatory cytokine could provide an effective, local treatment of psoriatic-like skin conditions. Applicants utilized a previously documented rodent model of psoriasis, induced via a single topical application of Imiquimod (IMQ) to the shaved back of rats. The therapeutic approach used for this initial investigation was delivery of plasmid DNA encoding rat interleukin- 10 (SEQ ID NO. 5-8) or human IL-10 (SEQ ID NO. 4-8) a non-viral gene therapy approach previously shown to be effective in suppressing neuroinflammatory disorders after localized delivery either intracerebrally or intrathecally. Translation of this CNS therapeutic for use in psoriatic-like skin disorders required reformulation to enable transcutaneous delivery. Toward that end, pDNA-rILlO was topically applied in a pharmaceutically acceptable transdermal carrier, and preferably a phospholipid bases, and more preferably Lipoderm® HMW™ (manufactured by co-applicant Professional Compounding Centers of America (“PCCA;” catalog # 30-4612). As used herein, Lipoderm® HMW™ describes a transdermal delivery system specifically designed to enhance the permeation of high molecular weight active pharmaceutical ingredients (APIs) through the skin. The formulation includes Amazonian and PEGylated oils, which form a stable oil-in-water emulsion. Upon contact with the skin, this emulsion breaks, promoting the solubilization and dispersion of APIs, thereby facilitating their permeation through the stratum corneum. The system's hydrogel polymer matrix provides structural integrity and ensures efficient delivery and absorption of the active ingredients while maintaining the stability of the formulation.

[0016] As described herein, Applicants demonstrate that a single topical application of pDNA- rlLlO in Lipoderm® HMW™ was effective in decreasing mRNA levels of pro-inflammatory cytokines as well as reducing the recruitment of T-cells to IMQ-treated skin. Furthermore, this transcutaneous IL- 10 gene therapy decreased signs of skin inflammation, reflected by reduced erythema. Moreover, the results provide an initial indication that IL 10 may stimulate hair regrowth in psoriatic-like skin.

[0017] Additional aspects of the invention may include one or more of the preferred embodiments set forth in the claims.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1. Illustration of the 2 x 2 cm area over which 40 mg IMQ was bilaterally applied across the rat's back (blue lines). This skin region included the 8-mm diameter skin punch areas at the center of each application side (yellow circles).

[0020] Figure 2A-D. Blinded assessments by 2 independent observers were done of the full 2 x 2 cm IMQ treatment area, at pre-treatment (baseline; BL) just prior to IMQ application, and across the time course beginning 48 h after IMQ; that is, from the time of pDNA-rILlO application 48 hr after IMQ. These timepoints (as shown in the X-axis) are 0, 48, 72, 120, 168 and 240 hr after pDNA-rILlO or vehicle, which temporally align as 48, 96, 120, 168, and 288 hr after IMQ application. Group sizes for IMQ treatment groups begin at n=20 rats at BL, 0 and 48 hr, decreasing by 4 rats at each timepoint as squads were successively euthanized for tissue collection. Panel A: Erythema, Panel B: Plaques; Panel C: Scabbing; Panel D: Hair loss. ** p<0.01, ***** p<0.0005

[0021] Figure 3A-C. Blinded assessments by 2 independent observers were done of the 8 cm skin punch area, so that these observations align with the tissue analyses undertaken with these samples. Scoring of each skin punch was done at baseline (BL; pretreatment) plus at the time of sample collection; that is, at the point of euthanasia. Hence, there are group sizes of n=20 at pretreatment BL and n=4 for each IMQ squad at the timepoint of tissue collection. For pooled controls, there are group sizes of n=8 both at pretreatment BL and when skin punches were collected at the last timepoint shown. Panel A: Erythema, Panel B: Plaques; Panel C: Hair loss. In these small samples, no scabbing was observed for any group, so no graph is included. ** p<0.01, ***p<0.005, ****p<0 0001, ***** p<0.0005

[0022] Figure 4A-B. Slices from tissue punches were analyzed by Immunohistochemistry to detect rat IL- 10 protein, pooling samples across timepoints of collection. Areas analyzed were hair follicles given their importance in protein production in skin and as a target for gene therapy (Panel A and top photomicrograph) and extra-follicular dermal regions (Panel B and bottom photomicrograph). Compared to pooled controls, elevation of IL-10 protein was detected in the 2 groups receiving pDNA-ILlO gene therapy. *p<0.05; ***p<0.005

[0023] Figure 5A-D. The central 8 mm skin punches were analyzed for pro-inflammatory cytokines by Real-time PCR. Timepoints (as shown in the X-axis) are 48, 72, 120, 168 and 240 hr after pDNA-rILlO or vehicle, which temporally align as 96, 120, 168, 216, and 288 hr after IMQ application. Group sizes for pooled controls are 8 (4 naive, 4 mannose) and 4 rats at each timepoint.

[0024] Figure 6A-B. IHC analysis of T-cell markers revealed an increase in CD3 in IMQ treated rats (blue dots) relative to IMQ + pDNA-rILlO (red dots). No change in CD4 was observed across groups.

[0025] Figure 7. pDNA-ILlO Plasmid constructs. (A): exemplary plasmid DNA encoding rat rat IL10 gene (pDNA-rILlO). (B) exemplary plasmid DNA encoding human IL-10 (pDNA-hlLlO. (C) exemplary plasmid DNA encoding human modified Jellyfish green fluorescent protein (pDNA-GFP). (D) plasmid DNA encoding enhanced Jellyfish green fluorescent protein (pDNA- eGFP). Key: Ampr, ampicillin resistance marker; ITR, AAV inverted terminal repeat; CMV enh, cytomegalovirus immediate early promoter enhancer; CMV pro, cytomegalovirus immediate early promoter; CB pro, chicken [3-acti n promoter; TVS, intervening sequence (intron); rl L-10, rat interleukin- 10 gene; hll-10, human interlukin- 10 gene; GFP, green fluorescent protein gene; eGFP, enhanced green fluorescent protein gene; pA, polyadenylation signal; TK pro, herpes simplex thymidine kinase promoter; neor, neomycin resistance marker.

[0026] DETAILED DESCRIPTION OF THE INVENTION

[0027] The present disclosure provides methods and compositions for treating diseases and conditions associated with inflammation by administering to a subject a vector including an expression construct expressing an interleukin- 10 (IL- 10) coding sequence. In preferred embodiments, the IL- 10 peptides are expressed from a single expression vector; however, in alternative embodiments, the IL- 10 coding sequences are expressed from different expression constructs. In some embodiments, the IL-10 / IL-10R1 expression vector(s) is administered transdermal to a subject in need.

[0028] In one embodiment, the present disclosure provides methods for treating inflammatory diseases of the skin, such as psoriasis, symptoms associated with inflammatory diseases of the skin, and slowing disease progression by transdermally administering to a subject a vector expressing a therapeutic interleukin- 10 (IL- 10) expression construct. In some embodiments, the IL- 10 expression construct is administered with a pharmaceutically acceptable transdermal carrier to form a pharmaceutical composition.

[0029] The IL-10 expression vector used in some embodiments of the present disclosure preferably include a bacterial backbone (plasmid DNA); an IL-10 coding sequence; and one or more DNA control sequences. In particular, the IL- 10 expression vectors of the present disclosure comprise at least one promoter driving transcription of the IL-10 coding sequences. In preferred embodiments, this promoter is a constitutive promoter. The term “constitutive” when made in reference to a promoter means that the promoter directs transcription of an operably linked nucleic acid sequence in the absence of a specific stimulus. Typically, constitutive promoters are capable of directing expression of a coding sequence in substantially any cell and any tissue. The promoters used to transcribe the IL- 10 peptides preferably are constitutive promoters, such as the promoters for ubiquitin, CMV, (P-actin, histone H4, EF-la or PGK genes controlled by RNA polymerase II, or promoter elements controlled by RNA polymerase I. In preferred embodiments, promoter elements controlled by RNA polymerase III are used, such as the U6 promoters (U6-1, U6-8, U6- 9), Hl promoter, 7SL promoter, the human Y promoters (hYl, hY3, hY4 and hY5), the human MRP-7-2 promoter, Adenovirus VAI promoter, human tRNA promoters, the 5s ribosomal RNA promoters, as well as functional hybrids and combinations of any of these promoters.

[0030] In alternative embodiments, the IL- 10 coding sequence may be under the control of an inducible promoter, such as tetracycline-controlled transcriptional activation where transcription is reversibly turned on (Tet-On) or off (Tet-Off) in the presence of the antibiotic tetracycline or a derivative thereof, such as doxycycline. In a Tet-Off system, expression of tetracycline response element-controlled genes can be repressed by tetracycline and its derivatives. Tetracycline binds the tetracycline transactivator protein, rendering it incapable of binding to the tetracycline response element sequences, preventing transactivation of tetracycline response element-controlled genes. In a Tet-On system on the other hand, the tetracycline transactivator protein is capable of initiating expression only if bound by tetracycline; thus, introduction of tetracycline or doxycycline initiates the transcription of IL- 10 peptide. Another inducible promoter system known in the art is the estrogen receptor conditional gene expression system. Compared to the Tet system, the estrogen receptor system is not as tightly controlled; however, because the Tet system depends on transcription and subsequent translation of a target gene, the Tet system is not as fast-acting as the estrogen receptor system.

[0031] The IL- 10 expression vector comprises at least one IL- 10 coding sequence. IL- 10 may be used in wild-type form, or the IL-10 may be a mutant IL-10. One mutant IL-10 of particular interest contains one or more mutations that cause amino acid substitutions, additions or deletions as compared to wildtype IL-10 in the “hinge” region of the IL-10 protein. The human IL-10 protein is a homodimer, where each monomer comprises six alpha helices A >F. the length of which are 21, 8, 19, 20, 12 and 23 amino acids, respectively. Helices A^D of one monomer non-covalently interact with helices E and F of a second monomer, forming a non-covalent V-shaped homodimer. The “hinge” region targeted for mutation according to the present invention comprises the amino acids between the D and E alpha helices on one or both monomers of wildtype IL- 10. For example, mutant rat and human IL-10 proteins have been described in which the phenylalanine at position 129 of the wildtype sequence has been replaced with a serine residue. (See, e.g., Sommer, et al., W02006 / 130580 and Milligan, et al., Pain, 126:294-308 (2006).) The resulting mutant IL-10 is referred to as IL-10F129S. Other substitutions for the wildtype phenylalanine at amino acid position 129 may be, e.g., threonine, alanine, or cysteine. Thus the present invention in yet another aspect encompasses one or more substitutions at amino acid position 129 or at other amino acids within the hinge region of the IL- 10 protein.

[0032] The present disclosure describes methods and compositions for treating an inflammatory skin disorder or a symptom thereof in a subject in need via the transdermal delivery of a nucleic acid encoding IL- 10 that is expressed in the affects cells. In a preferred aspect, the compositions and methods of the disclosure treat one more inflammatory skin disorders, such as psoriasis in a mammal, and more preferably in a human.. In another aspect, the compositions and methods of the disclosure treat one more symptoms associated with inflammatory skin disorders, such as psoriasis-induced hair loss in a mammal, and more preferably in a human. In one aspect, the aforementioned methods and compositions of the disclosure result in the localized downregulation of multiple proinflammatory cytokines, as well as suppression of CD3 T-cell recruitment and stimulation of hair regrowth.

[0033] In a preferred aspect, the present disclosure describes a pharmaceutical composition for treating an inflammatory skin disorder or a symptom thereof comprising a therapeutically effective amount of an isolated expression construct formed by a heterologous nucleic acid, operably linked to a promoter, encoding an interlukin-10 (IL-10) peptide, or functional fragment or variant thereof, and a pharmaceutically acceptable transdermal carrier that is applied to skin of a subject in need thereof, and the IL- 10 is expressed in one or more affected cells. In another aspect, the heterologous nucleic acid is operably linked to an endogenous or inducible promoter.

[0034] In a preferred aspect, the present disclosure describes a method of treating an inflammatory skin disorder or a symptom thereof in a subject. In this preferred aspect, the method can include administering to the skin of a subject a therapeutically effective amount of a pharmaceutical composition comprising an expression construct formed by a heterologous nucleic acid, operably linked to a promoter, encoding an interlukin-10 (IL- 10) peptide, or functional fragment or variant thereof, and a pharmaceutically acceptable transdermal carrier. As described below, the transdermal carrier assist in the transports uptake the nucleic acid to the affected cells where they are subject to an endogenous or inducible promoter and thereby expressed producing a therapeutically effective amount of IL- 10 peptide, or functional fragment or variant, in the affected cells thereof. In a preferred aspect, the expression construct of the disclosure includes a plasmid, preferably encoding comprises an endogenous IL- 10 peptide, or a functional fragment or variant thereof. In another preferred embodiment, the IL-10 peptide encoded by the plasmid is selected from SEQ ID NO. 1 -8, or a sequence having at least 80% or more sequence identity with SEQ ID NO. 1-8. In still further embodiments, the plasmid includes a pDNA-ILlO plasmid construct as incorporated herein by reference, , or a sequence having at least 80% or more sequence identity with pDNA-ILlO as incorporated herein by reference. .

[0035] In one preferred aspect, the pharmaceutically acceptable transdermal carrier of the disclosure includes a high-molecular weight transdermal carrier, and preferably Lipoderm® HMW™. In one preferred aspect, the composition and methods of the disclosure includes a nucleic acid adsorption enhancing excipient, which in a preferred embodiment include a sugar, such as D-mannose. In one embodiment, a high molecular weight molecule can refer to a molecule that at least 5500 Daltons or more. In other embodiment, a high molecular weight molecule can refer to a molecule that is at least 800 Daltons or more. In other embodiment, a high molecular weight molecule can refer to a molecule that is at least 500 Daltons or more.

[0036] Dosage ranges of the therapeutic anti-inflammatory compositions of the present disclosure vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated and the particular IL-lOexpression vector to be delivered, and the like. For example, dosage ranges include a therapeutically effective dose delivered transdermally to the subject at less than lOpg vector DNA per kg at 10-1000 pg vector DNA per kg, 20-500 pg vector DNA per kg, 25-250 pg vector DNA per kg, or 50-100 pg vector DNA per kg, or more than 1000 pg vector DNA per kg. Appropriate doses and timing for the same can be determined on one of ordinary skill in the art based on an evaluation of the subject severity of the condition or symptoms and subject reaction to treatment of the same, among other factors.

[0037] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0038] The term “IL- 10” or “Interleukin- 10” refers to a cytokine encoded by the IL- 10 gene. IL- 10 is a cytokine with pleiotropic effects in immunoregulation and inflammation. It is produced by mast cells, counteracting the inflammatory effect that these cells have at the site of an allergic reaction. While it is capable of inhibiting the synthesis of pro-inflammatory cytokines such as IFN- y, IL-2, IL-3, TNFa and GM-CSF, IL-10 is also stimulatory towards certain T cells and mast cells and stimulates B-cell maturation, proliferation and antibody production. IL- 10 can block NF-KB activity and is involved in the regulation of the JAK-STAT signaling pathway. It also induces the cytotoxic activity of CD8+ T-cells and the antibody production of B-cells, and it suppresses macrophage activity and tumor-promoting inflammation. The regulation of CD8+ T-cells is dosedependent, wherein higher doses induce stronger cytotoxic responses. Human IL-10 is a homodimer with a molecular mass of 37 kDa, wherein each 18.5 kDa monomer comprises 178 amino acids, the first 18 of which comprise a signal peptide, and two cysteine residues that form two intramolecular disulfide bonds. The IL-10 dimer becomes biologically inactive upon disruption of the non-covalent interactions between the two monomer subunits.

[0039] As used herein, the terms “IL- 10”, “IL- 10 polypeptide(s), “IL- 10 molecule(s)”, “IL- 10 agent(s)” and the like are intended to be broadly construed and include, for example, human and non-human IL-10-related polypeptides, including homologs, variants (including muteins), and fragments thereof, as well as IL- 10 polypeptides having, for example, a leader sequence (e.g., the signal peptide), and modified versions of the foregoing. In further particular embodiments, IL-10, IL-10 polypeptide(s), and IL-10 agent(s) are agonists. The present disclosure contemplates human IL-10 (SEQ ID NO: 1-4) and rat IL-10 (SEQ ID NOS: 5-8), which exhibit 80% homology, and use thereof. In addition, the scope of the present disclosure includes IL- 10 orthologs, and modified forms thereof, from other mammalian species, can include exemplary IL-10 sequences listed in paragraphs 0114-0136 of PCT / US2022 / 081736, the sequences and disclosure being incorporated here by reference.

[0040] IL- 10 further encompasses both the full-length molecule, as well modified and functional fragments of IL- 10 having deletions, additions and substitutions (either conservative or nonconservative in nature), to the native sequence, so long as the IL-10 peptide is therapeutically effective. Modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification. Accordingly, active proteins are typically between about 70 to 99% or more homologous to the parent sequence. The terms “functional fragment” means any portion of a polypeptide or nucleic acid sequence from which the respective full-length polypeptide or nucleic acid relates that is of a sufficient length and has a sufficient structure to confer a biological affect that is at least similar or substantially similar to the full-length polypeptide or nucleic acid upon which the fragment is based. In some embodiments, a functional fragment is a portion of a full-length or wild-type nucleic acid sequence that encodes any one of the nucleic acid sequences disclosed herein, and said portion encodes a polypeptide of a certain length and / or structure that is less than full-length but encodes a domain that still biologically functional as compared to the full-length or wild-type protein. In some embodiments, the functional fragment may have a reduced biological activity, about equivalent biological activity, or an enhanced biological activity as compared to the wildtype or full-length polypeptide sequence upon which the fragment is based. In some embodiments, the functional fragment is derived from the sequence of an organism, such as a human. In such embodiments, the functional fragment may retain 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity to the wild-type human sequence upon which the sequence is derived. In some embodiments, the functional fragment may retain 85%, 80%, 75%, 70%, 65%, or 60% sequence homology to the wild-type sequence upon which the sequence is derived.

[0041] As used herein “IL 10F129S” is described in U.S. Patent No. 7749490, and describes a mutant form of IL-10 wherein the residue present at a position corresponding to amino acid position 129 of the disclosed SEQ ID NOS: 2 and 3 of that disclosure is replaced with another amino acid. In preferred embodiments, the amino acid phenylalanine normally present at amino acid position 129 of rat and human IL-10 is replaced with the amino acid serine. This mutation is termed “F129S,” and can be expressed in a plasmid termed pDNA-IL10F129S, as described in U.S. Patent No. 7749490.

[0042] The term “pDNA-ILlO” refers to a circular vector or plasmid as provided herein which encodes human and rat IL- 10, or IL-10ri29Saccording to SEQ ID NOS. 1-8, or fragment of variants of the same. In one embodiment, “pDNA-rILlO” refers to a circular vector or plasmid as provided herein which encodes rat IL-10, or IL-10F129S. In one embodiment, the pDNA-ILlO includes both a rat (pDNA-ILlO) and human (pDNA-hILlO) vector as described in current Figure 7 by Watkins et al., Repeated intrathecal injections of plasmid DNA encoding interleukin- 10 produce prolonged reversal of neuropathic pain. Pain 126(l):p 294-308, December 2006, (the vector constructs and their corresponding sequences being incorporated herein reference). As described by Wtkins, teh plasmid vector pDNA-TLIO includes and expression cassete for adeno-associated virus-2 (AAV- 2) vector encoding rat IL-10 (pDNA-rILlO). The plasmid's transcriptional cassette is flanked by two AAV-2 viral elements, inverted terminal repeat sequences (ITRs), and consists of the cytomegalovirus enhancer / chicken b-actin promoter (CB-actin), an intronic region, the rat interleukin- 10 (IL-10) gene with a point mutation (F129S) and the SV40 polyadenylation signal region (Fig. 7A). The expression cassete encoding human IL-10 (pDNA-hILlO) (Fig. 7B) is similar to that for the rat expression cassette, with the exception that the ITR sequences were modified by deletion of 17bp and the CMV promoter directed the expression of human IL-10. The control plasmids are analogous plasmid cassettes in which the CMV enhancer / CB -actin promoter directs the expression of either the unmodified or the enhanced reporter gene, Jellyfish green fluorescent protein (GFP), and are noted as pDNA-GFP (Fig. 7C) and pDNA-eGFP (Fig. 7D), respectively. In another embodiment, pDNA-ILlO further includes plasmid in Figure 7 of PCT / US2022 / 081736 as XT-150, which is further incorporated herein by reference.

[0043] By “isolated” when referring to a nucleotide sequence, is meant that the expression construct is present in the substantial absence of other biological macromolecules of the same type. Thus, an “isolated nucleic acid molecule which encodes a particular polypeptide” refers to a nucleic acid molecule which is substantially free of other nucleic acid molecules that do not encode the subject polypeptide; however, the molecule may include some additional bases or moieties that do not deleteriously affect the basic characteristics of the composition.

[0044] As used herein, the terms “variants” and “homologs” are used interchangeably to refer to amino acid or DNA sequences that are similar to reference amino acid or nucleic acid sequences, respectively. The term encompasses naturally-occurring variants and non-naturally-occurring variants. Naturally-occurring variants include homologs (polypeptides and nucleic acids that differ in amino acid or nucleotide sequence, respectively, from one species to another), and allelic variants (polypeptides and nucleic acids that differ in amino acid or nucleotide sequence, respectively, from one individual to another within a species). Thus, variants and homologs encompass naturally occurring DNA sequences and proteins encoded thereby and their isoforms, as well as splice variants of a protein or gene. The terms also encompass nucleic acid sequences that vary in one or more bases from a naturally-occurring DNA sequence but still translate into an amino acid sequence that corresponds to the naturally-occurring protein due to degeneracy of the genetic code. Non-naturally-occurring variants and homologs include polypeptides and nucleic acids that comprise a change in amino acid or nucleotide sequence, respectively, where the change in sequence is artificially introduced (e.g., muteins); for example, the change is generated in the laboratory by human intervention. Therefore, non-naturally occurring variants and homologs may also refer to those that differ from the naturally-occurring sequences by one or more conservative substitutions and / or tags and / or conjugates.

[0045] As used herein, the terms “an inflammatory skin disorder or a symptom associated therewith”” mean any disease or medical condition associated with the skin, nails, or mucosal membranes displaying symptoms of redness, flushing, burning, scaling, acne, telangiectasis, sores, hair loss, surface irritation or pain, itching, and / or inflammation. Inflammatory dermatologic disorders include, but are not limited to, dermatitis, such as contact dermatitis, atopic dermatitis, seborrheic dermatitis, nummular dermatitis, generalized exfoliative dermatitis, statis dermatitis, lichen simplex chronicus; disorders of hair follicles and sebaceous glands, such as acne, rosacea and rhinophyma, perioral dermatitis, and pseudofolliculitis barbae; and inflammatory reactions, such as drug eruptions, erythema multiforme, erythema nodosum, and granuloma annulare.

[0046] As used herein, “psoriasis or a symptom associated therewith” is intended to encompass any type or classification of psoriasis and any symptom associated therewith. For example, the term “psoriasis or a symptom associated therewith” includes plaque psoriasis, pustular psoriasis, guttate psoriasis (small, drop like spots) and flexural psoriasis, nail psoriasis, psoriatic arthritis and erythrodermic psoriasis, and their associated symptoms, which can include hair loss in the affected area. The term also includes nonpustular psoriasis, such as psoriasis vulgaris (chronic stationary psoriasis, plaque-like psoriasis), psoriatic erythroderma (erythrodermic psoriasis); and pustular psoriasis, such as generalized pustular psoriasis (pustular psoriasis of von Zumbusch) (liquid-filled yellowish small blisters), pustulosis palmaris et plantaris (palmoplanar pustulosis (primarily affecting the palms and the soles), pustular psoriasis of the Barber type, pustular psoriasis of the extremities), annular pustular psoriasis, acrodermatitis continua and impetigo herpetiformis, and their associated symptoms. The term also encompasses drug- induced psoriasis, inverse psoriasis (in the folds like of the underarms, navel, and buttocks), napkin psoriasis, and seborrheic-like psoriasis, and their associated symptoms. In view of the present disclosure, a skin area affected by psoriasis or is prone to be affected by psoriasis can be identified using any diagnostic signs or means known in the art, and can be treated by methods according to embodiments of the present invention. As sued herein, an “affected cell” describes a cell or group of cells of a subject that is directly or indirectly affected by a an inflammatory skin disorder, a cell that exhibits or contributes to one or more symptoms or indications of an inflammatory skin disorder, or a cell that can be treated by the methods and compositions described herein to ameliorate one or more symptoms of an inflammatory skin disorder.

[0047] As used herein, the term “subject” refers to any mammal, and preferably a human at any age who suffers from an inflammatory skin disorder, and preferably psoriasis at any stage and / or degree.

[0048] The term “pharmaceutical composition”” as used herein refers to a composition having an active ingredient, such as an expression construct encoding an IL- 10 protein that has the ability treat a skin inflammation disorder, such as psoriasis, provide relief from symptoms caused by a skin inflammation disorder and / or prevent progression of skin and tissue damage due to a skin inflammation disorder as measured in any of the known animal models or by assessment performed in humans.

[0049] The terms “effective amount” or “therapeutically effective amount” of a therapeutic IL- 10 expression construct used in the methods of the invention refer to a nontoxic but sufficient amount of the IL- 10 expression construct to provide the desired response, such as a decrease in inflammation or symptoms caused by and inflammatory skin disorder and / or preventing progression of skin damage due to an inflammatory skin disorder. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, and the particular IL- 10 expression construct to be delivered. Dosage parameters for the present methods are provided herein; however, optimization of an appropriate “effective” amount in any individual case may be determined by one of ordinary skill in the art using the methods set forth herein and routine experimentation.

[0050] The term “nucleic acid adsorption enhancing excipient” of another agent, as used herein, refers to a pharmacological or immunological agent that modifies the effect of other agents. In the context of the present application, an excipient that increases the uptake of a vector is used to increase the efficacy of the therapeutic pDNA-IL-10 expression construct used in an appropriate dosing regimen. In some embodiments, such excipients include those that enhance the uptake or efficacy of the pDNA-IL- 10 expression construct. In some embodiments, such an excipient is selected from the group consisting of a sugar, calcium phosphate, a dendrimer, an oligonucleotide, a high molecular weight hyaluronic acid, and a lipid. In some embodiments, the excipient is D- mannose, sucrose, or glucose. In certain embodiments, the excipient is D-mannose.

[0051] The term “administer,” “administering,” or “administration” refers to introducing a composition described herein into a subject. In certain embodiments, “administering” a composition described herein refers to transdermally applying the composition into the skin, and preferably an area of the skin affected by an inflammatory skin disorder, such as psoriasis.

[0052] The compositions of the disclosure can have an anti-inflammatory effect, and further be used to treat inflammation or a symptom thereof in a subject in need. The term “anti-inflammatory” as used herein, refers to decreasing the action or production of one or more proinfl ammatory cytokines, which can include signaling molecules that are secreted from immune cells and promote inflammation, or proteins produced by other cell types.

[0053] The expression constructs of the invention can include one or more DNA control sequences. The term DNA “control sequences” refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites, enhancers, and the like, which collectively provide for the replication, transcription, and translation of a coding sequence in a recipient cell. The vectors provided herein contain one or more control sequence. Notably, not all types of control sequences need to be present, as long as the selected coding sequence is capable of being replicated, transcribed, and translated in an appropriate host cell.

[0054] The term “promoter” is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene that is capable of binding RNA polymerase and initiating transcription of a downstream (3'- direction) coding sequence. Transcription promoters can include “inducible promoters” (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), “repressible promoters” (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and “constitutive promoters.” In some embodiments, promoters may be chicken or human P-actin promoters, CAG hybrid promoter, cytomegalovirus immediate early promoters, glyceraldehyde 3 -phosphate dehydrogenase (GAPDH) promoters, elongation factor la (eFla) promoters, GFAP promoters, murine leukemia virus (MLV) promoters, herpes simplex virus thymidine kinase (TK) promoters, and woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) promoters. In certain embodiments, a promoter is a cytomegalovirus immediate early promoter. In some embodiments, the promoter (e.g., constitutive promoter) is selected from the group consisting of ubiquitin promoters, CMV promoters, P-actin promoters, cytomegalovirus enhancer / chicken beta-actin promoter, CAG hybrid promoters, histone H4 promoters, EF-la promoters, PGK gene promoters, promoter elements controlled by RNA polymerase II, promoter elements controlled by RNA polymerase I, promoter elements controlled by RNA polymerase III, U6 promoters (e.g., U6- 1 promoters, U6- 8 promoters, U6-9 promoters), Hl promoter, 7SL promoter, human Y promoters (e.g., hYl promoters, hY3 promoters, hY4 promoters, and hY5 promoters), human MRP-7-2 promoter, adenovirus VAI promoter, human tRNA promoters, and 5s ribosomal RNA promoters. In some embodiments the promotor is a chicken or human P-actin promoter, CAG hybrid promoter, cytomegalovirus immediate early promoter, or elongation factor la (eFla) promoter. In some embodiments the promotor is a chicken P-actin promoter. In some embodiments the promotor is a CAG hybrid promoter. In some embodiments the promotor is a CAG hybrid promoter. In some embodiments the promotor is a eFla promoter.

[0055] The terms “polynucleotide”, “nucleotide sequence”, “nucleic acid”, “nucleic acid molecule”, “nucleic acid sequence”, and “oligonucleotide” refer to a series of nucleotide bases (also called “nucleotides”) in DNA and RNA. The polynucleotides can be chimeric mixtures or derivatives or modified versions thereof, single stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, its hybridization parameters, etc. A nucleotide sequence typically carries genetic information, including the information used by cellular machinery to make proteins and enzymes. In some embodiments, a nucleotide sequence encodes IL- 10, or a fragment or variant thereof, as described herein. A nucleotide sequence (may be placed under the control of a promoter in an expression construct, such as a vector. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in cells. Expression of the sequence encoding IL- 10 can be facilitated by any promoter known in the art to act in mammalian, preferably human, cells. Such promoters can be inducible or constitutive. Any type of plasmid, cosmid, yeast artificial chromosome, or viral vector can be used to prepare the recombinant expression construct that can be introduced directly into the tissue site.

[0056] The polynucleotides of the disclosure may be flanked by natural regulatory (expression control) sequences or may be associated with heterologous sequences, including promoters, internal ribosome entry sites (TRES), and other ribosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5'- and 3 non-coding regions, and the like. In some embodiments, the polynucleotides are flanked by inverted terminal repeat (ITR) sequences. In some embodiments, an ITR sequence is an adeno- associated virus (AAV) ITR sequence.

[0057] As sued herein, “operably linked” refers to an arrangement of elements where the components so described are configured so as to perform their usual function. Thus, control sequences operably linked to a coding sequence are capable of effecting the expression of the coding sequence. The control sequences need not be contiguous with the coding sequence so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence, and the promoter.

[0058] The term “pharmaceutically acceptable carrier” refers to a substance added to a therapeutic composition to facilitate administration of the therapeutic IL-10 expression constructs, such as pDNA-ILlO. In certain embodiments, the excipient or diluent is inert. Examples, without limitation, of excipients include saline, calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, hyaluronic acid (optionally formulated with a surfactant), Plumoic F-68, vegetable oils, and polyethylene glycols.

[0059] As further used herein, a pharmaceutically acceptable transdermal carrier can include both pharmaceutically acceptable topical and / or transdermal carrier. Notably, The present invention is not limited to any specific type of pharmaceutically acceptable carrier, but is useful in connection with any pharmaceutically acceptable topical or transdermal carrier. Thus, the following is provided as general guidelines only, and is not intended to limit the scope of the invention in any way.

[0060] In some embodiments, the composition comprises a pharmaceutically acceptable topical carrier. Exemplary topical carriers include liquids, creams, lotions, salves, balms, pastes, gels and ointments.

[0061] Ointments are typically semisolid preparations that are often based on petrolatum or other petroleum derivatives. As with other carriers or vehicles, an ointment base may be inert, stable, nonirritating and nonsensitizing. As explained in Remington: The Science and Practice of Pharmacy, 19th Ed. (Easton, Pa.: Mack Publishing Co., 1995), at pages 1399- 1404, ointment bases may be grouped in four classes: oleaginous bases; emulsifiable bases; emulsion bases; and water- soluble bases.

[0062] Creams also are well known in the art, and include viscous liquids or semisolid emulsions, either oil-in-water or water-in-oil. Cream bases typically are water- washable, and usually contain an oil phase, an emulsifier and an aqueous phase. The oil phase, also called the "internal" phase, is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol. The aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant. The emulsifier in a cream formulation is generally a nonionic, anionic, cationic or amphoteric surfactant.

[0063] Gels are typically semisolid, suspension-type systems. Single-phase gels may contain organic macromolecules distributed substantially uniformly throughout the carrier liquid, which is typically aqueous, but also may contain an alcohol and / or an oil. Exemplary gelling agents include crosslinked acrylic acid polymers such as the "carbomer" family of polymers, e.g., carboxypolyalkylenes that may be obtained commercially under the Carbopol® trademark. Also known are hydrophilic polymers such as polyethylene oxides, polyoxyethylene- polyoxypropylene copolymers and polyvinylalcohol; cellulosic polymers such as hydroxypropyl cellulose, hydroxy ethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and methyl cellulose; gums such as tragacanth and xanthan gum; sodium alginate; and gelatin. In order to prepare a uniform gel, dispersing agents such as alcohol or glycerin can be added, or the gelling agent can be dispersed by trituration, mechanical mixing or stirring, or combinations thereof.

[0064] Lotions are typically preparations to be applied to the skin surface without friction, and are often liquid or semiliquid preparations in which solid particles, including the active agent, are present in a water or alcohol base. Lotions are usually suspensions of solids, and may comprise a liquid oily emulsion of the oil-in-water type. The insoluble matter in a lotion may be finely divided. Lotions may contain suspending agents to produce better dispersions, as well as compounds useful for localizing and holding the active agent in contact with the skin, e.g., methylcellulose, sodium carboxymethyl-cellulose, or the like.

[0065] Pastes are typically semisolid dosage forms in which the active agent is suspended in a suitable base. Depending on the nature of the base, pastes may be fatty pastes or may be made from single-phase aqueous gels. The base in a fatty paste is generally petrolatum or hydrophilic petrolatum or the like. The pastes made from single-phase aqueous gels generally incorporate carboxymethylcellulose or the like as a base.

[0066] In some embodiments, the composition include pharmaceutically acceptable transdermal carrier. In one embodiment, a transdermal carriers includes a solid compositions capable of conforming to the surface with which it comes into contact, and which is capable of maintaining the contact in such solid form so as to facilitate topical or transdermal application without adverse physiological response, and without being appreciably decomposed by aqueous contact during administration to a patient. Particular systems include polymer carriers such as the pressuresensitive adhesive matrix type in which the expression construct, and preferably plasmid construct is dispersed directly in the pressure-sensitive adhesive, or reservoir type carriers. Illustrative examples of suitable adhesives as matrix type flexible, finite delivery systems include those described in U.S. Pat. Nos. 5,474,783, and 5,656,386. Other systems include films, plasters, dressings, and bandages, as well as multilayer delivery systems in which the expression construct is solubilized or contained in one or more separate layers, and reservoir-type delivery systems in which the expression construct is solubilized or contained in a reservoir or depot separate from the adhesive which attaches directly to the skin or mucosa.

[0067] Thus, in some embodiments, the pharmaceutically acceptable transdermal carrier comprises an adhesive. Suitable adhesives are known in the art and include pressure- sensitive adhesives and bioadhesives. Bioadhesive materials useful in some embodiments include those described in U.S. Patent Number 6,562,363. For example, bioadhesive materials may include polymers, either water soluble or water insoluble, with or without crosslinking agents, which are bioadhesive. Exemplary bioadhesives include natural materials, cellulose materials, synthetic and semisynthetic polymers, and generally, any physiologically acceptable polymer showing bioadhesive properties, or mixtures of any two or more thereof. Pressure sensitive adhesives suitable for use in accordance with the invention include, but are not limited to, pressure-sensitive silicone adhesives, pressure-sensitive acrylic adhesives, and mixtures of any two or more thereof.

[0068] Exemplary pressure-sensitive silicone adhesives include polysiloxanes and other silicone adhesives as disclosed in U.S. Patent Nos. 4,591,622; 4,584,355; 4,585,836; 4,655,767; 5,958,446; in co-pending U.S. Patent Application Number 10 / 895,688; and in Sobieski, et al., "Silicone Pressure Sensitive Adhesives," Handbook of Pressure-Sensitive Adhesive Technology, 2nd ed., pp. 508-517 (D. Satas, ed.), Van Nostrand Reinhold, New York (1989). Suitable silicone pressure- sensitive adhesives are commercially available and include the silicone adhesives sold under the trademarks BIO-PSA X7-3027, BIO-PSA X7-4919, BIO- PSA X7-2685, and BIO-PSA X7-3122 by Dow Corning Corporation, Medical Products, Midland, Mich.

[0069] Suitable acrylic-based pressure-sensitive adhesives are also known in the art. Such acrylicbased polymers may be used as the primary pressure-sensitive adhesive (see, e.g., U.S. Patent Number 4,390,520), or may be used in combination with other polymers which may or may not be pressure-sensitive adhesives (see, e.g. U.S. Patent Number 4,994,267). Acrylic-based pressuresensitive adhesives may be polymerized with functional monomers to provide functional groups on the acrylic-based adhesive, such as may be desired to improve wear properties and expression construct delivery. Suitable polyacrylic acid polymers include polymers of acrylic acid crosslinked with polyalkenenyl ethers (genetically known as carbomers) or divinyl glycol (generically known as polycarbophils) and commercially available from B. F. Goodrich, Cincinnati, Ohio, under the trademark Carbopol® copolymers or resins such as Carbopol® 934 NF, 934P NF, 940 NF and 97 IP NF, Pemulen polymeric emulsifiers and Noveon polycarbophils. Other pressure-sensitive adhesive acrylic polymers are described in U.S. Patent Application Number 2006 / 0233870.

[0070] Polymer blends as described in U.S. Patent No. 5,958,446 may also be used as pharmaceutically acceptable carriers and adhesives in the transdermal compositions embodied herein.

[0071] In certain embodiments of the invention a plasticizer or tackifying agent is incorporated into the formulation to improve the adhesive characteristics of the composition. A tackifying agent is particularly useful in those embodiments in which the expression construct does not plasticize the polymer. Suitable tackifying agents are those known in the art including: (1) aliphatic hydrocarbons; (2) mixed aliphatic and aromatic hydrocarbons; (3) aromatic hydrocarbons; (4) substituted aromatic hydrocarbons; (5) hydrogenated esters; (6) polyterpenes; and (7) hydrogenated wood rosins. The tackifying agent employed is preferably compatible with the blend of polymers. In some embodiments, the tackifying agent is silicone fluid (e.g., 360 Medical Fluid, available from Dow Coming Corporation, Midland, Mich.) or mineral oil. Silicone fluid is useful for blends comprising polysiloxane as a major component. In other embodiments, where polyacrylate, for example, is a major component, mineral oil may be used as a tackifying agent.

[0072] Those skilled in the art will appreciate that suitable compositions may also contain agents known to accelerate the delivery of the expression construct through the skin. Such agents have been referred to as skin-penetration enhancers, accelerants, adjuvants, and sorption promoters, and are collectively referred herein as “enhancers.” This class of agents includes those with diverse mechanisms of action including those which have the function of improving the solubility and diffusibility of the expression construct within the multiple polymer and those which improve percutaneous absorption, for example, by changing the ability of the stratum corneum to retain moisture, softening the skin, improving the skin's permeability, acting as penetration assistants or hair-follicle openers or changing the state of the skin including the boundary layer. Some of these agents have more than one mechanism of action, but in essence they serve to enhance the delivery of the expression construct. Some exemplary agents are listed in U.S. Patent Nos. 5,958,446 and 6,562,363. Those skilled in the art will appreciate that the composition can contain other components, including other functional and inert components, that are known in the art for use in topical or transdermal compositions.

[0073] In some embodiments, a transdermal composition is applied to a substrate, to form a transdermal patch. In some embodiments, the substrate is laminated to one or more additional layers, such as a protective layer, a backing layer, a rate-controlling layer, a membrane layer, or one or more other types of layers known in the art.

[0074] In a preferred embodiment, a “pharmaceutically acceptable carrier” can include a transdermal or topical carrier, such as a stable oil-in water-emulsion. In another embodiment, a pharmaceutically acceptable carrier can include Lipoderm® HMW™, and / or one or more of its constituent parts, such as Amazonian and PEGylated oils as described herein.

[0075] The terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of an inflammatory skin disorder described herein. In some embodiments, treatment may be administered after one or more signs or symptoms of the disease have developed or have been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease. For example, treatment may be administered to a susceptible subject prior to the onset of symptoms. Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence. “Treatment” or “treating” inflammatory skin disorder may include: (1) decreasing inflammation of the skin or adjacent tissues or causing the inflammation to occur with less intensity in a subject that may be predisposed to skin inflammation but does not yet experience or display symptoms, or (2) inhibiting skin inflammation, which can include arresting the development of or reversing symptoms or physiological damage caused by inflammation. “Treatment” or “treating” skin inflammation may also refer to reducing pain associated with skin inflammation experienced by a patient, and / or improving the function of appearance of the skin. In some embodiments, the compositions and methods provided herein are methods for treating pain associated with an inflammatory skin disorder in a subject. In some embodiments, the compositions and methods provided herein are methods for treating hair loss associated with an inflammatory skin disorder in a subject.

[0076] The practice of the present invention will employ, unless otherwise indicated, conventional methods of pharmacology, chemistry, biochemistry, recombinant DNA techniques and immunology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell eds., Blackwell Scientific Publications); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.).

[0077] The terms “comprises”, “comprising”, are intended to have the broad meaning ascribed to them in U.S. Patent Law and can mean “includes”, “including” and the like.

[0078] While the invention has been particularly shown and described with reference to a number of embodiments, it would be understood by those skilled in the art that changes in the form and details may be made to the various embodiments disclosed herein without departing from the spirit and scope of the invention and that the various embodiments disclosed herein are not intended to act as limitations on the scope of the claims. All references cited herein are incorporated in their entirety by reference. The terminology used herein is for describing particular embodiments and is not intended to be limiting. As used herein, the singular forms “a,” “and” and “the” include plural referents unless the content and context clearly dictate otherwise. Thus, for example, a reference to “a” or “the” marker may include a combination of two or more such markers. Unless defined otherwise, all scientific and technical terms are to be understood as having the same meaning as commonly used in the art to which they pertain. For the purposes of the present invention, the following terms are defined above.

[0079] All publications, patents and patent applications cited herein are hereby incorporated by reference in their entireties.

[0080] The invention now being generally described will be more readily understood by reference to the following examples, which are included merely for the purposes of illustration of certain aspects of the embodiments of the present invention. The examples are not intended to limit the invention, as one of skill in the art would recognize from the above teachings and the following examples that other techniques and methods can satisfy the claims and can be employed without departing from the scope of the claimed invention. Indeed, while this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

[0081] EXAMPLES

[0082] Example 1 : Overview and Summary of Experimental Results.

[0083] The present disclosures provides an initial characterization of a new non-viral pDNA-ILlO gene therapy approach for treating psoriasis-like skin conditions. This approach utilized a single topical application of pDNA-rILlO, delivered in D-mannose to increase pDNA uptake plus the novel use of Lipoderm® HMW™ to enable pDNA penetration into skin. This combination successfully transduced cells in psoriatic-like skin lesions to produce IL-10 and downstream effects of IL-10 known to be therapeutic for psoriasis, including suppression of mRNA for IFN-y, TNF-oc, IL-ip and IL-6, decreased CD3 T-cell recruitment, decreased erythema and increased regrowth of hair. Based on this initial indication of therapeutic potential, future studies to optimize dosing and dosing regimen to define maximal attainable efficacy appear warranted.

[0084] Lipoderm® HMW™ was chosen as an exemplary transdermal delivery vehicle of pDNA as it is a base designed by PCCA to deliver high molecular weight compounds into the skin. It has previously been shown to be an effective transcutaneous delivery medium for a wide array of drugs as well as fluorescents probes. To our knowledge, this is the first demonstration that Lipoderm® HMW™ can provide an effective means of delivering plasmid DNA, opening this approach for topically applied gene therapy more generally.

[0085] In this initial embodiment, a single pDNA dose was tested via topical application. It was applied once at 48 hr after application of 40 mg IMQ, both to allow IMQ to induce a psoriasis-like state prior to pDNA-ILlO application and with the goal of avoiding the presence of IMQ cream on and in skin from physically interfering with pDNA-ILlO percutaneous delivery and uptake. Given the ease of using Lipoderm® HMW™ for plasmid delivery, this raises the likelihood that greater therapeutic efficacy may well be attainable, relative to that currently observed, by defining the optimal pDNA-ILlO dose and providing repeated topical dosing over time. The latter point is a notable advantage given the long duration of psoriasis symptom expression, with psoriasis flares lasting weeks to months.

[0086] The pDNA construct used in the present study for expressing IL- 10 has shown long-term, dose-dependent efficacy across species and routes of administration. The point mutation in a non- bindi[61-63]ng region of the transgene was a serendipitous event producing longer IL-10 actions than pDNA expression of native IL- 10. This pDNA-ILlO construct produces dose-dependent efficacy and reattainment of full therapeutic response following repeat dosing in rodents with intrathecal delivery for neuropathic pain. Additionally, it has demonstrated efficacy in both pet dogs and human patients with osteoarthritis following single and repeat dosing with intra-articular administration. The present disclosure provides initial evidence that efficacy can extend to topical administration as well.

[0087] Gene therapy is currently not approved for psoriasis in humans but is an active area of research. Gene therapy approaches for psoriasis are diverse, including viral DNA delivery as well as multiple RNA approaches. Defining a means for effective delivery remains a challenge for gene therapy, as is true for psoriasis therapeutics more generally. As one example, arrays (patches) of microneedles or dissolving microneedles are effective in passing the skin barriers for delivery of therapeutics and are being tested for use in psoriasis. However, the fact that psoriasis presents as an uneven surface of variable depth and can cover large areas of the body, even involving the entire body, makes it far more challenging for microneedle delivery than topical application with a carrier such as Lipoderm® HMW™.

[0088] The rationale for evaluating IL- 10 for the treatment of psoriasis-like skin conditions has support from the literature. Decreased skin and / or blood levels of IL-10 are associated with worsening of psoriasis and psoriasis-like IMQ symptoms. Further, inhibition of psoriasis-like effects of IMQ by estrogen, CDK8 inhibition, phosphodiesterase-4 (PDE4) inhibition, infliximab, ROCK2 inhibition, amongst other treatments, have each been linked to elevations of IL- 10.

[0089] Such observations led to testing IL- 10 protein in psoriasis patients. Positive results, including reports of decreased incidence of relapse and prolonged disease free interval have been reported following subcutaneous injection of recombinant human IL-10 protein (8 ug / kg once daily or 20 ug / kg three times per week) into psoriasis plaques. However, subcutaneous injections of recombinant IL-10 protein every day or even multiple days per week is not a practical solution for psoriasis treatment. Beyond expense and patient compliance with such a dosing regimen for weeks-to-months, administering IL-10 protein is constrained by its short half-life of approximately 3-5 hours following subcutaneous dosing, both in humans and rodents. Hence subcutaneous dosing, while minimizing unwanted systemic effects, leads to pulsatile exposure to IL- 10 followed by long IL- 10 free periods. In contrast, single doses of this pDNA-ILlO construct leads to multimonth symptom resolution in rats, dogs, and humans at least with the other routes of administration that have been tested to date.

[0090] Applicant’s data show that a single topical application of 40 mg IMQ to shaved back of rats is sufficient to induce a persistent state of skin inflammation, resembling human psoriatic skin lesions. IMQ, a TLR-7 / 8 agonist, triggers the activation of innate immune response, causing cutaneous inflammation with increased leukocyte influx to the skin. Activation of TLR-7 / 8 of leukocytes, induces the production of proinflammatory cytokines and chemokines such as IFNy, TNF-alpha, IL-ip and IL-6, an effect replicated here with IMQ. This pattern of effects supports the logic of targeting IL- 10, as IL- 10 represses several key proinflammatory cytokines, including IFNy, TNF-ot, IL-ip, and IL-6, as also shown in the current study.

[0091] Applicants also provide an initial analysis of the effect of topically delivered pDNA-rILlO on IMQ-induced T-cell recruitment. Psoriasis has been described as an organ-specific T cell-driven inflammatory disease, and T-cells play a dominant pathogenic role in the initiation and maintenance of psoriasis. The literature shows progressive accumulation of inflammatory CD3 and CD4 T cells in the skin, resulting in the typical epidermal inflammation. Applicants findings showed that a single topical application of pDNA-rILlO to the IMQ group was effective in reducing total T-cell recruitment, as showed by IHC for the pan T-cell marker CD3, while no change was found for CD4.

[0092] Lastly, the present disclosure provides data suggestive of a positive effect of IL-10 on enhancing hair regrowth in IMQ-treated skin. This appears to be the first observation of such an effect in psoriasis-like disorders. Psoriasis, especially on the scalp, can inhibit hair growth, with a higher proportion of follicles being in the telogen (resting) or catagen (follicle regression, hair detachment) phases. Additionally, it has been noted that multiple stressors and drugs that trigger or exaggerate psoriasis led to inhibition of hair growth. While IL- 10 does not appear to have previously been studied with regards to psoriasis, there are, however, several publications wherein suppression of IL-10 occurs in another related dermatological disorder, alopecia areata, which has characteristic inflammatory cell infiltrates surrounding hair bulbs. Therapeutic approaches that enhance IL-10 levels have been reported to be associated with increased hair growth in alopecia areata. Hence, further examination of the therapeutic potential of topical non-viral IL- 10 gene therapy as a stimulator of hair regrowth in dermatological diseases appears worthy of further consideration. It also supports the potential advantage of re-application of pDNA-ILlO in Lipoderm® HMW™ over time. This is because hair follicle cells are major protein producers in skin and if pDNA-ILlO protects follicles from being in telogen or catagen states, it predicts sustained ability to produce protein from applied transgenes.

[0093] In sum, the present disclosure provides initial insights into the therapeutic potential of non- viral pDNA-rILlO gene therapy for psoriatic-like skin disorders through transcutaneous (also sometimes referred to as transdermal) delivery using Lipoderm® HMW™. This approach was found to downregulate expression of multiple proinflammatory cytokines, as well as suppression of CD3 T-cell recruitment and stimulation of hair regrowth.

[0094] Example 2: A single imiquimod application induces a psoriasis-like skin response.

[0095] The literature describes diverse means of inducing a psoriasis-like condition with IMQ . A variety of dosages and dosing regimens have been reported, ranging from 80 to 120 mg for up to 10 successive days of topical IMQ application; only rarely was the size of the application area specified. Interestingly, Applicants found that a single topical application of 40 mg of IMQ spread across the 2 x 2 cm application site was sufficient to induce skin inflammation across measures and the time course examined (Fig. 1).

[0096] Using the blinded skin scoring system for erythema, plaques, scabbing and hair loss across the entire 2 x 2 cm skin application site (Figs. 1,2), all rats were assessed across time until euthanasia for tissue collection. For the pooled controls (n=4 naive plus n=4 Vehicle + D-mannose in Lipoderm® HMW™), euthanasia was at 168 hours, so these rats were scored throughout the time course. For the other groups, 4 rats per group were euthanized at each timepoint after skin scoring starting at 48 hr. Thus, these group sizes progressively lowered by n=4 per group at each timepoint thereafter. For the various treatment groups (other than the n=8 pooled controls), this yields group sizes at 0 hours (pre-treatment baseline; n=20), 48 hours (n=20), 72 hours (n=16), 120 hours (n=12), 168 hours (n=8), and 240 hours (n=4) after IMQ or vehicle application.

[0097] As anticipated, the pooled controls and the pDNA-IL-10 group that did not receive IMQ scored 0 for erythema, plaques, and scabbing (Fig. 2A-C) across all timepoints assessed and the pooled controls progressively regrew hair after shaving at time 0 (score of 5 due to shaved skin, Fig. 2D).

[0098] In contrast, the rats receiving IMQ only exhibited marked erythema (Figs. 2A), plaques (Fig. 2B), and scabbing (Fig. 2C) throughout the 10-day time course and failed to regrow hair (Fig. 2D), all signs of a psoriasis-like state.

[0099] Compared to the IMQ only group (Tukey’s posthoc test), rats receiving a single topical application of pDNA-ILlO at 48 hr after IMQ application (red filled circles, Fig. 2), exhibited reliably less erythema (pO.OOOl), trended toward fewer plaques (p=0.067), but showed no difference in scabbing. Notably, compared to the IMQ only group, rats receiving IMQ + pDNA- IL10 exhibited reliably faster hair re-growth, with reliably more hair returning by 120 hr (p=0.0059), 168 hr. (p=0.0005) and 240 hr. (p=0.0071), with hair re-growth in the IMQ + pDNA- IL10 group being no different than that of the pooled controls that received no IMQ.

[0100] Applicants applied the blinded skin scoring system described above to the skin punch area, an 8 mm circular punch collected from the center of the full skin application site (Fig. 1, yellow circles) on the day of the euthanasia. This scoring was done for all rats at baseline (prior to IMQ or vehicle application to the 2 x 2 cm sites; n=20 / treatment group plus n=8 pooled controls) and again on the day of euthanasia (n=4 / treatment group per timepoint; n=8 pooled controls at 240 hours) so that this skin score was representative for the tissue analyses that followed. Analysis of the 8 mm punch area was done as the psoriasis-like skin changes were not uniform across the 2 x 2 cm application area, so this targeted analysis provides an assessment of the tissue used in the analyses below. Given this variability in psoriasis-like characteristics across the skin surface, the results of this 8 mm punch analysis were anticipated to vary from the 2 x 2 cm analysis above. As done for the total skin application site (Fig. 2A-D), skin punches were scored for erythema (Fig. 3 A), plaques (Fig. 3B), scabbing, and hair loss (Fig. 3C). In contrast to the entire skin application site (Fig. 2C), scabbing was not observed within these small skin punches, with all subjects receiving a score of 0; hence, no graph is presented.

[0101] Pooled controls (green filled circles) and the pDNA-ILlO group that did not receive IMQ (open circles) all scored 0 for erythema (Fig. 3A), plaques (Fig. 3B), and scabbing within the circular skin punch at baseline and again at the point of tissue collection (euthanasia). In contrast, skin punches from rats that received IMQ only exhibited marked erythema (Fig. 3A) and plaques (Fig. 3B) throughout the entire 10 day experimental time course. Furthermore, only minor hair regrowth (Fig. 3C) was observed in the circular skin punches from the IMQ only rats. In contrast, pDNA-ILlO treatment led to milder IMQ-induced symptoms than IMQ alone. The skin punches from the rats in the IMQ group that received pDNA-ILlO exhibited less erythema (p=0.008 at 120 hr), less plaques (p=0.0004 at 240 hr) and far more robust hair growth (p=0.0051 at 75 hr; p=0.0075 at 168 hr; p=0.0046 at 240 hr) than IMQ alone. For all 3 measures (erythema, plaques, and hair loss), the IMQ + pDNA-ILlO group was statistically indistinguishable from either Pooled Controls or the Vehicle + pDNA-ILlO group by the end of the 10-day time course. Example 3 : Expression of interleukin- 10 in the dermis is achieved after a single topical application of pDNA-rILlO to the skin.

[0102] To determine whether topical delivery of pDNA-rILlO was successful in transducing cells to produce IL- 10, binding of anti-rat IL- 10 antibody was quantified by densitometry the skin punches described above, via diaminobenzidine (DAB) immunohistochemistry (IHC). Each skin punch sample was divided vertically into four equal parts and each sub-sample assigned to IHC for detection of rat IL-10 or to either histochemistry or PCR. As stable expression was observed across time, data within each group was collapsed across time. Two regions of interest were separately analyzed: (i) hair follicles, given prior literature supportive that cells within this structure support expression of transgenes (Fig. 4A) and (ii) the remainder of dermis outside of hair follicles (herein called “Dermis”; Fig. 4B). Epidermis was not able to be analyzed here or other IHC targets, as it exhibited intense non-specific DAB staining which obscured reliable imaging. The targeted question being addressed here was whether groups receiving topical pDNA- IL10 expressed higher levels of rat IL-10 than did the control group. Given this, one-tailed t-tests were used.

[0103] Within the hair follicles, a reliable increase in rat IL- 10 was detected, relative to pooled controls, for both groups receiving topical IL-10 gene therapy (Vehicle + pDNA-ILlO: p=0.0026; IMQ + pDNA-ILlO: p=0.0243), but not for the group receiving IMQ treatment without IL-10 gene therapy (IMQ + Vehicle: p=0.2116). In the dermis outside of the hair follicles, reliable increases in rat IL-10 were again observed, perhaps suggestive of IL-10 release to the extracellular space for both groups receiving topical IL-10 gene therapy (Vehicle + pDNA-ILlO: p=0.0018; IMQ + pDNA-IL 10: p=0.0273), but not for the group receiving IMQ treatment without IL- 10 gene therapy (IMQ + Vehicle: p=0.0918). Based on these findings, it is concluded that a single topical treatment with pDNA-ILlO in a base designed for transport into skin was able to induce rat IL- 10 protein still detectable 10 days later at study end. Example 4: Delivery of topical pDNA-rILlO decreases pro-inflammatory cytokine gene expression in the skin.

[0104] To explore the effects of pDNA-rILlO on mRNA levels of pro-inflammatory cytokines in the skin punches, tissues were analyzed for interferon-gamma (IFN-y; Fig. 5A), tumor necrosis factor alpha (TNF-oc; Fig. 5B), interleukin- Ibeta (IL-1J3; Fig. 5C), and interleukin-6 (IL-6; Fig. 5D). This analysis revealed that IMQ created a pro-inflammatory state in the skin within the first 48 hours after its topical application, as there was a significant increase in mRNA for IFN-y (p=0.0001), IL-ip (p=0.0001), and IL-6 (p=0.0001) relative to pooled controls (Fig. 5A, C, D).

[0105] Here, pDNA-rIL-10 gene therapy was effective at suppressing the inflammatory effects of IMQ, as significant decreases in mRNA levels of IFN-y (p=0.0001), IL-ip (p=0.0005) and IL-6 (p=0.0037) were found at 48 hours (Fig. 5A, C, D) comparing IMQ alone to IMQ+pDNA-rILlO. No changes were observed in any group for TNF-oc (p>0.6) mRNA levels at 48 hours (Fig.5 B).

[0106] At 72 hours following IMQ, only IFN-y (p=0.0001) and IL-6 (p=0.0001) were increased in the skin of IMQ rats when compared to pooled controls (Fig. 5A, D). pDNA-rILlO was again successful in suppressing the pro-inflammatory effects of IMQ, as IMQ+pDNA-rILlO lowered mRNA levels of both IFN-y (p=0.0001) and IL-6 (p=0.0001) relative to IMQ alone (Fig. 5A, D).

[0107] Similar results were observed for the pro-inflammatory cytokines at 120 hours following IMQ application. IFN-y (p=0.0001), IL-6 (p=0.0001), and IL-1 [3 (p=0.0001) mRNA levels were increased in the skin of IMQ rats when compared to pooled controls (Fig. 5 A, C, D). As observed for the previous time points, pDNA-rILlO, was again able to decrease the levels of pro- inflammatory cytokines IFN-y (p=0.0001), IL-6 (p=0.0001), IL-ip (p=0.0003) at time point 120 hours when comparing IMQ alone to IMQ+pDNA-rILlO (Fig. 5A, C, D). No statistical difference was observed for TNF-oc (p>0.4) comparing IMQ alone to pooled controls (Fig. 5B). In contrast, levels of TNF-oc mRNA (p=0.0065) were lowered in the IMQ+pDNA-rILlO when compared to IMQ alone (Fig. 5B).

[0108] At 168 hours following IMQ application, no group exhibited an increase in IFN-y(p>0.9) mRNA levels relative to pooled controls (Fig. 5A). In contrast, mRNA levels of TNF-oc (p=0.0001), IL-ip (p=0.0010), and IL-6 (p=0.0001) were again increased in the IMQ group compared to pooled controls (Fig. 5B-D). pDNA-rILlO gene therapy was effective in this time point as well, lowering mRNA expression of TNF-a (p=0.0001), IL-1J3 (p=0.0043), and IL-6 (p=0.0001) comparing IMQ alone to IMQ+pDNA-rIL 10 (Fig. 5B, C, D).

[0109] At time point 240 hours following IMQ application, all four pro-inflammatory cytokines mRNA decreased to normal values in the IMQ+pDNA-rILlO group relative to pooled controls (Fig. 5A-D). No statistical difference was observed between IMQ+pDNA-rILlO and pooled controls (IFN-y p=0.9390; TNF-a p=0.0686; IL-6 p=0.0692; IL-ip p=0.3850). In contrast, a continuous increase in pro-inflammatory cytokines mRNA levels for IFN-y (p=0.0018), TNF-a (p=0.0217), and IL-ip (p=0.0341) was observed in the IMQ group alone relative to pooled controls (Fig. 5A-C). No statistical difference was observed for IL-6 mRNA levels in the IMQ group alone compared to pooled controls at time point 240 hours (p=0.8076).

[0110] This analysis revealed that a single topical application of IMQ is able to consistently increase the levels of pro-inflammatory cytokines mRNA along the ten day experimental time course. Most importantly, our data revealed that topical pDNA-rILlO can exert antiinflammatory effects in inflamed skin. It was observed here that a single topical application of pDNA-rILlO was able to maintain decreased levels of mRNA of pro-inflammatory cytokines in the skin across the entire time course.

[0111] Example 5: Delivery of topical pDNA-ILlO decreases T-cell marker CD3 but not CD4 in the skin. As reported above, topical gene delivery of pDNA-rILlO applied to the skin of IMQ treated rats was able to significantly decrease IMQ-induced gene expression of pro-inflammatory cytokines. The role of T-cells in producing and secreting pro-inflammatory cytokines is well described in the literature, as is T-cell recruitment in response to topically applied IMQ. Applicants pDNA-rILlO gene therapy was effective in reducing total T-cell recruitment, as reflected by IHC for the pan-T cell marker CD3 (Fig. 6A), pooling across time (p=0.0228), while no change in T- helper cell (CD4) numbers was found (Fig. 6B).

[0112] Example 6: Materials and Methods.

[0113] Animals: Pathogen-free adult male Sprague-Dawley rats, 12 weeks of age at arrival (Envigo, Indianapolis, USA) were pair housed in an AAALAC approved animal facility on a 12- h light / dark cycle (lights on at 07:00 h). Given the exploratory nature of this initial pilot study, only one sex was able to be studied. Rats acclimated to the colony for 14 days prior to handling. Standard rat chow and water were available ad libitum. All procedures were in accordance with The National Institute of Health Guide for the Care and Use of Laboratory Animals and were approved by the University of Colorado - Boulder Institutional Animal Care and Use Committee. Health checks were conducted daily.

[0114] Topical imiquimod (IMQ) treatment: Rats had their backs bilaterally shaved in full under brief isoflurane anesthesia and an area of 2 x 2 square centimeters (4 cm2) was delineated to receive a single topical application of 40 mg (50 uL) of IMQ (Aldara™, 5% cream) applied bilaterally.

[0115] Topical delivery of IL-10 Rat Plasmid: The pDNA used included the expression cassette for adeno-associated virus-2 (AAV-2) vector encoding rat interleukin- 10 (rIL-10). This plasmid's transcriptional cassette is flanked by two AAV-2 viral elements, inverted terminal repeat sequences (ITRs), and consists of the cytomegalovirus enhancer / chicken beta-actin promoter, an intronic region, the rIL-10 gene with a point mutation outside of identified receptor-binding regions (F129S; pDNA-IL10F129S) and the SV40 polyadenylation signal region. This unencapsulated (naked) pDNA-rILlO plasmid has previously been demonstrated to be effective after intrathecal or intracerebral administration in resolving rodent models of neuropathic pain and opioid self-administration, respectively. For topical application, 240 pg of pDNA-rILlO (5 mg / ml in 0.3% sucrose) was added to 50 pL D-Mannose (3% in sterile water; Sigma #M6020), in keeping with prior publications supportive of enhanced pDNA efficacy with D-Mannose as an uptake enhancer. On the day of use, pDNA-rILlO was gently mixed into Lipoderm® HMW™ (PCCA; catalog # 30-4612), a base designed to deliver high molecular weight compounds into the skin. This resulted in a ratio of 240 pg pDNA-rILlO to 50 pL Lipoderm® HMW™. The control group received the same topical mixture, simply without pDNA added. Each rat received its appropriate mixture applied bilaterally (50 pL per side) to the shaved back as a single administration.

[0116] Skin score: Photos of the back of the rats were acquired daily from all groups for blinded analysis. Skin scores consisted of measures of skin inflammation based on erythema (generalized redness; scored 0-4), plaques (scored 0-4), scabbing (scored 0-3), and hair loss (scored 0-5). In this standard measurement system from the psoriasis literature, scoring reflects degree of change from healthy, normal skin where 0 = normal and progressing in intensity to the top number as extreme. Each score reflects agreement in assessment of two independent blinded observers.

[0117] Tissue Collection: Rats were transcardially perfused, under deep sodium pentobarbital anesthesia, for 5 min with ice-cold saline (0.9%) to remove blood. Bilateral 8 mm circular skin punches (Keyes Dermal Punch tool, Novo Surgical) were rapidly extracted, submersed in liquid nitrogen and stored at 80°C. Skin was processed for cytokine gene expression and i mmunohi stochemi stry .

[0118] Real time PCR: Total RNA was isolated from skin punch (epidermis plus dermis as these layers were not separable) utilizing a standard method of phenol: chloroform extraction. RNA isolation, cDNA synthesis, and PCR amplification protocols were as previously described in detail. cDNA sequences were obtained from GenBank at the National Center for Biotechnology Information (NCBI). Primer sequences were designed using the Qiagen Oligo Analysis and Plotting Tool, and tested for sequence specificity using the Basic Local Alignment Search Tool at NCBI. Rat primers (Table 1) were obtained from Invitrogen. Primer specificity was verified by melt curve analysis. PCR amplification of cDNA was performed using the Quantitect SYBR Green PCR Kit (Qiagen, Valencia, CA). Formation of PCR product was monitored in real time using the MyiQ Single-Color Real-Time PCR Detection System (BioRad, Hercules, CA). Relative gene expression was determined using the 2AACT. Mean CT of duplicate measures (C.V. <10%) was computed for each sample. Sample mean CT of GAPDH (internal control) was subtracted from the sample mean C of the respective gene of interest (AC ). The sample with the highest absolute ACT was selected as a calibrator and subtracted from the ACT of each experimental sample (AACT). 2A< / yields fold change in gene expression of the gene of interest normalized to the internal control gene expression.

[0119] Immunohistochemistry (IHC): For brightfield analysis of IL-10, CD3, and CD4 expression, 14 m skin sections were washed 3x10 minutes in PBS, blocked in 0.3% hydrogen peroxide in PBS for 15 minutes, blocked for 1 hour with 10% NGS, 0.3% Triton-X in PBS, and then incubated overnight at 4°C in 2% normal goat serum together with mouse monoclonal antirat IL-10 (1 :250; Invitrogen, cat# ARC9102), mouse monoclonal anti-CD 3 (1:250; Invitrogen, cat# 14-0030-82) and rabbit polyclonal anti-CD4 (1 :250; Bioss Antibodies, cat# bs-0766R). Slides were then washed 3x10 minutes in PBS and incubated in goat anti-animal biotin secondary antibody (1:200; Jackson ImmunoResearch, West Grove, PA) for 2 hours. Sections were washed, incubated in avidin biotin complex (ABC) solution (Vector Laboratories, Burlingame, CA) for 2 hours, washed again, and incubated in inactive Diaminobenzidine (DAB) (Sigma) for 8 minutes. DAB was then activated with 10 mg / dl B-D glucose (cat #100953. MP Biomedicals, Solon. OH). After incubation in active DAB for 5 minutes, slides were washed then dried overnight. Slides were then dehydrated in increasing concentrations of ethanol (50%, 70%, 95%, and 100%), cleared in Citrisolv, dried and covered with DPX mountant (Sigma). Images were acquired using a widefield Olympus BX61 microscope (Olympus, Center Valley, PA) with CellSens Dimension software (Olympus). Images from three sections per animal of treated skin were acquired in mosaic format with a mechanical stage at 40x magnification using 15 um extended depth of field at 3 um steps. Images were converted to 8-bit tifs. Image analysis was then conducted using python image library (PIL) and OpenCV libraries. Region of Interest for analysis were selected by an investigator who was blinded to treatment groups. Densitometry analysis was then performed in batch, using the same intensity threshold for all images. Data were expressed as total area positive for staining within the region of interest.

[0120] Statistics: All analyses were performed using GraphPad Prism (version 10.2). Data are presented as the mean and standard error (SE). For skin scoring, analysis of real time PCR and IHC data were by a one-way ANOVA followed by Tukey’s multiple comparison test. For IHC IL- 10 data analysis, one tailed t-tests were used as a specific direction of result was being analyzed. Given the absence of difference in data from the naive controls and mannose only groups across the study, these data were combined and used as a pooled control.

[0121] TABLES

[0122] Table 1. Primer sequences

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Claims

CLAIMSWhat is claimed is1. A pharmaceutical composition for treating an inflammatory skin disorder or a symptom thereof comprising:- a therapeutically effective amount of an isolated expression construct formed by a heterologous nucleic acid, operably linked to a promoter, encoding an interlukin-10 (IL- 10) peptide, or functional fragment or variant thereof; and- a pharmaceutically acceptable transdermal carrier;- wherein the composition is applied to skin of a subject in need thereof, and the IL-10 is expressed in one or more affected cells.

2. The composition of claim 1, wherein the inflammatory skin disorder is psoriasis.

3. The composition of claim 1, wherein the expression construct comprises a plasmid expression construct.

4. The composition of claim 1, wherein the IL-10 peptide comprises an endogenous IL-10 peptide, or a functional fragment or variant thereof.

5. The composition of claim 1, wherein the IL-10 peptide is selected from SEQ ID NO. 1-8, or a sequence having at least 80% or more sequence identity with any of SEQ ID NO. 1-8.

6. The composition of claim 1, wherein the expression construct comprises a DNA plasmid expression construct.

7. The composition of claim 6, wherein the DNA plasmid expression construct comprises the plasmid construct according to pDNA-ILlO, or a sequence having at least 80% or more sequence identity with pDNA-ILlO.

8. The composition of claim 1, wherein the pharmaceutically acceptable transdermal carrier facilitates permeation of high-molecular weight molecules across the skin.

9. The composition of any of claims 1 or 8, wherein the pharmaceutically acceptable transdermal carrier comprises a stable oil-in-water emulsion that solubilizes and disperses the expression construct upon contact with the skin.

10. The composition of claim 9, wherein the oil-in-water emulsion comprises a complex mixture of Amazonian and PEGylated oils.

11. The composition of any of claims 9 to 10, wherein the oil-in-water emulsion further includes a hydrogel polymer matrix.

12. The composition of any of claims 1 to 11, wherein the pharmaceutically acceptable transdermal carrier comprises Lipoderm® HMW™.

13. The composition of claim 1, further comprising a nucleic acid adsorption enhancing excipient.

14. The composition of claim 13, wherein the enhancing excipient comprises D-mannose.

15. The composition of claim 1, wherein the promoter comprises an endogenous promoter, or an inducible promoter.

16. The composition of claim 1, wherein the subject is a mammal.

17. The composition of claim 1, wherein the subject is a human.

18. A method of treating an inflammatory skin disorder or a symptom thereof in a subject, the method comprising:- administering to the skin of the subject a therapeutically effective amount of a pharmaceutical composition comprising:- an isolated expression construct formed by a heterologous nucleic acid, operably linked to a promoter, encoding an interlukin-10 (IL- 10) peptide, or functional fragment or variant thereof;- a pharmaceutically acceptable transdermal carrier; and- expressing the construct in one or more affected cells thereof.

19. The method of claim 18, wherein the inflammatory skin disorder is psoriasis.

20. The method of claim 18, wherein the IL-10 peptide comprises an endogenous IL-10 peptide, or a functional fragment or variant thereof.

21. The method of claim 18, wherein the IL-10 peptide is selected from SEQ ID NO. 1-8, or a sequence having at least 80% or more sequence identity with any of SEQ ID NO. 1-8.

22. The method of claim 18, wherein the expression construct comprises a DNA expression construct.

23. The method of claim 22, wherein the DNA plasmid expression construct comprises the plasmid construct according to pDNA-ILlO, or a sequence having at least 80% or more sequence identity with pDNA-ILlO.

24. The composition of claim 19, wherein the pharmaceutically acceptable transdermal carrier facilitates permeation of high-molecular weight molecules across the skin.

25. The composition of any of claims 18 or 24, wherein the pharmaceutically acceptable transdermal carrier comprises a stable oil-in-water emulsion that solubilizes and disperses the expression construct upon contact with the skin.

26. The composition of claim 25, wherein the oil-in-water emulsion comprises a complex mixture of Amazonian and PEGylated oils.

27. The composition of any of claims 25 to 26, wherein the oil-in-water emulsion further includes a hydrogel polymer matrix.

28. The method of any of claim 18-27, wherein the pharmaceutically acceptable transdermal carrier comprises Lipoderm® HMW™.

29. The method of claim 18, wherein the pharmaceutical composition further comprises a nucleic acid adsorption enhancing excipient.

30. The method of claim 29, wherein the enhancing excipient comprises D-mannose.

31. The method of claim 18, wherein the promoter comprises an endogenous promoter, or an inducible promoter.

32. The method of claim 18, wherein the subject is a mammal.

33. The method of claim 18, wherein the subject is a human.

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