WO2006035432A2 - Silençage genique s'utilisant en dermatologie - Google Patents

Silençage genique s'utilisant en dermatologie Download PDF

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WO2006035432A2
WO2006035432A2 PCT/IL2005/001031 IL2005001031W WO2006035432A2 WO 2006035432 A2 WO2006035432 A2 WO 2006035432A2 IL 2005001031 W IL2005001031 W IL 2005001031W WO 2006035432 A2 WO2006035432 A2 WO 2006035432A2
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seq
mrna
sirna
disorder
pharmaceutical composition
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WO2006035432A3 (fr
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Emanuel A. Yakobson
Yechezkel Sidi
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Tel Hashomer Medical Research Infrastructure And Services Ltd.
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/02—Pentosyltransferases (2.4.2)
    • C12Y204/0203—NAD+ ADP-ribosyltransferase (2.4.2.30), i.e. tankyrase or poly(ADP-ribose) polymerase
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    • C12N2310/14—Type of nucleic acid interfering N.A.
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    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • RNA interference RNA interference
  • siRNA small interference RNA
  • RNA interference i.e. gene silencing, or gene expression down- regulation is the process whereby a double-stranded RNA (dsRNA) induces the homology-dependent degradation of cognate mRNA. This resulted in subsequent reduction of the specific protein translated from the degraded mRNA.
  • the process may be initiated by introducing a 21 nucleotide dsRNA (siRNA - short interfering RNA) into cells.
  • RISC RNA-induced-silencing-complex
  • RISC uses an ATP molecule to unwind the siRNA molecule, leaving the complement (antisense) strand in the complex.
  • RISC binds through base paring to the appropriate mRNA sequence and cleaves it. The cleaved RNA strands are then digested by nucleases. The RISC applies this process repeatedly.
  • Cell hyper-proliferation and gross impairment of apoptotic pathways are one of the underlying features in cell biology of dermatological disorders and pathologies. These involved over-expression of Bcl-2 and BcI-XL genes.
  • the Bcl-2 protein and its pro-survival members, such as BcI-XL associate with the outer membrane of the mitochondria and with the endoplasmic reticulum and maintain their integrity. It has been established that the Bcl-2 gene malfunctioning in cell proliferation underlies the pathology of many diseases and disorders.
  • the present invention is based on empirical results showing that incubating siRNA duplex with two cell cultures derived from the epidermis, human keratinocytes and human melanoma cells, together with a transfection reagent (in the specific examples, lipid based transfection reagents) resulted in the inhibition of proliferation of proliferating (keratinocytes) or hyperproliferating (melanoma) cells. It was thus concluded that siRNA duplexes, as defined hereinbelow may be useful in treating pathological abnormal conditions of the skin.
  • the present invention provides the use of siRNA duplex that includes complementary sense and anti-sense sequences corresponding to at least part of a target mRNA sequence, or an alternative splice form, mutant or cognate thereof, for the preparation of a pharmaceutical composition for treating a dermatological disease or disorder.
  • the target mRNA is such that its expression is involved in the manifestation of a dermatological disease or disorder.
  • the mRNA is expressed in skin cells, as defined below.
  • a construct comprising a transfection vehicle and siRNA duplex that includes complementary sense and anti-sense sequences corresponding to at least part of a target mRNA sequence, or an alternative splice form, mutant or cognate thereof, for the preparation of a pharmaceutical composition for the treatment of a dermatological disease or disorder.
  • the invention also provides therapeutic methods of treatment of dermatological conditions.
  • a method for treating a dermatological disease or disorder comprises contacting skin cells with an amount of siRNA duplex that includes complementary sense and anti-sense sequences corresponding to at least part of a target mRNA sequence, or an alternative splice form, mutant or cognate thereof, the amount being effective to treat said disease or disorder.
  • a method for treating a dermatological disease or disorder comprises contacting skin cells with an amount of a construct comprising a transfection vehicle and siRNA duplex that includes complementary sense and anti-sense sequences corresponding to at least part of a target mRNA sequence, or an alternative splice form, mutant or cognate thereof, the amount being effective to treat said disease or disorder.
  • the skin cells are either diseased skin cells, i.e. exhibiting symptoms of the diseased or cells which are in disposition of developing a skin disease or disorder, as determined by considerations known to dermatologists.
  • the siRNA duplex is preferably combined with physiologically and pharmaceutically acceptable excipients, for facilitating the administration of the siRNA duplex to the target tissue or cells at a skin area.
  • the present invention also provides a pharmaceutical composition for treating a dermatological disease or disorder, the composition comprising a physiologically acceptable excipient and an amount of the siRNA duplex or a construct comprising a transfectant vehicle and said siRNA duplex, the amount being effective to treat or prevent said disease or disorder.
  • a pharmaceutical composition for the treatment of a dermatological disease or disorder comprising a physiologically acceptable excipient and an amount of siRNA duplex that includes complementary sense and anti-sense sequences corresponding to at least part of a target mRNA sequence, or an alternative splice form, mutant or cognate thereof or a construct comprising a transfectant vehicle and said siRNA duplex, the amount being effective to treat or prevent said disease or disorder.
  • Figures 1A-1D are optical microscope images showing growth inhibition of keratinocyte cells treated with Bcl-2 siRNA duplex loaded onto transit-TKO transfection reagent at two different concentrations (40OnM, Fig. 1C, and 120OnM, Fig. ID).
  • Non-treated cells Fig. IA
  • cells treated with empty transfection reagent Fig. IB
  • Figures 2A-2C are optical microscope images showing growth inhibition of keratinocyte cells treated with BcI-XL siRNA duplex loaded onto transit-TKO transfectant at two different concentrations, (40OnM, Fig. 2B, and 80OnM, Fig. 2C) cells.
  • Non-treated cells served as a control (Fig. 2A).
  • Figures 3A-3D are optical microscope images showing melanoma cell growth inhibition in the presence of 2OnM, 6OnM and 20OnM Bcl-2 siRNA duplex loaded TransIT-TKO transfectant (Fig. 3B, Fig. 3C and Fig. 3D, respectively).
  • Non-treated cells served as control (Fig. 3A).
  • RNA interference is a gene silencing phenomenon whereby double-stranded RNAs trigger the specific degradation of a homologous mRNA.
  • siRNA small interfering RNA
  • RISC RNA- induced silencing complex
  • siRNA annealed duplexes are potential agents for inhibiting skin cells growth, more specifically, for treating pathological conditions at the skin.
  • the invention concerns the use of siRNA duplexes for the above purposes.
  • siRNA used herein is well known in the art and should be understood as denoting any small (short) interfering RNA (-20-25) nucleotides, which induces a cellular machinery to cut up other single-stranded RNA having the same sequence as the siRNA.
  • siRNA to be used in accordance with the invention is preferably a siRNA duplex.
  • siRNA duplex denotes an ensemble of one or two oligonucleotides including a sense and an antisense sequences that can hybridize to form a dsRNA.
  • the sense and anti-sense sequences correspond to at least part of a target mRNA.
  • dsRNA denotes an RNA molecule that has a major portion that is double-stranded.
  • a dsRNA may be constructed out of two independent oligonucleotides that are hybridized to one another (e.g. sense and antisense sequences). Alternatively it may be comprised of a single oligonucleotide having complementary sequences that folds in a manner such that said complementary sequences hybridize to one another, typically to form a hairpin-type construct.
  • Such dsRNA may have terminal regions or hairpin loop regions in which the nucleotide sequence are not paired and are thus single-stranded.
  • the double-stranded portion of the dsRNA may contain one or more mismatched nucleotides that are not paired (hybridized) with a complementary one.
  • the sense and the antisense sequences correspond to at least part of a target mRNA sequence (the sequence, the transcription of which is inhibited by the siRNA).
  • the siRNA duplex in accordance with one embodiment, includes two independent oligonucleotides, one with a sense sequence and the other with an antisense sequence; in accordance with another embodiment it includes one oligonucleotide with both the sense and the antisense sequences, typically separated from one another by several nucleotides such that said one oligonucleotide can fold to hybridize with one another.
  • the sense or antisense sequences sufficiently correspond to at least part of the target mRNA sequence so as to activate RNA interference-based cleavage of the mRNA sequence.
  • the target mRNA is a sequence, the translation of which is involved in the development if the pathological condition in the skin. Thus, inhibition of its expression will result in the treatment or prevention of the disease.
  • the term "corresponding" when used to refer to the relationship between the siRNA duplex and the target mRNA means that the sense sequence and the anti-sense sequence have a high degree of identity or complementarity, respectively, to a selected sequence in the target mRNA. In some cases this means that one or both of the sense or the antisense sequence may be completely identical or complementary, respectively, to the selected sequence.
  • one or both of the sense and the anti-sense sequences may include one or more nucleotides that have no counterpart in the selected sequence.
  • siRNA sequences which may be employed according to the invention are those which correspond to at least part of the following mRNA sequences: Bcl-2, BcI-XL, integrin ⁇ l- ⁇ l, as well as other sequences of mRNA which involved in skin cell abnormalities such as hyper-proliferation, and in particular, with hyper-proliferation of keratinocytes.
  • mRNAs include, inter alia, signal transducer and activator of transcription 3 (STAT3), Insulin like growth factor receptor 1 (IGFRl), Poly ADP ribose polymerase 1 (PARPl), SMAD4 (a gene in TGF beta pathway important in keratinocyte proliferation control) the intracellular adhesion molecule ICAM-I; cytokines such as IL-2, IL-8, growth factors such as IGF-IL, EGF, VEGF; ornithine decarboxilase (ODC), and others.
  • STAT3 signal transducer and activator of transcription 3
  • IGFRl Insulin like growth factor receptor 1
  • PARPl Poly ADP ribose polymerase 1
  • SMAD4 a gene in TGF beta pathway important in keratinocyte proliferation control
  • ICAM-I intracellular adhesion molecule
  • cytokines such as IL-2, IL-8, growth factors such as IGF-IL, EGF, VEGF
  • ODC orn
  • genes are either expressed in skin cells (such as keratinocytes), or, like cytokines, affect proliferation of skin cells, and are usually expressed, but not always, from T and B- lymphocytes, which are prevalent in skin, especially in psoriatic lesions.
  • the siRNA duplex corresponds to at least part of a human mRNA so as to activate RNA interference based cleavage of the human mRNA. While the sequence may be a natural sequence, preferably, the siRNA duplex is a synthetic or semi ⁇ synthetic annealed sequence, as known to those in the art, which corresponds to at least part of a human mRNA.
  • the synthetic sequence may comprise naturally occurring nucleotides, chemically modified nucleotides as well as synthetic nucleotides. There are a variety of techniques available for the production of synthetic oligonucleotides, including various oligonucleotide synthesizers.
  • siRNA duplex sequences used in the specific examples below were synthesized, annealed and purified by HPLC to homogeneity by Dharmacon, by the use of 5'-Silyl-2'-Orthoester Protecting Groups as described in the art [Scaringe, S. A. et al. J Am. Chem. Soc. 120:11820-11821 (1998)].
  • siRNA duplexes When referring to alternative splice forms, mutants or cognates of the mRNA sequence, it is essential that the resulting siRNA duplex is functionally active. Functional activity is defined by the exhibition of cell growth inhibition in the presence of the siRNA duplex. Functionally active (i.e. capable of RNA interference) siRNA duplexes may be identified by appropriate assays known to those versed in the art. For example Santa Cruz Biotechnology (www.scbt.com) listed 2850 siRNA molecules proven to be active in suppressing gene activity and reducing the level of the respective protein product and this selective and specific reduction of protein levels was determined by immunofluorescent analysis and Western blotting assays, techniques well know in the art.
  • siRNA sequences which are applicable in the context of the present invention (when in the form of siRNA duplex), comprises: Sequences corresponding to part of the BCl-2 gene (Oligo RNA duplex (DHARMACON RNA Technologies):
  • siRNA sequences corresponding to at least part of the Integrin alpha- 1 gene available from DHARMACON siGENOME SMARTpool.
  • RNAi phenomena require penetration of the siRNA duplex via the cell membrane, into the cell, to achieve the biological effect.
  • a construct i.e. a construct comprising a transfection vehicle and the siRNA.
  • transfection vehicle denotes any biocompatible chemical or biological structure, which is capable of associating with the siRNA and facilitating its delivery to a target cell or tissue, preferably into the target cell or tissue.
  • Transfection vehicles may be roughly divided into viral (i.e. viral vectors), non- viral and hybrid (a combination of viral and non-viral) delivery vehicles.
  • the present invention preferably makes use of non-viral transfection reagents, preferably synthetic transfection reagents. Nonetheless, viral vectors, plasmids and other transcription systems known in the art and capable of being converted within the cells (e.g. by the RNAse III Dicer) into the siRNA duplex, are also applicable.
  • Synthetic transfection reagents are gaining increasing importance in gene delivery and therapy as an alternative to recombinant viruses.
  • lipid-based transfection vehicles such as bioadhesive liposomes and nanoliposomes
  • lipid-based transfection vehicles such as bioadhesive liposomes and nanoliposomes
  • cationic liposome based transfection vehicles [Sorensen, D. R., Leirdal, M. & Sioud, M.
  • cationic lipids are especially attractive as they can be prepared with relative ease and are extensively characterized. Further, each of their constituent parts can be modified, thereby facilitating the elucidation of structure-activity relationships.
  • cationic lipid denotes a molecule having an overall net positive charge.
  • Cationic lipids typically have a lipophilic moiety, such as a sterol, an acyl or diacyl chain and a head group, and the head group of the lipid carries the positive charge.
  • Cationic lipids may be divided into mono and polycationic lipids.
  • Monocationic lipids may include, without being limited thereto, 1,2-dimyristoyl-
  • DMTAP 3-trimethylammonium propane
  • DOTAP 3-trimethylammonium propane
  • DMRIE N-[l-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl-N- hydroxyethylammonium bromide
  • DORIE N-[l-(2,3,-dioleyloxy)propyl]-N,N- dimethyl-N-hydroxy ethyl- ammonium bromide
  • DORIE N-[l-(2,3-dioleyloxy) propyl] -N,N,N- trimethylammonium chloride (DOTMA); 3 ⁇ [N-(N',N'- dimethylaminoethane) carbamoly] cholesterol (DC-Choi); and dimethy 1-dioctadecylammonium (DDAB) .
  • DMRIE N-[l-(2,3,- ditetradecyloxy)propyl]-N,N-dimethyl-
  • Polycationic lipids may include, without being limited thereto, a similar lipophilic moiety as with the mono cationic lipids, to which polycationic moiety is attached.
  • Exemplary polycationic moieties include spermine or spermidine (as exemplified by DOSPA and DOSPER), or a peptide, such as polylysine or other polyamine lipids.
  • the neutral lipid can be derivatized with polylysine to form a cationic lipid
  • polycationic lipids include, without being limited thereto, N-[2-[[2,5-bis[3-aminopropyl)amino]-l-oxopentyl]amino]ethyl]-N,N-dimethyl- 2,3-bis[(l-oxo-9-octadecenyl)oxy]-l-propanaminium (DOSPA).
  • transfectant reagents which are not lipid-based include MPG Cage peptide based system [Deshayes S et al. Biochemistry 43(24):7698-706, (2004); Morris MC et al. Gene Ther. ll(9):757-64, (2004); Simeoni F et al. Nucleic Acids Res. 31(ll):2717-24, (2003)].
  • the transfection vehicle is associated with the siRNA duplex.
  • the combination of the transfection vehicle and the siRNA is termed herein, at times, "the construct" where the vehicle carries and facilitates the introduction of the siRNA into the cell.
  • the association between the siRNA and the transfection vehicle may be of various types, including loading, encapsulation, containment, or any other physical attachment/association between siRNA duplex and the transfection vehicle.
  • the physical attachment may be either containment or entrapment of the molecule within the vehicle e.g. when lipid vesicles are formed; non-covalent linkage of the siRNA duplex to the surface such vesicles, or embedment of the siRNA in the lipid layer forming the vesicle or containment within the internal space of the vesicle (e.g. intraliposomal space or in the internal core of a nanoparticle used as a vehicle).
  • the interaction may also include adsorption to the surface of the vehicle.
  • association may also be electrostatic, dipole, acid-base and/or by hydrophobic interactions, or any combination of these interactions.
  • Association of the siRNA duplex with the transfection vehicle may be determined according to any one of the technologies known in the art. These include, without being limited thereto, spectroflurometers, spectrophotometers, HPLC, confocal fluorescence microscopes.
  • the construct is preferably stable construct.
  • “Stable construct” as used herein denotes a construct being chemically and physically stable under storage conditions (e.g. at 4 0 C, in physiological medium) for at least one month.
  • the siRNA duplexes either introduced into the target cells directly (e.g. by the use of a transfection vehicle) or formed within the target cells (e.g. by the use of a transfection system in which the sequence is expressed by the cell), silence the expression of the target gene in the target cell.
  • the silencing of the expression of a target gene by the use of a siRNA duplex is usually transient and the target cells in such a case need to be repeatedly contacted with the respective active agent.
  • the silencing of expression by the use of a transfection system in which the siRNA duplex is expressed in the cell is typically longer lasting and at times also permanent and accordingly the target cells need to be contacted with the respective active agent with a lesser frequency, at times only once in the course of a treatment.
  • the artisan should be able to determine in appropriate animal or human trials, the proper treatment regimen in each case
  • the siRNA duplex is brought into contact with target cells, i.e. with skin cells, either in vivo or ex vivo under conditions which will permit the siRNA duplex to enter and transfect the target cells and silence the expression of the target gene in them.
  • skin denotes all types of skin cells and tissue, including cells and tissues forming or present at the epidermins, dermis and subcutaneous layers of the skin.
  • the skin is composed of living and dead layers.
  • the dead layers include the surface scale, outside hair and nail plate.
  • the living layers include the epidermis, dermis, subcutaneous and all of the structures found within those layers, including blood vessels, sweat glands, etc.
  • the Epidermis is the outermost layer of the skin. It is composed of a protective outer layer of nonliving keratin-derived scale cells (acting as an envelope or seal against the environment and exerts some control on the amount of moisture lost to the environment) and living cells including keratinocytes and melanocytes, as well as other specialized cells.
  • the dermis is the layer of the skin that lies just below the epidermis on most of the body. It is largely made up of collagen (fibrous or connective) tissue.
  • the dermis as a layer, makes up the bulk of the skin.
  • the dermis protects the body from mechanical injury, binds water, stores water, maintains temperature and carries nerves to detect sensation and feeling. Blood vessels, lymph vessels, nerves, sweat glands, oil glands, hair follicles, hair erecting muscles, and other structures reside in or course through the dermis.
  • the subcutaneous is the layer lying below or under the cutaneous (dermis) tissue.
  • This tissue is usually composed of fat cells and the structures that run through it, i.e., blood vessels, nerves, etc.
  • the sebaceous glands of the hair follicle arise in the subcutaneous layer.
  • the term “skin” includes structures as well as cells forming or residing in the skin, the latter including, inter alia, nerve cells, blood vessels, glands etc.
  • the siRNA duplex (alone or as part of a construct) is administered to the skin for the treatment of a dermatological/skin disease or disorder.
  • a dermatological/skin disease or disorder There are various pathological conditions associated with skin cells or tissue which may be treated in accordance with the invention.
  • Dermatological disease or disorder As used herein the terms "Dermatological disease or disorder”, “skin disease or disorder” and “skin condition” equally refer to any pathological condition that impairs the normal structure or function of the skin.
  • These terms refer to any condition which affects any of the cell-types present in the epidermis, dermis or subcutaneous (of the skin or the mucosal tissue), and more specifically a condition which impairs the function of epithelial cells, keratinocytes, lymphocytes, melanocytes, fibroblasts, mast cells, langerhans cells and other cell types of epidermis and dermis such as hair follicles, sebaceous glands, and sweat glands.
  • the condition may be the main manifestation of the disease, such is in the case of psoriasis, or may be merely the dermatological manifestation of another disease which is not necessarily dermatologic in origin, for example, a dermatological manifestation of rheumatoid arthritis (for example leg ulceration).
  • This term also refers to a disease impairing the epithelial cells of the mucosal tissue.
  • the local mucosal effect may be on the epithelial cells of the nose, mouth, ear, vagina, rectum, inner lid etc.
  • Hyper-proliferating state - where the dermatological condition is mainly due to hyper-proliferation (such as in cases of psoriasis, hypercarothosis, skin cancer such as melanoma, basal cell carcinoma and additional proliferative skin disorders) the mechanism is mainly believed to be due to reduced apoptosis, and/or modulation of cell cycle of the hyproliferative cells so as to increase the proportion of cells in the G0/G1 arrest stage;
  • hyper-proliferative skin conditions include psoriasis, epidermal hyperplasia, hyperkeratosis, acanthosis, papilloma (such as women's mucosal tissue of the vagina, as well as genital diseases, cervix carcinoma), scleroderma,
  • Nonmelanomas (usually basal cell and squamous cell cancers) are the most common cancers of the skin. They are called nonmelanoma because they develop from skin cells other than melanocytes. They rarely spread to sites elsewhere in the body. Melanoma is a cancer that begins in cells of the melanocyte system of the skin, and metastasis of melanoma is common. Although melanoma accounts for only about 4% of all skin cancer cases, it causes most skin cancer-related deaths.
  • the skin cancer may include, without being limited thereto, basal cell carcinoma, melanoma, squamous cell carcinoma, cutaneous T-cell lymphoma and
  • the therapeutic activity is believed to be caused by induction of T-cell activity, thereby increasing the production of cytokines resulting in an excessive inflammatory response.
  • Neutrophils, mast cells and lymphocytes orchestrate an inflammatory response that results in significant release of inflammatory mediators, and the creation of numerous free radicals.
  • Skin diseases in which inflammation is a significant component include and which may be treated in accordance with the invention, but are not limited to acne and rosacea, atopic dermatitis, contact dermatitis, drug eruptions, psoriasis, seborrheic dermatitis, connective tissue diseases (such as lupus, scleroderma, and rheumatoid arthritis), other autoimmune disorders such as the blistering disease bullous pemphigoid or pemphigus, pigmentary diseases (such as post-inflammatory hyperpigmentation, melasma and vitiligo), urticaria or hives, inflammation associated with skin infections such as tinea corporis or fungal infection of the finger or toenails, among others.
  • acne and rosacea atopic dermatitis, contact dermatitis, drug eruptions, psoriasis, seborrheic dermatitis, connective tissue diseases (such as lupus, scleroderma, and
  • alopecia areata which pathogenesis is pronounced of psoriasis.
  • Alopecia areata is a major (up to 3% of global population) unmet yet pharmacologically immunological disorder.
  • siRNA may be effective to treat skin conditions as defined above.
  • treatment refers to the providing of an amount of the siRNA to a subject in need of such treatment which is effective in one of the following: ameliorating undesired symptoms associated with the dermatological condition; effective in preventing the manifestation of such symptoms before they occur (for example to prevent remission of acute phase of autoimmune-associated dermatological conditions); effective in slowing down the progression of the disease or disorder (for example slowing progress of skin cancers); effective in slowing down the deterioration of the disease (for example effective in restricting the spreading of psoriasis to healthy region); effective to prolong the time period onset of remission period (especially in autoimmune diseases such as lupus); effective in slowing down the irreversible damage caused in the progressive chronic stage of the disorder; effective to delay the onset of said progressive stage (for example delaying the onset of melanoma stage IV); effective to lessen the severity or cure the disease or disorder; effective to
  • the siRNA duplex or construct comprising the siRNA duplex in association with the transfection vehicle are formulated with a pharmaceutically acceptable excipient to form a pharmaceutical composition.
  • siRNA duplex the particular transfection vehicle
  • properties and choice of excipient will be determined in part by the particular siRNA duplex, the particular transfection vehicle, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the siRNA duplex or the construct employed in accordance with the present invention, including, without being limited thereto, oral, intranasal, parenteral (subcutaneous, intravenous, intramuscular, interperitoneal), rectal, and vaginal administration. According to one preferred embodiment, the siRNA or construct comprising same is provided locally. According to yet another preferred embodiment, the siRNA duplex or construct comprising same is applied topically onto a skin area of a subject.
  • the term "locally” as used herein denotes the direct administration of the siRNA duplex or construct to the target tissue, e.g. by topical application, in case the target tissue or cell is at the epidermis layer of the skin, or, by injection, when the target tissue or cell is at the inner layers of the skin underneath the epidermis.
  • topically denotes application of the siRNA duplex or construct onto the epidermis, irrespective of whether the target tissue or cell is located at layers of the skin underneath the epidermis, i.e. at the dermis or subcutaneous layers, this being achieved by the use of excipients facilitating the delivery of the siRNA duplex or construct through the epidermis, as known in the art.
  • siRNA duplex or construct comprising the same, amounts and routes of administration to deliver an efficacious dosage of the siRNA duplex over time.
  • treatment regimens and appropriate dosage using the siRNA duplex or construct employed in accordance with the invention, inter alia, depending upon the level of control over release of the entrapped or encapsulated siRNA.
  • a preferred route of administration is by topical application of the siRNA duplex onto the diseased skin.
  • topically acceptable excipient refers to any adjuvant, excipient or diluent, which is known in the field of pharmacology for application onto the skin (or the epithelial layer of the mucosal tissue) and is approved for dermal/mucosal administration.
  • excipient will be determined by the particular active principle, i.e. the siRNA duplex or the construct, for example, its dissolution in that specific carrier (hydrophilic/hydrophobic), as well as by other criteria such as the size and the nature of the area to which it should be applied (for example in the scalp shampoos may be used while for small area a salve is more applicable, etc.).
  • the topical composition may be applied to the target skin cells according to any conventional mode of topical administration. This includes, without being limited thereto, ointment, cream, gel, solution, suspension, lotion, shampoo, foam, liposome formulation, paste, emulsion, salve. Additional formulations may be used where the pharmaceutical composition is to be applied locally to the treated cells for producing a local effect such as, suppositories, vaginal tablets, ocular salves or drops, otic drops, nasal spray, nasal drops.
  • Topical effectiveness of a pharmaceutical composition depends on two major factors; (a) bioavailability of the active principle in the topical preparation and (b) percutaneous absorption, penetration and distribution of the active principle to the target site in the skin.
  • a topical preparation containing 5% salicylic acid is therapeutically effective as a keratolytic, but that containing 5% sodium salicylate is not an effective product.
  • the reason for such difference is that salicylic acid is in bioavailable form and can penetrate the stratum corneum, but sodium salicylate is not in bioavailable form and cannot penetrate the stratum corneum of the skin.
  • the pharmaceutical composition may include other active principles, such as, without being limited thereto, agents that improve or eradicate age spots, keratoses and wrinkles; analgesics; anesthetics; antiacne agents; antibacterials; antiyeast agents; antifungal agents; antiviral agents; antidandruff agents; antidermatitis agents; antipruritic agents; antiemetics; antimotion sickness agents; antiinflammatory agents; antihyperkeratolytic agents; antidryskin agents; antiperspirants; antipsoriatic agents; antiseborrheic agents; hair conditioners and hair treatment agents; antiaging and antiwrinkle agents; antiasthmatic agents and bronchodilators; sunscreen agents; antihistamine agents; skin lightening agents; depigmenting agents; vitamins; corticosteroids; tanning agents; hormones; retinoids; topical cardiovascular agents and other dermatologicals.
  • active principles such as, without being limited thereto, agents that improve or eradicate age spots, keratoses and wrinkles;
  • Oligo RNA duplex (DHARMACON RNA Technologies, NY, USA):
  • TransIT-TKO ® Reagent MIRUS Corporation, WI, USA
  • siIMPORTER Upstate cell signaling, NY, USA
  • Bcl-2 (SEQ ID NO:1) and BcI-XL (SEQ ID NO:4) were loaded onto lipid assemblies by the use of the following two siRNA transfectant reagents: TransYT-TKO ® Reagent (MIRUS Corporation, WI, USA), or by siIMPORTER (Upstate cell signaling, NY, USA) according to manufacturers' protocol.
  • TransYT-TKO ® Reagent MIRUS Corporation, WI, USA
  • siIMPORTER Upstate cell signaling, NY, USA
  • siRNA dilutions [20-200 nM] in Universal Buffer (6mM HEPES-KOH, pH 7.5, 0.2mM MgCl 2 , 2OmM KCl) were added and incubated at room temperature for further 10 minutes. The final mixtures were added to the individual designated wells. Loading of the siRNA onto the transfection reagent was determined by fluorescent methods and showed to be greater than 80%, for both transfection reagents (data not shown).
  • Human keratinocyte HaCaT cell lines (5x10 3 cells per well) were plated in 96 well COSTAR plat in 10% fetal calf serum (FCS) containing DMEM medium with glutamine. Cells were incubated in 37°C incubator (TUTTNAUER, Brinkman
  • TransIT-TKO transfectant (40OnM or 120OnM), loaded onto TransIT-TKO transfectant, at a concentration of 0.5 ⁇ l per well (the amount of the mixture added to the well), prepared according to manufacturer's instructions. Untreated cells served as a control.
  • Figs. 1A-1D show images of growth inhibition of keratinocytes in the presence of TransIT-TKO transfectant-loaded Bcl-2 siRNA.
  • Fig. 1C shows the results of treatment with 40OnM Bcl-2 siRNA-loaded transfectant
  • Fig. ID shows the results of treatment with 120OnM Bcl-2 siRNA-loaded transfectant.
  • cells were either not treated (Fig. IA) or treated with an empty vehicle (Fig. IB Specifically, in the absence of transfectant-loaded Bcl-2, the cells reached full monolayer in the wells (Fig.
  • human keratinocyte HaCaT cell lines (5x10 3 cells per well) were treated with BcI-XL siRNA (SEQ ID NO:6, 40OnM or 80OnM), loaded onto
  • TransIT-TKO transfectant at a concentration of 0.5 ⁇ l per well (the amount of the mixture added to the well), prepared according to manufacturer's instructions. Untreated cells served as a control.
  • Figs. 2A-2C show images of growth inhibition of keratinocytes in the absence (Fig. 2A) or presence of transfectant-loaded BcI-XL siRNA (Fig. 2B-2C). Specifically, Fig. 2B shows the results of treatment with 40OnM BcI-XL siRNA-loaded transfectant, Fig. 2C shows the results of treatment with 80OnM BcI-XL siRNA-loaded transfectant. It is well observed that in the absence of transfectant-loaded BcI-XL, the cells reached full monolayer in the wells (Fig.
  • Human melanoma MB 001 cells (5x10 3 cells per well, Sheba Medical Center derived from melanoma patients) were treated with TransIT-TKO transfectant loaded Bcl-2 siRNA (SEQ ID NO:1, 2OnM) in the manner as described for human kerantinocytes.
  • Fig. 3B shows that in the presence of 2OnM siRNA loaded onto the TransIT- TKO transfectant resulted in over 50% inhibition of melanoma cell proliferation as compared to non-treated cells.
  • Basal Cell Carcinoma BCC
  • Actinic Keratosis AK
  • siRNA-transfectant Delivery of siRNA-transfectant is also tested on human psoriatic plaques. Specifically, psoriatic plaques (diameter of 10 mm) are treated overnight with the transfection reagent alone (silMPORTER) or the transfection reagent with 1-1OnM of Bcl-2 siRNA (SEQ ID NO:1) and Phosphate Buffer tissue culture grade. Digital photo images will be taken before and after topical treatment.

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Abstract

La présente invention est basée sur des résultats empiriques démontrant que, lorsqu'on combine des doubles hélices d'ARN (ARNsi) d'interférence courtes (petites) avec des réactifs de transfection à base de lipides et lorsqu'on les incube avec des cellules de la peau telles que les kératinocytes et des cellules de mélanome, on parvient à une inhibition importante de la prolifération des cellules. L'invention porte également sur l'utilisation des doubles hélices d'ARNsi comprenant des séquences complémentaires sens et antisens sur au moins une partie d'une séquence d'ARNm, ou sur une forme d'épissure alternative, un mutant ou un parent de celle-ci, dans la préparation d'une composition pharmaceutique pour traiter une maladie ou état dermatologique. L'invention porte également sur des méthodes thérapeutiques et sur des compositions pharmaceutiques destinées à traiter les maladies et états dermatologiques.
PCT/IL2005/001031 2004-09-27 2005-09-27 Silençage genique s'utilisant en dermatologie WO2006035432A2 (fr)

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WO2021130541A1 (fr) * 2019-12-24 2021-07-01 厦门大学 Composition d'administration transdermique d'arnsi et son utilisation

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WO2004046354A1 (fr) * 2002-11-20 2004-06-03 Beiersdorf Ag Oligoribonucleotides pour le traitement de maladies degeneratives de la peau par l'intermediaire de l'interference arn
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WO2003070969A2 (fr) * 2002-02-20 2003-08-28 Sirna Therapeutics, Inc. Inhibition de l'expression du gene bcl2 induite par interference arn au moyen de petits acides nucleiques interferents (sina)
US20040082534A1 (en) * 2002-08-21 2004-04-29 Martin Gleave Treatment of melanoma by reduction in clusterin levels
US20040096882A1 (en) * 2002-08-21 2004-05-20 Martin Gleave RNAi probes targeting cancer-related proteins
WO2004046354A1 (fr) * 2002-11-20 2004-06-03 Beiersdorf Ag Oligoribonucleotides pour le traitement de maladies degeneratives de la peau par l'intermediaire de l'interference arn
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021130541A1 (fr) * 2019-12-24 2021-07-01 厦门大学 Composition d'administration transdermique d'arnsi et son utilisation

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