HK1121778A - Anti-myosin va sirna and skin depigmentation - Google Patents

Abstract

The present invention relates to novel isolated siRNAs comprising a sense RNA strand and a complementary antisense RNA strand which together form an RNA duplex, characterized in that the sense RNA strand comprises a sequence which has at most one nucleotide that is distinct in relation to a fragment of 14 to 30 contiguous nucleotides of the nucleotide sequence of exon F of the gene encoding the myosin Va protein, and also to compositions comprising at least one such siRNA, and to the use of at least one such siRNA as a cosmetic or therapeutic agent for skin depigmentation.

Description

Anti-myosin Va siRNA and skin depigmentation

The present invention relates to novel isolated siRNAs comprising a sense RNA strand and a complementary antisense RNA strand together forming an RNA duplex, characterized in that the sense RNA strand comprises a sequence of at most one different nucleotide compared to a fragment of 14 to 30 consecutive nucleotides of the nucleotide sequence of exon F of the gene encoding myosin Va protein, compositions comprising at least one of the above siRNAs, and the use of at least one of the above siRNAs as a cosmetic or therapeutic agent in skin depigmentation.

Skin pigmentation is a complex process involving many steps. Melanocytes, which are located in the basal layer of the epidermis, can be activated by internal or external stimuli (e.g., exposure to Ultraviolet (UV) radiation) to produce melanin in specialized organelles called melanosomes associated with lysosomes. Following maturation, perinuclear melanosomes are transferred to the melanocyte dendritic periphery along the microtubule and actin cytoskeleton (Lambert J et al, Cell Mol Biol (Noisy-le-grand): 45: 905-18, 1999; Hara M et al, J Invest Dermatol.: 114: 438-43, 2000; Vancollie G et al, JInvest Dermatol.: 114: 421-9, 2000). Melanosomes, which include melanin, are then transferred from the distal ends of the dendrites to adjacent keratinocytes by a hitherto unknown mechanism.

Any alteration in steps in this complex process can lead to skin pigmentation disorders. In particular, an increase in the number of activated melanocytes, and/or an increase in melanin production, and/or an increase in the transfer of melanosomes from melanocytes to keratinocytes may result in hyperpigmentation (or dyschromatosis) of the skin. Such hyperpigmentation can be caused by a number of factors, such as drugs, sun exposure, metabolic or nutritional dysfunction, and by genetic or autoimmune diseases. This often implies serious aesthetic and psychological consequences for the patients who will in many cases seek appropriate treatment.

Accordingly, efforts have been made to develop effective treatments for hyperpigmentation. Most of the existing chemotherapies include depigmenting agents that target the melanin synthesis pathway, particularly by inhibiting the activity of tyrosinase, which is essential for melanin synthesis. Such depigmenting agents include, in particular, hydroquinone and its derivatives, retinol or tretinoin, ascorbic acid and its derivatives, placental extract, kojic acid, ferulic acid, arbutin, dihydroxybenzene derivatives (WO00/47045), guaiacol derivatives (WO00/47179), 4- (2, 3-dihydroxyphenyl) -cyclohexanol (WO00/56279), resorcinol derivatives (WO00/56702), phenol amides (WO 99/32077). These substances may have specific drawbacks. They may be unstable, need to be used at high concentrations, lack specificity in their mode of action, or be toxic or irritating.

Physical types of treatments have also been developed, such as medium depth exfoliation, dermabrasion or laser treatment. However, these treatments are often less effective and cause adverse side effects such as the risk of post-inflammatory hypopigmentation or hyperpigmentation, brown rot, or scarring, which may have carcinogenic consequences (Briganti S et al, Pigment Cell Res.: 16: 101-10, 2003; Halder RM, Noothti PK. J. Am Acad Dermatol.: 48: S143-48, 2003; Halder RM, Richards GM. skin thread Lett.: 9: 1-3, 2004).

Therefore, there is a need to develop new effective and reliable cosmetic and/or therapeutic treatments for hyperpigmentation.

In addition to chemical treatment, another possible way to inhibit gene expression is by using antisense nucleic acids or by using a method known as RNA interference (Dykxhoorn DM et al, Nat Rev Mol CellBiol: 4: 457-67, 2003; Southchek J et al, Nature: 432: 173-8, 2004).

RNA interference (hereinafter RNAi) is the process by which double-stranded RNA (dsrna) having a given sense nucleic acid sequence degrades all messenger RNA (mrna) comprising said nucleic acid sequence in a specific manner with respect to the nucleic acid sequence. Although the RNAi process was originally demonstrated in Caenorhabditis elegans, it is now clear that the RNAi process is a very general phenomenon, and inhibition of human genes by RNAi has been achieved.

The process of RNAi can be achieved using small interfering RNAs (or siRNAs). These sirnas are dsRNA sequences comprising a high degree of homology, preferably identical, less than 30 nucleotides in the sense sequence to a fragment of the target mRNA. When the siRNA crosses the plasma membrane, the cell responds by disrupting the siRNA and all sequences that include the same or highly homologous sequences. Thus, mRNA having a fragment identical or highly homologous to the siRNA sequence will be disrupted and expression of the gene will be inhibited.

For the treatment of hyperpigmentation, the use of antisense oligonucleotides directed against the gene of tyrosinase or TRP-1 protein (WO01/58918), or against the gene of PKC β 1 protein involved in the phosphorylation of tyrosinase (WO2005/073243) has been proposed in the prior art. The use of siRNAs targeting the tyrosinase gene has also been proposed (WO 2005/060536).

However, all these genes are involved in the melanin synthesis pathway, and no siRNA directed against a target gene involved not in the melanin synthesis pathway but in the transport and transfer of melanosomes in melanocytes to keratinocytes has been disclosed or suggested.

The present inventors have shown that myosin Va protein is involved in the transport of melanosomes in melanocytes (Lambert J et al, Cell Mol Biol (Noisy-le-grandd): 45: 905-18, 1999; Lambert et al, J Invest Dermatol.: 111: 835-40, 1998). More particularly, the myosin Va protein has several splice variants, and variants including exon F are essential for the transport of melanosomes (Westbrook W et al, J Jnvest German: 120: 465-75, 2003).

However, to date, there has been no attempt to inhibit variants of myosin Va protein including exon F using siRNA, or to apply this method to cosmetic or therapeutic treatment of hyperpigmentation.

The present inventors found that it is possible to induce a myosin Va protein exon F-specific RNAi process by synthesizing sirnas targeting myosin Va protein exon F and inducing these sirns. They also show that the siRNA is effective in inhibiting the expression of variants of the myosin Va protein including exon F in the absence of concentrations of siRNA that result in reduced cell viability, and that the effect is dose dependent. Furthermore, the inventors have found that the presence of siRNA targeting exon F of the myosin Va protein can inhibit the transfer of melanosomes from melanocytes to keratinocytes.

Thus, the present invention relates to novel isolated sirnas comprising a sense RNA strand and a complementary antisense RNA strand together forming an RNA duplex, characterized in that the sense RNA strand comprises a sequence of at most one different nucleotide compared to a fragment of 14 to 30, advantageously 15 to 29, 16 to 28, 17 to 27, 18 to 25, 18 to 23, or 18 to 21 consecutive nucleotides of the exon F nucleotide sequence of the gene encoding the myosin Va protein. The expression "comprising an exon F nucleotide sequence fragment of a myosin Va protein gene" as used throughout the specification of the invention is to be understood as meaning an RNA sequence corresponding to the gene sequence, i.e. a corresponding RNA sequence in which T is replaced by U in the gene sequence.

The sense strand of the siRNA of the invention therefore comprises a fragment of 14 to 30 consecutive nucleotides, advantageously 15 to 29, 16 to 28, 17 to 27, 18 to 25, 18 to 23, or 18 to 21 consecutive nucleotides, of the nucleotide sequence of exon F of the gene encoding myosin Va protein, or a sequence of one different nucleotide compared to such a fragment. This is because the RNAi process is sequence specific and high sequence homology is required for efficient RNAi. Advantageously, the sense strand of the RNA comprises a sequence identical to a fragment of 14 to 30 consecutive nucleotides, advantageously 15 to 29, 16 to 28, 17 to 27 or 18 to 25 consecutive nucleotides, of the nucleotide sequence of exon F of the gene encoding the myosin Va protein.

Myosin Va protein has been described in different species, particularly human, mouse and rat. In an advantageous embodiment, the nucleotide sequence of exon F of the gene encoding myosin Va protein is the sequence of exon F of the human gene encoding myosin Va protein represented by seq id NO: 1 is shown. The sequence of the human myosin Va protein is shown in FIG. 1A, with the portion corresponding to exon F (SEQ ID NO: 1) underlined.

All targeting sequences SEQ ID NO: 1 are included within the scope of the invention, advantageously 15 to 29, 16 to 28, 17 to 27, 18 to 25, 18 to 23 or 18 to 21 consecutive nucleotides. Advantageously, however, the targeted fragment of exon F of the myosin Va protein, i.e., the sequence included in the sense strand of the siRNA, is not a fragment having a characteristic structure in myosin Va protein mRNA or a fragment to which a regulatory protein can bind. The term "characteristic structure" as used in the present invention is understood to mean a stem or loop or hairpin-type structure that can be formed in the mRNA of the myosin Va protein. Several tissues provide different tools that are freely available on the internet (see table 1 below) and can be used to design sirnas targeting exon F of the human myosin Va protein.

TABLE 1 Internet site of siRNA selection tool

Tissue of	Internet address
		Ambion	http://www.ambion.com/techlib/misc/siRNA_design.html
Dharmacon	http://www.dharmacon.com/sidesign/
		Qiagen	http://www1.qiagen.com/Products/GeneSilencing/CustomSiRna/SiRnaDesigner.aspx
Emboss	http://bioweb.pasteur.fr/seqanal/interfaces/sirna.html
		Tuschl Laboratory	http://www.rockefeller.edu/labheads/tuschl/sirna.html
The Whitehead	http://jura.wi.mit.edu/bioc/siRNAext/

Most companies selling customized sirnas guarantee the effectiveness of the synthesized sirnas. Furthermore, the effectiveness of all sirnas targeting the exon F of the myosin Va protein can be demonstrated by different tests, in particular those detailed in examples 1 and 2.

The present inventors have synthesized and tested several specific sirnas targeting different fragments of exon F of the human myosin Va protein. In an advantageous embodiment of the siRNA according to the invention, the fragment of 14 to 30, advantageously 15 to 29, 16 to 28, 17 to 27, 18 to 25, 18 to 23 or 18 to 21 consecutive nucleotide sequences of the nucleotide sequence of exon F of the human myosin Va protein gene is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4. three sequences SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: the position of 4 is illustrated in detail in fig. 1B. In a particularly advantageous embodiment, a fragment of 14 to 30, advantageously 15 to 29, 16 to 28, 17 to 27, 18 to 25, 18 to 23 or 18 to 21 consecutive nucleotide sequences in the nucleotide sequence of exon F of the myosin Va protein gene consists of the nucleotide sequence SEQ ID NO: 3, and (3).

The siRNA according to the invention is a small double-stranded RNA having a sense strand and an antisense strand paired by Watson-Crick bonds, and wherein the sequence of the sense strand consists of or comprises 14 to 30, advantageously 15 to 29, 16 to 28, 17 to 27, 18 to 25, 18 to 23 or 18 to 21 consecutive nucleotide fragments of the nucleotide sequence of the myosin Va protein exon F, advantageously 15 to 29, 16 to 28, 17 to 27, 18 to 25, 18 to 23 or 18 to 21 consecutive nucleotide fragments of the nucleotide sequence of the myosin Va protein exon F.

Sirnas having a sequence consisting of 30 to 50% guanine and cytosine are known to be more effective than sequences having a higher proportion of guanine and cytosine. The siRNA according to the present invention therefore advantageously has a sequence of from 30 to 50% guanine and cytosine.

It is to be understood that an siRNA according to the present invention may equally comprise two complementary single-stranded RNA molecules, or a single-stranded RNA molecule, wherein the two complementary portions are paired by Watson-Crick bonds and covalently linked on one side by a hairpin structure (this is more specifically referred to as an shRNA, i.e., a "short hairpin RNA"), which may be considered a subclass of siRNA. Throughout the specification, the term siRNA should be understood in its broad sense, including shRNA, unless otherwise indicated. In an advantageous embodiment, the siRNA according to the invention comprises two complementary single stranded RNA molecules. In another advantageous embodiment, the siRNA according to the invention comprises or consists of a single molecule of single-stranded RNA, wherein the two complementary parts are paired by Watson-Crick bonds and covalently linked on one side by a hairpin structure, that is to say an shRNA.

In addition, the sense and/or antisense RNA strand may further comprise a 3' overhang of 2 to 4 nucleotides, particularly when the siRNA according to the invention comprises two complementary single stranded RNA molecules. The expression "3 'overhang fragment of 2 to 4 nucleotides" as used herein is understood to mean that 2 to 4 nucleotides are present in at least one strand of the RNA duplex without pairing with the complementary strand at the 3' distal end of said strand. The nucleotides used in the 3 ' overhang fragment may be natural nucleotides (ribonucleic acid or deoxyribonucleic acid), or modified nucleotides such as LNA (locked nucleic acid) which include a methylene bridge between the 2 ' and 4 ' positions of the ribose (Southchek J. et al, Nature.2004Nov 11; 432 (7014): 173-8). The 3' overhang fragment can also be subjected to various types of chemical modifications described in the following paragraphs for the sense RNA strand and/or antisense RNA strand of the siRNA according to the invention. Advantageously, the 3' overhang fragment consists of 2 nucleotides. In this case, the preferred sequence of the 3' overhang fragment is "TT" (where T represents deoxythymidine) or "UU" (where U represents uracil). Also advantageously, both complementary strands of the siRNA according to the invention include a 3' overhang. In this case, the length and sequence of the two 3' overhang fragments may be the same or different. Advantageously, the two complementary strands of the siRNA according to the invention each comprise a 3' overhang of 2 nucleotides having the sequence "TT". This includes the amino acid sequences of SEQ ID NOs: 2, SEQ ID NO: 3 or SEQ ID NO: 4 is shown in figure 1C and corresponds to a preferred embodiment of the invention. They correspond to:

-comprises the gene sequence SEQ ID NO: 2, siRNA MyoVa n °: 1, the strand having the following specific sequence:

sense strand: 5'-UGACCCUAAGAAGUAUCAATT-3' (SEQ ID NO: 9)

Antisense strand: 5'-UUGAUACUUCUUAGGGUCATT-3' (SEQ ID NO: 10);

-comprises the gene sequence SEQ ID NO: 3, siRNA MyoVa n °: 2, the strand having the following specific sequence:

sense strand: 5'-GUAUCAAUCAUAUCGGAUUTT-3' (SEQ ID NO: 11)

Antisense strand: 5'-AAUCCGAUAUGAUUGAUACTT-3' (SEQ ID NO: 12);

-comprises the gene sequence SEQ ID NO: 4, siRNA MyoVa n °: 3, the strand having the following specific sequence:

sense strand: 5'-UCAUAUCGGAUUUCCCUUUTT-3' (SEQ ID NO: 13)

Antisense strand: 5'-AAAGGGAAAUCCGAUAUGATT-3' (SEQ ID NO: 14).

When the siRNA according to the present invention is an shRNA consisting of two complementary parts paired by Watson-Crick bonds and covalently linked on one side by a hairpin structure, that is to say when the siRNA according to the present invention is an shRNA, said shRNA preferably comprises or consists of: 5' -CUCAAGAGA-3' (SEQ ID NO: 17), wherein the bold portions correspond to the two complementary portions paired by Watson-Crick bonds, corresponding to the sequences having the siRNA MyoVa n °: 2 sense strand and complementary strand thereof, and the gene sequence SEQ ID NO: 3, the portion that is not bold corresponds to a sequence having a hairpin structure linking two complementary strands.

Furthermore, in the siRNA according to the present invention, the sense RNA strand and/or the antisense RNA strand may further include at least one chemical modification in their sugar moiety, nucleobase or internucleotide backbone. Such modifications may be particularly useful in inhibiting degradation of the siRNA by ribozymes in vivo. Therefore, all chemical modifications that can improve the stability and in vivo bioavailability of the siRNA according to the present invention are included in the scope of the present invention. Among the advantageous modifications of the sugar moiety, particular mention must be made of modifications which occur at the 2 ' -position of the ribose, such as 2 ' -deoxy, 2 ' -fluoro, 2 ' -amino, 2 ' -thio or 2 ' -O-alkyl, in particular 2 ' -O-methyl, instead of the normal 2 on the ribonucleotide' -OH group, or the presence of a methylene bridge between the 2 ' and 4 ' ribose positions (LNA). As nucleobases, it is possible to use modified bases, such as in particular 5-bromo-uridine, 5-iodo-uridine, N³-methyl-uridine, 2, 6-Diaminopurine (DAP), 5-methyl-2 ' -deoxycytidine, 5- (1-propyne) -2 ' -deoxy-uridine (pdU), 5- (1-propyne) -2 ' -deoxycytidine (pdC), or a base conjugated to cholesterol. Finally, preferred modifications of the internucleotide backbone include the replacement of the phosphodiester groups in the backbone by phosphorothioate, methylphosphonate, phosphodiamide groups, or the use of a backbone (PNA, peptide nucleic acid) consisting of N- (2-aminoethyl) -glycine monomers linked by peptide bonds. It is clear that various modifications (bases, sugars, main chain) can be combined to obtain morpholino nucleic acids (bases fixed to the morpholino ring and linked by phosphodiamide groups) or PNAs (bases fixed to N- (2-aminoethyl) -glycine monomers linked by peptide bonds).

The sirnas according to the invention are "isolated", which means that they are not in the native state, but have been obtained by any method involving human intervention. Specifically, the siRNA according to the present invention is obtained by purifying an siRNA already present, by chemical synthesis, by amplifying a desired nucleotide sequence by Polymerase Chain Reaction (PCR), or by recombinant synthesis. Many companies also offer customized siRNA synthesis, particularly such companies as Eurogentec, Ambion, Dharmacon or Qiagen.

The invention also relates to the use of the siRNA according to the invention as a cosmetic. The expression "cosmetic" as used herein is understood to mean a substance that can maintain or improve the aesthetic appearance of the outside of the human or animal body.

The invention further relates to the use of the siRNA according to the invention as a medicament. Indeed, the use of siRNA according to the invention as a medicament is novel.

Another object of the invention is a composition comprising at least one siRNA according to the invention and an acceptable carrier. The term "acceptable carrier" as used herein means any cosmetically or pharmaceutically acceptable carrier known to those skilled in the art.

The compositions according to the invention are intended for the cosmetic and/or therapeutic treatment of the skin and are therefore preferably applied topically.

The compositions for topical application according to the invention may be formulated in any galenic form commonly used for topical application, such as, for example, aqueous solutions, white or colored creams, ointments, emulsions, lotions, gels, ointments, slurries, pastes, oil-in-water or water-in-oil emulsions, or foams. It can be applied to the skin in the form of an aerosol. It may also be in the form of a solid, powder or not, for example, in the form of a stick. It may also be in the form of a patch, pen, brush and applicator for topical application of print on the face or hands.

The composition according to the present invention may further comprise any carrier known to those skilled in the art, such that the delivery and bioavailability of the siRNA according to the present invention may be improved. Specific carriers that can be used with siRNA include, in particular, liposomes and peptides capable of crossing cell membranes (known as CPPs, i.e., "cell-penetrating peptides"). The term "liposome" as used herein is understood to mean an artificial lipid carrier having a membrane formed by one or several lipid bilayers and which has the ability to encapsulate and protect proteins and nucleic acids and to fuse with the cell membrane, thus allowing the encapsulated product to be transported into the cell. Thus the use of liposomes can protect the siRNA and facilitate their penetration into the cell. The term CPP as used herein is understood to mean a peptide capable of internalization and then reaching the cytoplasmic and/or nuclear compartment of a living cell. Examples of such CPPs include peptide permeants (penetraines), transporters (transportans), Tat, MAP, and SynB 1. Some particularly useful examples of CPPs are composed of virus-derived amphipathic peptides that interact directly with the nucleic acid to be delivered, so as to form nanoparticles that can diffuse through the plasma membrane. Thus conjugating the siRNA according to the invention to any CPP may also protect the siRNA and facilitate their entry into cells in vivo.

The composition according to the invention may also comprise one or more active substances aimed at improving the desired effect. In a particular embodiment, the composition according to the invention may further comprise, in particular, at least one other depigmenting agent. The term "depigmenting agent" as used herein is understood to mean any substance which acts directly on the melanocytes, or one of the steps of the melanin synthesis pathway, or the transport of melanosomes to the dendrites and transfer to keratinocytes by inhibiting activity. Among the known depigmenting agents suitable for incorporation into the composition according to the invention, mention may be made in particular of hydroquinone and its derivatives, retinol or tretinoin, ascorbic acid and its derivatives, placental extract, kojic acid, ferulic acid, arbutin, dihydroxybenzene derivatives (WO00/47045), guaiacol derivatives (WO00/47179), 4- (2, 3-dihydroxyphenyl) -cyclohexanol (WO00/56279), resorcinol derivatives (WO00/56702), phenol amides (WO 99/32077). Other depigmenting agents suitable for incorporation into the compositions according to the invention include antisense oligonucleotides or sirnas directed against genes other than the gene encoding the myosin Va protein: such as genes involved in coding for steps in the melanin synthesis pathway, or genes involved in the transport of melanosomes to dendrites and into keratinocytes, such as antisense oligonucleotides or siRNAs targeting tyrosinase (see WO01/58918 and WO2005/060536), the protein TRP-1 (see WO01/58918) or the protein PKC β 1 (see WO2005/073243), among others.

The compositions according to the invention may also comprise active substances or excipients commonly used in topical compositions for skin care, such as chemical and physical filters (e.g. octyl methoxycinnamate, butyl methoxydibenzoylmethane, titanium oxide and zinc oxide), anti-glycation and/or anti-oxidants (e.g. tocopherol and its derivatives, ergothioneine, thiotaurine, hypotaurine, aminoguanidine, thiamine pyrophosphate, pyridoxamine, lysine, histidine, arginine, phenylalanine, pyridoxine, adenosine triphosphate), anti-inflammatory agents (e.g. glycyrrhizic acid stearate), smoothing agents and mixtures thereof, preservatives (antibacterial or antifungal), moisturizers, pH regulators, keratolytic and/or keratolytic agents (e.g. salicylic acid and its derivatives), vitamins, thickeners, emollients, mineral water, surfactants, polymers, silicone oils, vegetable oils, essential oils, fragrances, colorants or pigments, and the like.

The invention also relates to the use of at least one siRNA or composition according to the invention as a cosmetic for depigmenting or bleaching the skin. The siRNA and composition according to the present invention can be used as cosmetics for depigmentation or bleaching of the skin, in order to reduce the pigmentation of the whole skin, or of hyperpigmented parts, regardless of the origin of the hyperpigmentation of said areas. This is because such use makes it possible to make the pigmentation of the skin more uniform, thus improving its appearance. Furthermore, some people want a lighter overall pigmentation even in the absence of hyperpigmented parts, which can be obtained by the cosmetic use according to the invention.

The invention further relates to the use of at least one siRNA or composition according to the invention for the preparation of a medicament for the treatment or prevention of skin hyperpigmentation, in particular epidermal hyperpigmentation, melanosis and post-inflammatory hyperpigmentation. Hyperpigmentation can result from a number of different diseases. Especially mentioned are melanosis of the face and neck, such as chloasma, Riehl's melanosis, reticular pigmentary dermatosis of the face and neck, or hereditary sclerosing dermatosis; freckle; cafe-au-lait plaques; sutton's nevus; hyperpigmentation due to metabolism, such as hemochromatosis, edison's disease, cushing's syndrome or hyperthyroidism; and hyperpigmentation due to extrinsic causes, such as hyperpigmentation caused by drugs (such as chlorpromazine, phenothiazine, hydantoin, inorganic arsenic, antimalarial drugs) or heavy metals (silver, gold, mercury); post-inflammatory hyperpigmentation induced following trauma, eczema, lichen simplex chronicus, lupus erythematosus and skin disorders including pityriasis rosea, psoriasis, dermatitis herpetiformis, fixed pigmented erythema and photo-contact dermatitis; Rothmund-Thomson syndrome; benign acanthosis nigricans or malignant acanthosis nigricans. The use of the siRNA or the composition according to the invention for the manufacture of a medicament makes it possible to treat these different pathologies and to make the pigmentation sites resulting from these diseases disappear. It can also prevent hyperpigmentation caused by these various diseases.

The invention also relates to a cosmetic treatment process for depigmenting and bleaching the skin, comprising the topical application of a composition according to the invention.

The invention further relates to a method for the therapeutic treatment of skin hyperpigmentation comprising the topical application of a composition according to the invention.

The following examples illustrate the invention but are not to be construed as limiting in any way.

Drawings

FIG. 1.3 design of siRNA targeting exon F of human myosin Va protein. A. Nucleotide coding sequence of the transcript of human myosin Va protein including exon F (including exon ADCDEF). The portion corresponding to exon F is underlined. B. Details of the sequence of exon F. The positions corresponding to the three selected siRNAs are underlined (siRNA MyoVa n °: 2(SEQ ID NO: 3)) or underlined (siRNAMYOYYVa n °: 1(SEQ ID NO: 2) and (MyoVa n °: 3(SEQ ID NO: 4)). C.3 Structure of siRNAs targeting the human myosin Va protein.

FIG. 2. MyoVa n ℃ with 2. mu. MsiRNA: 2(SEQ ID NO: 3) reduction in the amount of transcript products of myosin Va protein including exon F in electroporated PHEM cells. siRNA targeting GAPDH gene, or siRNA MyoVa n °: 2(SEQ ID NO: 3) electroporates PHEM cells. The effect of different sirnas at RNA level was measured by QPCR. A. Relative expression level of GAPDH gene as a function of electroporated siRNA. B. Expression level of the transcript of myosin Va protein including exon F as a function of electroporated siRNA.

FIG. 3. specificity of the amount of transcript of myosin Va protein including exon F was reduced, while the transcript not including exon F was not reduced. A. Relative expression levels of transcripts of myosin Va protein including exon F as a function of electroporated siRNA. B. Relative expression levels of all transcripts of the myosin Va protein detected by the sphere segment (GP) as a function of electroporated siRNA.

FIG. 4. for siRNA MyoVa n °: 1(SEQ ID NO: 2) and siRNA MyoVa n °: 2(SEQ ID NO: 3) determines the minimum concentration with the greatest efficacy. Different concentrations of siRNA targeting exon F of human myosin Va protein were determined, siRNA: MyoVa n °: 1(SEQ ID NO: 2) and B.MyoVa n °: 2(SEQ ID NO: 3). For MyoVa n °: 2, given as the mean with the standard deviation.

FIG. 5 Effect of SiRNA concentration on cell viability. For different concentrations of MyoVa n °: 2(SEQ ID NO: 3) the relative expression level and cell viability of the transcripts comprising exon F were determined in parallel. siRNA concentrations are given on the X-axis and relative expression levels and cell viability of transcripts including exon F are given on the Y-axis, with mean and standard deviation expressed as histograms (grey squares) and lines, respectively.

Figure 6. efficacy of siRNA targeting exon F of human myosin Va protein over time. Electroporation at 0.5. mu.M siRNAYVa n °: 1(SEQ ID NO: 2) or MyoVa n °: 2(SEQ ID NO: 3) at different times. MyoVa n °: 1(SEQ ID NO: 2). MyoVa n °: 2(SEQ ID NO: 3).

Figure 7. principle of in vitro testing of inhibition of melanosome transfer from melanocytes to keratinocytes by siRNA targeting exon F of human myosin Va protein.

FIG. 8. in the presence of 25nM or 10nM MyoVa n °: 2(SEQ ID NO: 3, exF) or BLOCK-iT^TMInhibition of the transcript including exon F (MyoVaexF transcript) and the total transcript of myosin Va protein detected in GP fraction (MyoVa GP transcript) in PHEM transfected with fluorescent oligonucleotides (sinEG) and 12 or 18. mu.l HiPerFect reagentAnd (6) evaluating. Myova exF transcript, 12 μ l HiPerFect reagent. Myovagp transcript, 12 μ l HiPerFect reagent. Myova exF transcript, 18 μ Ι hiperfect reagent. Myova GP transcript, 18 μ l HiPerFect reagent.

FIG. 9 evaluation of inhibition at RNA level and protein level during long-term experiments (D2, D4, D6, D8 after transfection). A. Analysis by quantitative real-time PCR at RNA level. B. Analysis at the protein level by western blot analysis using antibodies specific against the myosin Va protein subtype including exon F. C. Quantification was performed using Quantity One software, normalized by tubulin, all based on raw data.

FIG. 10. with siRNA MyoVa n °: 1(SEQ ID NO: 2, exF) or BLOCK-iT^TMEvaluation of inhibition of transcription products including exon F in PHEM transfected with fluorescent oligonucleotide (SinEG) and 18. mu.l HiPerFect reagent.

Examples

Example 1

Design, synthesis and research of 3 kinds of siRNA targeting human myosin Va protein exon F

1.13 design and Synthesis of siRNA

The sequence of the splice variant of the human myosin Va protein including exon F (actually including exon ABCDEF) is shown in figure 1A.

Within exon F, three sirnas targeting human myosin Va protein exon F were designed and synthesized using the siRNA design and synthesis service of Eurogentec. The portions of exon F specific for these sirnas are shown in fig. 1B.

These three siRNAs were synthesized with a 3' overhang of the sequence "TT" as shown in FIG. 1C. These three sirnas are referred to hereinafter as MyoVa n °: 1 (sense strand includes SEQ ID NO: 2), MyoVa n °: 2 (sense strand includes SEQ ID NO: 3), or MyoVa n °: 3 (sense strand includes SEQ ID NO: 4), the SEQ ID numbering shown in parentheses corresponds to the sequence of the human myosin Va protein exon F fragment included in the siRNA sense RNA strand.

1.2 testing of siRNA efficacy following electroporation into Primary Human Epidermal Melanocytes (PHEM)

1.2.1 selection of electroporation procedures

Primary Human Epidermal Melanocytes (PHEM) were grown in Ham's F10 medium (Gibco, Invitrogen Ltd, Paisley, UK) supplemented with 2.5% Fetal Calf Serum (FCS), 1% Ultroser, 5ng/ml basic fibroblast growth factor (bFGF), 10ng/ml endothelin-1 (ET-1), 0.33nM bile toxin (CT), 0.033mM isobutyl-methyl-xanthine (IBMX) and 5.3nM 12-O-tetracaprylphorbol-13-acetate (TPA). Cells were grown to 40-70% confluence and used between passages 2 to 5.

In the first case, plasmid pMAX-GFP (3 μ g) was used to test three different procedures for melanocyte electroporation, namely procedures U16, U20 and U24. Electroporation was performed using 500000 cells. The NHEM-NeoNucleofector kit for normal-neonatal human epidermal melanocytes (Amaxa, # VDP-1003) was used for all electroporation.

The electroporated cells in the T25 flasks were evaluated after 24-48 hours under an Arcturus microscope (LCM) equipped with a fluorescence filter capable of detecting the green fluorescence signal emitted by the pMAX-GFP plasmid.

Electroporated cells were also grown on glass slides. After 24 hours of electroporation, cells were fixed in ice-cold methanol and mounted on a slide with mounting fluid (DAKO). Cells involved in expressing the green fluorescent signal were analyzed under a zeiss fluorescent microscope.

The results obtained with live cells showed that an equal amount of dead cells was observed in any one electroporation procedure, but the most efficient electroporation was obtained using the U24 procedure. The results obtained using fixed cells confirmed that the most efficient electroporation was obtained by the U24 procedure. Therefore, the U24 procedure was used in all other experiments thereafter.

1.2.2siRNA MyoVan °: 2(SEQ ID NO: 3) preliminary analysis of efficacy

500000 cells were electroporated using a pMAX-GFP (3 μ g) plasmid and the following siRNA duplexes: positive control (3. mu.g or 2. mu.M, Ambion) targeting FAM-labeled GAPDH, negative control BLOCK-iT^TMFluorogenic Oligo (Invitrogen) and the exon F-specific siRNA MyoVan °: 2(SEQ ID NO: 3) duplex. The U24 procedure was used for electroporation and cells were collected 24 hours after electroporation.

The RNA was then analyzed by real-time quantitative RT-PCR (QPCR). Briefly, after RNA extraction, samples were treated with DNase and cDNA was synthesized using iScript reverse transcriptase (Biorad). QPCR primers (sense primer: 5 'CAGCCTGCAGCACGAGATC 3', SEQ ID NO: 5, antisense primer: 5 'TCTTAGGGTCATCTGCATATAATTCCT 3', SEQ ID NO: 6) and GAPDH for the myosin Va protein exon F were designed using PrimerExpress 2.0 software (Applied Biosystems) with only the minimum size limit (75bp) changed for the amplicon using Taqman default parameters. Two optimal step SYBR green | RT-PCR tests were used to determine the relative levels of gene expression. Ct comparison method was used for quantification. The PCR reaction was performed in an ABI Prism 7000 sequencing system (applied biosystem). To correct for differences in extracted RNA content and cDNA synthesis efficiency, the relative levels of gene expression were normalized to the geometric mean of the three housekeeping genes (RPL13a, SDHA and UBC) that have been used for melanocyte analysis (Vandesoppele J et al, Genome biol.: 3: research 34.001-research0034.001, 2002).

The results obtained show that the siRNA used leads to a reduction in RNA expression by the target gene after electroporation in PHEM cells. In fact, a 75% reduction in GAPDH was observed (fig. 2A) and a 62% reduction in the transcript of the myosin Va protein including exon F (fig. 2B) compared to the negative control.

1.2.3 with siRNAs MyoVa n °: 1(SEQ ID NO: 2), MyoVan °: 2(SEQ ID NO: 3) or Myo Van °: 3(SEQ ID NO: 4) expression of myosin Va protein Gene transcript in PHEM cells after electroporation

siRNA targeting GAPDH (1 μ M), negative control siRNA, or exon-specific three sirnas MyoVa n ° of the myosin Va protein gene with Nucleofector reagent alone (MOCK): 1(SEQ ID NO: 2), MyoVa n °: 2(SEQ ID NO: 3) or MyoVa n °: 3(SEQ ID NO: 4) of 500000PHEM cells were electroporated. Electroporation was performed using the U24 procedure, and cells were collected 48 hours after electroporation.

The RNA was then analyzed by PCR as described previously, with the transcript including exon F (exon F detection) or all transcripts detected by the Globular Portion (GP) of the myosin Va protein. QPCR primers for the Globular Portion (GP) of the myosin Va protein (sense primer: 5 'GCAGTCAATTTGATTCCAGGATT 3', SEQ ID NO: 7; antisense primer: 5 'TGATCATCATTCAGGTAGTCAGCAT 3', SEQ ID NO: 8) were designed as described previously using Primerexpress 2.0(Applied Biosystems) software.

For transcripts that include exon F of the myosin Va protein, for siRNAs MyoVan °: 1(SEQ ID NO: 2), MyoVa n °: 2(SEQ ID NO: 3) and MyoVa n °: 3(SEQ ID NO: 4) observed inhibition of 85%, 94% and 60%, respectively. MyoVa n °: 2(SEQ ID NO: 3) gave the greatest inhibition.

Also for all transcripts of the myosin Va protein detected using Globular Protein (GP), for siRNA MyoVa n °: 1(SEQ ID NO: 2), MyoVa n °: 2(SEQ ID NO: 3) and MyoVa n °: 3(SEQ ID NO: 4) were observed to inhibit 39%, 56% and 0%, respectively. This reduced inhibition can be explained in particular by the fact that: the siRNA used specifically targets exon F and therefore does not result in the breakdown of transcripts that do not include exon F.

1.2.4 conclusion

These preliminary results clearly show that the use of three synthetic sirnas can specifically and significantly reduce the content of human myosin Va protein transcripts that include exon F without affecting transcripts that do not include exon F.

Use siRNA MyoVa n °: 2(SEQ ID NO: 3) gave the most significant results.

1.3 optimization of siRNA concentration

1.3.1 determination of minimum concentration with maximum efficacy

To determine the minimum siRNA concentration for maximum efficacy, different concentrations ranging from 0.05 to 2 μ M were tested using the same experimental protocol as previously described. Negative control siRNA and GAPDH-targeting siRNA at 1 μ M concentration were used.

Cells were analyzed using the U24 procedure and 48 hours after electroporation.

The transcript including exon F was then analyzed by QPCR as described previously (specific exon F detection).

For sirnamyva n °: 1(SEQ ID NO: 2), vs 0.05; 0.1; 0.25; at concentrations of 0.5 and 1. mu.M, reductions of 47.5%, 31%, 73%, 73% and 79% of the transcript including exon F were observed, respectively (FIG. 4A). Thus the maximum effect was obtained with a concentration of 1 μ M, but almost the same effective inhibition was obtained with concentrations of 0.25 and 0.5 μ M.

For siRNA MyoVa n °: 2(SEQ ID NO: 3), a large decrease was observed at all concentrations tested (FIG. 4B). The greatest effect was observed for a concentration of 1 μ M, but the inhibition obtained with a concentration of 0.5 μ M was almost equally large.

Therefore, the optimal siRNA concentration is about 0.5-1. mu.M.

1.3.2 Effect of siRNA concentration on cell viability

siRNA MyoVa n °: 2(SEQ ID NO: 3) the effect of siRNA concentration on cell viability was analyzed. For this purpose, the same concentration range (0.05 to 2. mu.M) was tested in parallel using the MTS test (Promega Benelux), which determines the number of viable cells. Cells were analyzed 48 hours after electroporation using the U24 procedure.

The results are given in fig. 5 and show a significant decrease in cell viability (about 20%) only for siRNA concentrations of 1 or 2 μ M. Below 1 μ M, cell viability was greater than 90%.

1.3.3 conclusion

These results indicate that the effect of sirnas targeting exon F of the myosin Va protein is dose dependent and that maximal effect can be obtained for concentrations comprised between 0.5 and 1 μ M.

Furthermore, below 1 μ M, the presence of siRNA did not affect the viability of PHEM cells.

Therefore a concentration of 0.5 μ M appears to be the best, as this concentration combines the highest efficacy with the highest viability. This concentration was used in all experiments described below.

1.4 analysis over time

To analyze the effect of siRNA according to the invention over time, the results were analyzed by QPCR analysis with 0.5 μ M siRNA MyoVan °: 1(SEQ ID NO: 2) or siRNA MyoVa n °: 2(SEQ ID NO: 3) electroporated PHEM cells. Cells treated with positive or negative controls (0.5 μ M) were analyzed 48 hours after electroporation.

For siRNA MyoVa n °: 1(SEQ ID NO: 2), the reduction of the transcript including exon F was maximal at 24h (over 90%), then reduced at 48h (65%) and 72h (60%), and the expression level returned to normal at 96h (see FIG. 6A).

For siRNA MyoVa n °: 2(SEQ ID NO: 3), a more than 85% reduction in transcript was observed at 24 and 48 hours, then at 72 and 96 hours, while remaining significant compared to the negative control (FIG. 6B).

Thus, these results indicate that the maximum effect is obtained 24-48 hours after electroporation and siRNA MyoVa n °: 2(SEQ ID NO: 3) exceed the efficacy of siRNA MyoVan °: 1(SEQ ID NO: 2).

1.5 conclusion

Thus, the results presented above clearly indicate that the amount of transcription product of myosin Va protein including exon F can be significantly reduced using an siRNA targeting said exon F. Three different siRNAs were tested and each of these reduced the amount of transcript product of myosin Va protein including exon F. sirnamyva n °: 1(SEQ ID NO: 2), in particular siRNA MyoVa n °: 2(SEQ ID NO: 3), are particularly effective.

Furthermore, the effect of sirnas targeting exon F of the myosin Va protein was dose-dependent and the maximum effect could be obtained at concentrations that did not affect cell viability.

Efficacy varied with time and reached a maximum 24-48 hours after electroporation.

Example 2

Effect of the Presence of siRNA targeting exon F of human myosin Va protein on the transfer of melanosomes from melanocytes to keratinocytes in vitro

2.1 test principle

To test the effect of siRNA targeting exon F of the human myosin Va protein according to the invention on melanin transfer from melanocytes to keratinocytes, an in vitro test was developed.

The principle of this test is shown in fig. 7.

Using 0.1. mu.M; 0.25 μ M; and 0.5 μ M siRNA targeting exon F of the human myosin Va protein electroporates normal human Melanocytes (MC), or no electroporation. Then, the electroporated (siRNA MC) or non-electroporated (MC) melanocytes are grown in the presence of 2[2-14C]Thiouracil is introduced into melanin produced by MC in TFA-rich medium, so that a composition containing thiouracil can be obtained¹⁴C-labelled Melanin (MC)^*Or siRNA MC^*) The melanocyte of (1).

Mixing MC with^*Or siRNA MC^*Placed in a culture in serum-free keratinocyte medium (K-SFM, Gibco) at low calcium concentration, containing keratinocytes that have undergone 1 or 2 passages in this same medium. Mixing MC with^*Or siRNA MC^*Was placed in culture at a ratio of 1 to 3 with KC.

Optionally UV irradiation of the coculture is then used to stimulate melanosomes from the MC^*Or siRNA MC^*Transfer to KC. This step is performed because UV irradiation is known to stimulate the transfer of melanosomes from melanocytes to keratinocytes. Following co-culture and optional UV irradiation, keratinocytes¹⁴C radioactivity (KC)^*) Is and from MC^*Or siRNAMC^*The transferred melanin content is proportional.

KC was then purified by negative selection using MACS technology (Miltenyi Biotech)^*anti-CD 117PE antibody that binds melanocytes and anti-PE beads was used.

Finally, the purified KC is measured^*Is/are as follows¹⁴C, radioactivity.

2.2 results

The results obtained indicate that UV irradiation can effectively stimulate the transfer of melanin from melanocytes into keratinocytes without electroporation using siRNA targeting exon F of the human myosin Va protein.

In contrast, when electroporation was performed using siRNA targeting exon F of the myosin Va protein, UV irradiation was followed by transfer to keratinocytes¹⁴The content of C radioactive melanin is reduced.

2.3 conclusion

These results clearly demonstrate that the use of siRNA targeting exon F of the myosin Va protein makes it possible to reduce the transfer of melanin from melanocytes to keratinocytes, thus confirming the strategy used by the inventors to reduce skin pigmentation.

Example 3

Efficacy of siRNA targeting exon F of myosin Va protein after transfection into Primary Human Epidermal Melanocytes (PHEM)

3.1 transfection of PHEM with siRNA targeting myosin Va protein exon F

3.1.1 optimization of transfection with HiPerFect transfection reagent

Using siRNA MyoVa n °: 2(SEQ ID NO: 3) and BLOCK-iT as a negative control^TMFluorogenic oligo (Invitrogen) was transfected into 200000PHEM (in 0.5ml medium/6 well plates). For each transfection, 25nM or 10nM siRNA was used in combination with 12 or 18 μ l HiPerFect reagent (Qiagen).

Assessment of inhibition of RNA levels by quantitative real-time PCR as described in example 1.2.2 paragraph above, simply for transcripts that include exon F (MyoVa exF transcripts), or for all transcripts of myosin Va protein detected in the GP fraction (MyoVa GP transcripts).

The results obtained using 12 or 18 μ l of HiPerFect reagent for MyoVa exF or MyoVa GP transcripts are shown in figure 8 and indicate:

for MyoVa exF transcripts using 12 μ Ι HiPerFect reagent, observed for siRNA MyoVa n ° at 25nM and 10 nM: 2(SEQ ID NO: 3) were 80% and 85% inhibition, respectively (FIG. 8A).

For MyoVa GP transcripts using 12 μ l HiPerFect reagent, MyoVa n ° -for sirnas of 25nM and 10nM were observed: 2(SEQ ID NO: 3) were each 50% and 60% inhibition (FIG. 8B).

For MyoVa exF transcripts using 18 μ Ι HiPerFect reagent, we observed siRNAMyoVa n ° -for 25nM and 10 nM: 2(SEQ ID NO: 3) were 90% and 85% inhibition, respectively (FIG. 8C).

For MyoVa GP transcripts using 18 μ l HiPerFect reagent, MyoVa n ° -for sirnas of 25nM and 10nM were observed: 2(SEQ ID NO: 3) were 63% and 58% inhibition, respectively (FIG. 8D).

3.1.2 conclusion

These results show that by using siRNA MyoVa n °: 2(SEQ ID NO: 3) and HiPerFect reagent transfected PHEM significantly and specifically suppressed the amount of transcript of myosin Va protein including exon F. 25nM siRNAYoVa n °: 2(SEQ ID NO: 3) and 18. mu.l HiPerFect reagent gave the most effective inhibition.

These optimal conditions were used in all experiments in example 3 below.

3.2 with siRNA MyoVan °: 2(SEQ ID NO: 3) evaluation of inhibition obtained after transfection of PHEM

3.2.1 evaluation of inhibition at RNA and protein levels during Long-term experiments

With 18. mu.l HiPerFect reagent and 25nM siRNA MyoVa n °: 2(SEQ ID NO: 3) or BLOCK-iT^TMFluorecent oligo (negative control) was transfected with 800000PHEM (in 2.5ml medium/60 well plates). The effect of inhibition was evaluated over a period of 8 days. 1/3 for extracting RNA, 2/3 for purifying cell lysates.

Evaluation of inhibition at RNA level of transcripts comprising exon F (MyoVa exF transcripts) by real-time quantitative PCR measurements as described in example 1.2.2 paragraph above.

To correlate the inhibition observed at the RNA level with expression at the protein level, polyclonal antibodies specific for the subtype of myosin Va protein including exon F were developed by Eurogentec. Purified antibodies were tested for reactivity and specificity by western blot analysis and immunohistochemistry.

Cell lysates were then obtained during the course of the experiment by western blot analysis.

RNA level

The results are given in fig. 9A and show that for different times of MyoVa exF transcript a decrease of 72%, 81%, 78% and 82% was observed compared to the negative control (each at D2, D4, D6 and D8).

Protein level

The results of the analysis of the cell lysates by western blot are given in fig. 9B and C, each showing the raw results of the western blot analysis and the quantification results obtained using Quantity One software (Biorad), normalized with tubulin.

The results show that for different times of MyoVa exF transcript a 35%, 40%, 70% and 75% reduction was observed compared to the negative control (at D2, D4, D6 and D8 respectively).

3.2.2 measurement of MyoVa n °: 2(SEQ ID NO: 3) caused inhibitory Effect on the location/distribution of melanosomes in PHEM

The above shows that MyoVa exF transcripts are involved in the capture of melanosomes in the subcortical actin network. Thus, inhibition of MyoVa exF transcript may lead to a decrease in melanosome capture distal to the melanocyte dendrites and possibly to perinuclear accumulation of melanosomes.

To test this hypothesis, 25nM siRNA MyoVa exF n ° was performed on 300000PHEM coated on circular slides: 2(SEQ ID NO: 3) and 25nM negative control siRNA. PHEMs at different times post-transfection were fixed in 3% paraformaldehyde (day 3, day 6 and day 8).

Then, for tissue immunochemical experiments, antibodies specific for exon F of The myosin Va protein (dilution 1/500) (see above) were used in combination with The monoclonal antibody NKI-beteb (dilution 1/40) against (pre) -melanosome protein silver (sandwich b.v., Uden, The Netherlands). Analysis was then performed by confocal microscopy.

As a result:

3 days after transfection

For PHEM transfected with negative control siRNA and with siRNA MyoVa exF n °: 2(SEQ ID NO: 3), NO difference in melanosome distribution (present in the periphery and distal to the dendrites) was observed.

6 days after transfection

Co-labeling with anti-NKI-beteb antibodies and anti-myosin Va exon F antibodies showed treatment with sirnamyva exF n °: 2(SEQ ID NO: 3) Change in melanosome distribution in transfected PHEM: melanosomes are mainly located in the perikaryocyte region. Furthermore, no or weak markers for the myosin Va protein isoform including exon F are present at the distal end of the dendrites.

8 days after transfection

The same results as on day 6 were observed.

Conclusion

anti-NKI-beteb antibodies and anti-myosin Va exon F antibodies were used with sirnamyva exF n °: 2(SEQ ID NO: 3) or control siRNA transfected PHEM immunohistochemical analysis allowed visualization of melanosome intracellular transport (via NKI-beteb) and also detection of expression of isoforms of myosin Va protein including exon F.

The results obtained can be concluded as follows: inhibition of the myosin Va protein isoform including exon F resulted in an abnormal distribution of melanosomes, that is to say a perinuclear distribution, in PHEM 6 to 8 days after transfection, rather than the peripheral location and distal dendrites where the negative control was present.

3.3 confirmation of MyoVan °: 1(SEQ ID NO: 2) inhibitory Effect of MyoVaexF transcript after PHEM transfection

In these experiments, different sirnas, sirnamyva n °: 1(SEQ id no: 2) to demonstrate that inhibition is truly specific for the exon F of the myosin Va protein and not for specific artifacts.

3.3.1 evaluation of inhibition at RNA and protein levels during Long-term experiments

Using siRNA MyoVa n °: 1(SEQ ID NO: 2) replacement of siRNA MyoVa n °: 2(SEQ ID NO: 3) the same experiment as in paragraph 3.2.1 was repeated.

The results for RNA levels are given in fig. 10 and show a reduction of 58%, 59%, 41% and 51% at different time points (each at D2, D4, D6 and D8).

In addition, the results obtained at the protein level correlated with those obtained for RNA (data not shown).

3.3.2 measurement of MyoVa n °: 1(SEQ ID NO: 2) caused inhibitory Effect on the location/distribution of melanosomes in PHEM

Using siRNAYova n °: 1(SEQ ID NO: 2) substituted for siRNAYOYVa n °: 2(SEQ ID NO: 3) the same experiment as in paragraph 3.2.2 was repeated.

And sirnamyva n °: 2(SEQ ID NO: 3), as compared to a negative control, with a SiRNAMYOVA n °: the results obtained on day 8 after transfection with 1(SEQ ID NO: 2) indicated the perinuclear distribution of melanosomes in PHEM (data not shown).

3.3.3 conclusion

Although siRNA exF MyoVa n °: 1(SEQ ID NO: 2) inhibition of a subtype of myosin Va protein (MyoVa exF transcript) including exon F NO siRNA exF MyoVa n °: 2(SEQ ID NO: 3) but using a siRNAYOYoVa n °: 1(SEQ ID NO: 2) still significantly reduced the expression of MyoVa exF transcript, both at the RNA and protein levels.

Furthermore, immunohistochemical analysis of the fixed transfected PHEMs revealed the location distribution and transport of melanosomes in the PHEMs. Therefore, the following conclusions are drawn: from using sirnamyva exF n °: 1 and 2(SEQ ID NOS: 2 and 3) the inhibition of the subtype of myosin Va protein including exon F and the observed phenotypic effects are specific and not artifacts.

Example 4

Induction of phenotypic effects by long-term inhibition of the transcript of the myosin Va protein including exon F (Myo Va exF transcript)

To obtain long-term stable inhibition of the myosin Va protein subtype, including exon F, in PHEM, lentiviral vectors were synthesized that expressed short hairpin RNAs (or shrnas) against exon F of myosin Va protein.

4.1 materials and methods

From the most effective siRNA inhibiting MyoVa exF, i.e. siRNA MyoVa n °: 2 (including the RNA sequence having the gene sequence SEQ ID NO: 3), and a shRNA sequence was developed in the siRNA sequence. More specifically, a double-stranded oligonucleotide consisting of the following sequence was synthesized.

-sense strand: 5' -GATCCTCAAGAGA-3’(SEQ ID NO：15)

-the antisense strand: 5' -AGCTTCTCTTGAG-3’(SEQ ID NO：16)

Wherein:

bold portions correspond to the two shRNA complements paired by Watson-Crick bonds, corresponding to the sirnas MyoVa n °: 2 sense strand of SEQ ID NO: 3 and the complementary sequence thereof,

the normal-type sequence portion corresponds to a sequence having a hairpin structure connecting two complementary strands.

The underlined part corresponds to the 5' overhang fragment, so that the double-stranded oligonucleotide can be cloned in an expression vector, and

the italic part corresponds to the transcription termination sequence initiated by the rnapeliii type H1 promoter.

The double stranded oligonucleotide is then cloned into a lentiviral vector under the control of the rnapeliii H1 promoter.

This allows the production of shrnas with the following sequences in transfected cells: 5' -CUCAAGAGA-3' (SEQ ID NO: 17), wherein the bold portions correspond to the two complementary portions paired by Watson-Crick bonds, corresponding to the sequences having the siRNA MyoVa n °: 2 sense strand of SEQ ID NO: 3 and the complement thereof, the normal-type portion corresponding to a sequence having a hairpin structure connecting two complementary strands.

Lentiviral vectors expressing the hybrid shRNA were also generated and used as negative controls. In this case, the sequences of the sense and antisense strands of the oligonucleotides cloned in the lentiviral vector under the control of the rnapeliii type H1 promoter are as follows:

-sense strand: 5' -GATCCTCAAGAGA-3’(SEQ ID NO：18)

-the antisense strand: 5' -AGCTTCTCTTGAG-3’(SEQ ID NO：19)

Wherein:

bold portions correspond to the two shRNA complements paired by Watson-Crick bonds, corresponding to the sirnas MyoVa n °: 2 sense strand of SEQ ID NO: 3 and the complementary sequence thereof,

the normal-type sequence portion corresponds to a sequence having a hairpin structure connecting two complementary strands.

The underlined part corresponds to the 5' overhang fragment, so that the double-stranded oligonucleotide can be cloned in an expression vector, and

the italic part corresponds to the transcription termination sequence initiated by the rnapeliii type H1 promoter.

This makes it possible toTo produce shRNA in transfected cells with the following sequences: 5' -CUCAAGAGA-3' (SEQ ID NO: 20) where the bold part corresponds to the two shRNA complements paired by Watson-Crick bonds, (corresponding to the mismatched bases of the RNA sequence of the gene sequence SEQ ID NO: 3 with the siRNA MyoVan °: 2 sense strand and its complement), and the normal part corresponds to the sequence with the hairpin structure linking the two complementary strands.

4.2 transduction efficiency

Transduction efficiency in PHEM was determined by FACS analysis based on the presence of GFP marker in the lentiviral vectors described above.

Transduction of 100000PHEM with 10 multiplicity of infection (MOI) lentivirus resulted in a transduction efficiency of 90% after two rounds of infection.

4.3 evaluation of inhibition at RNA level and protein level

To evaluate the inhibitory effect at RNA level and protein level, the RNA level and protein level were evaluated using a pcr assay comprising shrnamyva exF n °: 2 or promiscuous shRNA (negative control) were transduced with 600000PHEM using a lentiviral vector at 10 MOI.

The results show that at RNA level and protein level, compared to negative controls, with a protein comprising shRNA MyoVa exF n °: 2 the subtype of myosin Va protein that includes exon F in cells transduced with the lentiviral vector was greatly suppressed.

4.4 inhibitory Effect on melanosome transport

Subsequently, a treatment with a composition comprising shRNA MyoVa exF n °: immunohistochemical analysis of lentiviral vector transduced PHEM with either 2 or a promiscuous shRNA (negative control). The results show that compared to negative controls, with a vector comprising shRNA MyoVa exF n °: 2 in PHEM transduced with the lentiviral vector of (a).

These results confirm the results previously observed in the inhibition of MyoVa exF transcripts using synthetic sirnas.

4.5 inhibitory Effect of MyoVa exF transcript on transfer of melanin from melanocytes to keratinocytes in a 3D model of reconstituted epidermis

With expression shRNA MyoVa exF n °: 2 transduces 50000 PHEM. These stably transduced PHEMs were introduced with 500000 keratinocytes into a reconstructed skin model. PHEM stably transduced with lentiviral vectors expressing promiscuous shRNA was used as a negative control. The reconstructed skin is then irradiated with UV light simulating sunlight or without irradiation.

Visual observation of the reconstituted skin showed that included the expression of shRNA MyoVa exF n °: 2, no increase in pigmentation was observed after irradiation, in contrast to skin comprising PHEMs transduced with lentiviral vectors expressing promiscuous shRNA. Quantitative assessment of pigmentation by Fontana-Masson staining and digital color imaging analysis then allowed these observations to be confirmed.

The distribution of melanosomes in the melanocytes and keratinocytes was then examined by electron microscopy. These analyses showed that the transfer of pigment from melanocytes to keratinocytes was stimulated in reconstituted skin irradiated with UV and comprising PHEM transduced with lentiviral vectors expressing hybrid shRNA, compared to the same skin without irradiation. In contrast, irradiated with UV and included MyoVa exF n ° -expressing shRNA: 2, only a reduction in melanin transfer was observed in reconstituted skin of PHEM transduced with lentiviral vector compared to non-irradiated control skin.

4.6 conclusion

Thus, the results indicate that stable transfection of PHEM with lentiviral vectors expressing shRNA specifically targeting human myosin Va protein exon F can significantly interfere with the number of MyoVa exF transcripts and myosin Va protein isoforms including exon F, as well as with the transport of melanosomes and transfer from melanocytes to keratinocytes.

Sequence listing

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Claims (15)

1. An isolated siRNA comprising a sense RNA strand and a complementary antisense RNA strand together forming an RNA duplex, wherein said sense RNA strand comprises a sequence of at most one different nucleotide compared to a fragment of 14 to 30 consecutive nucleotides of the exon F nucleotide sequence of the gene encoding myosin Va protein.

2. siRNA according to claim 1, characterized in that said sense RNA strand comprises a sequence identical to a fragment of 14 to 30 consecutive nucleotides of the exon F nucleotide sequence of the gene encoding the myosin Va protein.

3. siRNA according to claim 1 or 2, characterized in that the exon F nucleotide sequence of the myosin Va protein encoding gene is the human sequence SEQ ID NO: 1.

4. siRNA according to claim 3, characterized in that said 14 to 30 contiguous nucleotide fragments in the exon F nucleotide sequence of the human myosin Va protein-encoding gene are encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

5.siRNA according to claim 4, characterized in that said 14 to 30 consecutive nucleotide fragments in the exon F nucleotide sequence of the gene encoding myosin Va protein consist of the nucleotide sequence SEQ ID NO: 3, and (3).

6. siRNA according to claim 5, characterized in that said siRNA is shRNA consisting of a single-stranded RNA molecule in which two complementary parts are paired by Watson-Crick bonds and covalently linked on one side by a hairpin structure, wherein said shRNA comprises the sequence SEQ ID NO: 17 or by the sequence SEQ ID NO: 17.

7. siRNA according to any one of claims 1 to 5, characterized in that said sense and/or antisense RNA strand further comprises a 3' overhang of 2 to 4 natural or modified LNA type nucleotides.

8. siRNA according to any one of claims 1 to 7, characterized in that said sense RNA strand and/or said antisense RNA strand comprises at least one chemical modification in its sugar moiety, its nucleobase moiety or its internucleotide backbone.

9. siRNA according to any one of claims 1 to 8 for use as a cosmetic.

10. siRNA according to any one of claims 1 to 8 for use as a medicament.

11. A composition comprising at least one siRNA of any one of claims 1 to 8 and an acceptable carrier.

12. The composition according to claim 11, characterized in that it is for topical use.

13. The composition according to claim 11 or 12, characterized by further comprising at least one other depigmenting agent.

14. Use of at least one siRNA according to any of claims 1 to 8 or a composition according to any of claims 11 to 13 as a cosmetic for depigmentation or bleaching of the skin.

15. Use of at least one siRNA according to any one of claims 1 to 8 or a composition according to any one of claims 11 to 13 for the preparation of a medicament for the treatment or prevention of skin hyperpigmentation.