WO2022074579A1 - Arnsi dans le traitement du syndrome d'ectrodactylie-dysplasie ectodermique-fentes orofaciales (eec) - Google Patents

Arnsi dans le traitement du syndrome d'ectrodactylie-dysplasie ectodermique-fentes orofaciales (eec) Download PDF

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WO2022074579A1
WO2022074579A1 PCT/IB2021/059160 IB2021059160W WO2022074579A1 WO 2022074579 A1 WO2022074579 A1 WO 2022074579A1 IB 2021059160 W IB2021059160 W IB 2021059160W WO 2022074579 A1 WO2022074579 A1 WO 2022074579A1
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Mario Vincenzo DI IORIO
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Universita' Degli Studi Di Padova
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/34—Allele or polymorphism specific uses

Definitions

  • the invention concerns the pharmacological field, and in particular the treatment of diseases related to the Ectrodactyly-Ectodermal Dysplasia-Clefting Syndrome.
  • a small interfering RNA (siRNA) and use thereof as a medicament are described.
  • a pharmaceutical composition comprising this siRNA, specific for the R279H mutation of the p63 gene, is also described.
  • EEC Ectrodactyly-Ectodermal Dysplasia-Clefting Syndrome
  • EEC syndrome Patients affected by EEC syndrome often show ocular surface alterations, recurrent blepharitis and conjunctivitis, superficial microlesions, spontaneous perforations and ulcerations of the cornea, corneal epithelial dysfunction, and poor re-epithelialization following trauma or perforating keratoplasty.
  • Progressive keratopathy induces the formation of a vascularized corneal pannus which leads to a progressive decrease in visual capacity up to complete loss of vision.
  • EEC syndrome is caused by point mutations in the p63 gene, an important transcription factor during embryogenesis and responsible for the differentiation of stem cells in stratified epithelia.
  • EEC syndrome is an autosomal dominant genetic disease, mainly caused by heterozygous missense point mutations, i.e. the p63 gene is normally expressed (transcribed and translated), but only 50% of p63 proteins are normal while the remaining 50% show an amino acid change at specific points, but all located in the same domain of the protein, called the “DNA binding domain”.
  • the amino acid change consists in the replacement of the amino acid R (arginine) with H (histidine), while for the R304Q mutation the arginine is replaced with glutamine (Q).
  • Standard gene transfer methods aimed at allowing de novo expression of the protein are not advisable/optimal for the p63 gene, since they do not use “original-tissue-specific” promoters, able to guarantee the correct dosage and localization of the transgene, and therefore they would lead to an over-expression of the transgene, which unfortunately appears to be associated with various negative events including carcinoma.
  • additive gene therapy strategies are more suitable for recessive diseases, usually characterized by complete absence of a given gene expression, and it is therefore necessary to insert a de novo one. Therefore, gene therapy approaches for dominant-type diseases represent a demanding challenge, since they often require the selective silencing of the mutated allele, without this interfering with the expression of the wild-type allele.
  • the object of the present invention is therefore to provide a new drug for the treatment of EEC, for which there are no effective and non-invasive therapies to date.
  • the invention therefore concerns a siRNA (small interfering RNA) having the sequence of SEQ ID NO: 29 (also called “siRNAa”).
  • siRNAa small interfering RNA
  • the inventors have surprisingly developed a siRNA molecule specific for the R279H mutation of the p63 gene. This siRNA was shown to be active in the functional recovery of epithelial stem cells.
  • siRNA having the sequence described in SEQ ID NO: 29, for use as a medicament is described.
  • the present invention describes a siRNA having the sequence of SEQ ID NO: 29 for use in the treatment of Ectrodactyly-Ectodermal Dysplasia-Clefting Syndrome and of epithelial diseases related to Ectrodactyly-Ectodermal Dysplasia- Clefting Syndrome.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the siRNA having the sequence of SEQ ID NO: 29 and pharmacologically acceptable excipients.
  • Figure 1 Oral mucosa epithelial cells R279H-p63 (B-C) and R304Q-p63 (D-E) and control cells for p63 (A) plated on human corneal lenticule scaffolds (HKLs).
  • Figure 2 Analysis of p63/b-actin expression by real-time PCR. Box (A) shows data in healthy samples while box (B) shows data of affected subjects. The amplification curves of the housekeeping b-actin genes (O) show a lower CT than those of p63. The assay was performed on cultures with comparable p63expression: The delta of p63/b-actin CT are similar for both mutants and wild-type (C) samples.
  • FIG. 3 Expression of ANp63a during the lifespan of limbus/cornea (H-LESCs) cultures, oral mucosa (H-OMESCs) cultures and EEC patient cultures (OMESC R279H); in the latter an initial over-expression is highlighted (accumulation) and then a rapid decline with respect to normal cells.
  • H-LESCs limbus/cornea
  • H-OMESCs oral mucosa
  • OMESC R279H EEC patient cultures
  • Figure 4 (A) Relative qPCR showing the results after treatment with the different siRNAs (a-s) listed in Table 1. Strong down-regulation of ANp63a mRNA is observed with the R279H mutation, following treatment with the siRNAa corresponding to a variant of siRNA 10 of Table 1 , denoted with SEQ ID NO: 29.
  • U denotes the untreated sample
  • S denotes the control
  • the letters correspond to the siRNAs in Table 1.
  • FIG. 5 Organotypic cultures and tissue distribution of fluorescently labeled siRNAs.
  • Organotypic cultures prepared using a device capable of creating two different compartments at the level of a human corneal lenticule (corneal stroma (A) and (B) in the presence of siRNAa and in the presence of placebo (C)).
  • Confocal microscope analysis showed the presence of a well-organized and stratified epithelium, having 4-5 layers, in the treated area, while the adjacent region showed the typical phenotype of p63 (D) mutated cells.
  • FIG. 6 Photographs of basal epithelial cells anchored to the basement membrane and expressing different cell markers (laminin (33, Ck3, Ck12, DNp63alpha, 14-3-3G).
  • Figure 7 SiRNA-based therapeutic approach model for LSCD treatment in patients with Ectrodactyly-Ectodermal Dysplasia-Clefting Syndrome (EEC).
  • EEC Ectrodactyly-Ectodermal Dysplasia-Clefting Syndrome
  • Limbal stem cells (LESCs) with defective p63 have a reduced ability to repopulate the corneal epithelium, leading to progressive deterioration of vision culminating in complete bilateral blindness, associated with conjunctivalization.
  • gene silencing The inhibition of the expression of a gene is called gene silencing or knock down.
  • RNAi RNA interference
  • RNAi is a natural process used by cells to regulate gene expression, a phenomenon observed in various organisms (from protozoa to mammals), preserved during evolution as a defense mechanism against viruses.
  • RNAi mechanism is made possible as the cell can express RNAs that are not used to encode proteins but are processed by a series of enzymes to generate short RNAs (20-30 nt) called siRNA (small interfering RNA).
  • siRNA is complementary to the sequence of a specific mRNA. Its pairing leads to the specific degradation of the mRNA before it is translated. Therefore, the resulting gene silencing is a post-transcriptional process of gene expression control that exploits the ability of an “antisense” oligonucleotide sequence to hybridize with a specific mRNA target (“sense”), making it no longer accessible for translation into the corresponding protein and causing its degradation by specific nucleases.
  • ASO antisense oligonucleotides
  • siRNA small interfering RNA
  • RNA-induced silencing complex RNA-induced silencing complex
  • the antisense RNA strand remains protected by the RISC complex for a long time, thus creating a condition whereby it can silence gene expression even at a later time.
  • the discovery of this mechanism allowed C. Mello and A. Fire to win the Nobel Prize for Medicine in 2006. This physiological process immediately became object of intense studies to develop pharmacological approaches capable of targeting specific genes.
  • RNAi can also be allele-specific, i.e. mutationspecific.
  • Allele-specific silencing through RNAi consists in specifically inhibiting the expression of the dominant allele responsible for the disease, which differs from the normal one even for a single nucleotide. Specificity is crucial to minimize any potential effects also on the normal allele. For this approach it is necessary to identify the nucleotide variation, i.e. the point mutation of the single nucleotide, which characterizes the mutated allele. The effectiveness of this method was demonstrated in a fibroblast line of a patient suffering from Spinocerebellar Ataxia type 1 (SCA1 ), proving its possible therapeutic application in this disease (Fiszer et al. 2012). This type of method allows to keep intact the synthesis of the healthy protein, which essential in maintaining normal cellular functions.
  • SCA1 Spinocerebellar Ataxia type 1
  • the present invention relates to a siRNA (small interfering RNA) having the sequence of SEQ ID NO: 29 capable of silencing the mutated allele of the p63 gene and specific for the p63-R279H mutation.
  • Such silencing is modulated by introducing into the cell small synthetic double-stranded siRNA RNA molecules capable of recognizing and binding the specific RNA messenger sequence p63R279H, thus causing immediate degradation thereof.
  • LNA locked nucleic acids
  • LNAs blocked nucleic acids
  • Tm melting temperature
  • LNA oligonucleotides may be smaller than traditional DNA or RNA oligonucleotides and still maintain a high Tm. This is important when the oligonucleotide is small in size and is used to detect small or very similar targets.
  • LNA oligonucleotide offers substantially greater affinity for pairing with the corresponding complementary strand, when compared to traditional DNA or RNA oligonucleotides. This results in unprecedented sensitivity, specificity and reduced, if not completely absent, cytotoxicity, and makes LNA oligonucleotides ideal for the detection of small or very similar DNA or RNA targets.
  • the benefits and potential applications of LNAs include:
  • siRNA-based gene therapy strategy does not directly modify the genomic DNA, and therefore poses fewer problems both from a safety and an ethical point of view, avoiding the use of vectors of a viral nature able to integrate into DNA to express siRNAs in a constitutive way.
  • siRNA having the sequence of SEQ ID NO: 29, for use as a medicament is described.
  • siRNA having the sequence of SEQ ID NO: 29 could have clinical applications for both young and adult patients, depending on the severity of the stem cell deficiency affecting EEC patients.
  • the present invention describes siRNA having the sequence of SEQ ID NO: 29 for use in the treatment of Ectrodactyly-Ectodermal Dysplasia-Clefting Syndrome and diseases and conditions related to Ectrodactyly-Ectodermal Dysplasia- Clefting Syndrome.
  • Said diseases and conditions related to the Ectrodactyly-Ectodermal Dysplasia-Clefting Syndrome are selected from the group consisting of progressive keratopathies, blepharitis, conjunctivitis, superficial microlesions, spontaneous perforations and ulcerations of the cornea, and disfunctions of the corneal epithelium and mucous membranes.
  • the siRNA having the sequence of SEQ ID NO: 29 is for use in the treatment of diseases involving the cornea. In EEC patients these diseases manifest themselves towards the second-third decade of life, eventually leading to blindness. This course provides a therapeutic window to correct the genetic defect using the siRNA having the sequence of SEQ ID NO: 29 as preventive treatment. Treatment with the siRNA having the sequence of SEQ ID NO: 29 allows to maintain corneal transparency, prevent vision loss, and relieve pain and photophobia.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the siRNA having the sequence of SEQ ID NO: 29 (also referred to as siRNAa) and pharmacologically acceptable excipients.
  • composition according to the invention is administered by topical, enteral or parenteral routes.
  • said composition is administered by the topical route, preferably by ophthalmic, cutaneous or oral routes.
  • said composition is in a liquid form in the form of a suspension, emulsion, solution, eye drops or oral spray, or in a solid form of a cream or gel, powder, granules, tablet, capsule or pill.
  • the liquid form is preferred for use as eye drops, and the solid form as a cream or gel for the treatment of mucous membranes.
  • EEC patients often suffer from premature baldness and dry skin (very similar to psoriasis) and treatment with an ointment or gel comprising the siRNA having the sequence of SEQ ID NO: 29 could improve the patient’s condition.
  • the objectives of the present project were: (i) recruitment and genetic and clinical characterization of a cohort of patients affected by EEC syndrome in Europe; (ii) cellular, molecular and biochemical characterization of corneal stem cells obtained from EEC patients and determination of their regenerative capacity; (iii) development of gene silencing techniques for correcting the genetic defect of EEC corneal cells, through the use of specific RNA sequences (siRNAs) able to inactivate the mutated p63 allele and (iv) request submission for a gene therapy clinical trial of phase l/ll for transplantation of EEC genetically modified corneal stem cells.
  • siRNAs specific RNA sequences
  • Clinical diagnosis of the disease was carried out for each patient, by means of in-depth ophthalmological examinations (evaluation of the ocular surface by means of a slit lamp, Snellen test for measuring visual capacity, analysis of lacrimation capacity by Schirmer test, TBUT test and evaluation of the presence and functioning of meibomian glands, fluorescein staining for the assessment of the corneal epithelium integrity), and an accurate molecular diagnosis in order to identify the mutations in the p63 gene responsible for the syndrome.
  • Organotypic culture setup p63 mutant cells show defects in stratification and differentiation
  • epithelial stem cells isolated from the oral mucosa of two patients with EEC syndrome were analyzed, respectively.
  • the reason for selecting the oral mucosa instead of the limbo-corneal mucosa is based on the fact that recent clinical studies (Sotozono et al. 2013) suggest that the oral mucosa can represent a valid alternative to limbus transplantation, as demonstrated by the long-term data obtained from numerous patients with bilateral limbal deficit, and therefore lacking of healthy limbus areas, treated by autologous graft of oral mucosa stem cells.
  • Organotypic cultures were made using primary oral mucosa epithelial cells mutated for p63-R304Q (Figure 1 ): control oral mucosa epithelial cells, R279H-p63 and R304Q-p63 were plated on human corneal lenticuls (HKLs) and grown for 21 consecutive days.
  • the cell-generated epithelia, R279H-p63 (B-C) and R304Q-p63 (D- E) when compared to that obtained from control cells for p63 (A), appear thinner, consisting of only 1 -2 layers, some of them lacking cells or with irregular and terminally differentiated cells.
  • the thickness of the epithelium differs considerably when comparing those obtained with R279H-p63 cells (12.1 ⁇ 7 pm; 21.3 pm maximum) or R304Q-p63 cells (9.1 ⁇ 3 pm; 14.5 pm maximum) and that obtained with control cells (56.4 ⁇ 18 pm; 82.3 pm maximum).
  • the number of cell layers was higher in the hemicornea obtained from control cells (3.9 ⁇ 1.2 layers, 5.8 layers maximum) when compared to those obtained from R279H-p63 cells (1.2 ⁇ 1.2 layers, 2.2 layers maximum) or R304Q-p63 cells (1.1 ⁇ 1.0 layers, 1 .2 layers maximum).
  • Confocal laser scanning microscopy analyzes showed a different behavior, relative to cell differentiation processes, of the three hemicorneas.
  • the epithelium obtained from the control cells consisted of basal cells expressing the p63marker and its isoform ANp63a, suprabasal cells expressing 14-3-3-0 (an early differentiation marker for stratified epithelia) and terminally differentiated superficial cells expressing cytokeratin 3, an ocular surface marker.
  • Tissues generated from p63-defective cells showed defects in both stratification and differentiation, resulting in severe hypoplasia and loss of polarity. Abnormal clusters of terminally differentiated cells that strongly expressed cytokeratin 3 and involucrin, but not p63 and 14-3-3-0, thus indicating a premature terminal differentiation phenotype, were also observed.
  • p63 is essential for the formation of a properly stratified corneal epithelium; in the absence of this protein, the tissue assumes the characteristics of a simple epithelium, consisting of 1 -2 layers and characterized by irregular squamous cells.
  • siRNA small-interfering RNA
  • small RNA molecules capable of binding and silencing the messenger RNA produced by the mutated allele, but not that originating from the healthy allele.
  • siRNA molecules In order to develop a strategy for the design of nucleotide-specific siRNAs against the p63-R279H and R304Q mutants, the ability of siRNA molecules to discriminate between two RNA targets differing only for a single base was investigated. All possible siRNAs (21 +1 ) were designed and synthesized to analyze all possible target sequences containing the single nucleotide mutation, G953A, causing the p63-R279H mutation.
  • the first step was the design of two Taqman probes capable of specifically hybridizing to both wild-type and mutated allele, thus allowing for a competitive analysis. These probes were labeled with two different fluorophores, 6-FAM for the wild-type allele and
  • Table 2 Taqman Probes This approach has the advantage that the detection of the p63 mutation is independent of the amplification efficiency.
  • the presence of a probe against the wild-type sequence acts as an internal control to determine the quality of the RNA and also allows the quantification of the mutation rate by calculating the ratio between mutated and wildtype allele.
  • the intensities of the fluorescence signals were recorded and analyzed during PCR with the ABI Prism 7900 sequence detector, using the Applied Biosystems SDS software (version 2.4). The assay was validated for its accuracy by building standard curves for both wild-type and mutated probes.
  • the sensitivity of the assay was determined by carrying out 50% serial dilutions of the mutant RNA extracted from heterozygous patients, combined with 100% wild-type RNA, in order to obtain samples of the p63-R279H mutant allele present in a percentage of 50, 37.5, 25, 12.5, 10, 7.5, 5, 2.5 and 0. The test proved sensitive enough to detect up to 1 % mutated p63.
  • a low non-specific signal originating from the pairing of the mutated probe with wild-type sequences was also detected. This is due to cross-hybridization phenomenon, which commonly occurs when sequences differ only by a single base and is easily distinguishable from actual positive signals.
  • FIG. 2 shows an example of Real time PCR obtained from healthy and affected samples.
  • the curves obtained from the amplification of beta-actin show a lower Ct (Cycle Threshold), compared to that of p63 (highest curve).
  • the delta Ct p63/beta-actin obtained from mutated and wild-type samples are totally similar, indicating that the expression of p63 is not affected by the mutation.
  • This assay was developed in order to determine the gene silencing efficiency of the siRNAs and thus identify the best siRNA sequence for the subsequent construction of lentiviral vectors.
  • the first screenings were performed using two siRNA batteries (19-nucleotide Dharmacon and 25-nucleotide Invitrogen Stealth).
  • Each siRNA was used for transfection of primary oral mucosa cells R279H-p63, with a concentration range between 10 and 100 nM. 48 hours after transfection, the total RNA was isolated from the cells and analyzed by the allele-specific Real-Time PCR assay, for discrimination and quantification of wild-type and mutated messenger RNAs. Analysis of p63 total expression was carried out by quantitative Real-Time PCR (qPCR), comparing the amount of beta-actin/p63 transcripts. Analysis of the siRNA set demonstrated that some of them showed a higher total silencing rate of p63.
  • qPCR quantitative Real-Time PCR
  • LNA-siRNA having 19 nucleotides (Locked Nucleic Acid, Eqixon) directed against the mutated R279H-p63 allele.
  • LNA-siRNAs are siRNA molecules that were chemically modified in order to appropriately confer greater stability and binding specificity to the target sequences.
  • transfection of these lines was carried out using 19 LNA-siRNAs designed in such a way as to target the mRNA region of the R279H-p63 allele containing the nucleotide change G-A at position 953.
  • siRNAa (SEQ ID NO: 29) proved to be the most effective in reducing the expression of the mutated mRNA by about 90% ( Figure 4A).
  • Some chemical modifications were made to siRNAa in order to obtain a siRNA having the maximum efficiency in terms of specificity and stability.
  • This siRNA is the siRNA having the sequence of SEQ ID NO: 29, object of the present invention.
  • Western-blot analyzes demonstrated a significant and specific reduction of the mutated protein (45 ⁇ 4%) when siRNAa was used, while down-regulation with siRNAs b and c did not have consistent and reproducible effects.
  • siRNAa was able to (i) reduce the expression of the R279H-ANp63a-EGFP protein with concentration-dependent efficiency, and (ii) maintain the fluorescence of the WT- ANp63a-RFP protein ( Figure 4 B, C, D).
  • siRNAa was able to (i) reduce the expression of the R279H-ANp63a-EGFP protein with concentration-dependent efficiency, and (ii) maintain the fluorescence of the WT- ANp63a-RFP protein ( Figure 4 B, C, D).
  • transiently transfected HEK293T cell line in order to obtain an in vitro model of the EEC disease.
  • the HEK293T cell line was co-transfected, in a 1 :1 ratio, with both WT-ANp63a-RFP and mutant ANp63a-EGFP plasmids, in the presence of siRNAa.
  • Image analysis showed a specific and significant inhibition of the neo-synthesis of R279H -ANp63a -EGFP in about 80% of the total cells at 48 hours after transfection, when compared to cells treated with a siNCT negative control.
  • siRNAa was subsequently added to transient heterozygous cells, 18 hours after transfection, along with fluorescein-labeled siRNA (FITC-siRNA) to follow the distribution of siRNAs.
  • FITC-siRNA fluorescein-labeled siRNA
  • Z-stack analysis showed a specific silencing of R279H -ANp63a -EGFP. 48 hours after transfection, the maximum effect was observed in cells treated with siRNAa + FITC-siRNA. The next step was to validate the inhibitory and allelespecific potential of the siRNAs ex vivo.
  • R279H-OMESC cells were transfected using FITC-siRNA concentrations in the range of between 10 and 100 nM. 48 hours after transfection, the optimal transfection efficiency was 88 ⁇ 2% at 100 nM.
  • siRNAs a, b and c were transfected into R279H-OMESC cells at 100 nM, and the expression levels of wild-type and mutated p63 were measured by AS-qPCR.
  • a significant suppression of mutated mRNA was obtained with siRNAa, but also with siRNA b and c, with a mutated mRNA suppression rate of 80%, 72% and 65%, respectively.
  • siRNA concentrations equal to 0.1 , 10 and 100 nM. Titration of siRNAa abolished the R279H mRNA, showing a gradient of values related to the concentration used.
  • the 5’-3’ anti-sense (AS) sequence of siRNAa is as follows: /5Phos/rArUrUrGrGrArCrGrG +T+ GrGrUrUrCrArUrCrC+T+T (SEQ ID NO:29), where:
  • RNA bases are denoted as rA, rC, rG, rU;
  • T - DNA bases
  • siRNAa the sense strand (S), being for siRNAa as follows: rGrGrArUrGrArArCrCrArCrGrUrCrCrArUTT (SEQ ID NO:30), was also synthesized.
  • EEC the subsequent experimental phases were carried out directly on stem cells of treated EEC patients and not with SEQ ID NO:29.
  • ANp63a Since accumulation of p63, probably due to the increased stability of the mutant isoform, was previously observed in the tissues of EEC patients, the expression of ANp63a was quantified in OMESC R279H, using an absolute quantitative PCR (Ab-qPCR), at different passages in culture (I, III; V, VII and XI) and was then compared with OMESC control cells and limbal epithelial stem cells. As shown in Figure 3, ANp63a decreases during serial culture. The reduction rate resulted to be faster in OMESC R279H. The expression of ANp63a at the first passage is three times higher with respect to control OMESCs.
  • OMESCs greater self-renewal capacity explains why they are successfully used as a source of stem cells, enabling epithelial regeneration for a long time.
  • AS-qPCR quantitative allele-specific PCR
  • mutant p63 R279H
  • wild type mRNAs were 60 ⁇ 7% and 40 ⁇ 5%, respectively, while the ratios changed during aging, becoming 12 ⁇ 1.7% and 23 ⁇ 0.9%.
  • siRNAa was able to restore the functional phenotype in R279H - OMESC cells.
  • mutated cells treated with siRNAa exhibited a longer life-span equal to 7 passages in culture, compared to the 5-6 of the wild-type cells.
  • organotypic cultures were set up using a device capable of creating two different compartments at the level of a human corneal lenticule (corneal stroma, HKL, Figure 5A ), so that two organotypic cultures can be set up simultaneously with R279H- p63 cells, (i) in the presence of SEQ ID NO: 29 a and (ii) in the presence of placebo siRNA (Figure 5C).
  • a device capable of creating two different compartments at the level of a human corneal lenticule corneal stroma, HKL, Figure 5A
  • two organotypic cultures can be set up simultaneously with R279H- p63 cells, (i) in the presence of SEQ ID NO: 29 a and (ii) in the presence of placebo siRNA (Figure 5C).
  • transfections in the two compartments were performed by adding fluorescent siRNAs.

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Abstract

L'invention concerne le domaine pharmacologique, et en particulier le traitement de maladies liées au syndrome d'ectrodactylie-dysplasie ectodermique-fentes orofaciales. L'invention concerne un petit ARN interférent (ARNsi) et son utilisation en tant que médicament. L'invention concerne également une composition pharmaceutique comprenant cet ARNsi, spécifique pour la mutation R279H du gène p63.
PCT/IB2021/059160 2020-10-07 2021-10-06 Arnsi dans le traitement du syndrome d'ectrodactylie-dysplasie ectodermique-fentes orofaciales (eec) WO2022074579A1 (fr)

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EP21799330.2A EP4225918A1 (fr) 2020-10-07 2021-10-06 Arnsi dans le traitement du syndrome d'ectrodactylie-dysplasie ectodermique-fentes orofaciales (eec)

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IT102020000023647A IT202000023647A1 (it) 2020-10-07 2020-10-07 Sirna nel trattamento della sindrome ectrodattilia-displasia ectodermica-palatoschisi (eec)

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