Small interfering nucletides (sioligo) for the treatment of cole-carpenter syndrome (CCS)

Abstract

The present invention relates to small interfering nucleotides (siOligo), or derivatives or precursors thereof, complementary to the region comprising a point mutation in the mRNA of the mutated human P4HB gene, wherein said mutation is the point mutation A1178G causing the substitution of the amino acid Tyrosine (Y) for the amino acid Cysteine (C) at position 393 in humans (P4HB-Y393C), as well as pharmaceutical compositions comprising them and their use as a medicament, and in particular in the treatment of Cole-Carpenter Syndrome (CCS).

Description

[0001] SMALL INTERFERING NUCLETIDES (siOligo) FOR THE TREATMENT OF COLECARPENTER SYNDROME (CCS)

[0002] The present invention relates to small interfering nucleotides (siOligo), or derivatives or precursors thereof, complementary to the region comprising a point mutation in the mRNA of the mutated human P4HB gene, wherein said mutation is the point mutation A1178G causing the substitution of the amino acid Tyrosine (Y) for the amino acid Cysteine (C) at position 393 in the protein it encodes (P4HB-Y393C), which causes Cole-Carpenter Syndrome (CCS) in humans, as well as pharmaceutical compositions comprising them and their use as a medicament, and in particular in the treatment of CCS.

[0003] STATE OF THE ART

[0004] Cole-Carpenter Syndrome (CCS), discovered in 1987, especially affects bones, which become brittle and fracture many times. One of the genes whose mutation is responsible for CCS, known as P4HB, was identified in 2015. This gene encodes the p subunit of prolyl-4-hydroxylase, a multifunctional enzyme that is a member of the protein sulphide isomerase (PDI) family.

[0005] CCS is a rare genetic disease that primarily affects bone, which becomes brittle and fractures repeatedly. It was discovered in 1987 and affected individuals present with non-traumatic fractures, frontal protrusions, micrognathia, ocular proptosis, communicating hydrocephalus and craniosynostosis.

[0006] In addition, patients with CCS have skeletal deformities associated with so-called 'popcorn epiphyses' in the long bones; genetically, CCS has an autosomal dominant inheritance with a prevalence <1 :1,000,000. The heterozygous p.Y393C mutation in the P4HB gene causes CCS, leading to an amino acid substitution between tyrosine and cysteine in the protein chain. This gene encodes the p-subunit of prolyl 4- hydroxylase, a multifunctional enzyme belonging to the Protein Disulfide Isomerase (PDI) family, called PDIA1. PDIA1 is the most abundant protein in the Endoplasmic Reticulum (ER) and is involved in the formation of disulphide bridges in nascent polypeptide chains, playing a central role in protein formation and folding. Furthermore, this enzyme is involved in the hydroxylation of prolyl residues of procollagen 1 and acts as a chaperone, inhibiting the aggregation of misfolded proteins. For these reasons, global deletion of PDI is embryologically lethal.

[0007] Interestingly, the PDIA1 enzyme is widely expressed in organisms and has been linked to many common disease conditions, including cardiovascular, neurological and metabolic diseases. Indeed, PDIA1 is involved in the regulation of the renal receptor for angiotensin II type 1 (AT1) in the kidney and is also involved in the secretion of proinsulin by pancreatic p-cells.

[0008] To date, there are no studies showing how the PDIA1Y393C mutation that induces CCS affects skeletal and non-skeletal tissues, and the disease has no cure.

[0009] Due to its rare and neglected genetic nature, CCS has no cure. Patients grow into adulthood in a precarious state of health and present with symptoms such as short stature, poor bone density, numerous fractures, premature closure of skull sutures, eye protrusion, hydrocephalus, and facial deformities. It is therefore extremely important to develop therapies that can restore normal cell function.

[0010] Small interfering nucleotides (siOligo) are short single- or double-stranded oligonucleotides that can be used to regulate gene transcription and thus interfere with the expression of the molecules they encode. siOligos can include various types of small nucleic acid sequences, such as singlestranded oligonucleotides, double-stranded oligonucleotides, antisense oligonucleotides (ASOs), microRNAs (miRNAs) and small interfering RNAs (siRNAs). RNA interference is a mechanism that regulates gene expression at the post level.

[0011] Small interfering RNAs (siRNAs) are a class of double-stranded RNA molecules, between 19 and 21 nucleotides long, that have the ability to reduce gene expression in a very specific way. These are small RNA sequences used in the laboratory to modify cell function, which have revolutionised cell biology by enabling previously precluded manipulations.

[0012] The need therefore remains to find and then provide an effective product in the treatment of Cole-Carpenter Syndrome (CCS).

[0013] SUMMARY OF THE INVENTION

[0014] Small interfering oligonucleotides (siOligo) are small nucleotide sequences complementary to specific messenger RNA (mRNA) sequences, which induce their degradation resulting in reduced gene expression. siOligo sequences can therefore be designed and adapted to potentially any type of target mRNA.

[0015] The authors of the present invention have surprisingly found that certain siOligo molecules, which retain shared characteristics, are usable for the treatment of CCS, for which no cure has yet been identified. The data in the present description show that, in the absence of such shared features, even very similar siOligo molecules are not able to selectively silence the mutated mRNA. The authors provide, in the present description, siOligo capable of binding, the mRNA of the P4HB gene bearing the point mutation A1178G, which causes the substitution of the amino acid Tyrosine (Y) with the amino acid Cysteine (C), at position 393 in humans (P4HB-Y393C), the preferred forms of which are shown in Table 1.

[0016] Efficacy tests, carried out on stable cellular models bearing the human P4HB gene mutation, and in particular HEK293 cells transfected with a vector carrying the human wild-type (P4HB-WT) or mutated (P4HB-Y393C) construct associated with the GFP (Green Fluorescence Protein) sequence, showed that different sequences comprising shared features were able to selectively reduce the expression of the GFP mRNA associated with the mutated form of the P4HB gene, while the GFP transcript associated with the wild-type form remained unaffected (Figure 6B,F,I,J,K), whereas sequences that were very similar, but did not include such features, while reducing the amount of mutated protein, showed lower selectivity.

[0017] In order to provide data also in vivo, siOligo corresponding to human siOligo, comprising the same shared characteristics, were made and successfully tested on mouse models of CCS bearing the amino acid substitution Y395C, homologous to the human Y393C, as proof of concept (see examples).

[0018] The siOligos of the invention feature in some cases the addition of a nucleotide mismatch with respect to the corresponding target sequence of the mutant P4HB mRNA, in order to increase its specificity towards the mutated mRNA without affecting the expression of the wild-type (normal) one.

[0019] Therefore, objects of the present invention are a siOligo, a derivative or precursor thereof, complementary to the region encompassing the point mutation A1178G in the mutated human P4HB gene, having a nucleotide sequence of 15 to 25 nucleotides, wherein said nucleotide sequence comprises up to 17 nucleotides downstream or upstream of said mutation, and wherein said siOligo reduces the expression of the protein encoded by said mutated human gene but not of its wild type form, a pharmaceutical composition comprising one or more siOligo or a derivative or precursor thereof according to any of the forms of implementation described herein, and a pharmaceutically acceptable excipient and / or vehicle, a kit for evaluating the effectiveness of a therapy comprising the administration of a siOligo according to the present invention or a pharmaceutical composition according to the present invention, comprising a siOligo according to any of the embodiments described herein, and a diagnostic agent. GLOSSARY

[0020] In the present description, the term 'siOligo' refers to a small interfering oligonucleotide. The term 'small interfering RNAs (siRNAs)' in the invention refers to double-stranded (duplex) sequences, the first of which is called the 'guide' (or antisense) strand and the second the 'passenger' (or sense) strand. The guide (antisense) strand is the one complementary to the target RNA that is to be inhibited, silenced or degraded.

[0021] Since the sequence of the passenger strand is complementary to the guide strand, only the sequence of the guide strand is reported for all siRNAs of the invention disclosed in the present application. The siRNAs of the invention have a sequence comprising or consisting of a fragment comprising 15 to 25 nucleotides, e.g. 16, 17, 18, 19, 20, 21 , 22, 23, 25 or 25 nucleotides containing the point mutation. siRNAs or their derivatives can be used in the form of their precursors in vivo.

[0022] According to the present invention, the point mutation A1178G causes the amino acid Tyrosine (Y) to be replaced by the amino acid Cysteine (C) at position 393 in humans (P4HB-Y393C).

[0023] In the present description, the term 'derivatives' or 'chemical derivatives' refers to siOligos that have undergone specific chemical modifications, such as the substitution of one or more nucleotides or the addition of chemical groups, in order to improve the stability, specificity or activity of the siOligos themselves.

[0024] In the present invention, the term 'precursors' refers to shRNA (short hairpin RNA). By way of example, siRNAs can be replaced by corresponding shRNAs (short hairpin RNAs), particularly in the context of gene therapy. As is known to a person skilled in the art, shRNAs are short RNA sequences or transcripts, which consist of a doublestranded structure formed by the pairing of two complementary sequences of about 15- 29 nucleotides each, normally 19-25 or 15-20, connected by a loop of about 2-10 nucleotides, e.g. 4-9 or 5-6 nucleotides. When introduced and expressed in the cell, the shRNA-forming transcripts are processed by the DICER enzyme complex, which, by cutting the loop sequence, converts the shRNAs into the corresponding siRNAs directly in the cell. The latter perform their function of silencing or knocking down the target gene. Thus, in the context of gene therapy, the siRNAs of the invention can be replaced by the corresponding shRNAs.

[0025] In the present description, the mutated human P4HB gene with point mutation A1178G is the gene with genbank ID: NM_000918.4. The identification number refers to that of the gene in the GENBANK database updated to April 2025. In the present invention, the expression "complementary to the region comprising the A1178G point mutation in the mutated human P4HB gene" means a nucleotide sequence that is complementarily matched to the portion of the mutated human P4HB gene that contains the specific A1178G mutation, as defined in the nucleotide sequences provided.

[0026] In the present description, the expression 'downstream of said mutation' means the portion of nucleotide sequence located subsequent to the position of the mutation in the gene, as described in the sections on mutation localisation.

[0027] In the present description, the expression 'upstream of said mutation' means the portion of the nucleotide sequence located prior to the position of the mutation in the gene, as clarified in the description of nucleotide sequences and their arrangement with respect to the mutation.

[0028] In the present description, the term 'nucleotide mismatch' refers to a discrepancy in the nucleotide sequence between two strands of DNA or RNA, where a nucleotide in one strand does not correctly match its complementary in the other strand.

[0029] In the present description, the expression 'the ratio of efficacy of said siOligo in reducing the expression of the mutated human P4HB gene compared to the nonmutated gene is greater than 1.30' means that siOligo is more effective in reducing the expression of the mutated P4HB gene compared to the non-mutated gene, as demonstrated in the efficacy tests described.

[0030] DETAILED DESCRIPTION OF THE FIGURES

[0031] Figure 1. Generation of the Cole Carpenter syndrome (CCS) mouse model. (A) Schematic of the Ph4b allele recombined in heterozygosity. (B) Schematic of the SY23.5 TV2 targeting vector. (C) Southern blot analysis in embryonic stem (ES) cell clones to confirm homologous recombination with the P4hb locus. The 9.9 kb signal represents the correctly recombined (mutated) allele. (D) Genotyping of the CCS mouse model. Double bands indicate animals that are germline transmitters of the mutated P4hb allele. (E) Sanger sequencing used to detect the Y395C (A>G) mutation in exon 9 of the P4hb gene. (F) External appearance of generated WT and CCS mice. (G) Body weight of the mice. Images are representative and data in (G) are the mean±SD of 3 animals per group. Statistical analysis: Student's t-test.

[0032] Figure 2. Analysis of bone phenotype in CCS mice by micro-computer tomography (pCT). The pCT analysis was conducted on explanted tibiae from 1 -month-old WT and CCS mice, male and female separately. The analysis assessed (A) volume fraction (BV / TV%), (B) thickness (Tb.Th), (C) number (Tb.N) and (D) separation (Tb.Sp) of trabecular bone and (E) cortical bone volume. The analysis was conducted on 5 mice per group per condition and the statistic was performed using the student t-test.

[0033] Figure 3. Analysis of collagen type 1 in vivo and in vitro. (A) Gene expression analysis of collagen 1 was performed on femurs isolated from 1 -month-old WT and CCS mice using specific primers. Gene expression was normalised using murine Gapdh. "Rel. Expression. (B) Analysis of PINP1 levels was performed by ELISA on sera collected from 1 -month-old WT and CCS mice. Primary osteoblasts were isolated from 8-day-old wild-type (WT) and Cole Carpenter (CCS) mice, stimulated to produce collagen by adding ascorbic acid to the culture medium at a concentration of 50 pM for 72 h and proteins extracted using RIPA buffer. (C) Western blot for type 1 collagen. Quantification was performed for (D) the mature form of collagen 1 and (E) procollagen 1 by densitometric analysis of the bands obtained in the Western blot. Collagen expression was normalised using Ponceau. Data and figures are representative of 3 cultures or 3-5 animals per condition and statistics were performed using Student's t-test.

[0034] Figure 4. Generation of Cole Carpenter cell models. (A) HEK293 cells were transfected with the plasmid for the WT (P4HB-WT) and mutated (P4HB-Y393C) form of the P4HB gene bearing the EGFP tag. The expression of GFP was used to monitor the transfection by (B) immunofluorescence and (C) real-time PCR.

[0035] Figure 5. Screening of primers for PCR detection of the murine mutated allele (Y395C). (A) Representative agarose gel image showing the presence of signal only in the HT (mutated) lane and not in the WT lane. mGAPDH was used to normalise gene expression and to demonstrate the presence of mould cDNA in all samples analysed. (B) Electropherogram of Sanger sequencing to detect the Y395C mutation (A>G). (C) Alignment between the obtained sequences (P4HB SEQ) and the wild type (P4HB WT) present in the NCBI database.

[0036] Figure 6. In vitro screening of P4HB-Y393C siRNA sequences. HEK293 cells were transfected with wild-type (P4HBWT)- or mutated (P4HBY393C) vectors associated with the GFP reporter gene and treated for 48 hours with 100 nM P4HBY393C-specific siRNA or vehicle alone (Dharmafect). GFP mRNA expression was assessed by realtime RT-PCR, using GFP-specific primer pairs. Data represent the mean+s.d. of 3 (A- D) or 2 (E-L) independent experiments, normalised by GAPDH (Student's t-test). "Rel. Expression: Relative Expression. The figure shows the ratio of efficacy of the different siRNAs in reducing the expression of the mutated protein relative to WT, as can be seen, for siRNAs with SEQ ID 1 , 2, 3, 4, 6, 9, 10 and 11 this ratio is greater than 1. In particular, siRNAs with SEQ ID 2 and 9 show an efficacy ratio greater than 5. Figure 7. Dose response experiment of the P4HB-Y393C siRNA sequences #2, 9, 10. HEK293 cells transfected with mutated vector (P4 / - / B(Y393C) ) associated with the GFP reporter gene are treated for 48 hr with 50, 100 and 200 nM of P4HBY393C-specific siRNA or vehicle alone (Dharmafect). GFP mRNA expression was assessed by real time RT-PCR, using GFP-specific primer pairs. The data represent the mean+ s.d. of 3 independent experiments, normalised with GAPDH (Student's t-test). The results show that 100 nM is the dose that gives the maximum effect in vitro. "Rel. Expression: Relative Expression.

[0037] Figure 8. Analysis of the effect of the P4HB-Y393C siRNA pool #2, 9, 10. HEK293 cells transfected with mutated vector (P4 / - / B(Y393C) ) associated with the GFP reporter gene are treated for 48 hr with 10 nM of the indicated and specific P4HBY393CsiRNA pool or vehicle alone (Dharmafect). GFP mRNA expression was assessed by real time RT-PCR, using GFP-specific primer pairs. The data represent the mean+ s.d. of 3 independent experiments, normalised by GAPDH (Student's t-test). The results show that siRNAs #2, 9 and 10 are also effective when administered in combination (pooled). "Rel. Expression: Relative Expression.

[0038] Figure 9. In vitro screening of murine P4hb-Y395C siRNA sequences. Primary osteoblasts were isolated from the calvaria of WT and P4hbY395Cmice (CCS mice) and treated for 48 h with 100 nM P4nbY395C-specific siRNA or vehicle alone (Dharmafect). P4hb<Y395C) and P4hbWTmRNA expression was assessed by real time RT-PCR, using specific primer pairs. Expression data were normalised using the murine houskeeping gene Gapdh. The data represent the mean+ s.d. of 3 independent experiments (Student's t-test). The siRNAs selected for the Ex-vivo study were number 32 and number 35, which proved to be effective and specific in downregulating the expression of the mutated P4hb gene.

[0039] Figure 10. Ex-vivo testing of murine P4hb-Y395C siRNA sequences. Long bones (tibia and femur) were isolated from P4hbY395Cmice (CCS mice), placed in culture (organ culture) and treated for 48 h with 100 nM P4hbY395CsiRNA #32 and #35 or vehicle alone (Dharmafect). Expression of P4hbY395CmRNA was assessed by real time RT-PCR, using primer pairs specific for the mutated gene. Expression data were normalised using the murine houskeeping gene Gapdh. The data represent the mean+ s.d. of 5 bones per group (Student's t-test). siRNA #35 proved to be the most effective in down regulating the expression of the mutated P4hb gene in CCS mouse bones. DESCRIPTION OF SEQUENCES siRNA sequence for the human P4HB-Y393C gene, bold = mutated nucleotide; underlined = nucleotide mismatch to increase specificity siRNA sequence for the murine P4hb-Y395C gene, bold = mutated nucleotide; underlined = nucleotide mismatch to increase specificity. siOligo sequence for the human P4HB-Y393C gene, bold = mutated nucleotide; underlined = nucleotide mismatch to increase specificity siOligo sequence for the murine P4hb-Y395C gene, bold = mutated nucleotide; underlined = nucleotide mismatch to increase specificity.

[0040] DETAILED DESCRIPTION

[0041] It is known that the mRNA of the human P4HB gene can include the point mutation A1178G, which causes the amino acid Tyrosine (Y) to be replaced by the amino acid Cysteine (C) at position 393 in humans (P4HB-Y393C), pathogenic in CCS, and encodes the p-subunit of Prolyl 4-hydroxylase, a multifunctional enzyme belonging to the Protein Disulfide Isomerase (PDI) family, named PDIA1.

[0042] This mutation corresponds to the mutation in the corresponding murine P4hb gene Y395C.

[0043] The present invention relates to a siOligo, a derivative or precursor thereof, complementary to the region comprising the point mutation A1178G in the mutated human P4HB gene (NM_011032.3), having a nucleotide sequence of 15 to 25 nucleotides, wherein said nucleotide sequence comprises up to 17 nucleotides downstream or upstream of said mutation, and wherein said siOligo reduces the expression of the protein encoded by said mutated human gene but not of its wild type form.

[0044] In an embodiment, said siOligo is an siRNA, a single-stranded oligonucleotide, a double-stranded oligonucleotide, an antisense oligonucleotide (ASO), an miRNA, preferably said siOligo is an siRNA.

[0045] In order to further increase the selectivity and / or specificity of the siOligos of the invention towards mutated mRNA, the siOligo sequence may comprise one or more nucleotides that are not complementary (mismatch) to said mutated RNA sequence. Several siOligos showed significantly higher specificity for the mutated mRNA than for the W.T (wild-type) mRNA (Figure 6).

[0046] In an embodiment, said sequence comprises at least one nucleotide mismatch with respect to said region of the mutated human P4HB gene containing said mutation, wherein said at least one nucleotide mismatch is up to 7 nucleotides downstream of said mutation and / or at least 5 nucleotides upstream of said mutation.

[0047] In other words, reading the nucleotide sequence from 5' to 3', the nucleotide mismatch is placed 1, 2, 3, 4, 5, 6, or 7 nucleotides downstream of said mutation, and / or 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16 or 17 nucleotides upstream of said mutation.

[0048] The siOligos of the invention are selected for their ability to selectively bind to mRNA transcribed from the mutated allelic forms of the P4HB gene, reducing or cancelling the expression of the mutated PDIA1 protein. Due to their selectivity in silencing the mutated gene, their efficacy in reducing expression is greater for the mutated protein than for the normal protein. Thus, they have a PDIA1 mutated / PDIA1 normal efficacy ratio greater than one.

[0049] Furthermore, in order to increase the stability of the siOligos produced and to improve the efficiency of the effect produced, one or more nucleotides forming the siOligo sequence can be chemically modified so as to obtain siOligo derivatives of the invention.

[0050] All derivatives described below are therefore included in the scope of protection of this application.

[0051] Firstly, the siOligo sequence can be equipped with a protruding 3' terminal dTdT or dAdT sequence. The latter not only gives stability and improves efficiency, but also induces oligomerisation of the siRNA to mimic DNA (sticky siRNA). Sticky siRNA can therefore be combined with usual reagents that ensure efficient siRNA delivery in vivo and decrease the ability to provoke immune responses mediated by pro-inflammatory cytokines and interferon: e.g. the product jetPEI® which is a linear polyethylenimine derivative supplied by PolyPlus Transfection.

[0052] Other derivatives that improve the stability of siOligo of the invention in duplex form are

[0053] 2'-alkoxy (C1, C2, C3, C4) derivatives, e.g. 2'-methoxy- derivatives, (i.e. 2'-0Me derivatives) (Denise M Kenski, Gabor Butora, Aarron T Willingham, Abby J Cooper, Wenlang Fu, Ning Qi, Ferdie Soriano, Ian W Davies and W Michael Flanagan. "siRNA- optimized Modifications for Enhanced In Vivo Activity." Molecular Therapy Nucleic

[0054] Acids (2012) 1, e5; doi:10.1038 / mtna.2011.4) 2'-OMe-derivatives, normally present in rRNAs and tRNAs, are non-toxic derivatives of the siOligo of the invention, wherein the -OMe group is inserted at the 2' position of the ribose nucleus in the helix-sense or antisense helix or both.

[0055] The 2'-fluoro (i.e. 2'-F)-derivatives (Denise M. Kenski et al. above) are also compatible with the function performed by the siOligos of the invention and increase their duplex stability against degradation by necleases. Fluorine incorporation at the 2' position of the ribose core maintains the activity of the siOligos both in vitro and in vivo, increasing their stability. The combined use of 2'-F in pyrimidine nucleotides with 2'-0Me in purine nucleotides results in a siOligo duplex of extreme stability in serum and greatly enhanced efficacy.

[0056] 2'-O-(2-methoxyethyl) RNA (MOE-RNA) derivatives (Mark A. Behlke. "Chemical Modification of siRNAs for In Vivo Use". Oligonucleotides 18:305-320 (2008)) can also be used to increase the stability of the siOligos of the invention. MOE groups are frequently used in antisense oligonucleotides to give the oligonucleotide high nuclease resistance and to increase Tm.

[0057] Other siOligo derivatives, with improved function and stability, suitable for the present invention are 2'-O-benzyl and 2'-O-methyl-4-pyridine derivatives (see Denise M. Kenski et al. above), 2'-amino (2'-NH), 2'-aminoethyl (2'-AE), 2'-guanidinopropyl (2'-GP).

[0058] Of particular interest for the purposes of the present invention, due to their stability, are LNAs (locked nucleic acids) derivatives of siOligo (see Mark A. Behlke above). As is well known to the expert, these derivatives are characterised by a methylene bridge between the 2'-0 and 4'-C positions of the ribose. The methylene bridge blocks the saccharide unit in the 3'-endo configuration, thus offering a significant increase in Tm and resistance to nuclease.

[0059] In an embodiment of the invention, siOligos or their derivatives can be used in the form of their precursors in vivo. These are also an object of the present invention. As an example, considering siRNAs, these can be replaced by the corresponding shRNAs (short hairpin RNAs), particularly in the field of gene therapy. As is well known to the expert, shRNAs are short RNA sequences or transcripts consisting of a double helix structure formed by the pairing of two complementary sequences of about 15-29 nucleotides each, normally 19-25 or 15-20, linked by a loop of about 2-10 nucleotides, e.g. 4-9 or 5-6 nucleotides. When introduced and expressed in the cell, the transcripts forming the shRNAs are processed by the DICER enzyme complex, which cuts the loop sequence and converts the shRNAs into the corresponding siRNAs directly in the cell. These then perform their function of silencing or knockdown of the target gene. Thus, in the context of gene therapy, the siRNAs of the invention can be replaced by the corresponding shRNAs.

[0060] All the derivatives and precursors described above are within the scope of embodiments of the present invention.

[0061] In an embodiment, said siOligo has a sequence chosen from 37, 41, 44, 45, 46, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58. When siOligos are siRNAs, they have a sequence chosen from SEQ ID NO. 2, 6, 9, 10, 11 , 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23. Nucleotide synthesis methods for the preparation of short RNA sequences are known to the expert and described in the state of the art. The siRNAs of the invention were produced by chemical synthesis and are represented by duplexes of small oligonucleotides. These consist of 19 ribonucleotides with 2 'overhangs' of deoxyribonucleotides at the 3' end. Post-synthesis, the siRNAs underwent the following purification processes:

[0062] - Removal of salts by precipitation with ethanol or using C-type chromatography columns 18

[0063] - Removal of the 2'-ACE group present in RNA bases

[0064] - Association with the antisense sequence (synthesised in a separate reaction).

[0065] - Purification 1: The duplex siRNA is purified by acrylamide gel electrophoresis - Purification 2: The duplex siRNA, obtained through the above step, is further purified by ion exchange liquid chromatography (HPLC)

[0066] - Purification 3: The duplex siRNA, obtained via the step described above, is subjected to counterionic (Na+) exchange, desalted, sterilised by filtration and tested for the presence of endotoxins.

[0067] It is also an object of the present invention to provide a pharmaceutical composition comprising one or more siOligo or a derivative or precursor thereof according to any of the embodiments described herein, and a pharmaceutically acceptable excipient and / or carrier. The pharmaceutical composition of the present invention may therefore also comprise combinations of siOligos of the present invention, which may be administered sequentially or concomitantly.

[0068] The siOligos of the invention, their chemical derivatives and / or precursors may be administered systemically or locally. Pharmaceutical compositions suitable for the administration of the siOligos of the invention or their chemical derivatives are compositions containing a pharmaceutically effective amount of siOligo, a derivative thereof or a precursor thereof, in a suitable essentially liquid excipient. Such compositions are in the form of solutions, suspensions or emulsions. Any pharmaceutical excipient suitable for such applications may therefore be used. Suitable excipients are physiological parenteral solutions, hydroalcoholic solutions, glycol solutions, water / oil or oil / water emulsions, liposome or exosome emulsions / suspensions, oily solutions, micellar suspensions, vesicles or complexes with PEI (polyethyleneimine) or complexes with atelocollagen, all containing the usual pharmaceutical additives, diluents, stabilisers and pH adjusters to physiological values. Administration of the siOligo of the invention, its derivatives or precursors, may be by the parenteral route, e.g. intravenous, intraperitoneal, intramuscular, intradermal, subcutaneous, intraosseous, intra-articular administration. Alternatively, administration may be by the oral route, via pills, tablets, buccal or sublingual dissolution formulations, capsules, soft capsules, films, powders, granules; rectally or vaginally, via suppositories or ova; by inhalation, e.g. intrabronchially.

[0069] Local administration can be by any formulation suitable for local application, e.g. by topical application or direct application to or in the tissues to be treated, or by local administration of a siOligo precursor and in situ production of the siOligo of the invention. Compositions based on exosomes, liposomes, vesicles, micelles containing siOligo or their precursors are useful to achieve both a systemic and local effect.

[0070] In order to achieve a local effect, the siOligos of the invention or their derivatives or precursors can be administered via viral or non-viral vectors, or via DNA encoding siRNAs, or as isolated (naked) RNA (Pelled et al., 2010 Tissue Engineering: Part B, Volume 16, No.1 , 13-20) or via biocompatible three-dimensional matrices or implants, based, for example, on fibrinogen and thrombin polymers and localised at the site of application. In a specific embodiment, siOligos or their derivatives or precursors are ligated or combined or complexed with usual reagents that ensure efficient in vivo delivery of siOligo, e.g. polyethyleneimine (PEI) or derivatives thereof, such as polyethyleneimine-polyethylene glycol-N -acetylgalactosamine (PEI-PEG-GAL), or polyethyleneimine-polyethylene glycol-tri-N-acetylgalactosamine complex (PEI-PEG- triGAL). In a specific embodiment of the invention, the siRNAs are bound to the jetPEI® product, which is a linear polyethyleneimine derivative provided by PolyPlus Transfection.

[0071] Alternatively, considering the siRNAs of the invention, they may be administered locally as part of a gene therapy in the form of their shRNA precursor. For example, a shRNA, or DNA encoding a shRNA, can be transferred into a mammalian cell, using, for example, a suitable plasmid or an adenoviral vector as described by Egermann et al., Human Gene Ther. May 2006; 17(5):507-17. The shRNAs expressed and processed by the cell itself produce corresponding siRNAs capable of silencing the target gene.

[0072] In an alternative form of in vivo delivery to vectors, siOligos can be transferred into a cell by electroporation, ultrasonoporation, transfection mediated by cationic liposomes, microinjection, electropulsation. In another alternative form of local delivery, the siOligos of the invention, their derivatives or precursors, can be bound, adsorbed, immobilised also by covalent bonding to a matrix capable of releasing the genetic material (gene activated matrix (GAM)) as described by Luginbuehl et al., 2004, Eur J Pharm Biopharm 58: 197-208, and then implanted into the area of interest as described by Fang et al., 1996 (Proc Natl Acad Sci USA 93, 5753).

[0073] Transfection agents, although not necessary, can nevertheless be used to improve the internalisation of siOligo into osteoclasts. Suitable transfection agents for the present invention are: lipofectamine, Amaxa Nucleofector® nucleofection.

[0074] In an embodiment, the siOligo or a derivative or precursor thereof according to the present invention, or the pharmaceutical composition according to the present invention, are for use as a medicament. In particular, the siOligo or pharmaceutical composition is for use in the treatment of a disease or disorder caused by the A1178G mutation in the mRNA of the mutated human P4HB gene.

[0075] The disease or disorder caused by the A1178G mutation in the mRNA of the mutated human P4HB gene is Cole Carpenter Syndrome (CCS).

[0076] It is also an object of the present invention to provide a kit for evaluating the efficacy of a therapy against Cole Carpenter syndrome by administering a siOligo according to the present invention or a pharmaceutical composition according to the present invention, said kit comprising a siOligo according to any of the embodiments described herein, and at least one diagnostic agent.

[0077] In an embodiment, said diagnostic agent comprises the green fluorescent protein (GFP), and at least one forward primer for the detection of the mutated form of the P4HB gene having a sequence chosen from SEQ ID 78, 79, 80, 81 , 82 and 83 and at least one reverse primer specific for the detection of the mutated form of the P4HB gene having a sequence chosen from SEQ ID 84 and 85.

[0078] The siOligos described in the present invention can act by selectively reducing the expression of the mutated P4HB gene through a mechanism known as gene silencing. Specifically, siOligos bind to the mRNA containing the A1178G mutation and promote its degradation within the cell, preventing the production of the mutated PDIA1 -Y393C protein responsible for Cole-Carpenter Syndrome (CCS). The result is a specific reduction of the mutated mRNA, without interfering with normal (wild-type) mRNA, with the aim of limiting the pathological effects of the disease-causing mutation.

[0079] Finally, in jurisdictions where patentability is not excluded, the subject matter of the present invention is also a method of treating a disease or disorder caused by the A1178G mutation in the mRNA of the mutated human P4HB gene, and in particular where said disease is Cole Carpenter Syndrome (CCS), which includes the administration of a siOligo or pharmaceutical composition of the present invention.

[0080] In any part of the description and claims, the term "comprising" may be replaced by the term "consisting of". In any part of the description and claims, the term siOligo may be replaced by siRNA.

[0081] In compliance with Art. 170bis paragraph 2 of the C.P.I. and in accordance with Art. 21 paragraph 2 of the Regulation implementing the C.P.I. adopted by Ministerial Decree 13.1.2010 no. 33, it is hereby declared that the material of animal / vegetal origin, namely primary osteoblasts isolated from the mouse model of CCS and HEK293 cells, which form the basis of the invention which is the subject of the above-mentioned application are internally sourced respectively from the university's animal enclosure with regard to the mouse model and the laboratory's cell biobank with regard to the HEK293 cells. The mice were commissioned from the company PolyGene transgenetics (Rumlang, Switzerland).

[0082] With regard to the cells isolated from the mouse model, it is declared that these cells were obtained following the authorisations issued by the Ministry of Health for the use of animal models for experimental purposes (Aut. No. 321 / 2020-PR, No. 691 / 2023-PR. In compliance with Art. 170bis para 4 C.P.I., it is hereby declared that: with reference to the biological material, containing genetically modified microorganisms or organisms, which is the subject of or used in this application, the obligations arising from national or Community legislation, and in particular, from the provisions of paragraph 6 of Decree-Laws No 206 of 12 April 2001 and No 224 of 8 July 2003, concerning such modifications, have been complied with.

[0083] EXAMPLES

[0084] The mouse model of CCS bearing the Y395C amino acid substitution, homologous to the human Y393C amino acid substitution, was generated in collaboration with POLYGENE. Considering that the disease has an autosomal dominant inheritance, the mouse model was generated by introducing the heterozygous Y395C mutation into the P4hb gene locus via a knock-in strategy and using a neomycin cassette flanked by FRT sites (Figure 1A).

[0085] Briefly, to modify the P4hb gene locus, the SY23.5 TV2 targeting vector (Figure 1 B) was electroporated into embryonic stem cells (ES cells) derived from C57BI / 6 and selected for geneticin resistance. For confirmation of correct homologous recombination, a Southern blot analysis was performed using DNA digested with the restriction enzyme BstEII. The hybridisation results showed a signal of 8.0 kb for the wild-type (WT) allele and a signal of 9.9 kb for the correctly recombined (mutated) allele (Figure 1C). The selected ES cells were injected into blastocysts derived from C57BI / 6 mice and then transferred into CD-1 foster mice. Subsequently, the generated chimeras were mated with deleter Flp females for neomycin excision. The excised F1 offspring were mated for germline transmission of the recombinant (mutated) P4hb allele, which was subsequently verified by genotyping. Mice 1010, 1011 and 1015 were heterozygous (HT) for the Y395C mutation (Figure 1 D). To confirm the presence of the Y395C mutation in exon 9, the region surrounding the mutation was amplified and the PCR product was sequenced. Sequencing analysis confirmed the presence of the Y395C mutation in heterozygosity (Figure 1E). Finally, a preliminary evaluation of the first generation of mice revealed no obvious changes in body length (Figure 1 F) together with unchanged body weight (Figure 1G).

[0086] Subsequently, an analysis of the bone phenotype was performed in order to assess whether the generated model recapitulated human pathology. One-month-old male and female mice were selected for the analysis, given the paediatric nature of the pathology and the fact that the treatment will be carried out on growing animals. The results obtained show the presence of marked osteopenia (reduction in bone mass) in CCS mice compared to their WT counterparts. In fact, analyses conducted by microcomputer tomography (pCT) showed a significant reduction in bone mass levels at both trabecular (Figure 2 A-D) and cortical (Figure 2E) levels in both male and female 1- month-old CSS mice.

[0087] Subsequent analysis revealed reduced expression of type 1 collagen in bone tissue of CCS mice (Figure 3A) associated with reduced collagen deposition by osteoblasts (bone forming cells) as demonstrated by decreased serum levels of PINP1 (procollagen type I N-terminal propeptide) in CSS compared to WT mice (Figure 3B). Similar data were obtained from bone cells (osteoblasts) isolated from CCS mice, where there was a reduction in collagen 1 production associated with an accumulation of pro-collagen 1 (immature form of collagen) (Figure 3C-E).

[0088] In general, the animal model of CCS created by the inventors recapitulates the salient pathological features of Cole-Carpenter syndrome, characterised by marked osteopenia associated with an alteration in collagen type 1 biosynthesis. The mouse model was used for in vivo testing.

[0089] Generation of Cole Carpenter cell models and characterisation.

[0090] In addition to the primary bone cells we isolated from the CCS mouse, stable cell models bearing the human P4HB gene mutation were also generated by cell transfection and subsequent antibiotic selection. In particular, HEK293 cells transfected with a vector carrying the wild-type (P4HB-WT) or mutated (P4HB-Y393C) human construct associated with the GFP (Green Fluorescence Protein) sequence were used (Figure 4A)

[0091] Analyses showed that both generated cell lines were GFP-positive both from a protein perspective, by immunofluorescence assays (Figure 4B), and from a gene expression perspective (Figure 4C).

[0092] These cellular models were used to screen for human mutation-specific siRNAs sequences (Table 1 - SEQ ID 1-12).

[0093] Design of specific primers for the detection of mutated P4HB mRNA.

[0094] CCS is caused by the Y393C mutation of the P4HB gene expressed in heterozygosity. This means that the P4HB gene has one normal (WT) and one mutated allele, which is the target of our therapeutic approach. In order to monitor the expression of the mutated P4HB mRNA in both mouse (Y395C) and human (Y393) models, primers were designed to specifically complement the mutated P4HB mRNA by discriminating it from WT (Table 3 and 4).

[0095] Table 3. List of primers for PCR detection of the murine mutated allele (Y395C).

[0096] Table 4. List of primers for PCR detection of the mutated human allele (Y393C). For the primers directed against the murine form of the P4HB gene, sequences were identified that specifically complemented the mutated mRNA (Y395C) and discriminated it from the WT mRNA (Figure 5A). The specificity of these sequences was also confirmed by sequencing analysis, which confirmed that the selected primers only amplified the mutated form of P4hb and not the WT form (Figure 5B-C).

[0097] Screening of siRNAs sequences for the P4HB-Y393C gene.

[0098] The siRNAs for the mutated form of the P4HB gene (P4HB-Y393C siRNAs) listed in Table 1 were assayed in vitro in human HEK293 cells transfected with a vector carrying the wild-type (P4HB-WT) or mutated (P4HB-Y393C) construct associated with the GFP (Green Fluorescence Protein) sequence.

[0099] The test results show that the SEQ ID siRNA sequences NO. 2, 6, 9, 10 and 11 were able to reduce the expression of the GFP mRNA associated with the mutated form of the P4HB gene, while the GFP transcript associated with the wild-type form remained unaffected (Figure 6B,F,I,J,K).

[0100] The other siRNAs assayed were either ineffective (Figure 6E,G,H) or non-specific for the mutated form of the P4HB gene (Figure 6A,C,D,L) under the conditions used in the test.

[0101] As is evident from the results, the laboratory tests performed gave positive results, allowing the identification of sequences to be used in subsequent preclinical analyses.

Claims

CLAIMS1. A siOligo, a derivative or precursor thereof, complementary to the region comprising the point mutation A1178G in the mutated human P4HB gene, having a nucleotide sequence of 15 to 25 nucleotides, wherein said nucleotide sequence comprises up to 17 nucleotides downstream or upstream of said mutation, and wherein said siOligo reduces the expression of the protein encoded by said mutated human gene but not of its wild type form.

2. The siOligo or derivatives or precursors thereof according to claim 1, wherein said siOligo is an siRNA, a single-stranded oligonucleotide, a double-stranded oligonucleotide, an antisense oligonucleotide (ASO) or a miRNA.

3. The siOligo or derivatives or precursors thereof according to any one of claims 1 or 2, wherein said sequence comprises at least one nucleotide mismatch with respect to said region of the mutated human P4HB gene comprising said mutation, wherein said at least one nucleotide mismatch is up to 7 nucleotides downstream of said mutation and / or at least 5 nucleotides upstream of said mutation.

4. The siOligo or derivative or precursor thereof according to any one of claims 1 to 3, wherein said derivative is a siOligo comprising one or more chemically modified nucleotides.

5. The derivative according to claim 4 wherein said one or more chemically modified nucleotides are selected from the group consisting of: 2'-alkoxy derivatives, 2'-methoxy derivatives, 2'-methoxy derivatives, 2'-fluoro derivatives, 2'-O-(2-methoxyethyl)- derivatives, 2'-O-benzyl-derivatives, 2'-O-methyl-4-pyridinyl-derivatives, 2'-amino- derivatives, 2'-aminoethyl-derivatives, 2'-guanidinopropyl-derivatives or LNA- derivatives.

6. The siOligo or derivative or precursor thereof according to any one of claims 1 to 5, wherein said siOligo has a sequence selected from SEQ ID NO. 37, 41, 44, 45, 46, 48, 49, 50,51 , 52, 53, 54, 55, 56, 57, 58.

7. The siOligo or derivative or precursor thereof according to claim 6, wherein said siOligo is an siRNA having a sequence chosen from SEQ ID NO. 2, 6, 9, 10, 11 , 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,8. A pharmaceutical composition comprising one or more siOligo or derivative or precursor thereof according to any one of claims 1 to 7 and a pharmaceutically acceptable excipient and / or carrier.

9. The pharmaceutical composition according to claim 8, in a form suitable for intravenous, intraperitoneal, intramuscular, intradermal, subcutaneous, intraosseous, intracartilaginous, intraarticular, oral, oral with buccal dissolution, oral with sublingual dissolution, rectal, vaginal, intrabronchial administration, by inhalation or by administration via electroporation, ultrasound-induced poration, cationic liposome- mediated transfection, microinjection, electropulsation, or via viral or non-viral vectors, or via siOligo-encoding DNA or as isolated (naked) RNA or via three-dimensional, biocompatible matrices or implants.

10. The siOligo or derivative or precursor thereof according to any one of claims 1 to 7, or the pharmaceutical composition according to claim 8 or 9, for use as a medicament.

11. The siOligo or pharmaceutical composition for use according to claim 10, for use in the treatment of a disease or disorder caused by the A1178G mutation in the mRNA of the mutated human P4HB gene.

12. The siOligo or pharmaceutical composition for use according to claim 11 , wherein said disease or disorder is Cole Carpenter Syndrome (CCS).

13. A kit for evaluating the effectiveness of a therapy against Cole Carpenter syndrome by administering a siOligo according to any one of claims 1 to 7 or a pharmaceutical composition according to any one of claims 8 or 9, said kit comprising a siOligo according to any one of claims 1 to 7 and at least one diagnostic agent.

14. The kit according to claim 13, wherein said diagnostic agent comprises the green fluorescent protein (GFP) and at least one forward primer specific for the detection of the mutated form of the P4HB gene having a sequence chosen from SEQ ID 78,79, 80,81 , 82 and 83 and at least one reverse primer specific for the detection of the mutated form of the P4HB gene having a sequence chosen from SEQ ID 84 and 85.

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