EP4288536A1 - Small interfering rna (sirna) for therapy of mmihs caused by actg2 gene mutation - Google Patents

Small interfering rna (sirna) for therapy of mmihs caused by actg2 gene mutation

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Publication number
EP4288536A1
EP4288536A1 EP22700444.7A EP22700444A EP4288536A1 EP 4288536 A1 EP4288536 A1 EP 4288536A1 EP 22700444 A EP22700444 A EP 22700444A EP 4288536 A1 EP4288536 A1 EP 4288536A1
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seq
sirna
derivative
precursor
derivatives
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German (de)
French (fr)
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Riccardo PAONE
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Dante Labs Srl
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Dante Labs Srl
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    • 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
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Definitions

  • SIRNA SMALL INTERFERING RNA
  • the present invention belongs to the sector of the molecules known as "small interfering RNA " with therapeutic applications.
  • SiRNAs have the ability to reduce the expression of genes in a very specific way. These are small double-stranded RNA sequences normally used in the laboratory to modify cell function, which have revolutionized cell biology by allowing previously precluded molecular manipulations.
  • Disorders related to the ACTG2 gene represent a subgroup of visceral myopathy with variable involvement of the bladder and bowel.
  • Disorders affecting the bladder can manifest in more severe forms, such as neonatal megacysts and megaureter (with its most severe form of plum belly syndrome), or in milder forms, such as recurrent urinary tract infections and dysfunction of the bladder.
  • Chronic bladder dysfunction carries a high risk of urinary tract infections, dilation of the upper urinary tract, and impaired kidney function.
  • disorders affecting the gut include malrotation, neonatal manifestations of microcolon, megacystic-microcolon intestinal hypoperistalsis syndrome (MMIHS), and chronic intestinal pseudo-obstruction (CIPO) in infants, children and adults.
  • MMIHS megacystic-microcolon intestinal hypoperistalsis syndrome
  • CIPO chronic intestinal pseudo-obstruction
  • Chronic bladder dysfunction typically requires the intervention of a urologist and may involve the use of a routine urinary catheter or diversion to reduce the risk of upper urinary tract dilation as well as the associated risk of urinary tract infection and impairment of the urinary tract, kidney function.
  • CIP generally requires the intervention of a gastroenterologist and / or a nutritionist experienced in intestinal motility disorders.
  • Diagnosis of an ACTG2-related disorder is established on the basis of the presence, in a proband, of a heterozygous pathogenic variant in ACTG2. Normally, an analysis of the ACTG2 sequence is performed first; a deletion / duplication analysis may also be considered if no pathogenic variant can be identified. However, all the pathogenic variants identified to date are missense variants, while deletions I duplications of exons or entire genes have not been identified as the cause of ACTG2-related disorders.
  • ACTG2 intestinal pseudo-obstruction
  • This condition reproduces a state of physical blockage of the intestine, without a real obstruction.
  • Individuals with this disorder often have problems related to bladder emptying.
  • Mutations in the ACTG2 gene responsible for intestinal pseudo-obstruction are thought to inhibit the formation of actin filaments in the cytoskeleton and reduce the ability to contract smooth muscle in the intestine and bladder. As a result, intestinal peristalsis is compromised and the bladder is less able to contract and excrete urine, leading to the onset of signs and symptoms of the disease.
  • MMIHS megacystal-microcolon intestinal hypoperistalsis syndrome
  • the mutations responsible for MMIHS are not inherited; instead they occur as a random event (de novo ) during the formation of reproductive cells (eggs or sperm) or during early embryonic development.
  • These alterations result in variations of single amino acids in the y- 2 actin protein (ACTG2).
  • ACTG2 y- 2 actin protein
  • These changes inhibit the formation of actin filaments and reduce the ability to contract smooth muscle in the bladder and intestines.
  • the bladder cannot empty normally, leading to an enlarged bladder (megacysts) and painful abdominal swelling (distension).
  • partially digested food can accumulate in the intestine, which can also contribute to the phenomenon of distention. Poor digestion can lead to malnutrition in individuals with MMIHS.
  • de novo mutations in the gene are believed to be responsible for the more severe forms of the disorders associated with ACTG2. Due to the reduced significance of the disease, only some of the causative mutations have been investigated. A significant number of the pathogenic variations in the ACTG2 gene is represented by a non-synonymous mutation of the arginine residues (among them R40H, R40C; R63G; R178C, R135C; R178H, R135H; R178L, R135L; R257C, R214C).
  • SiRNAs small interfering RNA are small sequences of RNA complementary to specific sequences of messenger RNA (mRNA), which induce their degradation. Although some siRNAs have been shown to block the expression of the mutated allele to some extent, their selectivity for the mutated allele and ability to discriminate between mutant and wildtype (WT) allele remains an open challenge.
  • the purpose of the present invention is to provide novel siRNAs optimized for the treatment of ACTG2-dependent MMIHS disease.
  • the invention object of the present application is based on the finding made by the present inventors that the complementarity, albeit total, to the mRNA sequence comprising the point mutation, is not sufficient alone to obtain effective and selective siRNAs, i.e. effective in silencing the expression of the mutated protein but inactive on the expression of the WT protein.
  • the optimal conjugation of effectiveness and selectivity depends on several factors such as the mutation itself on the mRNA, the length of the sequences that flank the mutation, therefore the position of the mutated nucleotide in the siRNA sequence, the presence or absence of one or more nucleotides mismatch with respect to the WT mRNA sequence and the position of said mismatches in the siRNA sequence: in short words from the design of the siRNA sequence itself.
  • the gene is also known as ACT; ACTE; VSCM; ACTA3; ACTL3; ACTSG.
  • the gene is predominantly expressed in the adult, in the bladder, in the prostate, in the esophagus, in the appendix, in the colon, in the duodenum, in the endometrium and in the gallbladder.
  • a first object of the present application are small interfering RNA (siRNA), complementary to the region comprising a point mutation in the messenger RNA (mRNA) of the mutated human ACTG2 gene or derivative or precursor thereof.
  • the siRNAs object of the application are characterized in that (i) said mutations are reflected in the corresponding mutations of the ACTG2 protein: R40H, R40C; R63G; R178C, R135C; R178H, R135H; R178L, R135L; R257C, R214C; that (ii) the siRNAs have a nucleotide sequence comprising a fragment of 15 to 25 nucleotides, comprising the point mutation; that (iii) the siRNAs selectively reduce the expression of the mutated ACTG2 protein and that (iv) the ratio of effectiveness of the siRNAs of the invention in reducing the expression of the mutated ACTG2 protein relative to the normal protein is greater than one.
  • sequence of the small interfering RNA (siRNA) of the invention comprises, in addition to the mutated nucleotide, one or more nucleotides mismatches compared to the corresponding target sequence of the mRNA containing the mutation.
  • the small interfering RNA (siRNA) sequence of the invention also comprises a short sticky sequence at the 3 'end composed of dA and dT nucleotides.
  • a second object of the invention is represented by the aforementioned siRNAs for use in a therapeutic treatment, specifically in the therapeutic treatment of megacyst-microcolon intestinal hypoperistalsis syndrome (MMIHS) caused by mutation of the ACTG2 gene.
  • MMIHS megacyst-microcolon intestinal hypoperistalsis syndrome
  • a third object of the invention is a method for preparing the siRNAs seen above.
  • a fourth object of the invention is represented by pharmaceutical compositions comprising, as active ingredient, one or more siRNAs and a pharmacologically acceptable excipient. Such compositions are preferably for parenteral administration.
  • a further object of the invention is represented by the same compositions for use in the therapeutic treatment of the megacyst-microcolon-intestinal hypoperistalsis syndrome (MMIHS), also in association with a second active ingredient.
  • MMIHS megacyst-microcolon-intestinal hypoperistalsis syndrome
  • siRNAs according to the invention designed and tested in isolated cells and in animal models, proved highly specific for the mutated gene. They selectively eliminate up to 95% of the mutated gene transcript, improving disease symptoms.
  • siRNAs of the invention also offer the further advantages of being internalized by the cells of interest, in particular by the smooth muscle cells, by simple incubation, without the need for any transfection agent, and to remain in the cell for a long time.
  • Human ACTG2 mRNA is known to include mutations, pathogenic in intestinal megacystic- microcolon-hypoperistalsis syndrome (MMIHS), which generate mutated proteins as indicated in Table 1 :
  • the position of the mutation is given with reference to the amino acid position of the wild type protein reported in uniprot as P63267 and in the present description as SEQ ID NO 4.
  • RNAs complementary to the region comprising a point mutation in human ACTG2 messenger RNA have been designed and produced for all mutations in the gene known to be pathogenetic for intestinal megacystic-microcolon-hypoperistalsis syndrome (MMIHS) , reported in Table 1.
  • the small interfering RNAs (siRNAs) of the invention are double-stranded (duplex) sequences, the first of which is called “guide” (or antisense) and the second "passenger” (or sense).
  • the guide helix (antisense) is the one complementary to the target RNA that is to be inhibited, silenced or degraded.
  • siRNAs of the invention have a sequence comprising or consisting of a fragment composed of 15 to 29 nucleotides, for example 16, 17, 18, 19, 20, 21 , 22, 23 or 24, 25, 26, 27 or 28 containing the point mutation.
  • the siRNAs of the invention are selected for their ability to selectively bind to the mRNA transcribed from the mutated allelic forms of the ACTG2 gene by reducing or nullifying the expression of the mutated ACTG2 protein. Thanks to their selectivity in silencing the mutated gene, their effectiveness in reducing expression is greater for the mutated protein than for the normal protein. Therefore they have a mutated ACTG2/normal ACTG2 effectiveness ratio greater than one.
  • the siRNA sequence may comprise one or more non-complementary nucleotides (mismatch) to said mutated RNA sequence so as to have a significantly greater specificity for mutated mRNA with respect to the WT mRNA.
  • one or more nucleotides that form the siRNA sequence can be chemically modified in order to obtain derivatives of the siRNAs of the invention. All derivatives described below are therefore included in the scope of protection of this application.
  • the siRNA sequence can be endowed with a protruding 3 'terminal dTdT or dAdT sequence.
  • the latter in addition to providing stability and improving efficiency, induces the oligomerization of the siRNA in order to mimic the DNA (sticky siRNA).
  • the sticky siRNAs can therefore be associated with usual reagents that ensure an efficient distribution of siRNA in vivo and decrease the ability to provoke immune responses mediated by pro-inflammatory cytokines and interferon: for example the jetPEI® product which is a linear derivative of polyethyleneimine provided by PolyPlus Transfection.
  • siRNAs of the invention not modified, or otherwise modified, as described below, can equally be effectively used.
  • siRNAs of the invention are the 2'-alkoxy (C1, C2, C3, C4) derivatives, for example the 2'-methoxy-derivatives, (i.e. 2'-OMe derivatives) ( Denise M Kenski, Gabor Butora, Aarron T Willingham, AbbyJ 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 Acids ( 2012) 1, e5; doi: 10.1038 / mtna.2011.4 ).
  • 2'-OMe-derivatives normally present in rRNA and tRNAs, are non-toxic derivatives of the siRNAs of the invention, wherein the -OMe group is inserted in position 2 'of the ribose nucleus in the sense-helix or anti- sense or both.
  • the 2'-fluorine (ie 2'-F) -derivatives are also compatible with the function performed by the siRNAs of the invention and increase the stability of the duplex against nuclease degradation.
  • the incorporation of fluorine in the 2 'position of the ribose nucleus maintains the activity of siRNAs both in vitro and in vivo, increasing their stability.
  • the combined use of 2'-F in pyrimidine nucleotides with 2'-OMe in purine nucleotides results in an extremely stable duplex siRNA in serum and greatly improved effectiveness.
  • MOE-RNA 2'-O- (2-methoxyethyl) RNA
  • Oligonucleotides 18: 305-320 (2008) can equally be used for increasing the stability of the siRNAs of the invention.
  • MOE groups are frequently used in antisense oligonucleotides to give the oligonucleotide high resistance to nucleases and to increase Tm.
  • siRNA derivatives with improved function and stability, suitable for the present invention are the 2'-O-benzyl derivatives and the 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).
  • LNAs locked nucleic acids
  • siRNAs see Mark A. Behlke above.
  • these derivatives are characterized by a methylene bridge between the 2'-0 and 4'-C positions of ribose.
  • the methylene bridge blocks the saccharide unit in the 3'-endo configuration, thus offering a significant increase in Tm and resistance to nucleases.
  • the siRNA of the invention comprises one or more chemically modified nucleotides which increase its stability and/or effectiveness.
  • siRNAs or derivatives thereof can be used in the form of their precursors in vivo.
  • the latter also form object of the present invention.
  • siRNAs can be replaced by corresponding short hairpin RNAs (shRNAs), particularly in the context of gene therapy.
  • 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, for example 4-9 or 5-6 nucleotides.
  • the transcripts that form the shRNAs are processed by the DICER enzyme complex which by cutting the loop sequence directly converts the shRNAs into the corresponding siRNAs in the cell. These will then perform their function of silencing or knockdown of the target gene. Therefore, in the context of gene therapy, the siRNAs of the invention can be replaced by the corresponding shRNAs.
  • siRNA sequences were provided with a protruding dTdT sequence at the 3 'end to improve their stability and effectiveness.
  • the dTdT sequence is replaced by the dAdT sequence which further improves its stability and efficiency and allows its binding to any vehicles that allow a better in vivo distribution of the siRNA and reduce any immune responses.
  • siRNA sequences highlighted in-silico show the mutated nucleotide in bold underlined (referring to the sequence encoding the human protein Actin, gamma-enteric smooth muscle reported in uniprot as P63267) and in bold italics the additional nucleotide (s) mismatch (the).
  • preferred siRNAs of the invention are: AUCAUGUGCAUGGACUUGGCU (SEQ ID NO: 1)
  • AUCAUGCUCCUUGACUUGGCU (SEQ ID NO: 3).
  • the preferred siRNAs of the invention are characterized by a mutated nucleotide and an additional nucleotide mismatch, wherein said additional mismatch is located at a distance between 3 and 6 nucleotides from the mutated nucleotide.
  • siRNAs are:
  • siRNAs of the invention were produced by chemical synthesis, and are represented by duplexes of small oligonucleotides. These consist of 19 ribonucleotides with 2 deoxy-ribonucleotides "overhangs" at 3'. After synthesis, the siRNAs underwent the following purification processes:
  • compositions and dosages are provided.
  • siRNAs of the invention can be administered systemically or locally.
  • compositions suitable for administering the siRNAs of the invention or their chemical derivatives are compositions containing a pharmaceutically effective amount of siRNA, derivates or precursors 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 can then be used.
  • Suitable excipients are physiological solutions for parenteral use, hydroalcoholic solutions, glycol solutions, water/oil or oil/water emulsions, liposome or exosome emulsions/suspensions, oil solutions, micellar suspensions, vesicles, or complexes with PEI (polyethylene imine) or complexes with atelocollagen, all containing the usual pharmaceutical additives, diluents, stabilizers and pH regulators at physiological values.
  • the administration of the siRNAs of the invention, derivatives or precursors thereof can occur parenterally, e.g. intravenous, intraperitoneal, intramuscular, intradermal, subcutaneous, intraosseous, intracartilaginous, intraarticular administration.
  • the administration can be orally, through pills, tablets, oral or sublingual dissolving formulations, capsules, soft capsules, films, powders, granules; rectally or vaginally through suppositories or pessaries; by inhalation, e.g. intrabronchial.
  • Local administration can take place through any formulation suitable for local application, for example through topical application or direct application on or in the tissues to be treated, or by local administration of a siRNA precursor and in situ production of the siRNA of the invention.
  • Compositions based on exosomes, liposomes, vesicles, micelles containing siRNAs or precursors thereof can be used to achieve both a systemic and local effect.
  • the siRNAs of the invention or their derivatives or precursors can be administered through viral or non-viral vectors, or through the DNA encoding the siRNAs or as isolated (nacked) RNA (Pelled et al., 2010 Tissue Engineering: Part B, Volume 16, No.1, 13-20) or through three-dimensional biocompatible matrices or implants based e.g. on fibrinogen and thrombin polymers and placed at the point of application.
  • the siRNAs, derivatives or precursors thereof are bonded or associated or complexed with usual reagents that ensure efficient distribution of the siRNA in vivo , for example, polyethyleneimine (PEI) or its derivatives such as the polyethyleneimine complex, polyethylene glycol-N-acetylgalactosamine (PEI-PEG-GAL), or the polyethyleneiminepolyethylene glycol-tri-N-acetylgalactosamine complex (PEI-PEG-triGAL).
  • PI polyethyleneimine
  • PEG-GAL polyethylene glycol-N-acetylgalactosamine
  • PEI-PEG-triGAL polyethyleneiminepolyethylene glycol-tri-N-acetylgalactosamine complex
  • the siRNAs are linked to the jetPEI® product which is a linear derivative of the polyethyleneimine supplied by PolyPlus Transfection.
  • the siRNAs of the invention can be locally administered in the form of their shRNA precursor as part of a gene therapy.
  • an shRNA or the DNA encoding an shRNA
  • the shRNAs expressed and processed by the cell itself produce the corresponding siRNAs capable of silencing the target gene.
  • siRNAs can be transferred into a cell through electroporation, ultrasound, cationic liposome-mediated transfection, microinjection, electrical pulsation.
  • the siRNAs of the invention can be bonded, adsorbed, immobilized also through covalent bonds 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 in the area of interest as described by Fang et al., 1996 (Proc Natl Acad Sci USA 93, 5753).
  • GAM gene-activated matrix
  • transfection agents are not needed, they can nevertheless be used to enhance siRNA internalization in the cells of interest, preferably in smooth muscle cells.
  • Suitable transfection agents for the present invention are: lipofectamine, nucleofection by Amaxa Nucleofector® method (Lonza, Cologne, Germany) using specific kit (Lonza, code VPA-1007).
  • the siRNA treatment regimen of the invention may include once-a-day to once-a-week administrations, for example 1, 2, 3, 4, 5, 6 or 7 administrations/week.
  • the treatment can be performed with a daily administration or every 2, 3, 4, 5, 6, 7 days.
  • the duration of treatment depends on the severity of the disease and varies from a treatment lasting a few weeks to a chronic treatment.
  • the tests performed by the present inventors are aimed at demonstrating that the siRNAs of the invention are effective in restoring the functionality of the cells of interest, preferably smooth muscle cells, in a broad spectrum of dosages ranging from about 1 ng/kg of body weight to about 100 mg/kg of body weight of the subject to be treated or subject in which the symptoms of the progression of the megacyst-microcolon intestinal hypoperistalsis syndrome have manifested.
  • the dosages will be from about 1 pg/Kg to 20 mg/Kg of body weight, preferably from about 1 mg/Kg to about 10mg/Kg.
  • siRNAs of the invention can be used in association with other active ingredients.
  • the term "in combination” means either a co-therapy or combination therapy, or a co-formulation in a single pharmaceutical form, or in a single commercial package, for example a kit or a blister of two or more active ingredients.
  • Example 1 Generation of the vector bearing the ACTG2 gene construct
  • the WT-ACTG2 construct was obtained by cloning the complete human ACTG2 cDNA sequence into the pEGFP-C1 expression vector, using the Hindi 11 and Xhol restriction enzymes.
  • Example 2 Cell models and cell lines
  • HEK293 cells transfected with the empty vector or with the vector WT-, p.R178C, p.R178H, and P.R178L-EGFP were employed.
  • the relative expression of ACTG2 is quantified by Real-Time RT-PCR.
  • HEK293 cells were transfected with vectors carrying the WT-, p.R178C, p.R178H, and p.R178L- EGFP constructs, then the expression and localization of the fluorescent fusion protein EGFP was detected by confocal microscopy.
  • Example 3 Primary lines, Isolation of intestinal stem cells
  • Crypt cells were isolated from the small intestine (jejunum) resulting from surgical resection by enzymatic / mechanical method and cultured in IESC medium for intestinal epithelial stem cells. Crypt-derived cells were then differentiated by culture in mTeSRTM medium on Matrigel-coated plates. Differentiation into mesodermal lineage cells was initiated at time 0 by culturing cells with CHIR99021 (5 pM) and BMP-4 (10 ng I mL) in RPMI1640 medium and 2% B27. Differentiation into contractile Smooth Muscle Cells (SMCs) began on day 3.
  • Synthetic SMCs were produced by culturing cells with 25 ng I mL of VEGF-A and FGFp in RPMI1640 and 2% of B27 from day 3 to day 7, with 25 ng / mL VEGF-A and FGFp in RPMI1640 and 2% B27 from day 7 to day 9, and with 10 ng / mL PDGF ⁇ and 3 ng / mL TGF
  • the Differentiated cells were maintained in RPMI1640 metabolic medium enriched with 4 mM lactate for 4-6 days. Stem cells were transfected with the blank EGFP vector (control) or with the vector p.R178C, p.R178H, and p.R178L-EGFP.
  • siRNAs having SEQ ID sequences. Nos. 1-3 and 5-7 were tested in silico for their effectiveness on WT mRNA or mutated mRNA (Table 2). The siRNAs tested showed significantly greater effectiveness in reducing mutant mRNA compared to WT mRNA. With an effectiveness greater than 98% in reduction of mutated mRNA, siRNAs having SEQ ID N 1-3 were found to be highly more effective than siRNAs having SEQ ID No. 5-7.
  • a murine model was created by humanization strategy in ROSA26 for over-expression of the human ACTG2 gene under control of the murine ACTG2 promoter.
  • the coding region (Coding DNA sequence, CDS) of the mutated human ACTG2 gene was inserted into the ROSA26 locus preceded by a Neomycin resistance cassette flanked by 2 FRT sites and by the 6KB region identified on Ensembl as Promoter Murino by ACTG2.

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Abstract

The present invention belongs to the sector of molecules known as "small interfering RN " with therapeutic applications. SiRNAs have the ability to reduce the expression of genes in a very specific way. These are small double-stranded RNA sequences normally used in the laboratory to modify cell function, which have revolutionized cell biology by allowing previously precluded molecular manipulations

Description

SMALL INTERFERING RNA (SIRNA) FOR THERAPY OF MMIHS CAUSED BY ACTG2 GENE MUTATION
Technical field of the invention
The present invention belongs to the sector of the molecules known as "small interfering RNA " with therapeutic applications. SiRNAs have the ability to reduce the expression of genes in a very specific way. These are small double-stranded RNA sequences normally used in the laboratory to modify cell function, which have revolutionized cell biology by allowing previously precluded molecular manipulations.
State of the Prior Art
Disorders related to the ACTG2 gene (Actin, gamma-enteric smooth muscle uniprot P63267) represent a subgroup of visceral myopathy with variable involvement of the bladder and bowel. Disorders affecting the bladder can manifest in more severe forms, such as neonatal megacysts and megaureter (with its most severe form of plum belly syndrome), or in milder forms, such as recurrent urinary tract infections and dysfunction of the bladder. Chronic bladder dysfunction carries a high risk of urinary tract infections, dilation of the upper urinary tract, and impaired kidney function.
Disorders affecting the gut include malrotation, neonatal manifestations of microcolon, megacystic-microcolon intestinal hypoperistalsis syndrome (MMIHS), and chronic intestinal pseudo-obstruction (CIPO) in infants, children and adults.
Infants affected by these conditions (with or without evidence of bowel malrotation) often show feeding intolerance and signs of non-mechanical intestinal obstruction that persist even after surgical correction of malrotation. Individuals who develop manifestations of CIP in late childhood or adulthood often experience an episodic increase and decline in intestinal motility. They may undergo frequent abdominal surgery (possibly related to malrotation or adhesions causing mechanical obstruction) resulting in resection of dilated segments of the intestine, often becoming dependent on total parenteral nutrition (TPN).
Management of these disorders requires a multidisciplinary approach involving primary care providers, gastroenterologists, surgeons, urologists, nephrologists, dieticians and social workers. Chronic bladder dysfunction typically requires the intervention of a urologist and may involve the use of a routine urinary catheter or diversion to reduce the risk of upper urinary tract dilation as well as the associated risk of urinary tract infection and impairment of the urinary tract, kidney function. CIP generally requires the intervention of a gastroenterologist and / or a nutritionist experienced in intestinal motility disorders.
Diagnosis of an ACTG2-related disorder is established on the basis of the presence, in a proband, of a heterozygous pathogenic variant in ACTG2. Normally, an analysis of the ACTG2 sequence is performed first; a deletion / duplication analysis may also be considered if no pathogenic variant can be identified. However, all the pathogenic variants identified to date are missense variants, while deletions I duplications of exons or entire genes have not been identified as the cause of ACTG2-related disorders.
Several inherited mutations in the ACTG2 gene have been identified in individuals with intestinal pseudo-obstruction, a condition that impairs smooth muscle contractions responsible for the movement of food through the digestive tract (peristalsis). This condition reproduces a state of physical blockage of the intestine, without a real obstruction. Individuals with this disorder often have problems related to bladder emptying. Mutations in the ACTG2 gene responsible for intestinal pseudo-obstruction are thought to inhibit the formation of actin filaments in the cytoskeleton and reduce the ability to contract smooth muscle in the intestine and bladder. As a result, intestinal peristalsis is compromised and the bladder is less able to contract and excrete urine, leading to the onset of signs and symptoms of the disease.
Approximately 22 mutations of the ACTG2 gene have been identified capable of causing the megacystal-microcolon intestinal hypoperistalsis syndrome (MMIHS), which is characterized by impaired peristalsis and the ability to empty the bladder. The mutations responsible for MMIHS are not inherited; instead they occur as a random event (de novo ) during the formation of reproductive cells (eggs or sperm) or during early embryonic development. These alterations result in variations of single amino acids in the y- 2 actin protein (ACTG2). These changes inhibit the formation of actin filaments and reduce the ability to contract smooth muscle in the bladder and intestines. As a result, the bladder cannot empty normally, leading to an enlarged bladder (megacysts) and painful abdominal swelling (distension). Furthermore, partially digested food can accumulate in the intestine, which can also contribute to the phenomenon of distention. Poor digestion can lead to malnutrition in individuals with MMIHS.
Approximately 25 mutations in the ACTG2 gene are currently deposited in NCBI's Clinvar database, although it was not known that variants in the same gene could be responsible for the development of ACTG2- associated disorders.
As previously mentioned, de novo mutations in the gene are believed to be responsible for the more severe forms of the disorders associated with ACTG2. Due to the reduced significance of the disease, only some of the causative mutations have been investigated. A significant number of the pathogenic variations in the ACTG2 gene is represented by a non-synonymous mutation of the arginine residues (among them R40H, R40C; R63G; R178C, R135C; R178H, R135H; R178L, R135L; R257C, R214C).
A molecular model of the sporadic pathogenic variants R178L and R178C was investigated in the study by Thorson et al. " De novo ACTG2 mutations cause congenital distended bladder, microcolon, and intestinal hypoperistalsis " Hum. Genet 133, 737-742 (2014). According to this study, the replacement of arginine with a cysteine or a leucine suppresses the interaction with the side chain of a histidine residue (H74). This weak interaction is likely critical to the integrity of the actin structure for polymerization.
Furthermore, similar non-synonymous variants have been observed in the aortic smooth muscle isoform (R179 of the ACTA4 gene). These mutations are responsible for multisystem smooth muscle dysfunction syndrome as well as familial thoracic aortic aneurysm.
SiRNAs (small interfering RNA) are small sequences of RNA complementary to specific sequences of messenger RNA (mRNA), which induce their degradation. Although some siRNAs have been shown to block the expression of the mutated allele to some extent, their selectivity for the mutated allele and ability to discriminate between mutant and wildtype (WT) allele remains an open challenge.
The purpose of the present invention is to provide novel siRNAs optimized for the treatment of ACTG2-dependent MMIHS disease.
Summary of the Invention
The invention object of the present application is based on the finding made by the present inventors that the complementarity, albeit total, to the mRNA sequence comprising the point mutation, is not sufficient alone to obtain effective and selective siRNAs, i.e. effective in silencing the expression of the mutated protein but inactive on the expression of the WT protein.
On the contrary, the optimal conjugation of effectiveness and selectivity depends on several factors such as the mutation itself on the mRNA, the length of the sequences that flank the mutation, therefore the position of the mutated nucleotide in the siRNA sequence, the presence or absence of one or more nucleotides mismatch with respect to the WT mRNA sequence and the position of said mismatches in the siRNA sequence: in short words from the design of the siRNA sequence itself.
In the present invention reference is made to the mutations of the wild type y -2 actin protein (Actin, gamma enteric smooth muscle), encoded by the ACTG2 gene, reported in uniprot as P63267 and in the present application as SEQ ID NO 4.
The gene is also known as ACT; ACTE; VSCM; ACTA3; ACTL3; ACTSG.
The gene is predominantly expressed in the adult, in the bladder, in the prostate, in the esophagus, in the appendix, in the colon, in the duodenum, in the endometrium and in the gallbladder.
For this reason, a first object of the present application are small interfering RNA (siRNA), complementary to the region comprising a point mutation in the messenger RNA (mRNA) of the mutated human ACTG2 gene or derivative or precursor thereof. The siRNAs object of the application are characterized in that (i) said mutations are reflected in the corresponding mutations of the ACTG2 protein: R40H, R40C; R63G; R178C, R135C; R178H, R135H; R178L, R135L; R257C, R214C; that (ii) the siRNAs have a nucleotide sequence comprising a fragment of 15 to 25 nucleotides, comprising the point mutation; that (iii) the siRNAs selectively reduce the expression of the mutated ACTG2 protein and that (iv) the ratio of effectiveness of the siRNAs of the invention in reducing the expression of the mutated ACTG2 protein relative to the normal protein is greater than one.
Optionally, the sequence of the small interfering RNA (siRNA) of the invention comprises, in addition to the mutated nucleotide, one or more nucleotides mismatches compared to the corresponding target sequence of the mRNA containing the mutation.
Optionally, the small interfering RNA (siRNA) sequence of the invention also comprises a short sticky sequence at the 3 'end composed of dA and dT nucleotides.
A second object of the invention is represented by the aforementioned siRNAs for use in a therapeutic treatment, specifically in the therapeutic treatment of megacyst-microcolon intestinal hypoperistalsis syndrome (MMIHS) caused by mutation of the ACTG2 gene.
A third object of the invention is a method for preparing the siRNAs seen above.
A fourth object of the invention is represented by pharmaceutical compositions comprising, as active ingredient, one or more siRNAs and a pharmacologically acceptable excipient. Such compositions are preferably for parenteral administration.
A further object of the invention is represented by the same compositions for use in the therapeutic treatment of the megacyst-microcolon-intestinal hypoperistalsis syndrome (MMIHS), also in association with a second active ingredient.
The siRNAs according to the invention, designed and tested in isolated cells and in animal models, proved highly specific for the mutated gene. They selectively eliminate up to 95% of the mutated gene transcript, improving disease symptoms.
The siRNAs of the invention also offer the further advantages of being internalized by the cells of interest, in particular by the smooth muscle cells, by simple incubation, without the need for any transfection agent, and to remain in the cell for a long time.
DETAILED DESCRIPTION OF THE INVENTION
Human ACTG2 mRNA is known to include mutations, pathogenic in intestinal megacystic- microcolon-hypoperistalsis syndrome (MMIHS), which generate mutated proteins as indicated in Table 1 :
Table 1
The position of the mutation is given with reference to the amino acid position of the wild type protein reported in uniprot as P63267 and in the present description as SEQ ID NO 4.
SiRNAs
Small interfering RNAs complementary to the region comprising a point mutation in human ACTG2 messenger RNA (mRNA) have been designed and produced for all mutations in the gene known to be pathogenetic for intestinal megacystic-microcolon-hypoperistalsis syndrome (MMIHS) , reported in Table 1.
The small interfering RNAs (siRNAs) of the invention are double-stranded (duplex) sequences, the first of which is called "guide" (or antisense) and the second "passenger" (or sense). The guide helix (antisense) is the one complementary to the target RNA that is to be inhibited, silenced or degraded.
Since the sequence of the passenger helix is complementary to the guide helix, for all siRNAs of the invention indicated in the present application, only the sequence of the guide helix is reported. The siRNAs of the invention have a sequence comprising or consisting of a fragment composed of 15 to 29 nucleotides, for example 16, 17, 18, 19, 20, 21 , 22, 23 or 24, 25, 26, 27 or 28 containing the point mutation.
The siRNAs of the invention are selected for their ability to selectively bind to the mRNA transcribed from the mutated allelic forms of the ACTG2 gene by reducing or nullifying the expression of the mutated ACTG2 protein. Thanks to their selectivity in silencing the mutated gene, their effectiveness in reducing expression is greater for the mutated protein than for the normal protein. Therefore they have a mutated ACTG2/normal ACTG2 effectiveness ratio greater than one.
In order to further increase the selectivity and/or specificity of the siRNAs towards the mutated mRNA, the siRNA sequence may comprise one or more non-complementary nucleotides (mismatch) to said mutated RNA sequence so as to have a significantly greater specificity for mutated mRNA with respect to the WT mRNA.
SiRNA derivatives
Furthermore, in order to increase the stability of the siRNAs produced and improve the efficiency of the effect produced, one or more nucleotides that form the siRNA sequence can be chemically modified in order to obtain derivatives of the siRNAs of the invention. All derivatives described below are therefore included in the scope of protection of this application.
First, the siRNA sequence can be endowed with a protruding 3 'terminal dTdT or dAdT sequence. The latter, in addition to providing stability and improving efficiency, induces the oligomerization of the siRNA in order to mimic the DNA (sticky siRNA). The sticky siRNAs can therefore be associated with usual reagents that ensure an efficient distribution of siRNA in vivo and decrease the ability to provoke immune responses mediated by pro-inflammatory cytokines and interferon: for example the jetPEI® product which is a linear derivative of polyethyleneimine provided by PolyPlus Transfection.
However, the siRNAs of the invention not modified, or otherwise modified, as described below, can equally be effectively used.
Other derivatives which improve the stability of the siRNAs of the invention in duplex form are the 2'-alkoxy (C1, C2, C3, C4) derivatives, for example the 2'-methoxy-derivatives, (i.e. 2'-OMe derivatives) ( Denise M Kenski, Gabor Butora, Aarron T Willingham, AbbyJ 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 Acids ( 2012) 1, e5; doi: 10.1038 / mtna.2011.4 ). 2'-OMe-derivatives, normally present in rRNA and tRNAs, are non-toxic derivatives of the siRNAs of the invention, wherein the -OMe group is inserted in position 2 'of the ribose nucleus in the sense-helix or anti- sense or both.
The 2'-fluorine (ie 2'-F) -derivatives (Denise M. Kenski et al) are also compatible with the function performed by the siRNAs of the invention and increase the stability of the duplex against nuclease degradation. The incorporation of fluorine in the 2 'position of the ribose nucleus maintains the activity of siRNAs both in vitro and in vivo, increasing their stability. The combined use of 2'-F in pyrimidine nucleotides with 2'-OMe in purine nucleotides results in an extremely stable duplex siRNA in serum and greatly improved effectiveness.
The 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 equally be used for increasing the stability of the siRNAs of the invention. MOE groups are frequently used in antisense oligonucleotides to give the oligonucleotide high resistance to nucleases and to increase Tm.
Other siRNA derivatives, with improved function and stability, suitable for the present invention are the 2'-O-benzyl derivatives and the 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).
Particularly interesting for the purposes of the present invention, for their stability, are the LNAs (locked nucleic acids) derivatives of siRNAs (see Mark A. Behlke above). As well known to the skilled person, these derivatives are characterized by a methylene bridge between the 2'-0 and 4'-C positions of ribose. The methylene bridge blocks the saccharide unit in the 3'-endo configuration, thus offering a significant increase in Tm and resistance to nucleases.
In a preferred embodiment, the siRNA of the invention, derivative or precursor thereof, comprises one or more chemically modified nucleotides which increase its stability and/or effectiveness.
Precursors
In a specific embodiment of the invention, siRNAs or derivatives thereof can be used in the form of their precursors in vivo. The latter also form object of the present invention.
As an example, siRNAs can be replaced by corresponding short hairpin RNAs (shRNAs), particularly in the context of gene therapy. As 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, for example 4-9 or 5-6 nucleotides. When introduced and expressed in the cell, the transcripts that form the shRNAs are processed by the DICER enzyme complex which by cutting the loop sequence directly converts the shRNAs into the corresponding siRNAs in the cell. These will then perform their function of silencing or knockdown of the target gene. Therefore, in the context of gene therapy, the siRNAs of the invention can be replaced by the corresponding shRNAs.
All derivatives and precursors described above are included in the scope of protection of this application.
As part of this work, various siRNAs specific for the mRNA of alleles of the human ACTG2 gene containing the mutations indicated in Table 1 were designed and produced. Subsequently, the effectiveness of the individual RNA fragments reported below has been analysed (Table 2) in inhibiting the expression of the ACTG2-WT protein with respect to proteins mutated in the positions: p.R178C, p.R178H, p.R178L.
In the experimental work described in the present application, all the reported siRNA sequences were provided with a protruding dTdT sequence at the 3 'end to improve their stability and effectiveness. For in vivo use the dTdT sequence is replaced by the dAdT sequence which further improves its stability and efficiency and allows its binding to any vehicles that allow a better in vivo distribution of the siRNA and reduce any immune responses.
The results obtained on siRNAs tested in silico in terms of their effectiveness on WT mRNA or mutated mRNA are listed in Table 2 below.
Table 2
The siRNA sequences highlighted in-silico show the mutated nucleotide in bold underlined (referring to the sequence encoding the human protein Actin, gamma-enteric smooth muscle reported in uniprot as P63267) and in bold italics the additional nucleotide (s) mismatch (the). According to an embodiment, preferred siRNAs of the invention are: AUCAUGUGCAUGGACUUGGCU (SEQ ID NO: 1)
AUCAUGCACCUUGACUUGGCU (SEQ ID NO: 2)
AUCAUGCUCCUUGACUUGGCU (SEQ ID NO: 3).
The preferred siRNAs of the invention are characterized by a mutated nucleotide and an additional nucleotide mismatch, wherein said additional mismatch is located at a distance between 3 and 6 nucleotides from the mutated nucleotide.
According to a further aspect of the invention, further preferred siRNA are:
GGGACGGGGUACGGUAGUACA (SEQ ID NO:8)
GGACGGGGUACGGUAGUACAC (SEQ ID NO:9)
GACGGGGUACGGUAGUACACG (SEQ ID NO: 10)
ACGGGGUACGGUAGUACACGG (SEQ ID NO: 12)
CGGGGUACGGUAGUACACGGA (SEQ ID NO: 13)
GGGGUACGGUAGUACACGGAC (SEQ ID NO: 14)
GGGUACGGUAGUACACGGACC (SEQ ID NO: 15)
GGUACGGUAGUACACGGACCU (SEQ ID NO: 16)
GUACGGUAGUACACGGACCUG (SEQ ID NO: 17)
UACGGUAGUACACGGACCUGA (SEQ ID NO: 18)
ACGGUAGUACACGGACCUGAA (SEQ ID NO: 19)
ACGGUACUACACGGACCUGAA (SEQ ID NO: 20)
ACGGUUGUACACGGACCUGAA (SEQ ID NO: 21)
ACGGUACUACACGGACCUGAA (SEQ ID NO: 22)
CGGUAGUACACGGACCUGAAC (SEQ ID NO: 23)
GGUAGUACACGGACCUGAACC (SEQ ID NO: 24)
GUAGUACACGGACCUGAACCG (SEQ ID NO: 25)
UAGUACACGGACCUGAACCGA (SEQ ID NO: 26)
AGUACACGGACCUGAACCGAC (SEQ ID NO: 27)
GUACACGGACCUGAACCGACC (SEQ ID NO: 28)
UACACGGACCUGAACCGACCG (SEQ ID NO: 29)
ACACGGACCUGAACCGACCGG (SEQ ID NO: 30)
CACGGACCUGAACCGACCGGC (SEQ ID NO: 31)
ACGGACCUGAACCGACCGGCA (SEQ ID NO: 32) In a preferred embodiment of the invention, all of the above listed siRNAs are provided, preferably for in vivo use, of the short protruding sequence dAdT or dTdT at the 3 'end.
SiRNAs preparation method
Nucleotide synthesis methods for the preparation of short RNA sequences are known to those skilled in the art and described in the prior 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 deoxy-ribonucleotides "overhangs" at 3'. After synthesis, the siRNAs underwent the following purification processes:
• Removal of salts by precipitation in ethanol or using chromatographic columns of the type 018
• Removal of the 2'-ACE group present in RNA bases
• Pairing with the antisense sequence (synthesized in a separate reaction).
• Purification 1: Duplex siRNA is purified by acrylamide gel electrophoresis
• Purification 2: the duplex siRNA obtained from the above step is further purified with ion exchange liquid chromatography (HPLC)
• Purification 3: the duplex siRNA obtained from the step described above is subjected to ion exchange (Na+), desalted, sterilized by filtration and tested for the presence of endotoxins.
Compositions and dosages
The siRNAs of the invention, their chemical derivatives and/or precursors can be administered systemically or locally.
Tests conducted in vitro on cell cultures and in vivo on animal models are aimed at demonstrating that the siRNAs of the invention are effectively internalized in the cell without the need for any transfection agent, but by simple incubation with the cell in solution.
Therefore, pharmaceutical compositions suitable for administering the siRNAs of the invention or their chemical derivatives are compositions containing a pharmaceutically effective amount of siRNA, derivates or precursors 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 can then be used. Suitable excipients are physiological solutions for parenteral use, hydroalcoholic solutions, glycol solutions, water/oil or oil/water emulsions, liposome or exosome emulsions/suspensions, oil solutions, micellar suspensions, vesicles, or complexes with PEI (polyethylene imine) or complexes with atelocollagen, all containing the usual pharmaceutical additives, diluents, stabilizers and pH regulators at physiological values. The administration of the siRNAs of the invention, derivatives or precursors thereof, can occur parenterally, e.g. intravenous, intraperitoneal, intramuscular, intradermal, subcutaneous, intraosseous, intracartilaginous, intraarticular administration. Alternatively, the administration can be orally, through pills, tablets, oral or sublingual dissolving formulations, capsules, soft capsules, films, powders, granules; rectally or vaginally through suppositories or pessaries; by inhalation, e.g. intrabronchial.
Local administration can take place through any formulation suitable for local application, for example through topical application or direct application on or in the tissues to be treated, or by local administration of a siRNA precursor and in situ production of the siRNA of the invention. Compositions based on exosomes, liposomes, vesicles, micelles containing siRNAs or precursors thereof can be used to achieve both a systemic and local effect.
To obtain a local effect, the siRNAs of the invention or their derivatives or precursors can be administered through viral or non-viral vectors, or through the DNA encoding the siRNAs or as isolated (nacked) RNA (Pelled et al., 2010 Tissue Engineering: Part B, Volume 16, No.1, 13-20) or through three-dimensional biocompatible matrices or implants based e.g. on fibrinogen and thrombin polymers and placed at the point of application.
In a specific embodiment, the siRNAs, derivatives or precursors thereof, are bonded or associated or complexed with usual reagents that ensure efficient distribution of the siRNA in vivo , for example, polyethyleneimine (PEI) or its derivatives such as the polyethyleneimine complex, polyethylene glycol-N-acetylgalactosamine (PEI-PEG-GAL), or the polyethyleneiminepolyethylene glycol-tri-N-acetylgalactosamine complex (PEI-PEG-triGAL). In a specific embodiment of the invention the siRNAs are linked to the jetPEI® product which is a linear derivative of the polyethyleneimine supplied by PolyPlus Transfection.
Alternatively, the siRNAs of the invention can be locally administered in the form of their shRNA precursor as part of a gene therapy. For example, an shRNA, or the DNA encoding an shRNA, can be transferred into a mammalian cell, e.g. human, using e.g. a suitable plasmid or adenoviral vector as described by Egermann et al., Human Gene Ther . May 2006; 17 (5): 50717. The shRNAs expressed and processed by the cell itself produce the corresponding siRNAs capable of silencing the target gene. In an alternative in vivo delivery form to vectors, siRNAs can be transferred into a cell through electroporation, ultrasound, cationic liposome-mediated transfection, microinjection, electrical pulsation.
In another alternative local administration form, the siRNAs of the invention, their derivatives or precursors can be bonded, adsorbed, immobilized also through covalent bonds 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 in the area of interest as described by Fang et al., 1996 (Proc Natl Acad Sci USA 93, 5753).
Although transfection agents are not needed, they can nevertheless be used to enhance siRNA internalization in the cells of interest, preferably in smooth muscle cells. Suitable transfection agents for the present invention are: lipofectamine, nucleofection by Amaxa Nucleofector® method (Lonza, Cologne, Germany) using specific kit (Lonza, code VPA-1007).
Dosage
In vitro and in vivo tests are aimed at demonstrating that the siRNAs internalized in the cells of interest, preferably smooth muscle cells, maintain their integrity and therefore their functionality for a period of several days.
Therefore, the siRNA treatment regimen of the invention may include once-a-day to once-a-week administrations, for example 1, 2, 3, 4, 5, 6 or 7 administrations/week. Alternatively, the treatment can be performed with a daily administration or every 2, 3, 4, 5, 6, 7 days.
The duration of treatment depends on the severity of the disease and varies from a treatment lasting a few weeks to a chronic treatment.
The tests performed by the present inventors are aimed at demonstrating that the siRNAs of the invention are effective in restoring the functionality of the cells of interest, preferably smooth muscle cells, in a broad spectrum of dosages ranging from about 1 ng/kg of body weight to about 100 mg/kg of body weight of the subject to be treated or subject in which the symptoms of the progression of the megacyst-microcolon intestinal hypoperistalsis syndrome have manifested. In a preferred embodiment, the dosages will be from about 1 pg/Kg to 20 mg/Kg of body weight, preferably from about 1 mg/Kg to about 10mg/Kg.
In order to test the in vivo effectiveness of siRNAs as potential drugs, the following methodology is being adopted:
1. Verify that siRNA for normal ACGT2 mRNA is effective in reducing normal gene expression in WT mice. 2. Verify that siRNA against mutated ACGT2 mRNA does not alter normal gene expression.
3. Verify that siRNA against mutated ACGT2 mRNA is effective in reducing mutated mRNA and improving the phenotype of MMIHS mice.
Combined therapy
The siRNAs of the invention can be used in association with other active ingredients. The term "in combination" means either a co-therapy or combination therapy, or a co-formulation in a single pharmaceutical form, or in a single commercial package, for example a kit or a blister of two or more active ingredients.
Sequence Listing:
SEQ ID NO 1- siR178C
AUCAUGUGCAUGGACUUGGCU
SEQ ID NO 2- siR178H
AUCAUGCACCUUGACUUGGCU
SEQ ID NO 3 - siR178L
AUCAUGCUCCUUGACUUGGCU
SEQ ID NO 4
SEQ ID NO 5- siR178C.1
GGCAUGCUCCUUGACUUGGCU
SEQ ID NO 6- siR178H.1
AUCAUGCACCUyGACUUGUUU
SEQ ID NO 7- siR178L.1
AUCAUGCUCCUGGACUUAGAU
SEQ ID NO 8 - siR178C.1a
GGGACGGGGUACGGUAGUACA
SEQ ID NO 9 - siR178C.2 GGACGGGGUACGGUAGUACAC
SEQ ID NO 10 - siR178C.3
GACGGGGUACGGUAGUACACG
SEQ ID NO 11 - Actin, gamma-enteric smooth muscle ACTG2
MCEEETTALVCDNGSGLCKAGFAGDDAPRAVFPSIVGRPRHQGVMVGMGQKDSYVGDEAQ
SKRGILTLKYPIEHGIITNWDDMEKIWHHSFYNELRVAPEEHPTLLTEAPLNPKANREKM
TQIMFETFNVPAMYVAIQAVLSLYASGRTTGIVLDSGDGVTHNVPIYEGYALPHAIMRLD
LAGRDLTDYLMKILTERGYSFVTTAEREIVRDIKEKLCYVALDFENEMATAASSSSLEKS
YELPDGQVITIGNERFRCPETLFQPSFIGMESAGIHETTYNSIMKCDIDIRKDLYANNVL
SGGTTMYPGIADRMQKEITALAPSTMKIKIIAPPERKYSVWIGGSILASLSTFQQMWISK
PEYDEAGPSIVHRKCF
SEQ ID NO 12 - siR1780.4
ACGGGGUACGGUAGUACACGG
SEQ ID NO 13 - siR178C.5
CGGGGUACGGUAGUACACGGA
SEQ ID NO 14 - siR178C.6
GGGGUACGGUAGUACACGGAC
SEQ ID NO 15 - siR178C.7
GGGUACGGUAGUACACGGACC
SEQ ID NO 16 - siR178C.8
GGUACGGUAGUACACGGACCU
SEQ ID NO 17 - siR178C.9
G UACGG UAG UACACGGACCUG
SEQ ID NO 18 - siR178C.1O
UACGG UAG UACACGG ACC U GA
SEQ ID NO 19 - siR178C.11A ACGGUAGUACACGGACCUGAA
SEQ ID NO 20 - siR178C.110
ACGGUACUACACGGACCUGAA
SEQ ID NO 21 - SIR178C.11 D
ACGGUUGUACACGGACCUGAA
SEQ ID NO 22 - siR178C.11 E
ACGGUACUACACGGACCUGAA
SEQ ID NO 23 - siR178C.12
CGGUAGUACACGGACCUGAAC
SEQ ID NO 24 - siR178C.13
GGUAGUACACGGACCUGAACC
SEQ ID NO 25 - siR178C.14
GUAGUACACGGACCUGAACCG
SEQ ID NO 26 - siR178C.15
U AG U ACACGGACC U GAACCGA
SEQ ID NO 27 - siR178C.16
AGUACACGGACCUGAACCGAC
SEQ ID NO 28 - siR178C.17
G U ACACGGACC U G AACCG ACC
SEQ ID NO 29 - siR178C.18
UACACGGACCUGAACCGACCG
SEQ ID NO 30 - siR178C.19
ACACGGACCUGAACCGACCGG
SEQ ID NO 31 - siR178C.2O
CACGGACCUGAACCGACCGGC SEQ ID NO 32 - siR178C.21
ACGGACCUGAACCGACCGGCA
Experimental part
Example 1 : Generation of the vector bearing the ACTG2 gene construct
The WT-ACTG2 construct was obtained by cloning the complete human ACTG2 cDNA sequence into the pEGFP-C1 expression vector, using the Hindi 11 and Xhol restriction enzymes.
Example 2: Cell models and cell lines
HEK293 cells transfected with the empty vector or with the vector WT-, p.R178C, p.R178H, and P.R178L-EGFP were employed. The relative expression of ACTG2 is quantified by Real-Time RT-PCR.
HEK293 cells were transfected with vectors carrying the WT-, p.R178C, p.R178H, and p.R178L- EGFP constructs, then the expression and localization of the fluorescent fusion protein EGFP was detected by confocal microscopy.
Example 3: Primary lines, Isolation of intestinal stem cells
Crypt cells were isolated from the small intestine (jejunum) resulting from surgical resection by enzymatic / mechanical method and cultured in IESC medium for intestinal epithelial stem cells. Crypt-derived cells were then differentiated by culture in mTeSRTM medium on Matrigel-coated plates. Differentiation into mesodermal lineage cells was initiated at time 0 by culturing cells with CHIR99021 (5 pM) and BMP-4 (10 ng I mL) in RPMI1640 medium and 2% B27. Differentiation into contractile Smooth Muscle Cells (SMCs) began on day 3. Synthetic SMCs were produced by culturing cells with 25 ng I mL of VEGF-A and FGFp in RPMI1640 and 2% of B27 from day 3 to day 7, with 25 ng / mL VEGF-A and FGFp in RPMI1640 and 2% B27 from day 7 to day 9, and with 10 ng / mL PDGFβ and 3 ng / mL TGF|3 in RPMI1640 and 2% B27 from day 10 to day 14. The Differentiated cells were maintained in RPMI1640 metabolic medium enriched with 4 mM lactate for 4-6 days. Stem cells were transfected with the blank EGFP vector (control) or with the vector p.R178C, p.R178H, and p.R178L-EGFP.
Example 4 - In silico experiments
SiRNAs having SEQ ID sequences. Nos. 1-3 and 5-7 were tested in silico for their effectiveness on WT mRNA or mutated mRNA (Table 2). The siRNAs tested showed significantly greater effectiveness in reducing mutant mRNA compared to WT mRNA. With an effectiveness greater than 98% in reduction of mutated mRNA, siRNAs having SEQ ID N 1-3 were found to be highly more effective than siRNAs having SEQ ID No. 5-7.
Example 5: In vivo experiments
In order to test the in vivo effectiveness of siRNAs, a murine model was created by humanization strategy in ROSA26 for over-expression of the human ACTG2 gene under control of the murine ACTG2 promoter. By homologous recombination, the coding region (Coding DNA sequence, CDS) of the mutated human ACTG2 gene, was inserted into the ROSA26 locus preceded by a Neomycin resistance cassette flanked by 2 FRT sites and by the 6KB region identified on Ensembl as Promoter Murino by ACTG2.
For the molecular and functional characterization of humanized R178C ACTG2 mice, a number of animals equal to at least 7 males and 7 females from litters other than the F2 generation are being used. This is the number of animals to be used normally for an assessment of suffering as per Corrigendum of 23/01/2013 and which in this case is sufficient for a molecular and functional characterization of the models. Therefore, 66 animals are being employed of which 9 animals are WT and 57 GM (ACTG2 humanized R178C in heterozygosity and in homozygosity). Therefore, we are using the couplings described in the following diagram:

Claims

1. Small interfering RNA (siRNA) complementary to the region comprising a point mutation in the messenger RNA (mRNA) of the mutated human gene ACTG2, or derivative or precursor thereof, characterized in that:
• the mutation is selected from one of the mutations resulting in the following mutations of the corresponding protein ACTG2: R40H, R40C; R63G; R178C, R135C; R178H, R135H; R178L, R135L; R257C, R214C;
• that the siRNA has a nucleotide sequence comprising a fragment of 15 to 25 nucleotides, containing the point mutation; that the siRNA reduces the expression of the mutated protein ACTG2;
• and that the ratio of efficacy of the siRNA in reducing the expression of the mutated protein ACTG2 compared to the normal protein is greater than 1.
2. The small interfering RNA (siRNA), derivative or precursor thereof, according to claim 1 , characterized in that the sequence of the siRNA comprises, in addition to the mutated nucleotide, one or more nucleotide mismatches compared to the corresponding target sequence of the mRNA containing the mutation and/or characterized in that the sequence of the siRNA comprises a short sequence dTdT or dAdT protruding to the 3’ end.
3. The small interfering RNA (siRNA), derivative or precursor thereof, according to any one of claims 1 or 2, characterized in that the derivative is a siRNA comprising one or more chemically modified nucleotides.
4. The small interfering RNA (siRNA), derivative or precursor thereof, according to claim 3, wherein the derivative is selected from the group comprising: 2’-alcoxy derivatives, 2’-metoxy- derivatives, 2’-ethoxy-derivatives, 2’-fluorine-derivatives, 2’-O-(2-metoxyethyl)-derivatives, 2’-O- benzyl-derivatives, 2’-O-methyl-4-pyridinil-derivatives, 2’-amino-derivatives, 2’-aminoethyl- derivatives, 2’-guanidinopropyl-derivatives or LNAs-derivatives.
5. The small interfering RNA (siRNA), derivative or precursor thereof, according to any one of claims 1 to 4, wherein the siRNA or derivative or precursor thereof is linked or associated or complexed to polyethyleneimine (PEI) or to a derivative thereof selected from the polyethyleneimine-polyethylene-glycol-N-acetyl galactosamine (PEI-PEG-GAL) complex, or the polyethyleneimine-polyethylene glycol-tri-N-acetyl galactosamine (PEI-PEG-triGAL) complex.
6. The small interfering RNA (siRNA), derivative or precursor thereof, according to any one of claims 1-5, characterized in that the precursor is a shRNA.
7. The small interfering RNA (siRNA) according to any one of claims 1 to 6 selected from the sequences:
AUCAUGUGCAUGGACUUGGCU (SEQ ID NO:1)
AUCAUGCACCUUGACUUGGCU (SEQ ID NO:2)
AUCAUGCUCCUUGACUUGGCU (SEQ ID NO:3)
GGGACGGGGUACGGUAGUACA (SEQ ID NO:8)
GGACGGGGUACGGUAGUACAC (SEQ ID NO:9)
GACGGGGUACGGUAGUACACG (SEQ ID NO: 10)
ACGGGGUACGGUAGUACACGG (SEQ ID NO: 12)
CGGGGUACGGUAGUACACGGA (SEQ ID NO: 13)
GGGGUACGGUAGUACACGGAC (SEQ ID NO: 14)
GGGUACGGUAGUACACGGACC (SEQ ID NO: 15)
GGUACGGUAGUACACGGACCU (SEQ ID NO: 16)
GUACGGUAGUACACGGACCUG (SEQ ID NO: 17)
UACGGUAGUACACGGACCUGA (SEQ ID NO: 18)
ACGGUAGUACACGGACCUGAA (SEQ ID NO: 19)
ACGGUACUACACGGACCUGAA (SEQ ID NO: 20)
ACGGUUGUACACGGACCUGAA (SEQ ID NO: 21)
ACGGUACUACACGGACCUGAA (SEQ ID NO: 22)
CGGUAGUACACGGACCUGAAC (SEQ ID NO: 23)
GGUAGUACACGGACCUGAACC (SEQ ID NO: 24)
GUAGUACACGGACCUGAACCG (SEQ ID NO: 25)
UAGUACACGGACCUGAACCGA (SEQ ID NO: 26)
AGUACACGGACCUGAACCGAC (SEQ ID NO: 27)
GUACACGGACCUGAACCGACC (SEQ ID NO: 28)
UACACGGACCUGAACCGACCG (SEQ ID NO: 29)
ACACGGACCUGAACCGACCGG (SEQ ID NO: 30)
CACGGACCUGAACCGACCGGC (SEQ ID NO: 31)
ACGGACCUGAACCGACCGGCA (SEQ ID NO: 32) all sequences optionally having a short dTdT or dAdT sequence protruding to the 3’ end or derivatives or precursors thereof.
8. The small interfering RNA (siRNA), derivative or precursor thereof, according to any one of claims 1 to 7, for use in a therapeutic treatment.
9. The small interfering RNA (siRNA), derivative or precursor thereof, according to any one of claims 1 to 8, for use in the therapeutic treatment of megacystis microcolon intestinal hypoperistalsis (MMIHS).
10. The small interfering RNA (siRNA), derivative or precursor thereof, according to claim 9, for use in the therapeutic treatment of MMIHS caused by a mutation of the gene ACTG2 and/or wherein said therapeutic treatment provides for a daily administration, or an administration every 2, 3, 4, 5, 6, or 7 days and/or, wherein said therapeutic treatment provides dosages for single administration from about 1 ng/kg of body weight to about 100 mg/kg of body weight, or dosages from about 1 pg/Kg to 20 mg/Kg of body weight, or dosages from about 1 mg/Kg to about 10mg/Kg.
11. The small interfering RNA (siRNA), derivative or precursor thereof, according to any one of claims 8 to 10, wherein said therapeutic treatment provides for intravenous, intraperitoneal, intramuscular, intradermal, subcutaneous, intraosseous, intracartilagineous, intraarticular, oral, oral with buccal dissolution, oral with sublingual dissolution, rectal, vaginal, intrabronchial administration or by inhalation and/or, wherein said therapeutic treatment provides for administration through electroporation, ultrasound-induced poration, cationic liposome-mediated transfection, microinjection, electropulsation and/or wherein said therapeutic treatment provides for administration through viral or non-viral vectors, or through the DNA encoding the siRNA or as isolated (naked) RNA or through three-dimensional, biocompatible matrices or implants.
12. The small interfering RNA (siRNA), derivative or precursor thereof, according to any one of claims 8 to 11, wherein the siRNA or derivative or precursor thereof is bonded or associated or complexed to polyethyleneimine (PEI) or to a derivative thereof selected from the polyethyleneimine-polyethylene-glycol-N-acetyl galactosamine (PEI-PEG-GAL) complex, or the polyethyleneimine-polyethylene glycol-tri-N-acetyl galactosamine (PEI-PEG-triGAL) complex.
13. The small interfering RNA (siRNA), derivative or precursor thereof, according to any one of claims 8 to 12, wherein the therapeutic treatment provides for the administration of siRNA, derivative or precursor thereof, associated with one or more additional active principles.
14. A method for the preparation of the siRNA, or derivatives or precursors thereof, according to any one of the claims 1 to 7, characterized in that the siRNA have been prepared by chemical synthesis and subsequent purification.
15. A pharmaceutical composition comprising, as active ingredient, one or more small interfering RNA (siRNA), derivative or precursor thereof, according to any one of claims 1 to 13 and a pharmacologically acceptable excipient and optionally comprising an additional active principle.
16. The pharmaceutical composition according to claim 15 in the form of solution, suspension, emulsion and/or comprising exosomes, liposomes, vesicles, micelles.
17. The pharmaceutical composition according to any one of claims 15 or 16, for use in the therapeutic treatment of megacystis microcolon intestinal hypoperistalsis (MMIHS) caused by a mutation of the gene ACTG2.
EP22700444.7A 2021-02-05 2022-01-18 Small interfering rna (sirna) for therapy of mmihs caused by actg2 gene mutation Withdrawn EP4288536A1 (en)

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IT102021000002573A IT202100002573A1 (en) 2021-02-05 2021-02-05 SMALL INTERFERING RNA (SIRNA) FOR THE THERAPY OF MEGACYSTI-MICROCOLON-INTESTINAL HYPERYSTALSIS SYNDROME (MMIHS) CAUSED BY MUTATION OF THE ACTG2 GENE
PCT/IB2022/050376 WO2022167877A1 (en) 2021-02-05 2022-01-18 Small interfering rna (sirna) for therapy of mmihs caused by actg2 gene mutation

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