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WO2023199039A1 - Molécule d'acides nucléiques fonctionnelle - Google Patents

Molécule d'acides nucléiques fonctionnelle Download PDF

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Publication number
WO2023199039A1
WO2023199039A1 PCT/GB2023/050956 GB2023050956W WO2023199039A1 WO 2023199039 A1 WO2023199039 A1 WO 2023199039A1 GB 2023050956 W GB2023050956 W GB 2023050956W WO 2023199039 A1 WO2023199039 A1 WO 2023199039A1
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Prior art keywords
nucleotides
sequence
nucleic acid
acid molecule
functional nucleic
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Inventor
Mathieu LATREILLE
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Transine Therapeutics Ltd
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Transine Therapeutics Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y306/00—Hydrolases acting on acid anhydrides (3.6)
    • C12Y306/05—Hydrolases acting on acid anhydrides (3.6) acting on GTP; involved in cellular and subcellular movement (3.6.5)
    • C12Y306/05005—Dynamin GTPase (3.6.5.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3519—Fusion with another nucleic acid

Definitions

  • the present invention relates to functional nucleic acid molecules that upregulate the expression of OPA1 by increasing protein translation. Also included are methods of enhancing OPA1 translation, and methods of treating diseases or disorders associated with OPA1 using the functional nucleic acid molecules of the invention.
  • ADOA Autosomal Dominant Optic Atrophy
  • the human OPA1 protein is a ubiquitously expressed dynamin-related GTPase, which localizes in the inner mitochondrial membrane (IMM). OPA1 regulates mitochondrial morphology by mediating the equilibrium between mitochondrial fusion and fission events and is functionally important in mitochondrial homeostasis. OPA1 is one of the main factors that controls mitochondrial fusion, mitochondrial DNA (mtDNA) maintenance, bioenergetics, and cristae integrity. These cellular processes are implicated in several diseases.
  • IMM inner mitochondrial membrane
  • OPA1 controls apoptosis through cristae remodelling and cytochrome c release independently from mitochondrial fusion (Frezza et al. (2006) Cell 126(1):177-89). OPA1 is highly expressed in the brain, retina, and heart.
  • OPA1 under-expression As seen in ADOA patients, and over-expression have deleterious consequences, with both of these alterations in OPA1 levels leading to elevated apoptosis (Chen et al. (2009) Cardiovasc Res. 84(1):91-9).
  • Data supporting the in vivo effects of OPA1 expression variance are complex. Whilst transgenic mice with moderate OPA1 over-expression appear healthy, fertile, and exhibit protection against insults to specific tissue such as the liver and brain, prolonged overexpression in the SV129 mouse strain increases incidence of spontaneous cancer and reduces lifespan (Varanita et al. (2015) Cell Metab. 21(6): 834-44). High expression of OPA1, and other mitochondrial proteins that promote fusion, is linked to cancerous cell proliferation, survival and invasion. 0PA1 is highly expressed in lung adenocarcinoma cells and is associated with cisplatin resistance and poor prognoses (Fang et al. (2012) Hum. Pathol. 43(1):105-14).
  • SINEUPs are able to selectively enhance the translation of their target proteins by binding to the mRNAs which encode them and promoting translational upregulation.
  • SINEUP activity relies on the combination of two domains: the overlapping region, or binding domain (BD), that confers target specificity, and an embedded inverted SINE B2 element, or effector domain (ED), that promotes enhanced translation of the target mRNA.
  • BD binding domain
  • ED effector domain
  • WO 2012/133947 and WO 2019/150346 which are incorporated herein by reference in their entirety, disclose functional nucleic acid molecules including SINEUPs.
  • Another class of IncRNAs use effector domains comprising an internal ribosome entry site (IRES) sequence to provide trans-acting functional nucleic acid molecules and are described in WO 2019/058304, which is incorporated herein by reference in its entirety.
  • IRS internal ribosome entry site
  • the aim of the invention is to provide target specific functional nucleic acids that can increase or restore OPA1 protein levels by increasing translation of OPA1 mRNAs.
  • Such functional nucleic acids would have particular utility in treating diseases or disorders associated with OPA1, such as ADOA.
  • SINEUP functional nucleic acids that are both SINEUPs and non- SINE containing IncRNAs (i.e. , which contain IRES effector domains or regulatory domains).
  • SINEUP may be used to encompass both traditional SINEUPs containing a SINE element as well as corresponding and functionally analogous functional nucleic acids containing an IRES.
  • the functional nucleic molecule disclosed herein targets OPA1 for translational upregulation.
  • a functional nucleic acid molecule comprising:
  • target binding sequences comprising a sequence reverse complementary to an OPA1 mRNA sequence
  • regulatory sequences comprising a SINE B2 element or a functionally active fragment of a SINEB2 element, or an internal ribosome entry site (IRES) or a functionally active fragment of an IRES.
  • the target binding sequence of the functional nucleic acid herein is coupled to the effector functionality of either a SINE B2 sequence or an IRES sequence, a sequence with appropriate percentage identity thereto such that functionality is retained, or a functionally active fragment thereof.
  • a DNA molecule encoding the functional nucleic acid molecule as defined herein.
  • an expression vector comprising the functional nucleic acid molecule, or the DNA molecule, as defined herein.
  • composition comprising the functional nucleic acid molecule, the DNA molecule or the expression vector, as defined herein.
  • composition as defined herein, comprising the functional nucleic acid molecule, the DNA molecule or the expression vector, as defined herein.
  • nucleic acid molecule for enhancing translation of OPA1 mRNA.
  • the functional nucleic acid molecule the DNA molecule, the expression vector, the composition or the pharmaceutical composition, as defined herein, for use in medicine.
  • a method of treating a disease associated with 0PA1 comprising administering the functional nucleic acid molecule, the DNA molecule, the expression vector, the composition or the pharmaceutical composition, as defined herein, to a subject.
  • an in vitro or in vivo method for increasing the translation of OPA1 in a cell comprising administering the functional nucleic acid molecule, the DNA molecule, the expression vector, the composition or the pharmaceutical composition, as defined herein, to the cell.
  • Figure 1 Schematic representation of sequence-based 0PA1 target binding domain design.
  • BD Functional nucleic acid molecule binding domains
  • BDs were designed against the 5’UTR and exon 1 of human OPA1 (hOPA1) using a ‘tiling’ approach whereby BDs of a given length, e.g., 18, 33, 44, or 72 nucleotides, are complementary to target sequences that are each one nucleotide downstream (i.e. , 3’) of the previous target sequence.
  • hOPA1 mRNA is illustrated with the approximate region encompassed by SEQ ID NO:1, comprising the 5’UTR and part of exon 1, shown in expanded view.
  • the schematic mRNA sequence is annotated with uORF and M1 (or Met1) sites shown as sticks.
  • Structural regulatory elements within the 5’UTR are shown as grey arrows.
  • BD sequences are shown as black lines beneath the mRNA schematic.
  • Figure 2 Schematic representation of structure-based OPA1 target binding domain design.
  • Functional nucleic acid molecule binding domain sequences may be designed and classified on the basis of the proximity to, or participation of, their corresponding target mRNA sequence in RNA structures, or structural regulatory elements.
  • Predicted RNA structures within the hOPA1 5’UTR are illustrated, with base pairing probabilities colour-coded in a gradient from 0 to 1 (blue to red).
  • RNAfold v2.4.18 http://rna.tbi.univie.ac.at/cgi- bin/RNAWebSuite/RNAfold.cgi ) was used to predict the secondary structures of human 0PA1 5'IITR, using standard parameters.
  • 5’IITR sequences were obtained from the Ensembl genome browser.
  • the upstream open reading frame (uORF) AUG codon is circled.
  • Illustrative target sequences are shown as black lines and labelled A - L. The sequence displayed corresponds to SEQ ID NO: 1599.
  • Figure 3 Schematic representation of RNA structure within hOPA1 exon 1 and 5’UTR.
  • RNAfold v2.4.18 http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi ) was used to predict the secondary structures of human 0PA1 exon 1 and 5'UTR, using standard parameters.
  • the M1 (or Met1) initiator methionine is shown circled.
  • a functional nucleic acid molecule comprising one or more target binding sequence comprising a sequence reverse complementary to an 0PA1 mRNA sequence and one or more regulatory sequence comprising a SINE B2 element, or functionally active fragment thereof, or an internal ribosome entry site (IRES), or functionally active fragment thereof, which act post- transcriptionally to increase target protein levels, i.e. 0PA1 levels.
  • the functional nucleic acid molecule of the invention may be utilised for the targeted upregulation of 0PA1 protein, e.g., in diseases or disorders characterised by a lack of 0PA1 protein, without affecting mRNA levels. Further, the functional nucleic acid molecule of the invention may increase protein levels within a normal physiological range, i.e., not promote protein over-expression above normal physiological upper limits, and in so doing enhance the translation of target 0PA1 mRNA sequences without inducing negative side-effects associated with increasing expression of the target above normal physiological levels.
  • a functional nucleic acid molecule of the present invention comprises one or more target binding sequence, wherein each target binding sequence comprises a sequence reverse complementary to an 0PA1 mRNA sequence for which protein translation is to be enhanced; one or more regulatory sequence comprising a SINE B2 element or a functionally active fragment of a SINEB2 element, or an internal ribosome entry site (IRES) or a functionally active fragment of an IRES.
  • each target binding sequence comprises a sequence reverse complementary to an 0PA1 mRNA sequence for which protein translation is to be enhanced
  • regulatory sequence comprising a SINE B2 element or a functionally active fragment of a SINEB2 element, or an internal ribosome entry site (IRES) or a functionally active fragment of an IRES.
  • the “functional nucleic acid molecule” referred to herein is a synthetic molecule of the invention.
  • the term “functional nucleic acid molecule” describes a nucleic acid molecule (e.g. DNA or RNA) that is capable of enhancing translation of a target OPA1 mRNA.
  • the term “functional RNA molecule” refers to instances wherein the functional nucleic acid molecule is formed of RNA and said RNA molecule is capable of enhancing the translation of a target OPA1 mRNA.
  • the functional nucleic acid molecule of the invention is an RNA molecule.
  • the functional nucleic acid molecule further comprises one or more spacer sequences between the target binding sequences, i.e. , where there are more than one, and the regulatory sequence (where there are more than one), and/or between any target binding sequence and any regulatory sequence.
  • SEQ ID NO: 1581 is a non-limiting example of the spacer/linker sequence which may be used in the present invention.
  • the target binding sequence(s) and the effector domain sequence(s) within the functional nucleic acid molecule are separated by spacer sequences or “spacers”.
  • a non-limiting example of a suitable spacer/linker sequence is: AUCUGCAGAAUUC (SEQ ID NO: 1581)
  • the spacer/linker sequence comprises SEQ ID NO: 1581.
  • the spacer/linker sequence consists of SEQ ID NO: 1581.
  • the functional nucleic acid molecule provided herein may trans-acting such that it functionally modulates sequences present on other RNA molecules (i.e. OPA mRNA).
  • the functional nucleic acid molecule provided herein is a trans-acting functional nucleic acid molecule.
  • the functional nucleic acid molecule is single stranded. In one embodiment, the functional nucleic acid molecule comprises RNA nucleotides.
  • the functional nucleic acid molecule of the present invention preferably comprises RNA nucleotides.
  • the functional nucleic acid molecule consists of RNA nucleotides.
  • the functional nucleic acid molecule of the present invention preferably consists of RNA nucleotides.
  • the functional nucleic acid molecule is RNA.
  • the functional nucleic acid molecule of the present invention preferably is RNA.
  • the functional nucleic acid molecule comprises DNA nucleotides.
  • the functional nucleic acid molecule consists of DNA nucleotides.
  • the functional nucleic acid molecule is DNA.
  • the invention also encompasses a DNA molecule encoding the functional nucleic acid molecule of the invention.
  • the functional nucleic acid molecule comprises one or more modifications or chemical modifications.
  • modification refers to a structural change in, or on, the most common, natural ribonucleotides: adenosine, guanosine, cytidine, thymidine, or uridine ribonucleotides.
  • the chemical modifications described herein may be changes in or on a nucleobase (i.e. a chemical base modification), or in or on a sugar (i.e. a chemical sugar modification).
  • the chemical modifications may be introduced co-transcriptionally (e.g. by substitution of one or more nucleotides with a modified nucleotide during synthesis), or post-transcriptionally (e.g. by the action of an enzyme).
  • the chemical modification is a chemical base modification.
  • the chemical base modification may be selected from a modification of an adenine, cytosine and/or uracil base.
  • the chemical base modification is selected from methylation and/or isomerisation.
  • the chemical base modification is selected from the group consisting of: Pseudouridine ( ⁇ P), N1 -Methylpseudouridine (Nlm ⁇ P), 5-Methylcytidine (m5C) and N6-Methyladenosine (m6A).
  • the chemical base modification is selected from the group consisting of: Pseudouridine, N1- Methylpseudouridine and N6-Methyladenosine.
  • the chemical modification is a chemical sugar modification.
  • the chemical sugar modification is methylation.
  • the chemical sugar modification is a 2’ modification, such as a 2'-O-Methyl modification.
  • the chemical sugar modification is 2'-O-Methyladenosine (Am).
  • the functional nucleic acid molecule comprises a 3’-polyadenylation (polyA) tail.
  • a “3’-polyA tail” refers to a long chain of adenine nucleotides added to the 3’-end of the functional nucleic acid which provides stability to the RNA molecule and can promote translation.
  • the functional nucleic acid molecule comprises a 5’-cap.
  • a “5’-cap” refers to an altered nucleotide at the 5’-end of the transcript which provides stability to the molecule, particularly from degradation from exonucleases, and can promote translation.
  • the 5’-cap may be a 7-methylguanylate cap (m7G), i.e. a guanine nucleotide connected to the RNA via a 5' to 5' triphosphate linkage and methylated on the 7 position.
  • m7G 7-methylguanylate cap
  • the functional nucleic acid molecule described herein may constitute a miniSINEUP or microSINEUP, as defined in WO 2019/150346 and PCT/GB2021/052502, which are incorporated herein by reference in their entirety.
  • miniSINEUP there is intended a functional nucleic acid molecule comprising (or consisting of) one or more target binding domains (i.e. complementary sequences to target mRNAs), optionally a spacer sequence, and any SINE or IRES sequence as the effector domain (Zucchelli et al., Front Cell Neurosci., 9: 174, 2015).
  • microSINEUP there is intended a functional nucleic acid molecule comprising (or consisting of) one or more target binding domains (i.e. complementary sequences to target mRNAs), optionally a spacer sequence, and a functionally active fragment of the SINE or IRES sequence.
  • the functional nucleic acid molecule may be circular.
  • sequences that are represented herein as being implicitly DNA are to be understood as also representing a corresponding RNA molecule in which each T nucleotide in the sequence is replaced with a II nucleotide.
  • sequences according to the invention may be either DNA sequences or RNA sequences.
  • the representation of a sequence as either one or the other herein is enacted solely for simplicity and does not imply a sequence is restricted to being a DNA or RNA sequence, unless expressly indicated in the text.
  • a target sequence is a sequence within a naturally occurring mRNA, it will be understood that said sequence would implicitly comprise uracil nucleotides even where said sequence is disclosed herein as a target sequence comprising thymine nucleotides.
  • sequences depicted in Tables 3 and 4 herein comprise U nucleotides but are, in accordance with the foregoing, represented in the corresponding sequence listing as DNA molecules comprising T nucleotides in place of each U nucleotide.
  • the target binding sequence (also referred to as the target determinant sequence or overlapping region) is the portion of the functional nucleic acid molecule that binds to the target mRNA and therein confers target specificity.
  • target binding sequence may equally refer in the singular to embodiments in which the functional nucleic acid molecule contains one target binding sequence or in the plural, to functional nucleic acid molecule which contain more than one target binding sequence.
  • the target binding sequence is the portion of the functional RNA molecule that binds to the target mRNA.
  • the functional nucleic acid molecule of the invention comprises one or more target binding sequence comprising a sequence reverse complementary to an OPA1 mRNA sequence.
  • the functional nucleic acid molecule comprises one, two, three, four, or five target binding sequences.
  • the functional nucleic acid molecule comprises one target binding sequence.
  • the functional nucleic acid molecule comprises two target binding sequences.
  • the functional nucleic acid molecule comprises three target binding sequences.
  • target sequence is used herein to denote the sequence to which the target binding sequence binds.
  • target sequence is an OPA1 sequence.
  • the OPA1 sequence may be an OPA1 sequence derived from any suitable animal, wherein the animal is preferably a mammal, and is further preferably a mouse, primate, or human.
  • OPA1 isoforms may be targeted by the functional nucleic acids of the present invention.
  • the OPA1 target sequence comprises a sequence within the 5’IITR of OPA1.
  • the OPA1 target sequence consists of a sequence within the 5’IITR of OPA1.
  • the OPA1 target sequence comprises a sequence within exon 1 of OPA1. In one embodiment the OPA1 target sequence consists of a sequence within exon 1 of OPA1.
  • the OPA1 target sequence comprises a sequence within the 5’IITR and exon 1 of OPA1.
  • the OPA1 target sequence consists of a sequence within the 5’IITR and exon 1 of OPA1.
  • the target sequence may comprise a sequence within the 5’IITR and exon 1 region of OPA1 , wherein said region is shown in the following sequence:
  • the OPA1 target sequence comprises a sequence set forth in SEQ ID NO: 1 , or a fragment thereof.
  • the OPA1 target sequence consists of a sequence set forth in SEQ ID NO: 1 , or a fragment thereof.
  • the target binding sequence comprises a sequence reverse complementary to a sequence within the 5’IITR of OPA1 , within exon 1 of OPA1 , or within both the 5’IITR and exon 1 of OPA1.
  • the binding sequence comprises a sequence reverse complementary to a sequence within SEQ ID NO: 1.
  • the target binding sequence is 18 nucleotides in length. In one embodiment the target binding sequence is reverse complementary to any contiguous 18 nucleotide sequence within SEQ ID NO: 1.
  • the target binding sequence is reverse complementary to any contiguous nucleotide sequence of between 10 and 100 nucleotides in length, that is between nucleotides 32 and -192 relative to M1 in OPA1 (+32/-192 M1).
  • the target binding sequence is reverse complementary to any contiguous 18 nucleotide sequence between nucleotides 32 and -192 relative to M1 in OPA1 (+32/-192 M1).
  • the target binding sequence is 18 nucleotides in length and is reverse complementary to any contiguous 18 nucleotide sequence within SEQ ID NO: 1.
  • the target binding sequence comprises a sequence as set forth in SEQ ID NOs: 2 - 208, or a fragment thereof.
  • the target binding sequence consists of a sequence as set forth in SEQ ID NOs: 2 - 208, or a fragment thereof.
  • the target sequence comprises a sequence as set forth in SEQ ID NOs: 753 - 959.
  • the target sequence consists of a sequence as set forth in SEQ ID NOs: 753 - 959.
  • the target binding sequence is 33 nucleotides in length.
  • the target binding sequence is reverse complementary to any contiguous 33 nucleotide sequence within SEQ ID NO: 1.
  • the target binding sequence is reverse complementary to any contiguous 33 nucleotide sequence between nucleotides 32 and -192 relative to M1 in OPA1 (+32/-192 M1).
  • the target binding sequence is 33 nucleotides in length and is reverse complementary to any contiguous 33 nucleotide sequence within SEQ ID NO: 1.
  • the target binding sequence comprises a sequence as set forth in SEQ ID NOs: 209 - 400, or a fragment thereof.
  • the target binding sequence consists of a sequence as set forth in SEQ ID NOs: 209 - 400, or a fragment thereof
  • the target sequence comprises a sequence as set forth in SEQ ID NOs: 960 - 1151.
  • the target sequence consists of a sequence as set forth in SEQ ID NOs: 960 - 1151.
  • the target binding sequence is 44 nucleotides in length.
  • the target binding sequence is reverse complementary to any contiguous 44 nucleotide sequence within SEQ ID NO: 1.
  • the target binding sequence is reverse complementary to any contiguous 44 nucleotide sequence between nucleotides 32 and -192 relative to M1 in OPA1 (+32/-192 M1).
  • the target binding sequence is 44 nucleotides in length and is reverse complementary to any contiguous 44 nucleotides within SEQ ID NO: 1.
  • the target binding sequence comprises a sequence as set forth in SEQ ID NOs: 401- 581, or a fragment thereof.
  • the target binding sequence consists of a sequence as set forth in SEQ ID NOs: 401- 581, or a fragment thereof.
  • the target sequence comprises a sequence as set forth in SEQ ID NOs: 1152 - 1332.
  • the target sequence consists of a sequence as set forth in SEQ ID NOs: 1152 - 1332. In one embodiment the target binding sequence is 72 nucleotides in length.
  • the target binding sequence is reverse complementary to any contiguous 72 nucleotide sequence within SEQ ID NO: 1.
  • the target binding sequence is reverse complementary to any contiguous 72 nucleotide sequence between nucleotides 32 and -192 relative to M1 in OPA1 (+32/-192 M1).
  • the target binding sequence is 72 nucleotides in length and is reverse complementary to any contiguous 72 nucleotide sequence within SEQ ID NO: 1.
  • the target binding sequence comprises a sequence as set forth in SEQ ID NOs: 582 - 734, or a fragment thereof.
  • the target binding sequence consists of a sequence as set forth in SEQ ID NOs: 582 - 734, or a fragment thereof.
  • the target sequence comprises a sequence as set forth in SEQ ID NOs: 1333 - 1485.
  • the target sequence consists of a sequence as set forth in SEQ ID NOs: 1333 - 1485.
  • the target binding sequence comprises a sequence as set forth in any one of SEQ ID NOs: 2 - 752, or a fragment thereof.
  • the target binding sequence consists of a sequence as set forth in any one of SEQ ID NOs: 2 - 752, or a fragment thereof.
  • the target sequence comprises a sequence as set forth in any one of SEQ ID NOs: 1 , and 753 - 1503.
  • the target sequence consists of a sequence as set forth in any one of SEQ ID NOs: 1 , and 753 - 1503.
  • the nucleotides of the 5’UTR sequence are numbered sequentially using decreasing negative numbers approaching the AUG site on the target mRNA (e.g. -3, -2, -1).
  • the nucleotides of the CDS sequence are numbered sequentially using increasing positive numbers (e.g. +1 , +2, +3) from the AUG site, such that the A of the AUG site is numbered +1.
  • the region bridging the 5’UTR and the CDS will therefore be numbered -3, -2, -1 , +1 , +2, +3, with the A of the AUG site numbered +1. It should be noted that according to this numbering scheme there is no nucleotide “0”.
  • a target binding sequence needs to have only about 60% similarity with a sequence reverse complementary to the target mRNA. In fact, the target binding sequence can even display a large number of mismatches and retain activity.
  • the target OPA1 mRNA is encoded by the genomic DNA sequence as set forth in SEQ ID NO: 1582
  • the target sequence is a sequence encoded by SEQ ID NO: 1582, or a fragment thereof.
  • the target binding sequence is reverse complementary to a sequence encoded by SEQ ID NO: 1582, or a fragment thereof
  • the target binding sequences of the functional nucleic acid molecule of the invention may display about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity with a sequence reverse complementary to the target OPA1 mRNA.
  • the target binding sequences of the functional nucleic acid molecule of the invention may display about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity with a sequence reverse complementary to SEQ ID NO: 1.
  • the one or more target binding sequence comprises a sequence with about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity with a sequence as set forth in SEQ ID NOs: 2 - 752.
  • the one or more target binding sequence consists of a sequence with about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity with a sequence as set forth in SEQ ID NOs: 2 - 752.
  • the target binding sequences is reverse complementary to a sequence having at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to an OPA1 sequence.
  • the target binding sequences is reverse complementary to a sequence having at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to a sequence of any one of SEQ ID NOs: 1 , and 753 - 1503.
  • polypeptide or polynucleotide sequences are said to be the same as or “identical” to other polypeptide or polynucleotide sequences, if they share 100% sequence identity.
  • Residues in sequences are numbered from left to right, i.e. from N- to C- terminus for polypeptides; from 5’ to 3’ terminus for polynucleotides. If closely related sequences are not identical they may be similar, i.e., they may possess a certain, quantifiable, degree of sequence identity, e.g., a sequence may have 50%, 60%, 70%, 80%, 90%, 95% or 99% sequence identity to another sequence.
  • any quoted sequence identity will be understood as being calculated across the residue range over which the two sequences are aligned.
  • the aligned residue range may represent the entirety of one or more of the input sequences or a contiguous section of sequence of one or more of the input sequences, and is typically determined by standard tools known in the art, e.g., NCBI BLAST.
  • the “% sequence identity” between a first nucleotide sequence and a second nucleotide sequence may be calculated using NCBI BLAST, using standard settings for nucleotide sequences (BLASTN).
  • the “% sequence identity” between a first polypeptide sequence and a second polypeptide sequence may be calculated using NCBI BLAST, using standard settings for polypeptide sequences (BLASTP).
  • a “difference” between sequences refers to an insertion, deletion or substitution of a single nucleotide in a position of the second sequence, compared to the first sequence. Insertions, deletions or substitutions in a second sequence which is otherwise identical (100% sequence identity) to a first sequence result in reduced % sequence identity.
  • “Complementarity” relates to the Watson-Crick base pairing principle that ‘A’ nucleotides will hydrogen bond with T (or ‘U’) nucleotides, and ‘G’ nucleotides with ‘C’ nucleotides to form double stranded structures that associate via said “complementary” nucleotides.
  • a “complementary” sequence is a sequence closely-related to another sequence such that such base pairing can occur.
  • Complementary sequences may be 100% complementary (i.e. , identical) such that they may base pair across their entire length, or they may be e.g., 99%, 90%, 80%, 70%, or 60% complementary etc., such that they base pair across portions of their sequence.
  • a complementary sequence may also be called a “reverse complementary” sequence.
  • the target binding sequence comprises a sequence which is sufficient in length to bind to the target mRNA transcript. Therefore, the target binding sequence may be at least about 10 nucleotides in length, such as at least about 14 nucleotides in length, such as at least about 15 nucleotides in length, such as at least about 16 nucleotides in length, such as at least about 17 nucleotides in length, such as least 18 nucleotides in length.
  • the target binding sequence may be less than about 250 nucleotides in length, preferably less than about 200 nucleotides in length, less than about 150 nucleotides in length, less than about 140 nucleotides in length, less than about 130 nucleotides in length, less than about 120 nucleotides in length, less than about 110 nucleotides in length less than about 100 nucleotides in length, less than about 90 nucleotides in length, less than about 80 nucleotides in length, less than about 70 nucleotides in length, less than about 60 nucleotides in length or less than about 50 nucleotides in length.
  • the target binding sequence is between about 4 and about 100 nucleotides in length, such as between about 18 and about 72 nucleotides in length, between about 18 and about 44 nucleotides in length, between about 18 and about 33 nucleotides in length.
  • the target binding sequence is 18 nucleotides in length.
  • the target binding sequence is 33 nucleotides in length.
  • the target binding sequence is 44 nucleotides in length.
  • the target binding sequence is 72 nucleotides in length.
  • the target binding sequence may hybridise with the 5’-untranslated region (5’ UTR) of the target OPA1 mRNA sequence.
  • the sequence is reverse complementary to a sequence that is 0 to 72 nucleotides, such as 0 to 70, 0 to 60, 0 to 50, 0 to 45, 0 to 44, 0 to 43, 0 to 42, 0 to 41 , 0 to 40, 0 to 39, 0 to 38, 0 to 37, 0 to 36, 0 to 35, 0 to 34, 0 to 33, 0 to 32, 0 to 31 , 0 to 30, 0 to 29, 0 to 28, 0 to 27, 0 to 26, 0 to 25, 0 to 24, 0 to 23, 0 to 22, 0 to 21 O to 20, O to 19, O to 18, O to 17, O to 16, O to 15, O to 14, O to 13, 0 to 12, 0 to 11 , 0 to 10, 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, or 0 to 4
  • the target binding sequence may hybridise to exon 1 of the coding sequence (CDS) of the target OPA1 mRNA sequence.
  • the sequence is reverse complementary to a sequence that is 0 to 72 nucleotides in length, such as 0 to 70, 0 to 60, 0 to 50, 0 to 45, 0 to 44, 0 to 43, 0 to 42, 0 to 41 , 0 to 40, 0 to 39, 0 to 38, 0 to 37, 0 to 36, 0 to 35, 0 to 34, 0 to 33, 0 to 32, 0 to 31 , 0 to 30, 0 to 29, 0 to 28, 0 to 27, 0 to 26, 0 to 25, 0 to 24, 0 to 23, 0 to 22, 0 to 21 0 to 20, 0 to 19, 0 to 18, 0 to 17, 0 to 16, 0 to 15, 0 to 14, 0 to 13, 0 to 12, 0 to 11, 0 to 10, 0 to 9, 0 to 8, 0 to
  • the target binding sequence may hybridise to the coding sequence (CDS) of the target OPA1 mRNA sequence.
  • the sequence is reverse complementary to a sequence that is 0 to 72 nucleotides in length, such as 0 to 70, 0 to 60, 0 to 50, 0 to 45, 0 to 44, 0 to 43, 0 to 42, 0 to 41 , 0 to 40, 0 to 39, 0 to 38, 0 to 37, 0 to 36, 0 to 35, 0 to 34, 0 to 33, 0 to 32, 0 to 31, 0 to 30, 0 to 29, 0 to 28, 0 to 27, 0 to 26, 0 to 25, 0 to 24, 0 to 23, 0 to 22, 0 to 21 0 to 20, 0 to 19, 0 to 18, 0 to 17, 0 to 16, 0 to 15, 0 to 14, 0 to 13, 0 to 12, 0 to 11 , 0 to 10, 0 to 9, 0 to 8, 0 to 7, 0 to
  • the target binding sequence may hybridise to a region upstream of an AUG site (start codon), such as a start codon within the CDS, of the target OPA1 mRNA sequence.
  • the binding sequence is reverse complementary to a sequence that is 0 to 80 nucleotides in length, such as 0 to 70, 0 to 60, 0 to 50, 0 to 40, 0 to 39, 0 to 38, 0 to 37, 0 to
  • the target binding sequence may hybridise to the target OPA1 mRNA sequence downstream of said AUG site.
  • the binding sequence is reverse complementary to a sequence that is 0 to 80 nucleotides in length, such as 0 to 70, 0 to 60, 0 to 50, 0 to 40, 0 to 39, 0 to 38, 0 to 37, 0 to 36, 0 to 35, 0 to 34, 0 to 33, 0 to 32, 0 to 31, 0 to 30, 0 to 29, 0 to 28, 0 to 27, 0 to 26, 0 to 25, 0 to 24, 0 to 23, 0 to 22, 0 to 21 , O to 20, O to 19, O to 18, O to 17, O to 16, O to 15, O to 14, O to 13, O to 12, O to 11, O to 10, or 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, or 0 to 4 nucleotides in length, and is downstream of the AUG site.
  • the target binding sequence may bind sequences both upstream and downstream of an AUG site.
  • a target binding sequence that is “-40/+4 of M1” refers to a target binding sequence that is reverse complementary to the 40 nucleotides within the 5’ UTR upstream of the AUG site (-40) and the 4 nucleotides within the CDS downstream of the AUG site (+4).
  • the nucleotides of the 5’UTR sequence are numbered sequentially using decreasing negative numbers approaching the AUG site on the target mRNA (e.g. -3, -2, -1).
  • the nucleotides of the CDS sequence are numbered sequentially using increasing positive numbers (e.g. +1 , +2, +3) from the AUG site, such that the A of the AUG site is numbered +1.
  • the region bridging the 5’UTR and the CDS will therefore be numbered -3, -2, -1 , +1 , +2, +3, with the A of the AUG site numbered +1. It should be noted that according to this numbering scheme there is no nucleotide “0”.
  • the target binding sequence is 18 nucleotides in length and comprises SEQ ID NO: 30, which targets the M1 -14/+4 region of OPA1.
  • the target binding sequence is 33 nucleotides in length and comprises SEQ ID NO: 222, which targets the M1 -14/+19 region of OPA1.
  • the target binding sequence is 44 nucleotides in length and comprises SEQ ID NO: 429, which targets the M1 -40/+4 region of OPA1.
  • the target binding sequence is 72 nucleotides in length and comprises SEQ ID NO: 582, which targets the M1 -40/+32 region of OPA1.
  • the target binding sequence refers independently to each of the target binding sequences of the invention in embodiments when there are more than one target binding sequences present in a functional nucleic acid.
  • one of the “at least one target binding sequences” may be 20nt long and have 80% sequence identity to its target mRNA whilst another may be 30nt long and have 95% sequence identity to its target mRNA.
  • the target binding sequences may be separated from one another and from other functional elements by nucleic acid sequences comprising “spacers”. In embodiments wherein there are more than one target binding sequences present, the target binding sequences may be separated from one another by spacers.
  • the target binding sequences are separated from one another by a spacer.
  • the target binding sequence is separated from the regulatory sequence by a spacer.
  • the target binding sequences are separated from one another or from the regulatory sequence by a spacer, wherein the spacer is 19 nucleotides in length.
  • the spacer may comprise SEQ ID NO: 1581.
  • RNAs may be described in terms of secondary and tertiary structures that are known in the art.
  • a functional nucleic acid molecule according to the invention may target a binding site that is within proximity of a region of RNA secondary or tertiary structure, comprises a region of RNA secondary or tertiary structure, or consists of a region of RNA secondary or tertiary structure.
  • the target binding sequence binds to a target sequence that possesses secondary or tertiary RNA structure.
  • the target binding sequence binds to a target sequence within proximity of a sequence that possesses secondary or tertiary RNA structure.
  • a target sequence may be within about 5 nucleotides, within about 10 nucleotides, within about 15 nucleotides, within about 20 nucleotides, within about 25 nucleotides, within about 30 nucleotides, within about 35 nucleotides, within about 40 nucleotides, within about 45 nucleotides, within about 50 nucleotides, within about 55 nucleotides, within about 60 nucleotides, within about 65 nucleotides, within about 70 nucleotides, within about 75 nucleotides, within about 80 nucleotides, within about 85 nucleotides, within about 90 nucleotides, within about 95 nucleotides, within about 100 nucleotides, within about 110 nucleotides, within about 120 nucleotides, within about 130 nucle
  • suitable target sequences or the target binding sequences that are targeted thereby, may be classified or grouped on the basis of the proximity of the target sequence to RNA structures, or indeed the presence of an RNA structure within a target sequence.
  • RNA structures may be present within the OPA1 5’IITR and may comprise: G- quadruplexes, internal loops, stems, stem-loops, double internal loops, multi-branched loops, and triple internal loops.
  • the target sequence comprises a G-quadruplex, internal loop, stem, stem-loop, double internal loop, multi-branched loop, and/or triple internal loop. In another embodiment, the target sequence is within proximity of a G-quadruplex, internal loop, stem, stem-loop, double internal loop, multi-branched loop, and/or triple internal loop.
  • RNA secondary structures can be predicted using online prediction tools such as RNAfold V2.4.18, which is available
  • RNA structures within the OPA1 mRNA region defined by SEQ ID NO: 1 can be found in Table 1. It will be understood that by ’’participating sequence” it is meant that the fragment or sequence of mRNA is predicted to form the indicated structure. Structures may be overlapping.
  • the target sequence comprises a sequence that comprises any one or more of SEQ I D NOs: 1583 - 1598, or a fragment thereof.
  • Target sequences may also be classified by their inclusion (or not) of the M1 start codon of OPA1.
  • the target binding sequence comprises a sequence reverse complementary to a portion of OPA1 mRNA that comprises the M1 AUG codon.
  • this target binding sequence may be selected from the group consisting of SEQ ID NOs: 15 - 30, 209 - 238, 401 - 430, 582 - 611 , 736 - 743, 748, and 750.
  • the target binding sequence comprises a sequence reverse complementary to a portion of OPA1 mRNA that does not comprises the M1 AUG codon.
  • this target binding sequence may be selected from the group consisting of SEQ ID NOs: 2 - 14, 31 - 208, 239 - 400, 431 - 581, 612 - 735, 744 - 747, 749, 751 and 752.
  • the target binding sequence is selected from the group consisting of: SEQ ID NO: 16, 20, 24, 28, 30, 31, 34, 38, 42, 51, 73, 83, 91, 111, 133, 152, 168, 182, 185, 405, 411 , 417, 423, 429, and 735 - 752.
  • the target sequence comprises the OPA1 M1 AUG codon.
  • the target sequence may be selected from the group consisting of SEQ ID NOs: 1 , 766 - 781 , 960 - 989, 1152 - 1181, 1333 - 1362, 1487 - 1494, 1499, and 1501.
  • the target sequence does not comprises the OPA1 M1 AUG codon.
  • this target sequence may be selected from the group consisting of SEQ ID NOs: 753 - 765, 782 - 959, 990 - 1151, 1182 - 1332, 1363 - 1486, 1495 - 1498, 749, 1500, 1502, and 1503.
  • the target sequence is selected from the group consisting of: SEQ ID NO: 767, 771, 775, 779, 781 , 782, 785, 789, 793, 802, 824, 834, 842, 862, 884, 903, 919, 933, 936, 1156, 1162, 1168, 1174, 1180, and 1486 - 1503.
  • Target sequences may also be classified by broader sequence based classifications, e.g., as being within exon 1 or within the 5’UTR.
  • target binding sequences may be combined with any suitable regulatory sequence as defined in the following section.
  • the target binding sequence comprises any one or more of a sequence selected from SEQ ID NOs 2 - 752, or a fragment thereof
  • the one or more regulatory sequences comprises any one or more of a sequence selected from SEQ ID NOs 1504 - 1573, or a fragment thereof.
  • the target binding sequence is 18 nucleotides in length
  • the regulatory sequence comprises any one or more of a sequence selected from SEQ ID NOs: 1504 - 1506, 1510, 1514, 1544, and 1555, or a fragment thereof.
  • the target binding sequence is any one or more of a sequence selected from SEQ ID NOs 2 - 208, or a fragment thereof
  • the regulatory sequence comprises any one or more of a sequence selected from SEQ ID NOs: 1504 - 1506, 1510, 1514, 1544, and 1555, or a fragment thereof.
  • the target binding sequence is 33 nucleotides in length
  • the regulatory sequence comprises any one or more of a sequence selected from SEQ ID NOs: 1504 - 1506, 1510, 1514, 1544, and 1555, or a fragment thereof.
  • the target binding sequence is any one or more of a sequence selected from SEQ ID NOs 209 - 400, or a fragment thereof
  • the regulatory sequence comprises any one or more of a sequence selected from SEQ ID NOs: 1504 - 1506, 1510, 1514, 1544, and 1555, or a fragment thereof.
  • the target binding sequence is 44 nucleotides in length
  • the regulatory sequence comprises any one or more of a sequence selected from SEQ ID NOs: 1504 - 1506, 1510, 1514, 1544, and 1555, or a fragment thereof.
  • the target binding sequence is any one or more of a sequence selected from SEQ ID NOs 401 - 581 , or a fragment thereof
  • the regulatory sequence comprises any one or more of a sequence selected from SEQ ID NOs: 1504 - 1506, 1510, 1514, 1544, and 1555, or a fragment thereof.
  • the target binding sequence is 72 nucleotides in length, and the one or more regulatory sequences comprises any one or more of a sequence selected from SEQ ID NOs: 1504 - 1506, 1510, 1514, 1544, and 1555, or a fragment thereof.
  • the target binding sequence is any one or more of a sequence selected from SEQ ID NOs 582 - 734, or a fragment thereof, and the regulatory sequence comprises any one or more of a sequence selected from SEQ ID NOs: 1504 - 1506, 1510, 1514, 1544, and 1555, or a fragment thereof.
  • the target binding sequence is 18 nucleotides in length
  • the regulatory sequence comprises any one or more of a sequence selected from SEQ ID NOs: 1504 - 1506, 1510, 1514, 1544, and 1555, or a fragment thereof.
  • the target binding sequence is any one or more of a sequence selected from SEQ ID NOs 2 - 208, or a fragment thereof
  • the regulatory sequence comprises any one or more of a sequence selected from SEQ ID NOs: 1504 - 1506, 1510, 1514, 1544, and 1555, or a fragment thereof.
  • the target binding sequence is 33 nucleotides in length
  • the regulatory sequence comprises any one or more of a sequence selected from SEQ ID NOs: 1504 - 1506, 1510, 1514, 1544, and 1555, or a fragment thereof.
  • the target binding sequence is any one or more of a sequence selected from SEQ ID NOs 209 - 400, or a fragment thereof
  • the regulatory sequence comprises any one or more of a sequence selected from SEQ ID NOs: 1504 - 1506, 1510, 1514, 1544, and 1555, or a fragment thereof.
  • the target binding sequence is 44 nucleotides in length
  • the regulatory sequence comprises any one or more of a sequence selected from SEQ ID NOs: 1504 - 1506, 1510, 1514, 1544, and 1555, or a fragment thereof.
  • the target binding sequence is any one or more of a sequence selected from SEQ ID NOs 401 - 581 , or a fragment thereof
  • the regulatory sequence comprises any one or more of a sequence selected from SEQ ID NOs: 1504 - 1506, 1510, 1514, 1544, and 1555, or a fragment thereof.
  • the target binding sequence is 72 nucleotides in length, and the one or more regulatory sequences comprises any one or more of a sequence selected from SEQ ID NOs: 1504 - 1506, 1510, 1514, 1544, and 1555, or a fragment thereof.
  • the target binding sequence is any one or more of a sequence selected from SEQ ID NOs 582 - 734, or a fragment thereof, and the regulatory sequence comprises any one or more of a sequence selected from SEQ ID NOs: 1504 - 1506, 1510, 1514, 1544, and 1555, or a fragment thereof.
  • the target binding sequence is 18 nucleotides in length
  • the regulatory sequence comprises any one or more of a sequence selected from SEQ ID NOs: 1574 - 1580, or a fragment thereof.
  • the target binding sequence is any one or more of a sequence selected from SEQ ID NOs 2 - 208, or a fragment thereof
  • the regulatory sequence comprises any one or more of a sequence selected from SEQ ID NOs: 1574 - 1580, or a fragment thereof.
  • the target binding sequence is 33 nucleotides in length
  • the regulatory sequence comprises any one or more of a sequence selected from SEQ ID NOs: 1574 - 1580, or a fragment thereof.
  • the target binding sequence is any one or more of a sequence selected from SEQ ID NOs 209 - 400, or a fragment thereof
  • the regulatory sequence comprises any one or more of a sequence selected from SEQ ID NOs: 1574 - 1580, or a fragment thereof.
  • the target binding sequence is 44 nucleotides in length
  • the regulatory sequence comprises any one or more of a sequence selected from SEQ ID NOs: 1574 - 1580, or a fragment thereof.
  • the target binding sequence is any one or more of a sequence selected from SEQ ID NOs 401 - 581 , or a fragment thereof
  • the regulatory sequence comprises any one or more of a sequence selected from SEQ ID NOs: 1574 - 1580, or a fragment thereof.
  • the target binding sequence is 72 nucleotides in length, and the one or more regulatory sequences comprises any one or more of a sequence selected from SEQ ID NOs: 1574 - 1580, or a fragment thereof.
  • the target binding sequence is any one or more of a sequence selected from SEQ ID NOs 582 - 734, or a fragment thereof, and the regulatory sequence comprises any one or more of a sequence selected from SEQ ID NOs: 1574 - 1580, or a fragment thereof.
  • Functional nucleic acid molecules of the invention may be designed in view of preferable combinations of target binding sequences and regulatory sequences that may be selected on the basis of preferred characteristics, e.g., increased activity, size, and/or stability.
  • target sequences and target binding sequences are disclosed in Table 2.
  • the sequences disclosed in Table 2 were designed using a ‘tiling’ approach as illustrated in Figure 1. It will be understood that target sequences and target binding sequences according to the invention my comprise or consist of sequences with a certain percentage identity to the sequences disclosed in Table 2, or be fragments thereof (i.e. , be shorter sequences consisting of a contiguous stretch of one of the longer sequences disclosed in Table 2). Additionally, whilst Table 2 discloses sequences comprising T nucleotides, it is to be understood that a sequence with any or all T nucleotides substituted for II nucleotides are also encompassed thereby.
  • the functional nucleic acid molecule of the invention comprises one or more regulatory sequences comprising a SINE B2 element or a functionally active fragment thereof, or an internal ribosome entry site (IRES) or a functionally active fragment thereof.
  • regulatory sequences may also be known as effector domains (EDs).
  • the functional nucleic acid molecule comprises one, two, three, four, or regulatory sequences.
  • the functional nucleic acid molecule comprises one regulatory sequence.
  • the functional nucleic acid molecule comprises two regulatory sequences.
  • the functional nucleic acid molecule comprises three regulatory sequences.
  • the regulatory sequence has translation enhancing activity such that protein production is increased.
  • Increased or enhanced protein translation activity indicates that the efficiency or activity of translation is increased as compared to a case where the functional nucleic acid molecule according to the present invention is not present in a system.
  • the regulatory sequence has protein translation enhancing activity.
  • the regulatory sequence increases or enhances translation of target mRNA.
  • the functional nucleic acid molecule of the invention is applicable to uses and methods for enhancing translation of one or more target OPA1 mRNA sequences.
  • enhancing translation of one or more target OPA1 mRNA sequences it is meant that translation of the entire protein-coding region of the target mRNA will be enhanced such that synthesis of the protein encoded thereby is increased.
  • expression of the protein encoded by the target mRNA is increased by at least 1.2 fold, such as at least 1.5 fold, in particular at least 2 fold.
  • expression of the protein encoded by the target mRNA is increased between 1.5 to 3 fold, such as between 1.6 and 2.2 fold.
  • protein expression it is meant the level of protein present in a system as determined by the translational activity within that system.
  • increasing protein expression will be understood to mean ultimately increasing the amount of a given protein in the system.
  • the expression of the protein encoded by the target mRNA is increased by at least about 1.1 fold, at least about 1.2 fold, at least about 1.3 fold, at least about 1.4 fold, at least about 1.5 fold, at least about 1.6 fold, at least about 1.7 fold, at least about 1.8 fold, at least about 1.9 fold, at least about 2.0 fold, at least about 2.1 fold, at least about 2.2 fold, at least about 2.3 fold, at least about 2.4 fold, at least about 2.5 fold, at least about 2.6 fold, at least about 2.7 fold, at least about 2.8 fold, at least about 2.9 fold, or at least about
  • the expression of the protein encoded by the target mRNA is increased about 1.1 fold, about 1.2 fold, about 1.3 fold, about 1.4 fold, about 1.5 fold, about 1.6 fold, about 1.7 fold, about 1.8 fold, about 1.9 fold, about 2.0 fold, about 2.1 fold, about 2.2 fold, about 2.3 fold, about 2.4 fold, about 2.5 fold, about 2.6 fold, about 2.7 fold, about 2.8 fold, about 2.9 fold, or about 3.0 fold.
  • the expression of the protein encoded by the target mRNA is increased by less than about 1.2 fold, less than about 1.3 fold, less than about 1.4 fold, less than about
  • the target mRNA it is meant the target OPA1 mRNA.
  • the regulatory sequence is located within the functional nucleic acid molecule 3’ of the target binding sequence.
  • the regulatory sequence may be in a direct or inverted orientation relative to the 5’ to 3’ orientation of the functional nucleic acid molecule.
  • Reference to “direct” refers to the situation in which the regulatory sequence is embedded (inserted) with the same 5’ to 3’ orientation as the functional nucleic acid molecule.
  • “inverted” refers to the situation in which the regulatory sequence is 3’ to 5’ oriented relative to the functional nucleic acid molecule.
  • the regulatory sequence is located 3’ of the target binding sequence within the functional nucleic acid molecule.
  • the regulatory sequence comprises a SINE B2 element or a functionally active fragment thereof.
  • the SINE B2 element is preferably in an inverted orientation relative to the 5’ to 3’ orientation of the functional nucleic acid molecule, i.e. an inverted SINE B2 element.
  • the regulatory sequence comprises a SINE B2 element, or a functionally active fragment thereof. Said sequence enhances translation of the target mRNA sequence.
  • the regulatory sequence consists of a SINE B2 element or a functionally active fragment of a SINE B2 element.
  • the SINE B2 element, or functionally active fragment thereof, may be in the direct or inverted orientation relative to the functional nucleic acid molecule.
  • the regulatory sequence comprises a SINE B2 element or a functionally active fragment thereof, wherein the regulatory sequence is orientated, within the functional nucleic acid molecule, in the direct orientation relative to the 5’ to 3’ orientation of the functional nucleic acid molecule.
  • the regulatory sequence comprises a SINE B2 element or a functionally active fragment thereof, wherein the regulatory sequence is orientated, within the functional nucleic acid molecule, in the inverted orientation relative to the 5’ to 3’ orientation of the functional nucleic acid molecule
  • SINE Short Interspersed Nuclear Element refers to an interspersed repetitive sequence: (a) that encodes a protein having neither reverse-transcription activity nor endonuclease activity or the like; and (b) whose complete or incomplete copy sequences exist abundantly in genomes of living organisms.
  • SINE B2 element is defined in WO 2012/133947, where specific examples are also provided (see table starting on page 69 of the PCT publication) and which is incorporated herein by reference in its entirety. The term is intended to encompass both SINE B2 elements in direct orientation and in inverted orientation relative to the 5’ to 3’ orientation of the functional nucleic acid molecule.
  • SINE B2 elements may be identified, for example, using programs like RepeatMask as published (Bedell et al. Bioinformatics. 2000 Nov; 16(11): 1040-1. MaskerAid: a performance enhancement to RepeatMasker).
  • a sequence may be recognizable as a SINE B2 element by returning a hit in a Repbase database with respect to a consensus sequence of a SINE B2, with a Smith-Waterman (SW) score of over 225, which is the default cutoff in the RepeatMasker program.
  • SW Smith-Waterman
  • a SINE B2 element is not less than 20 bp and not more than 400 bp.
  • the SINE B2 element is derived from tRNA.
  • a SINE B2 element By the term “functionally active fragment of a SINE B2 element” there is intended a portion of sequence of a SINE B2 element that retains protein translation enhancing activity. This term also includes sequences that are mutated in one or more nucleotides with respect to the wild-type sequences, but retain protein translation enhancing activity. The term is intended to encompass both SINE B2 elements in direct orientation and in inverted orientation relative to the 5’ to 3’ orientation of the functional nucleic acid molecule.
  • Short fragments of the regulatory sequence are particularly useful when providing functional RNA molecules for use as a nucleic acid therapeutic.
  • RNA molecules are highly unstable in living organisms, therefore stability provided by the chemical modifications as described herein, is more effective for shorter RNA molecules.
  • the regulatory sequence comprises a functionally active fragment that is less than 250 nucleotides, such as less than 240 nucleotides, less than 230 nucleotides, less than 220 nucleotides, less than 210 nucleotides, less than 200 nucleotides, less than 190 nucleotides, less than 180 nucleotides, less than 170 nucleotides, less than 160 nucleotides, less than 150 nucleotides, less than 140 nucleotides, less than 130 nucleotides, less than 120 nucleotides, less than 110 nucleotides, less than 100 nucleotides, less than 90 nucleotides, less than 80 nucleotides, less than 70 nucleotides, less than 60 nucleotides, less than 50 nucleotides, less than 40 nucleotides, less than 30 nucleotides, less than 20 nucleotides, or less than 10 nucleotides.
  • a functionally active fragment
  • the regulatory sequence comprises or consists of a functionally active fragment of a sequence selected from the group consisting of SEQ ID NO: 1504 - 1555, wherein the fragment is about is about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250 or more nucleotides in length.
  • the functional nucleic acid molecule comprises a SINE B2 element, wherein said SINE B2 element comprises a sequence selected from the group consisting of SEQ ID NO: 1504 - 1555, or a functionally active fragment thereof.
  • the functional nucleic acid molecule comprises a SINE B2 element, wherein said SINE B2 element consists of a sequence selected from the group consisting of SEQ ID NO: 1504 - 1555, or a functionally active fragment thereof.
  • the functional nucleic acid molecule comprises a SINE B2 element, wherein said SINE B2 element comprises or consists of a sequence which has at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1504 - 1555, or a functionally active fragment thereof.
  • the regulatory sequence comprises a sequence with at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1504 - 1555.
  • the regulatory sequence consists of a sequence with at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1504 - 1555.
  • the regulatory sequence comprises a functionally active fragment of a SINE B2 element according to the foregoing, wherein the fragment comprises a sequence with at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to a fragment or region of a sequence selected from the group consisting of SEQ ID NO: 1504 - 1555.
  • the regulatory sequence consists of a functionally active fragment of a SINE B2 element according to the foregoing, wherein the fragment comprises or consists of a sequence with at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1504 - 1555.
  • SEQ ID NO: 1504 (the 167 nucleotide variant of the inverted SINE B2 element in AS LlchU) and SEQ ID NO: 1505 (the 77 nucleotide variant of the inverted SINE B2 element in AS LlchU that includes nucleotides 44 to 120), as well as sequences with a suitable percentage identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity) to these sequences are particularly preferred.
  • a suitable percentage identity e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%,
  • SEQ ID NO: 1506 - 1555 Other inverted SINE B2 elements and functionally active fragments of inverted SINE B2 elements are SEQ ID NO: 1506 - 1555. Experimental data showing the protein translation enhancing activity of these sequences is not explicitly shown in the present patent application, but is disclosed in e.g., WO 2019/150346, which is incorporated herein by reference in its entirety. SEQ ID NO: 1506 - 1555 can therefore also be used as regulatory sequences in the functional nucleic acid molecule of the present invention.
  • SEQ ID NOs: 1506 - 1509, 1511 - 1514, and 1521 are functionally active fragments of inverted SINE B2 transposable element derived from AS LlchU .
  • the use of functional fragments reduces the size of the regulatory sequence, which is advantageous if used in an expression vector (e.g. viral vectors which may be size-limited) because this provides more space for the one or more target sequences and/or expression elements.
  • SEQ ID NO: 1510 is a full-length 183 nucleotide (nt) inverted SINE B2 transposable element derived from AS LlchU.
  • SEQ ID NOs: 1515 - 1520, 1522, 1523, and 1542 - 1545 are mutated functionally active fragments of inverted SINE B2 transposable element derived from AS LlchU.
  • SEQ ID Nos: 1524 - 1528, and 1531 - 1541 are different SINE B2 transposable elements.
  • the regulatory sequence comprises an IRES sequence, or functionally active fragment thereof.
  • the regulatory sequence may also comprise a fragment, such as a functionally active fragment, of an IRES sequence. Therefore, in one embodiment, the regulatory sequence comprises an IRES sequence or a functionally active fragment of an IRES sequence. Said sequence enhances translation of the target mRNA.
  • the regulatory sequence comprises an IRES sequence, or a functionally active fragment thereof. Said sequence enhances translation of the target mRNA sequence.
  • IRES sequences recruit the 40S ribosomal subunit and promote cap-independent translation of a subset of protein coding mRNAs. IRES sequences are generally found in the 5’ untranslated region (5’IITR) of cellular mRNAs coding for stress-response genes, thus stimulating their translation in c/s.
  • IITR 5’ untranslated region
  • an IRES sequence is a nucleotide sequence capable of promoting translation of a second cistron in a bicistronic construct.
  • a dual luciferase (Firefly luciferase [Flue], Renilla Luciferase [Rluc]) encoding plasmid is used for experimental tests.
  • Said test may be considered “The Standard Bicistronic Plasmid Test for Cellular mRNA IRESs” used to test putative IRES sequences.
  • the foregoing is a functional test wherein the putative IRES sequence is inserted between RLuc and FLuc, e.g., as described in Jasckson, Cold Spring Harb Perspect Biol 2013; 5:a011569, wherein the translational function of the putative IRES sequence is determined by the Fluc/RLuc value, thus measuring c/s-acting activity.
  • the regulatory sequence of the functional nucleic acid molecule of the invention may be trans-acting.
  • the functional nucleic acid molecule comprises an IRES regulatory sequence that is trans-acting.
  • IRESite A major database exists, namely IRESite, for the annotation of nucleotide sequences that have been experimentally validated as IRES, using dual reporter or bicistronic assays (http://iresite.org/IRESite_web.php).
  • a web-based tool is available to search for sequence-based and structure-based similarities between a query sequence of interest and the entirety of annotated and experimentally validated IRES sequences within the database.
  • the output of the program is a probability score for any nucleotide sequence to be able to act as IRES in a validation experiment with bicistronic constructs.
  • Additional sequence-based and structurebased web-based browsing tools are available to suggest, with a numerical predicting value, the IRES activity potentials of any given nucleotide sequence (http://rna.informatik.uni- frburg.de/; http://regrna.mbc.nctu.edu.tw/index1.php).
  • HCV Hepatitis C Virus
  • poliovirus IRESs e.g. SEQ ID NO: 1558 and 1559
  • human encephalomyocarditis (EMCV) virus e.g. SEQ ID NO: 1560 and 1561
  • human cricket paralysis (CrPV) virus e.g. SEQ ID NO: 1562 and 1563
  • human Apaf-1 e.g. SEQ ID NO: 1564 and 1565
  • ELG-1 e.g.
  • the regulatory sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 1556 - 1573, or a functionally active fragment thereof.
  • the regulatory sequence consists of a sequence selected from the group consisting of SEQ ID NOs: 1556 - 1573, or a functionally active fragment thereof.
  • the regulatory element has at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 86% sequence identity, at least about 87% sequence identity, at least about 88% sequence identity, at least about 89% sequence identity, at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 1556 - 1573.
  • the regulatory sequence consists of a sequence with at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 86% sequence identity, at least about 87% sequence identity, at least about 88% sequence identity, at least about 89% sequence identity, at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1556 - 1573.
  • the regulatory sequence comprises or consists of a functionally active fragment of any one of SEQ ID NOs: 1556 - 1573, wherein the fragment is about is about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about
  • the functionally active fragment retains IRES activity within the definition provided above.
  • the functionally active fragment retains protein translation enhancing activity.
  • a “functionally active fragment” of an IRES might also be considered an IRES perse.
  • “functionally active fragment” of an IRES is utilised to delineate IRES sequences that are shorter in length as compared with ‘parental’ IRES sequences from which they are designed or derived.
  • any regulatory sequence according to the invention may be suitably paired with any binding sequence according to the invention.
  • a DNA molecule encoding a functional nucleic acid molecule of the invention.
  • an expression vector comprising said DNA molecule.
  • Exemplary expression vectors are known in the art and may include, for example, plasmid vectors, viral vectors (for example adenovirus, adeno-associated virus, retrovirus or lentivirus vectors), phage vectors, cosmid vectors and the like.
  • viral vectors for example adenovirus, adeno-associated virus, retrovirus or lentivirus vectors
  • phage vectors for example adenovirus, adeno-associated virus, retrovirus or lentivirus vectors
  • cosmid vectors cosmid vectors and the like.
  • the choice of expression vector may be dependent upon the type of host cell to be used and the purpose of use.
  • the following plasmids have been used for expression of functional nucleic acid molecule:
  • Viral vectors pAAV (an Adeno-Associated Virus vector) rcLV -TetOne-Puro (a 3 rd generation Lentivirus vector) pLPCX-link (a 3 rd generation Retrovirus vector)
  • the mammalian expression plasmid is pCDNA3.1 (-).
  • the mammalian expression plasmid is pDUAL-eGFPA.
  • Plasmids of the invention may comprise any one of more features selected from the list comprising: a CMV promoter, a H1 promoter, and/or a BGH poly(A) terminator.
  • the viral vector is pAAV.
  • the viral vector is rcLV -TetOne-Puro.
  • the viral vector is pLPCX-link.
  • Vectors of the invention may comprise any one of more features selected from the list comprising: a CAG promoter, a CMV enhancer, SV40 late poly(A) terminator, a LTR-TREt (Tre-Tight) promoter, and/or a BGH poly(A) terminator.
  • any promoter may be used in the vector. Since the activity of the functional nucleic acids of the invention is independent of the promoter it is envisaged that these will work just as well as those exemplified above.
  • the present invention also relates to compositions comprising the functional nucleic acid molecule, the DNA molecule or the expression vector according to the invention.
  • composition may comprise components which enable delivery of said functional nucleic acid molecule by viral vectors (AAV, lentivirus and the like) and non-viral vectors (nanoparticles, lipid particles and the like).
  • viral vectors AAV, lentivirus and the like
  • non-viral vectors nanoparticles, lipid particles and the like.
  • the functional nucleic acid molecule of the invention may be administered as naked or unpackaged RNA.
  • composition may comprise components that are known in the art to aid the stability of the nucleic acid molecule, e.g., salts (such as those providing Mg 2+ ions).
  • the functional nucleic acid molecule may be administered as part of a composition, for example a composition comprising a suitable carrier.
  • the carrier is selected based upon its ability to facilitate the transfection of a target cell with one or more functional nucleic acid molecule.
  • composition comprising the functional nucleic acid molecule, the DNA molecule, or the expression vector as described herein.
  • a pharmaceutical composition comprising at least one functional nucleic acid molecule, at least one DNA molecule, or at least one expression vector according to the present invention.
  • a pharmaceutical composition may comprise at least one functional nucleic acid molecule, at least one DNA molecule, or at least one expression vector according to the present invention with a suitable pharmaceutical excipient, diluent, carrier, and/or salt.
  • the suitable pharmaceutical excipient, diluent, carrier, and/or salt may depend on the intended route of administration and standard pharmaceutical practice.
  • a suitable carrier may include any of the standard pharmaceutical carriers, vehicles, diluents or excipients known in the art and which are generally intended for use in facilitating the delivery of nucleic acids, such as RNA.
  • Liposomes, exosomes, lipidic particles or nanoparticles are examples of suitable carriers that may be used for the delivery of RNA.
  • the carrier or vehicle delivers its contents to the target cell such that the functional nucleic acid molecule is delivered to the appropriate subcellular compartment, such as the cytoplasm.
  • a method for enhancing translation of a target mRNA comprising administering the functional nucleic acid molecule, DNA molecule, expression vector, composition or pharmaceutical composition as defined herein to the cell.
  • the cell is a mammalian cell, such as a human or mouse cell.
  • an in vitro method for increasing the synthesis of OPA1 protein in a cell or cell-free system comprising administering the functional nucleic acid molecule, DNA molecule, expression vector, composition or pharmaceutical composition described herein, to the cell or cell-free system.
  • an in vivo method for increasing the synthesis of OPA1 protein in a cell comprising administering the functional nucleic acid molecule, DNA molecule, expression vector, composition or pharmaceutical composition described herein, to the cell.
  • a method for increasing the synthesis of OPA1 protein in a cell comprising administering the functional nucleic acid molecule, DNA molecule, expression vector, composition or pharmaceutical composition described herein, to the cell.
  • the cell is a mammalian cell, such as a human or a mouse cell.
  • a method for increasing the protein synthesis efficiency of OPA1 in a cell comprising administering the functional nucleic acid molecule, DNA molecule, expression vector, composition or pharmaceutical composition described herein, to the cell.
  • the cell is a mammalian cell, such as a human or a mouse cell.
  • Methods of the invention result in increased levels of OPA1 protein in a cell and therefore find use, for example, in methods of treatment of diseases or disorders which are associated with gene defects (e.g. one or more gene defects which result in reduced OPA1 protein levels and/or loss-of-function mutations of the encoding gene).
  • Methods of the invention find particular use in diseases caused by a quantitative decrease in the predetermined, normal OPA1 protein level, such as haploinsufficiency.
  • OPA1 may be considered a therapeutic target.
  • therapeutic target or “therapeutic target mRNA sequence” refers to a target which may be used to treat a disease or condition in a subject when its translation is enhanced, such as enhanced by using a functional nucleic acid molecule according to the present invention.
  • a therapeutic target when expressed in a subject (such as in a cell of a subject), may: replace a protein that is deficient or abnormal in a cell; augment an existing pathway in a cell; and/or provide a novel function or activity in a cell; thereby treating a disease or condition of said subject.
  • target binding domains which enhance translation of 0PA1 can be used to treat diseases or disorders in which 0PA1 protein is affected, e.g., wherein 0PA1 is associated with the disease phenotype, such as in ADOA.
  • Methods of the invention can be performed in vitro, ex vivo or in vivo.
  • the methods described herein may comprise transfecting into a cell the functional nucleic acid molecule, DNA molecule, expression vector, composition or pharmaceutical composition as defined herein.
  • the functional nucleic acid molecule, DNA molecule, expression vector, composition or pharmaceutical composition may be administered to target cells using methods known in the art and include, for example, microinjection, lipofection, electroporation, using calcium phosphate, self-infection by the vector or viral transduction.
  • the functional nucleic acid molecules of the invention find use in increasing the level of OPA1 protein within a cell.
  • OPA1 has primary functions in mitochondrial homeostasis and therefore, according to a further aspect of the invention, there is provided the functional nucleic acid molecule, DNA molecule, expression vector composition or pharmaceutical composition for use in the treatment of a disease-associated with mitochondrial defects.
  • the functional nucleic acid molecules, DNA molecules, compositions and/or pharmaceutical compositions can be used as medicaments, preferably for treating Autosomal Dominant Optic Atrophy (ADOA) and in particular promoting the recovery of disease-associated mitochondrial defects.
  • ADOA Autosomal Dominant Optic Atrophy
  • RGCs Retinal ganglion cells
  • mutated OPA1 and RGC- specific OPA1 deficient mice have been shown to play a role in autophagy in ADOA pathogenesis (Zaninello et al. (2020) Nat. Comm. 11(1): 4029).
  • the disease associated with mitochondrial defects is ADOA.
  • the main symptoms of the disease are a bi-lateral degeneration of Retinal Ganglion Cells (RGCs) and optic nerve atrophy with possible muscular and neurodegenerative symptoms associated.
  • RRCs Retinal Ganglion Cells
  • Various forms of ADOA have been reported, such as ADOA plus, which also displays muscular defects and neurosensory deafness, and ADOAC, which leads to cataract.
  • nucleic acid molecule DNA molecule
  • expression vector composition or pharmaceutical composition as defined herein for the manufacture of a medicament.
  • OPA1 protein expression may be therapeutic for additional diseases.
  • Civiletto et al. (2015) Cell Metab. 21(6): 845-854 showed that moderate OPA1 overexpression ameliorates the phenotype of two mitochondrial disease mouse models that had defects in the Ndufs4 or Cox15 genes.
  • mutations in NDLIFS4 are associated with early-onset fatal Leigh syndrome due to severe Complex I (Cl) deficiency, while mutations in COX15 have been reported in children with severe isolated cardiomyopathy, encephalopathy, or cardioencephalomyopathy.
  • Opa1‘ 9 mice (a model overexpressing OPA1 about 1.5 fold) are protected from muscular atrophy and myocardial infarction, and are less susceptible to Fas-induced liver damage, and have mitochondria that are resistant to cristae remodelling and cytochrome C release.
  • OPA1 is potentially toxic (Cipolat et al. (2004) PNAS 101(45): 15927-15932); hence, the methods provided herein are particularly well suited to treating diseases associated with OPA1 deficiencies since they may avoid unwanted side effects associated with overexpression that elevated protein levels too much above physiologically normal levels.
  • the disease is a disease associated with mitochondrial defects is a neurological disease.
  • the neurological disease is selected from: Alzheimer’s disease, Huntington’s disease and Parkinson’s disease.
  • the disease associated with mitochondrial defects is a prion disease.
  • Wu et al. (2019) Cell Death Dis. 10(10): 710 describe that downregulation of OPA1 is observed in prion disease models in vitro and in vivo, and this occurs concomitantly to mitochondria structure damage and dysfunction, loss of mtDNA, and neuronal apoptosis. These symptoms were alleviated by increasing OPA1 expression.
  • nucleic acid molecule DNA molecule
  • expression vector composition or pharmaceutical composition for use in therapy.
  • nucleic acid molecule DNA molecule
  • expression vector composition or pharmaceutical composition for use as a medicament.
  • the functional nucleic acid molecule of the invention finds use in increasing the level of OPA1 protein. Said increase in OPA1 is preferably within a cell, such as the cell of a subject.
  • the functional nucleic acid molecule, DNA molecule, expression vector, composition or pharmaceutical composition may be administered to a subject having an existing disease or condition in order to lessen, reduce or improve at least one symptom associated with the disease and/or to slow down, reduce or block the progression of the disease.
  • Said subject preferably has a disease or disorder associated with a reduction in OPA1 protein levels or activity.
  • the functional nucleic acid molecule, DNA molecule, expression vector, composition or pharmaceutical composition for use in the treatment of a disease associated with one or more gene defects, wherein the gene defects affect the level of OPAI protein.
  • gene defect refers to one or more abnormalities in a gene which results in reduced protein levels and/or loss-of-function mutations of the encoding gene.
  • a gene defect may be caused by a mutation in a single gene, mutations in multiple genes, chromosomal abnormality, or mutation(s) in mitochondrial DNA or in nuclear genes.
  • the gene defect or defects reduce the levels or impair the function of OPA1.
  • a disease associated with one or more gene defects may be ADOA.
  • nucleic acid molecule DNA molecule
  • expression vector composition or pharmaceutical composition for use in the treatment of ADOA.
  • a method of treating a disease or disorder associated with one or more gene defects comprising administering a therapeutically effective amount of the functional nucleic acid molecule, DNA molecule, expression vector, composition or pharmaceutical composition, to a subject.
  • a method of treating a disease or disorder associated with one or more gene defects comprising administering a therapeutically effective amount of the functional nucleic acid molecule, DNA molecule, expression vector, composition or pharmaceutical composition, to a subject, wherein the one or more gene defects is in the OPA1 gene.
  • a method of treating a disease or disorder associated with OPA1 comprising administering a therapeutically effective amount of the functional nucleic acid molecule, DNA molecule, expression vector, composition or the pharmaceutical composition, to a subject.
  • the functional nucleic acid molecule, DNA molecule expression vector, composition or pharmaceutical composition as defined herein, for use in medicine.
  • nucleic acid molecule for use in the treatment or prevention of a disease or disorder associated with OPA1.
  • the disease or disorder is a neurological disease or disorder, or a mitochondrial disease or disorder.
  • the disease or disorder is any one or more of: Autosomal Dominant Optic Atrophy (ADOA), Alzheimer’s disease, Huntington’s disease and Parkinson’s disease.
  • ADOA Autosomal Dominant Optic Atrophy
  • Alzheimer’s disease Huntington’s disease
  • Parkinson’s disease any one or more of: Autosomal Dominant Optic Atrophy (ADOA), Alzheimer’s disease, Huntington’s disease and Parkinson’s disease.
  • the disease or disorder is Autosomal Dominant Optic Atrophy (ADOA).
  • ADOA Autosomal Dominant Optic Atrophy
  • defects of or within the OPA1 gene e.g., within the coding sequence thereof
  • defects outside of the OPA1 gene may affect the levels of OPA1 protein and thus be susceptible to targeted protein upregulation using the functional nucleic acid of the invention.

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Abstract

La présente invention concerne des molécules d'acides nucléiques fonctionnelles qui ciblent l'ARNm d'OPA1 et augmentent les niveaux de protéine OPA1. L'invention concerne également des procédés d'amélioration de la traduction de la protéine OPA1, et des procédés de traitement ou de soulagement de maladies ou de troubles associés à des défauts géniques qui affectent OPA1, à l'aide des molécules d'acides nucléiques fonctionnelles de l'invention.
PCT/GB2023/050956 2022-04-12 2023-04-11 Molécule d'acides nucléiques fonctionnelle Ceased WO2023199039A1 (fr)

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