WO2023199039A1

WO2023199039A1 - Functional nucleic acid molecule

Info

Publication number: WO2023199039A1
Application number: PCT/GB2023/050956
Authority: WO
Inventors: Mathieu LATREILLE
Original assignee: Transine Therapeutics Ltd
Current assignee: Transine Therapeutics Ltd
Priority date: 2022-04-12
Filing date: 2023-04-11
Publication date: 2023-10-19
Anticipated expiration: 2024-10-12
Also published as: GB202205423D0

Abstract

The present invention relates to functional nucleic acid molecules that target OPA1 mRNA and increase OPA1 protein levels. The invention also encompasses methods of enhancing OPA1 protein translation, and methods of treating or ameliorating diseases or disorders associated with gene defects that affect OPA1, using the functional nucleic acid molecules of the invention.

Description

FUNCTIONAL NUCLEIC ACID MOLECULE

FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

Autosomal Dominant Optic Atrophy (ADOA) is the most common inherited optic neuropathy, which in 75% of cases is caused by heterozygous mutations in the OPA1 gene. ADOA is an early-onset autosomal dominant haploinsufficient disorder, with prevalence ranging from 1 in 12,000 to 1 in 50,000 births. ADOA is characterized by degeneration of the retinal ganglion cells, which leads to optic nerve atrophy and blindness.

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.

Additionally, 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.

Both 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).

A class of functional long non-coding RNAs (IncRNAs), known as SINEUPs, has recently been described. 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. 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.

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.

SUMMARY OF THE INVENTION

Herein, the inventors provide for functional nucleic acids that are both SINEUPs and non- SINE containing IncRNAs (i.e. , which contain IRES effector domains or regulatory domains). However, herein the term “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.

According to a first aspect of the invention, there is provided a functional nucleic acid molecule comprising:

- one or more target binding sequences comprising a sequence reverse complementary to an OPA1 mRNA sequence; and - one or more 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.

According to a further aspect of the invention, there is provided a DNA molecule encoding the functional nucleic acid molecule as defined herein.

According to a further aspect of the invention, there is provided an expression vector comprising the functional nucleic acid molecule, or the DNA molecule, as defined herein.

According to a further aspect of the invention, there is provided a composition comprising the functional nucleic acid molecule, the DNA molecule or the expression vector, as defined herein.

According to a further aspect of the invention, there is provided a pharmaceutical composition as defined herein, comprising the functional nucleic acid molecule, the DNA molecule or the expression vector, as defined herein.

According to a further aspect of the invention, there is provided use of the functional nucleic acid molecule, the DNA molecule, the expression vector, the composition or the pharmaceutical composition, as defined herein, for enhancing translation of OPA1 mRNA.

According to a further aspect of the invention, there is provided the functional nucleic acid molecule, the DNA molecule, the expression vector, the composition or the pharmaceutical composition, as defined herein, for use in medicine.

According to a further aspect of the invention, there is provided the functional nucleic acid molecule, the DNA molecule, the expression vector, the composition or the pharmaceutical composition, as defined herein, for use in the treatment or prevention of a disease or disorder associated with OPA1. According to a further aspect of the invention, there is provided 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.

According to a further aspect of the invention, there is provided the functional nucleic acid molecule, the DNA molecule, the expression vector, the composition or the pharmaceutical composition, as defined herein, for use in the manufacture of a medicament.

According to a further aspect of the invention, there is provided 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.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 - Schematic representation of sequence-based 0PA1 target binding domain design.

Functional nucleic acid molecule binding domains (BD) 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.

Predicted RNA structures within the hOPA1 exon 1 and 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 exon 1 and 5'UTR, using standard parameters. The M1 (or Met1) initiator methionine is shown circled.

DETAILED DESCRIPTION OF THE INVENTION

It is an object of the present invention to provide 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.

Functional Nucleic Acid Molecule

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.

The “functional nucleic acid molecule” referred to herein is a synthetic molecule of the invention. In particular, 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.

In a preferred embodiment, the functional nucleic acid molecule of the invention is an RNA molecule.

In one embodiment, 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.

In one embodiment, 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)

In one embodiment, the spacer/linker sequence comprises SEQ ID NO: 1581.

In one embodiment, 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). In one embodiment, the functional nucleic acid molecule provided herein is a trans-acting functional nucleic acid molecule.

In one embodiment, 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.

In one embodiment, the functional nucleic acid molecule consists of RNA nucleotides.

The functional nucleic acid molecule of the present invention preferably consists of RNA nucleotides.

In one embodiment, the functional nucleic acid molecule is RNA.

The functional nucleic acid molecule of the present invention preferably is RNA.

In one embodiment, the functional nucleic acid molecule comprises DNA nucleotides.

In one embodiment, the functional nucleic acid molecule consists of DNA nucleotides.

In one embodiment, the functional nucleic acid molecule is DNA.

The invention also encompasses a DNA molecule encoding the functional nucleic acid molecule of the invention.

In one embodiment, the functional nucleic acid molecule comprises one or more modifications or chemical modifications.

The term “modification” or "chemical modification" refers to a structural change in, or on, the most common, natural ribonucleotides: adenosine, guanosine, cytidine, thymidine, or uridine ribonucleotides. In particular, 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).

Chemical modifications are known in the art, for example as described in The RNA Modification Database provided by The RNA Institute (https://mods.rna.albany.edu/mods/). Examples of chemical modifications which may be useful in the present invention are described in PCT/GB2021/052607, which is incorporated herein by reference in its entirety. In one embodiment, 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.

In one embodiment, the chemical base modification is selected from methylation and/or isomerisation. In a further embodiment, the chemical base modification is selected from the group consisting of: Pseudouridine (^P), N1 -Methylpseudouridine (Nlm^P), 5-Methylcytidine (m5C) and N6-Methyladenosine (m6A). In a further embodiment, the chemical base modification is selected from the group consisting of: Pseudouridine, N1- Methylpseudouridine and N6-Methyladenosine.

In one embodiment, the chemical modification is a chemical sugar modification. In one embodiment, the chemical sugar modification is methylation. In one embodiment, the chemical sugar modification is a 2’ modification, such as a 2'-O-Methyl modification. In a further embodiment, the chemical sugar modification is 2'-O-Methyladenosine (Am).

In one embodiment, 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.

In one embodiment 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. Most commonly, 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.

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. By the term “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). By the term “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.

In one embodiment the functional nucleic acid molecule may be circular.

It will be understood that the functional nucleic acid molecule of the present invention may be a DNA or an RNA molecule, as such, sequences that are represented herein as being implicitly DNA (i.e., by containing T rather than II) 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. Hence, 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. For example, where 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.

The 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.

Target binding sequences

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.

It will be understood that the term “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. In preferred embodiments wherein the functional nucleic acid molecule is a functional RNA molecule, the target binding sequence is the portion of the functional RNA molecule that binds to the target mRNA.

In one aspect, 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.

In one embodiment, the functional nucleic acid molecule comprises one, two, three, four, or five target binding sequences.

In one embodiment, the functional nucleic acid molecule comprises one target binding sequence.

In one embodiment, the functional nucleic acid molecule comprises two target binding sequences.

In one embodiment, the functional nucleic acid molecule comprises three target binding sequences.

The term “target sequence” is used herein to denote the sequence to which the target binding sequence binds. Herein the 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.

It will be understood that as well as OPA1 orthologues, OPA1 isoforms may be targeted by the functional nucleic acids of the present invention.

In one embodiment the OPA1 target sequence comprises a sequence within the 5’IITR of OPA1.

In one embodiment the OPA1 target sequence consists of a sequence within the 5’IITR of OPA1.

In one embodiment 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.

In one embodiment the OPA1 target sequence comprises a sequence within the 5’IITR and exon 1 of OPA1.

In one embodiment 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:

GGACTGAGTACGGGTGCCTGTCAGGCTCTTGCGGAAGTCCATGCGCCATTGGGAGGG CCTCGGCCGCGGCTCTGTGCCCTTGCTGCTGAGGGCCACTTCCTGGGTCATTCCTGGA CCGGGAGCCGGGCTGGGGCTCACACGGGGGCTCCCGCGTGGCCGTCTCGGCGCCTG CGTGACCTCCCCGCCGGCGGGATGTGGCGACTACGTCGGGCCGCTGTGGCCTG (SEQ ID NO: 1); and wherein the bold and underlined codon is the start codon, M1.

In one embodiment the OPA1 target sequence comprises a sequence set forth in SEQ ID NO: 1 , or a fragment thereof.

In one embodiment the OPA1 target sequence consists of a sequence set forth in SEQ ID NO: 1 , or a fragment thereof.

In one embodiment, 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.

In one embodiment, the binding sequence comprises a sequence reverse complementary to a sequence within SEQ ID NO: 1.

In one embodiment 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.

In one embodiment 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).

In one embodiment 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).

In one embodiment the target binding sequence is 18 nucleotides in length and is reverse complementary to any contiguous 18 nucleotide sequence within SEQ ID NO: 1.

In one embodiment the target binding sequence comprises a sequence as set forth in SEQ ID NOs: 2 - 208, or a fragment thereof.

In one embodiment the target binding sequence consists of a sequence as set forth in SEQ ID NOs: 2 - 208, or a fragment thereof.

In one embodiment the target sequence comprises a sequence as set forth in SEQ ID NOs: 753 - 959.

In one embodiment the target sequence consists of a sequence as set forth in SEQ ID NOs: 753 - 959.

In one embodiment the target binding sequence is 33 nucleotides in length.

In one embodiment the target binding sequence is reverse complementary to any contiguous 33 nucleotide sequence within SEQ ID NO: 1.

In one embodiment 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).

In one embodiment the target binding sequence is 33 nucleotides in length and is reverse complementary to any contiguous 33 nucleotide sequence within SEQ ID NO: 1. In one embodiment the target binding sequence comprises a sequence as set forth in SEQ ID NOs: 209 - 400, or a fragment thereof.

In one embodiment the target binding sequence consists of a sequence as set forth in SEQ ID NOs: 209 - 400, or a fragment thereof

In one embodiment the target sequence comprises a sequence as set forth in SEQ ID NOs: 960 - 1151.

In one embodiment the target sequence consists of a sequence as set forth in SEQ ID NOs: 960 - 1151.

In one embodiment the target binding sequence is 44 nucleotides in length.

In one embodiment the target binding sequence is reverse complementary to any contiguous 44 nucleotide sequence within SEQ ID NO: 1.

In one embodiment 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).

In one embodiment the target binding sequence is 44 nucleotides in length and is reverse complementary to any contiguous 44 nucleotides within SEQ ID NO: 1.

In one embodiment the target binding sequence comprises a sequence as set forth in SEQ ID NOs: 401- 581, or a fragment thereof.

In one embodiment the target binding sequence consists of a sequence as set forth in SEQ ID NOs: 401- 581, or a fragment thereof.

In one embodiment the target sequence comprises a sequence as set forth in SEQ ID NOs: 1152 - 1332.

In one embodiment 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.

In one embodiment the target binding sequence is reverse complementary to any contiguous 72 nucleotide sequence within SEQ ID NO: 1.

In one embodiment 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).

In one embodiment the target binding sequence is 72 nucleotides in length and is reverse complementary to any contiguous 72 nucleotide sequence within SEQ ID NO: 1.

In one embodiment the target binding sequence comprises a sequence as set forth in SEQ ID NOs: 582 - 734, or a fragment thereof.

In one embodiment the target binding sequence consists of a sequence as set forth in SEQ ID NOs: 582 - 734, or a fragment thereof.

In one embodiment the target sequence comprises a sequence as set forth in SEQ ID NOs: 1333 - 1485.

In one embodiment the target sequence consists of a sequence as set forth in SEQ ID NOs: 1333 - 1485.

In one embodiment, the target binding sequence comprises a sequence as set forth in any one of SEQ ID NOs: 2 - 752, or a fragment thereof.

In one embodiment, the target binding sequence consists of a sequence as set forth in any one of SEQ ID NOs: 2 - 752, or a fragment thereof.

In one embodiment, the target sequence comprises a sequence as set forth in any one of SEQ ID NOs: 1 , and 753 - 1503.

In one embodiment, the target sequence consists of a sequence as set forth in any one of SEQ ID NOs: 1 , and 753 - 1503. In accordance with conventional numbering, 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”.

In WO 2012/133947, which is incorporated herein by reference in its entirety, it was shown that 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.

In one embodiment, the target OPA1 mRNA is encoded by the genomic DNA sequence as set forth in SEQ ID NO: 1582

GGACTGAGTACGGGTGCCTGTCAGGCTCTTGCGGAAGTCCATGCGCCATTGGGAGGG

CCTCGGCCGCGGCTCTGTGCCCTTGCTGCTGAGGGCCACTTCCTGGGTCATTCCTGGA

CCGGGAGCCGGGCTGGGGCTCACACGGGGGCTCCCGCGTGGCCGTCTCGGCGCCTG

CGTGACCTCCCCGCCGGCGGGATGTGGCGACTACGTCGGGCCGCTGTGGCCTGTGA

GGTCTGCCAGTCTTTAGTGAAACACAGCTCTGGAATAAAAGGAAGTTTACCACTACAAA

AACTACATCTGGTTTCACGAAGCATTTATCATTCACATCATCCTACCTTAAAGCTTCAAC

GACCCCAATTAAGGACATCCTTTCAGCAGTTCTCTTCTCTGACAAACCTTCCTTTACGTA

AACTGAAATTCTCTCCAATTAAATATGGCTACCAGCCTCGCAGGAATTTTTGGCCAGCA

AGATTAGCTACGAGACTCTTAAAACTTCGCTATCTCATACTAGGATCGGCTGTTGGGGG

TGGCTACACAGCCAAAAAGACTTTTGATCAGTGGAAAGATATGATACCGGACCTTAGTG

AATATAAATGGATTGTGCCTGACATTGTGTGGGAAATTGATGAGTATATCGATTTTGAGA

AAATTAGAAAAGCCCTTCCTAGTTCAGAAGACCTTGTAAAGTTAGCACCAGACTTTGACA

AGATTGTTGAAAGCCTTAGCTTATTGAAGGACTTTTTTACCTCAGGTTCTCCGGAAGAAA

CGGCGTTTAGAGCAACAGATCGTGGATCTGAAAGTGACAAGCATTTTAGAAAGGGTCT

GCTTGGTGAGCTCATTCTCTTACAACAACAAATTCAAGAGCATGAAGAGGAAGCGCGCA

GAGCCGCTGGCCAATATAGCACGAGCTATGCCCAACAGAAGCGCAAGGTGTCAGACAA

AGAGAAAATTGACCAACTTCAGGAAGAACTTCTGCACACTCAGTTGAAGTATCAGAGAA

TCTTGGAACGATTAGAAAAGGAGAACAAAGAATTGAGAAAATTAGTATTGCAGAAAGAT

GACAAAGGCATTCATCATAGAAAGCTTAAGAAATCTTTGATTGACATGTATTCTGAAGTT

CTTGATGTTCTCTCTGATTATGATGCCAGTTATAATACGCAAGATCATCTGCCACGGGTT

GTTGTGGTTGGAGATCAGAGTGCTGGAAAGACTAGTGTGTTGGAAATGATTGCCCAAG

CTCGAATATTCCCAAGAGGATCTGGGGAGATGATGACACGTTCTCCAGTTAAGGTGACT

CTGAGTGAAGGTCCTCACCATGTGGCCCTATTTAAAGATAGTTCTCGGGAGTTTGATCT

TACCAAAGAAGAAGATCTTGCAGCATTAAGACATGAAATAGAACTTCGAATGAGGAAAA

ATGTGAAAGAAGGCTGTACCGTTAGCCCTGAGACCATATCCTTAAATGTAAAAGGCCCT

GGACTACAGAGGATGGTGCTTGTTGACTTACCAGGTGTGATTAATACTGTGACATCAGG

CATGGCTCCTGACACAAAGGAAACTATTTTCAGTATCAGCAAAGCTTACATGCAGAATC

CTAATGCCATCATACTGTGTATTCAAGATGGATCTGTGGATGCTGAACGCAGTATTGTTA

CAGACTTGGTCAGTCAAATGGACCCTCATGGAAGGAGAACCATATTCGTTTTGACCAAA

GTAGACCTGGCAGAGAAAAATGTAGCCAGTCCAAGCAGGATTCAGCAGATAATTGAAG

GAAAGCTCTTCCCAATGAAAGCTTTAGGTTATTTTGCTGTTGTAACAGGAAAAGGGAAC AGCTCTGAAAGCATTGAAGCTATAAGAGAATATGAAGAAGAGTTTTTTCAGAATTCAAAG

CTCCTAAAGACAAGCATGCTAAAGGCACACCAAGTGACTACAAGAAATTTAAGCCTTGC

AGTATCAGACTGCTTTTGGAAAATGGTACGAGAGTCTGTTGAACAACAGGCTGATAGTT

TCAAAGCAACACGTTTTAACCTTGAAACTGAATGGAAGAATAACTATCCTCGCCTGCGG

GAACTTGACCGGAATGAACTATTTGAAAAAGCTAAAAATGAAATCCTTGATGAAGTTATC

AGTCTGAGCCAGGTTACACCAAAACATTGGGAGGAAATCCTTCAACAATCTTTGTGGGA

AAGAGTATCAACTCATGTGATTGAAAACATCTACCTTCCAGCTGCGCAGACCATGAATT

CAGGAACTTTTAACACCACAGTGGATATCAAGCTTAAACAGTGGACTGATAAACAACTT

CCTAATAAAGCAGTAGAGGTTGCTTGGGAGACCCTACAAGAAGAATTTTCCCGCTTTAT

GACAGAACCGAAAGGGAAAGAGCATGATGACATATTTGATAAACTTAAAGAGGCTGTTA

AGGAAGAAAGTATTAAACGACACAAGTGGAATGACTTTGCGGAGGACAGCTTGAGGGT

TATTCAACACAATGCTTTGGAAGACCGATCCATATCTGATAAACAGCAATGGGATGCAG

CTATTTATTTTATGGAAGAGGCTCTGCAGGCTCGTCTCAAGGATACTGAAAATGCAATT

GAAAACATGGTGGGTCCAGACTGGAAAAAGAGGTGGTTATACTGGAAGAATCGGACCC

AAGAACAGTGTGTTCACAATGAAACCAAGAATGAATTGGAGAAGATGTTGAAATGTAAT

GAGGAGCACCCAGCTTATCTTGCAAGTGATGAAATAACCACAGTCCGGAAGAACCTTG

AATCCCGAGGAGTAGAAGTAGATCCAAGCTTGATTAAGGATACTTGGCATCAAGTTTAT

AGAAGACATTTTTTAAAAACAGCTCTAAACCATTGTAACCTTTGTCGAAGAGGTTTTTATT

ACTACCAAAGGCATTTTGTAGATTCTGAGTTGGAATGCAATGATGTGGTCTTGTTTTGGC

GTATACAGCGCATGCTTGCTATCACCGCAAATACTTTAAGGCAACAACTTACAAATACTG

AAGTTAGGCGATTAGAGAAAAATGTTAAAGAGGTATTGGAAGATTTTGCTGAAGATGGT

GAGAAGAAGATTAAATTGCTTACTGGTAAACGCGTTCAACTGGCGGAAGACCTCAAGAA

AGTTAGAGAAATTCAAGAAAAACTTGATGCTTTCATTGAAGCTCTTCATCAGGAGAAATA

AATTAAAATCGTACTCATAATCAGCTCTGCATACATCTGAAGAACAAAAACATCAACGTC

TTTTGTCCAGCCTCTTTTTCTTCTGCTGTTCCACCTTTCTAAACATACAATAAAGTCATGG

GATAAAAATAATCGATGTATGTTACGGGCGCTTTAACCATCAGCTGCCTCTCGAATGGA

AGAACAGTGGTAATGGATTAACATCCTATTTTGTTGTACTAAAGTGACAAATCGGAATAA

TATAATTGGTATGGCCATTAGGTTCAGTCCTTGAAGATAAGAAACTTGTTCTCTGTTTGT

TGTCTTATTTGTGGTGGCACTCGTTTAATGGATTAACTGAGGTTGCTCAATGTTCAGTTT

CTTTTCCAGAAATACAATGCTAGGTGTTTTGAAATAAAACTTATATAGCAATTGTTTAAAG

TTATCAATTGTATATAAAATCACAGTAGCCTGCTAAATCATTGTATGTGTCTGTAGTATTC

TATTCCCAGAAACTATTTGACCATGATAATTCAGTTTATATTCACCACATGAAAGAAAAAT

GGGTAACAGAAGAACCCTTAAAACAGGTTAATTTGGATTGTAACGTTCAGTGAAAGAAA

TTTCAACCCTTCATAGCCAGCGAAGAAATTTGCCTTGGAAGCCAAGTCAGTACCAGCTT

ACCTATTTGATTCAGTTGCTGTTTTCTCACTCTCTATATCCATTTGAAATTGATTTATTTTA

GATGTTGTATACTTACGTTAGGCTTTCTGTTAATAGTGGTTTTTCTCCTGTTGACAGAGC

CACCGGATTATGACACAGGATGAGGAAGATTAAGGATAATCAATTGACTAATTTCATTTA

GAATATTATCAAACATTTCAACTAGGTATCAGAAAAAGGCTTTCTTTCATAAGACTATTTT

AAATAGAAATTATTTCAACAATTAAAGTAATGTTGACCATCCCCCTCTCAGCTGAATAAA

GAAAAATTTAGTTCAATTTATTGCAATTTAATTACAATACTACCTTCACAACATTTTCATGT

GTTTTAAATAAATATTTTTTAATTGGCTAAAGGACATTCAAGCAAAGAAATGCTTTCTTTA

CTTAAAATGTCTATCTCATTTGCTGCCTTTTCACTAAGCCTTTACTTTGTTAATAAAAGTG

TCCATTGTGTGATGTTTTTGATTTTACAGTTTGCTAAATCTTATTTTCTTGGAGTTGCTTT

TTGGTAACAGCCCCATTGCTACTCCCCATTTTATTGTTTTACATCAATGCATGCTTCGTT

GTGATCCCTCAAGATGTAACACTTGGTATGCTCGGTTGAGGATATGAAAAAATACTTCC

GAAACCAGGAATTCAATGTATGTTTGTTTTATACTGTTTGATAAGAAAAGTAGGTCCAGC

CTTAAGCAGCACAGATGCGCTGGTAGATGCATAGTCAGGAACTTTTTTTATTTCTTTTAG

GTCTAGGGACAGGAGTGAATAGAAAGGGAGGAGAGCTCTATTATGTTCTATACACAGAT

TAGGAGATGACCTTACTGGGTACACCCCTCTAACCAGTGCTTACAGGTTAATGCATGTT

AATGAATATTTTTGCAGTTGTAAAGCATAACAATTACAACTACACATCTATTTCTAAAGAA

TAAAACAGGACCATATTTATTTACTTCTGTCAACTATAGAAAGAAAGACCTTCAGCTGTA

TTTCCACAGATTTCTCCCAAGGAAAAGGCTAATATTAGTCACTACTGTTATCACATCCCT

TTGTATAAGTTTTAAAAAGAGATGGAGGGAGATCTTCATTTCTTTGAGGAGATCAGTATT

GTAACGTATGTGAATAGATGATAACAATTAATATTACTAAAAGTCCCACATGAGAGTCCT

GACGCCCTCTCCATGCCCCACAGTAATGTGGCTTCTTTCATGGGTTTTTTTTTCTTCTTT TTAGCTGATCTCATCCTAAGCATGCTTTATTTTTCCTTGAAAGCTAGGTATTTATCAACTG

CAGATGTTATTGAAAGAAAATAAAATTCAGTCTCAAGAGTAAACCCTGTGTCTTGTGTCT

GTAGTTCAAAAGTCAGAAATGATTCTAATTTAAACAAAAAGATACTAAATATACAGAAGTT

AAATTCGAACTAGCCACAGAATCATTTGTTTTTATGTCAGAATTTGCAAAGAGTGGAGTG

GACAAAGCTCTGTATGGAAGACTGAACAACTGTAAATAGATGATATCCAAACTTAATTTG

GCTAGGACTTCAATTTTAAAAATCAGTGTACCTAGGCAGTGCACAGCACGAAATAAGTG

GCCCTTGCAGCTTCCCCGTTTAACCCACTGTGCTATAGTTGCGGGTGGAACAGTCAAC

CTTTCTAGTAGTTTATGATATTGCCCTCTTTGTATTCCCATTTTCTACAGTTTTTTCCGCA

GACTTCTTTCTGCAAATTATTCAGCCTCCAAATGCAAATGAATGATATAAAAATAAGTAG

GGAACATGGCAGAGAGTGGTGCTTCCCAGCCTCACAATGTGGGAATTTGACATAGGAT

GAGAGTCAGAGTATAGGTTTAAAAGATAAAATCTTTAGTTAATAATTTTGTATTTATTTAT

TCTAGATGTATGTATCTGAGGAAAGAAATCTGGTATTTTTGCTTTCCAATAAAGGGGATC

AAAGTAATGGTTTTTCTCTCAGTTCTCTAAGCTGGTCTATGTTATAGCTCTAGCAGTATG

GAAATGTGCTTTAAAATATGCTTACCTTTTGAATGATCATGGCTATATGTTGTTGAGATAT

TTGAAACTTACCTTGTTTTCACTTGTGCACTGTGAATGAACTTTGTATTATTTTTTTAAAA

CCTTCACATTACGTGTAGATATTATTGCAACTTATATTTTGCCTGAGCTTGATCAAAGGT

CATTTGTGTAGATGAGTAATTAAAAAATATTTAAATCACATTATAATTCTATTATTGGAGA

GCATCTTTTAAATTTTTTTCTGTTTTAACGAGGGAAAGAGAAACCTGTATACCTAGGGTC

ATTATTTGACCCCATAGTATAACCAGATTCATGGTCTAACAAGCTCTCAGTGTGGCTTTT

CTCTGAATGCTTGAATTTCACATGCCTTGCATTTCACAGTTGTACTCCATGGTCAACCGG

TGCTTTTTTTCACATCGTGGTACTTGTCAAAACATTTTGTTATTTTCCTTGGTAAAATATA

TAAAAAAGGTTTTCTAATTTCA

(SEQ ID NO: 1582)

In one embodiment, the target sequence is a sequence encoded by SEQ ID NO: 1582, or a fragment thereof.

In another embodiment, 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.

In one embodiment, 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. In one embodiment, 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.

In one embodiment, 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.

In one embodiment 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.

In one embodiment 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.

Herein, 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. Unless a specific reference range is given, e.g., with respect to the nucleotide positions, 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.

For the purposes of comparing two closely-related polynucleotide sequences, 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). For the purposes of comparing two closely-related polypeptide sequences, 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. Herein, 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. Here, as is common in the art, 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. Furthermore, 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. In one embodiment, 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.

In one embodiment, the target binding sequence is 18 nucleotides in length.

In one embodiment, the target binding sequence is 33 nucleotides in length.

In one embodiment, the target binding sequence is 44 nucleotides in length.

In one embodiment, 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. In one embodiment, 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 nucleotides in length within the 5’ UTR.

Alternatively, or in combination, the target binding sequence may hybridise to exon 1 of the coding sequence (CDS) of the target OPA1 mRNA sequence. In one embodiment, 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 6, 0 to 5, or 0 to 4 nucleotides in length, within exon 1.

Alternatively, or in combination, the target binding sequence may hybridise to the coding sequence (CDS) of the target OPA1 mRNA sequence. In one embodiment, 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 6, 0 to 5, or 0 to 4 nucleotides in length within the CDS. 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. In one embodiment, 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 , 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, 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 upstream of the AUG site.

Alternatively, or in combination, the target binding sequence may hybridise to the target OPA1 mRNA sequence downstream of said AUG site. In one embodiment, 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.

Hence, the target binding sequence may bind sequences both upstream and downstream of an AUG site.

It will be understood that an AUG and ATG site are synonymous as used herein, since the target gene perse will comprise an ATG codon but the target mRNA, which is the target of the functional nucleic acid of the invention, will comprise an AUG.

In the context of referencing a sequence reverse complementary to a region in the 5’ UTR and the CDS, this may be anchored around the AUG site, i.e. the region in the 5’ UTR is directly upstream of the AUG site of the target mRNA. For example, reference to 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).

In accordance with conventional numbering, 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”.

In one embodiment, the target binding sequence is 18 nucleotides in length and comprises SEQ ID NO: 30, which targets the M1 -14/+4 region of OPA1.

In one embodiment, the target binding sequence is 33 nucleotides in length and comprises SEQ ID NO: 222, which targets the M1 -14/+19 region of OPA1.

In one embodiment, the target binding sequence is 44 nucleotides in length and comprises SEQ ID NO: 429, which targets the M1 -40/+4 region of OPA1.

In one embodiment, the target binding sequence is 72 nucleotides in length and comprises SEQ ID NO: 582, which targets the M1 -40/+32 region of OPA1.

It is to be understood that “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. For example, 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.

In one embodiment the target binding sequences are separated from one another by a spacer.

In one embodiment the target binding sequence is separated from the regulatory sequence by a spacer.

In one embodiment 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.

In a further embodiment, the spacer may comprise SEQ ID NO: 1581. It will be understood that, in additional to their primary structure (i.e., sequence), RNAs may be described in terms of secondary and tertiary structures that are known in the art. To this end, 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.

In one embodiment, the target binding sequence binds to a target sequence that possesses secondary or tertiary RNA structure.

In one embodiment, the target binding sequence binds to a target sequence within proximity of a sequence that possesses secondary or tertiary RNA structure. By “within proximity” it is meant that 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 nucleotides, within about 140 nucleotides, within about 150 nucleotides, within about 160 nucleotides, within about 170 nucleotides, within about 180 nucleotides, within about 190 nucleotides, within about 200 nucleotides, within about 210 nucleotides, within about 220 nucleotides, within about 230 nucleotides, of the start or end of an RNA structure.

As such, it will be understood that 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.

Such 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.

In one embodiment, 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

An example of predicted 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.

Table 1. Predicted RNA secondary structures within OPA1 mRNA

In one embodiment, 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. In one embodiment, the target binding sequence comprises a sequence reverse complementary to a portion of OPA1 mRNA that comprises the M1 AUG codon.

In one embodiment 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.

In one embodiment, the target binding sequence comprises a sequence reverse complementary to a portion of OPA1 mRNA that does not comprises the M1 AUG codon.

In one embodiment 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.

In one embodiment, 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.

In one embodiment, the target sequence comprises the OPA1 M1 AUG codon.

In one embodiment 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.

In one embodiment, the target sequence does not comprises the OPA1 M1 AUG codon.

In one embodiment 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.

In one embodiment, 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.

The target binding sequences according to the foregoing may be combined with any suitable regulatory sequence as defined in the following section. In one embodiment the target binding sequence comprises any one or more of a sequence selected from SEQ ID NOs 2 - 752, or a fragment thereof, and 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.

In a preferred embodiment, the target binding sequence is 18 nucleotides in length, 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.

In a preferred embodiment, the target binding sequence is any one or more of a sequence selected from SEQ ID NOs 2 - 208, 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.

In a preferred embodiment, the target binding sequence is 33 nucleotides in length, 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.

In a preferred embodiment, the target binding sequence is any one or more of a sequence selected from SEQ ID NOs 209 - 400, 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.

In a preferred embodiment, the target binding sequence is 44 nucleotides in length, 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.

In a preferred embodiment, the target binding sequence is any one or more of a sequence selected from SEQ ID NOs 401 - 581 , 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.

In a preferred embodiment, 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. In a preferred embodiment, 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.

In a preferred embodiment, the target binding sequence is 18 nucleotides in length, and the regulatory sequence comprises any one or more of a sequence selected from SEQ ID NOs: 1574 - 1580, or a fragment thereof.

In a preferred embodiment, the target binding sequence is any one or more of a sequence selected from SEQ ID NOs 2 - 208, 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.

In a preferred embodiment, the target binding sequence is 33 nucleotides in length, and the regulatory sequence comprises any one or more of a sequence selected from SEQ ID NOs: 1574 - 1580, or a fragment thereof.

In a preferred embodiment, the target binding sequence is any one or more of a sequence selected from SEQ ID NOs 209 - 400, 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.

In a preferred embodiment, the target binding sequence is 44 nucleotides in length, and the regulatory sequence comprises any one or more of a sequence selected from SEQ ID NOs: 1574 - 1580, or a fragment thereof.

In a preferred embodiment, the target binding sequence is any one or more of a sequence selected from SEQ ID NOs 401 - 581 , 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.

In a preferred embodiment, 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. In a preferred embodiment, 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.

Exemplary 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.

Table 2. Exemplary target sequences and target binding sequences

Regulatory Sequences

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.

In accordance with SINEIIP nomenclature or by analogy to SINEUPs (where IRES are used), regulatory sequences may also be known as effector domains (EDs).

In one embodiment, the functional nucleic acid molecule comprises one, two, three, four, or regulatory sequences.

In one embodiment, the functional nucleic acid molecule comprises one regulatory sequence.

In one embodiment, the functional nucleic acid molecule comprises two regulatory sequences.

In one embodiment, 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.

It will be understood that by “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. In one embodiment, 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.

In a further embodiment, 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.

It will be understood that by “protein expression”, it is meant the level of protein present in a system as determined by the translational activity within that system. For example, “increasing protein expression” will be understood to mean ultimately increasing the amount of a given protein in the system.

In one embodiment 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

3.0 fold.

In one embodiment 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.

In one embodiment 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

1.5 fold, less than about 1.6 fold, less than about 1.7 fold, less than about 1.8 fold, less than about 1.9 fold, less than about 2.0 fold, less than about 2.1 fold, less than about 2.2 fold, less than about 2.3 fold, less than about 2.4 fold, less than about 2.5 fold, less than about

2.6 fold, less than about 2.7 fold, less than about 2.8 fold, less than about 2.9 fold, or less than about 3.0 fold.

These increases in protein expression are within physiological ranges. It is envisaged that increasing protein expression within these ranges will allow the treatment of diseases associated with one or more gene defects, such as cancer or neurodegenerative diseases, without leading to negative side effects associated with increasing expression of the target above non-disease state or ‘wild-type’ physiological levels.

It is understood that by “the target mRNA” it is meant the target OPA1 mRNA.

In one embodiment, 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. Alternatively, “inverted” refers to the situation in which the regulatory sequence is 3’ to 5’ oriented relative to the functional nucleic acid molecule.

In a further embodiment, the regulatory sequence is located 3’ of the target binding sequence within the functional nucleic acid molecule.

SINE B2

In one embodiment, 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.

In one embodiment, 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.

In one embodiment 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. In one embodiment 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

The term “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.

The term “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. Generally, a SINE B2 element is not less than 20 bp and not more than 400 bp. Preferably, the SINE B2 element is derived from tRNA.

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 (such as a SINE B2 element) 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. Therefore, in one embodiment, 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.

In some embodiments 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.

In one embodiment, 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.

In one embodiment, 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.

In one embodiment, 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.

Preferably, 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.

In one embodiment, 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.

In one embodiment, 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.

In one embodiment, 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. 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.

IRES

Alternatively, 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.

In one embodiment, the regulatory sequence comprises an IRES sequence, or a functionally active fragment thereof. Said sequence enhances translation of the target mRNA sequence.

The terms “internal ribosome entry site (IRES) sequence” is defined in WO 2019/058304, which is incorporated herein by reference in its entirety. 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. The person skilled in the art would know that an IRES sequence is a nucleotide sequence capable of promoting translation of a second cistron in a bicistronic construct. Typically, 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. Thus, in one embodiment the functional nucleic acid molecule comprises an IRES regulatory sequence that is trans-acting.

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).

Within the IRESite, 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- freiburg.de/; http://regrna.mbc.nctu.edu.tw/index1.php).

Several IRESs having sequences ranging from 48 to 576 nucleotides have been tested with success, e.g. human Hepatitis C Virus (HCV) IRESs (e.g. SEQ ID NO: 1556 and 1557), human 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), human ELG-1 (e.g. SEQ ID NO: 1566 and 1567), human c-MYC (e.g. SEQ ID NO: 1568 - 1571) and human dystrophin (DMD) (e.g. SEQ ID NO: 1572 and 1573). In some embodiments the regulatory sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 1556 - 1573, or a functionally active fragment thereof.

In some embodiments the regulatory sequence consists of a sequence selected from the group consisting of SEQ ID NOs: 1556 - 1573, or a functionally active fragment thereof.

Such sequences have been disclosed, defined and exemplified in WO 2019/058304, which is incorporated herein by reference in its entirety.

In one embodiment 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.

In one embodiment, 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.

In some embodiments 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

180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about

260, about 270, about 280, about 290, about 300, about 310, about 320, about 330, about

340, about 350, about 360, about 370 or more nucleotides in length. In some embodiments, the functionally active fragment retains IRES activity within the definition provided above.

In some embodiments, the functionally active fragment retains protein translation enhancing activity.

It will be understood that, owing to the functional nature of The Standard Bicistronic Plasmid Test for Cellular mRNA IRESs, a “functionally active fragment” of an IRES might also be considered an IRES perse. Herein, “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.

Further, it will be understood that any regulatory sequence according to the invention may be suitably paired with any binding sequence according to the invention.

Table 3. Exemplary regulatory sequences

Table 4. Exemplary regulatory/linker sequences

DNA molecules and vectors

According to a further aspect of the invention, there is provided a DNA molecule encoding a functional nucleic acid molecule of the invention.

According to a further aspect of the invention, there is provided 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. The choice of expression vector may be dependent upon the type of host cell to be used and the purpose of use. In particular, and without limitation, the following plasmids have been used for expression of functional nucleic acid molecule:

Mammalian expression plasmids:

- pCDNA3.1 (-) pDUAL-eGFPA (modified from peGFP-C2)

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) In one embodiment the mammalian expression plasmid is pCDNA3.1 (-).

In another embodiment 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.

In one embodiment the viral vector is pAAV.

In one embodiment the viral vector is rcLV -TetOne-Puro.

In one embodiment 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.

It should be noted that 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.

Compositions and methods

The present invention also relates to compositions comprising the functional nucleic acid molecule, the DNA molecule or the expression vector according to the invention.

The 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). Alternatively, the functional nucleic acid molecule of the invention may be administered as naked or unpackaged RNA.

The 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 Mg²⁺ ions).

The functional nucleic acid molecule may be administered as part of a composition, for example a composition comprising a suitable carrier. In certain embodiments, the carrier is selected based upon its ability to facilitate the transfection of a target cell with one or more functional nucleic acid molecule.

Therefore, according to a further aspect of the invention, there is provided a composition comprising the functional nucleic acid molecule, the DNA molecule, or the expression vector as described herein.

In one embodiment, there is provided 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.

Suitably, 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. In a preferred embodiment, 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.

Methods, methods of treatment and medical uses

In one aspect of the present invention, there is provided a method for enhancing translation of a target mRNA, such as a therapeutic target mRNA, in a cell comprising administering the functional nucleic acid molecule, DNA molecule, expression vector, composition or pharmaceutical composition as defined herein to the cell. Preferably, the cell is a mammalian cell, such as a human or mouse cell.

According to a further aspect of the invention, there is provided 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.

According to a further aspect of the invention, there is provided 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.

According to a further aspect of the invention, there is provided 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. Preferably, the cell is a mammalian cell, such as a human or a mouse cell.

According to a further aspect of the invention, there is provided 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. Preferably, 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.

As used herein, “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.

For example when expressed in a subject (such as in a cell of a subject), a therapeutic target 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. Herein, the use of 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.

It will be understood that 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. Retinal ganglion cells (RGCs) expressing 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).

Therefore, in a further embodiment, 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. 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. Furthermore, intra-familial and inter-familial variations in disease severity among patients with the same mutation have been reported. It is intended that the functional nucleic acid molecule, DNA molecule, expression vector, composition or pharmaceutical composition of the invention may be used to treat any of these forms of ADOA.

According to a further aspect of the invention, there is provided the use of the functional nucleic acid molecule, DNA molecule, expression vector, composition or pharmaceutical composition as defined herein for the manufacture of a medicament.

According to another aspect of the invention, there is provided a therapeutically effective amount of the functional nucleic acid molecule, DNA molecule, expression vector, composition or pharmaceutical composition defined herein for use in the manufacture of a medicament for the treatment of a disease associated with mitochondrial defects or decreased OPA1 , such as ADOA.

In addition to ADOA, some reports propose that a mild increase of OPA1 protein expression may be therapeutic for additional diseases. For example, 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. In humans, 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. As another example, Varanita et al. (2015) showed that 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.

Excessive overexpression of 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.

Diseases associated with mitochondrial defects are well known in the art, for example as described in Gorman et al. (2016) Nat. Rev. Disease Primers, 2, 16080. They may be characterised by defects in oxidative phosphorylation due to mutations in nuclear or mitochondrial DNA that result in mutated/dysfunctional mitochondrial proteins. Mitochondrial defects have been associated with neural disease and development. Therefore, in one embodiment, the disease is a disease associated with mitochondrial defects is a neurological disease. Caglayan et al. (2020) iScience 23: 101154, described genetically modified human embryonic and patient-derived induced pluripotent stem cells with OPA1 haploinsufficiency led to aberrant nuclear DNA methylation and significantly altered the transcriptional circuitry in neural progenitor cells (NPCs). In particular, OPA1+/- NPCs could not develop into GABAergic interneurons. Changes to normal OPA1 expression have also been linked to Alzheimer’s disease, Huntington’s disease and Parkinson’s disease (see Wang et al. (2009) J. Neurosci. 29(28): 9090-9103; Costa et al. (2010) EMBO Mol. Med. 2(12): 490-503; Santos et al. (2015) Mol. Neurobiol. 52(1): 573-86; Ramonet et al. (2013) Cell Death Diff. 20(1): 77-85; lannielli et al. (2018) Cell Rep. 22(8): 2066-2079; and lannielli et al. (2019) Cell Rep. 29(13): 4646-4656). In one embodiment, the neurological disease is selected from: Alzheimer’s disease, Huntington’s disease and Parkinson’s disease.

In one embodiment, the disease associated with mitochondrial defects is a prion disease. For example, 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.

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 therapy.

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 as a medicament.

It will be understood that 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.

Thus 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.

In one embodiment 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 one or more gene defects, wherein the gene defects affect the level of OPAI protein.

As used herein, “gene defect” or “gene defects”, refer to one or more abnormalities in a gene which results in reduced protein levels and/or loss-of-function mutations of the encoding gene. For example, 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. In one embodiment, the gene defect or defects reduce the levels or impair the function of OPA1.

For example, a disease associated with one or more gene defects may be ADOA.

In one embodiment there is provided the functional nucleic acid molecule, DNA molecule, expression vector, composition or pharmaceutical composition for use in the treatment of ADOA.

In one embodiment, there is provided 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.

In one embodiment, there is provided 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.

In one aspect, there is provided 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. According to a further aspect of the invention, there is provided the functional nucleic acid molecule, DNA molecule expression vector, composition or pharmaceutical composition, as defined herein, for use in medicine.

According to a further aspect of the invention, there is provided the functional nucleic acid molecule, DNA molecule, expression vector, composition or pharmaceutical composition, as defined herein, for use in the treatment or prevention of a disease or disorder associated with OPA1.

In one embodiment, the disease or disorder is a neurological disease or disorder, or a mitochondrial disease or disorder.

In one embodiment, 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.

In one embodiment, the disease or disorder is Autosomal Dominant Optic Atrophy (ADOA).

It will be understood that both defects of or within the OPA1 gene (e.g., within the coding sequence thereof) and 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.

Herein instances of the plural form of words should be taken to cover also the singular form of the word and vice versa, unless the context clearly dictates otherwise.

The invention will now be illustrated with reference to the following non-limiting examples.

Claims

1. A functional nucleic acid molecule comprising:

- one or more target binding sequences comprising a sequence reverse complementary to an OPA1 mRNA sequence; and

- 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.

2. The functional nucleic acid molecule of claim 1, wherein 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.

3. The functional nucleic acid molecule of claim 1 or claim 2, wherein the target binding sequence comprises a sequence reverse complementary to a sequence within the SEQ ID NO: 1.

4. The functional nucleic acid molecule of any one of claims 1 to 3, wherein the OPA1 mRNA sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 753 - 1485.

5. The functional nucleic acid molecule of any one of claims 1 - 4, wherein the target binding sequence comprises a sequence as set forth in any one of SEQ ID NOs: 2 - 734.

6. The functional nucleic acid molecule of any one of claims 1 - 5, wherein the target binding sequence comprises a sequence that is:

(a) 18 nucleotides in length;

(b) 33 nucleotides in length;

(c) 44 nucleotides in length; or

(d) 72 nucleotides in length.

7. The functional nucleic acid molecule of any one of claims 1 - 6, wherein the target binding sequence comprises a sequence that is:

(a) selected from the group consisting of: SEQ ID NOs: 2 - 208;

(b) selected from the group consisting of: SEQ ID NOs: 209 - 400;

(c) selected from the group consisting of: SEQ ID NOs: 401 - 581 ;

(d) selected from the group consisting of: SEQ ID NOs: 582 - 743; or (e) a functionally active fragment of any one of (a) to (d).

8. The functional nucleic acid molecule of claim 7, wherein the functionally active fragment is suitably: 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 nucleotides, 49 nucleotides, 50 nucleotides, 51 nucleotides, 52 nucleotides, 53 nucleotides, 54 nucleotides, 55 nucleotides, 56 nucleotides, 57 nucleotides, 58 nucleotides, 59 nucleotides, 60 nucleotides, 61 nucleotides, 62 nucleotides, 63 nucleotides, 64 nucleotides, 65 nucleotides, 66 nucleotides, 67 nucleotides, 68 nucleotides, 69 nucleotides, 70 nucleotides, or 71 nucleotides in length.

9. The functional nucleic acid molecule of any one of claims 1 - 8, wherein the target binding sequence is reverse complementary to an OPA1 mRNA sequence that comprises the M1 AUG codon.

10. The functional nucleic acid molecule of claim 8, wherein the target binding sequence is selected from the group consisting of: SEQ ID NOs: 15 - 30, 209 - 238, 401 - 430, and 582 - 611 , or a functionally active fragment thereof.

11. The functional nucleic acid molecule of any one of claims 1 - 8, wherein the target binding sequence is reverse complementary to an OPA1 mRNA sequence that does not comprises the M1 AUG codon.

12. The functional nucleic acid molecule of claim 11 , wherein the target binding sequence is selected from the group consisting of: SEQ ID NOs: 2 - 14, 31 - 208, 239 - 400, 431 - 581 , and 612 - 734, or a functionally active fragment thereof.

13. The functional nucleic acid molecule of claim 1 , wherein the OPA1 mRNA sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 1, 753 - 1503 and 1583 - 1598.

14. The functional nucleic acid of claim 1 or claim 13, wherein the target binding sequence comprises a sequence as set forth in any one of SEQ ID NOs: 2 - 752, or a functionally active fragment thereof.

15. The functional nucleic acid molecule of any one of claims 1 , 13, or 14, wherein the target binding sequence is reverse complementary to an OPA1 mRNA sequence that comprises the M1 AUG.

16. The functional nucleic acid molecule of claim 15, wherein the target binding sequence is selected from the group consisting of: SEQ ID NOs 15 - 30, 209 - 238, 401 - 430, 582 - 611, 736 - 743, 748, and 750, or a functionally active fragment thereof.

17. The functional nucleic acid molecule of any one of claims 1, 13, or 14, wherein the target binding sequence is reverse complementary to an OPA1 mRNA sequence that does not comprise the M1 AUG.

18. The functional nucleic acid molecule of claim 17, wherein the target binding sequence is selected from the group consisting of: 2 - 14, 31 - 208, 239 - 400, 431 - 581, 612 - 734, 735, 744 - 747, 749, 751 and 752 or a functionally active fragment thereof.

19. The functional nucleic acid molecule according to any one of claims 14 - 18, wherein the functionally active fragment is suitably: less than 134 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 in length.

20. The functional nucleic acid molecule according to any one of claims 1 - 19, wherein the regulatory sequence comprises a sequence with at least 75% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 1504 - 1573, or a functionally active fragment thereof.

21. The functional nucleic acid molecule according to any one of claims 1 - 19, wherein the regulatory sequence comprises a sequence with at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 1504 - 1573, or a functionally active fragment thereof.

22. The functional nucleic acid molecule according to any one of claims 1 - 19, wherein the regulatory sequence comprises a sequence as set forth in the group consisting of SEQ ID NOs: 1504 - 1573, or a functionally active fragment thereof.

23. The functional nucleic acid molecule according to any one of claims 20 - 22, wherein the functionally active fragment is less than 250 nucleotides, 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 in length.

24. The functional nucleic acid molecule according to any one of claims 1 - 23, further comprising at least one spacer or linker sequence.

25. The functional nucleic acid molecule according to claim 24, wherein the spacer or linker is between:

(a) any two target binding sequences; and/or

(b) any two regulatory sequences; and/or

(c) any target binding sequence and any regulatory sequence.

26. The functional nucleic acid molecule of any one of claims 1 - 25, 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.

27. The functional nucleic acid molecule of any one of claims 1 - 25, 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.

28. The functional nucleic acid molecule of any one of claims 1 - 27, wherein the regulatory sequence is located 3’ of the target binding sequence.

29. The functional nucleic acid molecule of any one of claims 1 - 28, wherein the functional nucleic acid molecule is a trans-acting functional nucleic acid molecule.

30. The functional nucleic acid molecule according to any one of claims 1 - 29, wherein the functional nucleic acid molecule is circular.

31. The functional nucleic acid molecule of any one of claims 1 - 30, wherein the functional nucleic acid molecule comprises RNA nucleotides or modified RNA nucleotides.

32. The functional nucleic acid molecule of claim 31 , wherein the functional nucleic acid molecule consists of RNA nucleotides or modified RNA nucleotides.

33. The functional nucleic acid molecule of any one of claims 1 - 32, wherein the functional nucleic acid molecule is single stranded.

34. A DNA molecule encoding the functional nucleic acid molecule of any one of claims 1 - 33.

35. An expression vector comprising the DNA molecule of claim 34.

36. A composition comprising the functional nucleic acid molecule of any one of claims 1 - 33, the DNA molecule of claim 34 or the expression vector of claim 35.

37. A pharmaceutical composition comprising the functional nucleic acid molecule of any one of claims 1 - 33, the DNA molecule of claim 34 or the expression vector of claim 35, and a pharmaceutically acceptable excipient, diluent, carrier, and/or salt.

38. Use of a functional nucleic acid molecule of any one of claims 1 - 33, the DNA molecule of claim 34, the expression vector of claim 35, the composition of claim 36, or the pharmaceutical composition of claim 37 for enhancing translation of OPA1 mRNA.

39. The functional nucleic acid molecule of any one of claims 1 - 33, the DNA molecule of claim 34, the expression vector of claim 35, the composition of claim 36, or the pharmaceutical composition of claim 37 for use in medicine.

40. The functional nucleic acid molecule of any one of claims 1 - 33, the DNA molecule of claim 34, the expression vector of claim 35, the composition of claim 36, or the pharmaceutical composition of claim 37 for use in the treatment or prevention of a disease or disorder associated with OPA1.

41. The functional nucleic acid molecule, the DNA molecule, the expression vector, the composition, or the pharmaceutical composition for use according to claim 40, wherein the disease or disorder is a neurological disease or disorder, or a mitochondrial disease or disorder.

42. The functional nucleic acid molecule, the DNA molecule, the expression vector, the composition, or the pharmaceutical composition for use according to claim 41, wherein the disease or disorder is selected from the group consisting of: Autosomal Dominant Optic Atrophy (ADOA), Alzheimer’s disease, Huntington’s disease and Parkinson’s disease.

43. The functional nucleic acid molecule, the DNA molecule, the expression vector, the composition, or the pharmaceutical composition for use according to claim 41 or claim 42, wherein the disease or disorder is Autosomal Dominant Optic Atrophy (ADOA).

44. A method of treating a disease associated with OPA1 comprising administering the functional nucleic acid molecule of any one of claims 1 - 33, the DNA molecule of claim 34, the expression vector of claim 35, the composition of claim 36, or the pharmaceutical composition of claim 37 to a subject.

45. The functional nucleic acid molecule of any one of claims 1 - 33, the DNA molecule of claim 34, the expression vector of claim 35, the composition of claim 36, or the pharmaceutical composition of claim 37 for use in the manufacture of a medicament.

46. An in vitro or in vivo method for increasing the translation of OPA1 in a cell comprising administering the functional nucleic acid molecule according to any one of claims 1 - 33, the DNA molecule of claim 34, the expression vector of claim 35, the composition of claim 36, or the pharmaceutical composition of claim 37, to the cell.

47. The method according to claim 46, wherein the cell is a mammalian cell.

48. The method according to claim 47, wherein the cell is a human or mouse cell.

49. The method according to any one of claims 46 - 48, wherein the functional nucleic acid molecule is administered as naked RNA.

50. The method according to any one of claims 46 - 49, wherein the cell is OPA1 haploinsufficient.