Functional nucleic acid molecules

Abstract

The present invention relates to functional nucleic acid molecules for use in upregulating OPA1 expression. The functional nucleic acid molecules typically contain at least one target binding sequence that is reverse complementary to the OPA1 mRNA sequence and at least one regulatory sequence that includes a SINE B2 element or an internal ribosome entry site (IRES) sequence. Therapeutic methods using the functional nucleic acids are also described.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to functional nucleic acid molecules for use in upregulating OPA1 expression. [Background technology]

[0002] BACKGROUND OF THE INVENTION Autosomal dominant optic atrophy (ADOA) is the most common hereditary optic neuropathy, caused by heterozygous mutations in the OPA1 gene in 75% of cases. ADOA is an early-onset, autosomal dominant, haploinsufficient disorder with a prevalence ranging from 1:12,000 to 1:50,000 live births. It is characterized by retinal ganglion cell degeneration, leading to optic nerve atrophy and blindness. Human OPA1 is a ubiquitously expressed dynamin-related GTPase protein that plays a key role in mitochondrial homeostasis. It localizes to the inner mitochondrial membrane (IMM), with highest expression levels in the brain, retina, and heart.

[0003] Data indicate that both underexpression and overexpression of OPA1, as seen in ADOA patients, have deleterious consequences, and both of these changes in OPA1 levels lead to increased apoptosis (Chen et al. (2009) Cardiovasc Res. 84(1):91-9). In vivo, the data are mixed. Transgenic mice with mild OPA1 overexpression appear healthy and fertile and even exhibit protection against injury to specific tissues, such as the liver and brain, whereas long-term overexpression in the SV129 mouse strain has been observed to increase the incidence of spontaneous cancers and shorten lifespan (Varanita et al. (2015) Cell Metab. 21(6):834-44). High expression of OPA1 and other mitochondrial proteins that promote fusion are associated with cancer cell proliferation, survival, and invasion. OPA1 is highly expressed in lung adenocarcinoma cells and is associated with cisplatin resistance and poor prognosis (Fang et al. (2012) Hum. Pathol. 43(1):105-14).

[0004] A new class of long non-coding RNAs (lncRNAs) known as SINEUPs have previously been described that can selectively enhance the translation of their targets. SINEUP activity relies on the combination of two domains: an overlapping region, or binding domain (BD), that confers specificity, and an embedded inverted SINE B2 element, or effector domain (ED), that enhances target mRNA translation. WO 2012 / 133947 and WO 2019 / 150346 disclose functional nucleic acid molecules containing SINEUPs. Another class of lncRNAs that uses an effector domain containing an internal ribosome entry site (IRES) sequence to provide a functional nucleic acid molecule that acts in trans is described in WO 2019 / 058304.

[0005] The aim of the present invention is to provide the first gene-specific technology that targets OPA1 translation, specifically for use in the treatment of ADOA. Summary of the Invention

[0006] (Summary of the Invention) According to a first aspect, there is provided a functional nucleic acid molecule comprising: at least one target binding sequence comprising a sequence reverse complementary to an OPA1 mRNA sequence; and at least one regulatory sequence comprising a SINE B2 element or a functionally active fragment of a SINE B2 element or an RNA comprising an internal ribosome entry site (IRES) sequence or an IRES-derived sequence; The functional nucleic acid molecule is provided, comprising:

[0007] According to a further aspect of the present invention there is provided a DNA molecule encoding a functional nucleic acid molecule as defined herein. According to a further aspect of the present invention there is provided an expression vector comprising a functional nucleic acid molecule as defined herein.

[0008] According to a further aspect of the present invention there is provided a composition comprising a functional nucleic acid molecule, DNA molecule or expression vector as defined herein.

[0009] According to a further aspect of the present invention, there is provided a method for increasing the efficiency of OPA1 protein synthesis in a cell, the method comprising administering to the cell a functional nucleic acid molecule, DNA molecule, expression vector or composition as defined herein.

[0010] According to a further aspect of the present invention there is provided a method of treating a disease associated with a mitochondrial defect, such as ADOA, comprising administering a therapeutically effective amount of a functional nucleic acid molecule, DNA molecule, expression vector or composition as defined herein.

[0011] According to a further aspect of the present invention there is provided a therapeutically effective amount of a functional nucleic acid molecule, DNA molecule, expression vector or composition for use in the manufacture of a medicament for the treatment of a disease associated with a mitochondrial defect, such as ADOA. [Brief explanation of the drawings]

[0012] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] FIG. 1: Schematic representation of SINEUP functional domains and the human OPA1 gene, and examples of target binding domains of functional nucleic acids according to the invention. [Figure 2]Figure 2: HEK 293T cells were transfected with mini-SINEUP lacking the BD (ΔBD) and mini-SINEUP-OPA1 mutants. (A) Total cell lysates were analyzed by Western blotting using anti-OPA1 and anti-β-actin antibodies. One representative experiment is shown. The graph shows real-time PCR analysis of OPA1 mRNA and mini-SINEUP RNA expression in transfected cells. Columns represent the mean ± SEM of n ≥ 3 independent experiments. (B) Average fold change in OPA1 protein levels. For each sample, values ​​are reported as two separate columns representing the long (left) and short (right) isoforms, respectively. Columns represent the mean ± SEM of n ≥ 4 independent experiments; ns, p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 (one-way ANOVA followed by Dunnett's post-hoc test). [Figure 3] Figure 3: Mouse Neruo2A cells were transfected with mini-SINEUP lacking the BD (ΔBD) and the mini-SINEUP-OPA1 mutant. (A) Whole cell lysates were analyzed by Western blotting using anti-OPA1 and anti-β-actin antibodies. One representative experiment is shown. The graph shows real-time PCR analysis of OPA1 mRNA and mini-SINEUP RNA expression in transfected cells. Columns represent the mean ± SEM of n ≥ 3 independent experiments. (B) Average fold change in OPA1 protein levels. For each sample, values ​​are reported as two separate columns representing the long (left) and short (right) isoforms, respectively. Columns represent the mean ± SEM of n ≥ 3 independent experiments; ns, p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 (one-way ANOVA followed by Dunnett's post-hoc test). [Figure 4]Figure 4: Mouse astrocytes were transfected with mini-SINEUP lacking the BD (ΔBD) and the mini-SINEUP-OPA1 mutant. (A) Whole cell lysates were analyzed by Western blotting using anti-OPA1 and anti-β-actin antibodies. One representative experiment is shown. The graph shows real-time PCR analysis of OPA1 mRNA and mini-SINEUP RNA expression in transfected cells. Columns represent the mean ± SEM of n ≥ 3 independent experiments. (B) Average fold change in OPA1 protein levels. For each sample, values ​​are reported as two separate columns representing the long (left) and short (right) isoforms, respectively. Columns represent the mean ± SEM of n ≥ 3 independent experiments; ns, p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 (one-way ANOVA followed by Dunnett's post-hoc test). [Figure 5] Figure 5: HEK293T cells were transfected with mini-SINEUP (ΔBD), mini-SINEUP-OPA1 (-14 / +4-M1-AUG), and micro-SINEUP-OPA1 (-14 / +4-M1-AUG) mutants lacking the BD. (A) Whole cell lysates were analyzed by Western blotting using anti-OPA1 and anti-β-actin antibodies. One representative experiment is shown. The graph shows real-time PCR analysis of OPA1 mRNA and mini-SINEUP RNA expression in transfected cells. Columns represent the mean ± SEM of n≥3 independent experiments. (B) Average fold change in OPA1 protein levels. For each sample, values ​​are reported as two separate columns representing the long (left) and short (right) isoforms, respectively. Columns represent the mean ± SEM of n=4 independent experiments; ns, p>0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 (one-way ANOVA followed by Dunnett's post-hoc test). [Figure 6]Figure 6: HEK 293T cells were transfected with mini-SINEUP (ΔBD) lacking the binding domain and nano2SINEUP-OPA1 (-14 / +4-M1-AUG). (A) Whole cell lysates were analyzed by Western blotting using anti-OPA1 and anti-β-actin antibodies. One representative experiment is shown. (B) Real-time PCR analysis of OPA1 mRNA (left panel) and nano2SINEUP RNA expression (right panel). NT = not transfected. [Figure 7] Figure 7: Mini-SINEUP in two different vector backbones showing OPA1-nanoluc luminescence in vitro. [Figure 8] Figure 8: Results of synthetic nano2SINEUP on OPA1 protein and mRNA levels. (A) Whole cell lysates were analyzed by Western blotting using anti-OPA1 and anti-β-actin antibodies. (B) Average fold change in OPA1 protein levels. Real-time PCR analysis of OPA1 mRNA (C) and nano2SINEUP RNA (D). DETAILED DESCRIPTION OF THE INVENTION

[0013] (Detailed explanation) The objective of the present invention is to provide functional nucleic acid molecules that increase OPA1 protein expression without compromising physiological levels, target OPA1 expression in a highly gene-specific manner across all isoforms, and limit side effects.

[0014] We utilized SINEUP technology to express and target OPA1 to increase endogenous levels of all OPA1 protein isoforms in human, mouse, and patient-derived cell lines. In vitro screening demonstrated that OPA1-specific SINEUPs could selectively increase both human and mouse OPA1 protein. Importantly, when expressed in ADOA patient-derived fibroblasts, SINEUPs-OPA1 increased OPA1 protein levels by twofold, demonstrating sufficient activity for functional rescue without the negative side effects associated with increasing OPA1 expression above physiological levels. Furthermore, SINEUPs-OPA1 did not disrupt the ratio of the long / short forms of OPA1 protein, which is crucial for rescuing mitochondrial network morphology.

[0015] definition By "functional nucleic acid molecule" it is generally intended that the nucleic acid molecule is capable of enhancing translation of a target mRNA of interest, in this particular case OPA1 mRNA.

[0016] By "OPA1 mRNA sequence" is intended any mRNA sequence of at least 10 nucleotides in length contained in the mRNA of the corresponding OPA1 gene. Alternative splicing of the OPA1 transcript generates eight different isoforms, which share a common 5'UTR and are widely expressed. The resulting OPA1 protein also undergoes cleavage to generate both long (l) and short (s) OPA1 forms. OPA1 gene sequences are known in the art; see, for example, Gene ID: 4976 or Ensembl ID: ENSG00000198836.

[0017] The OPA1 gene encodes the OPA1 mitochondrial dynamin-like GTPase (also known as mitochondrial dynamin-like 120 KDa protein), herein referred to as the "OPA1 protein." The OPA1 protein sequence is known in the art, see, for example, UniProt ID: O60313.

[0018] The term "SINE" (Short Interspersed Nuclear Element), sometimes referred to as a non-LTR (long terminal repeat) retrotransposon, refers to an interspersed repeat element that (a) encodes a protein with no reverse transcription or endonuclease activity, and (b) is abundant in the genome of an organism, with perfect or imperfect copies.

[0019] The term "SINE B2 element" is defined in WO 2012 / 133947, which also provides specific examples (see the table beginning on page 69 of the PCT publication). This term is intended to encompass both forward and reverse SINE B2 elements relative to the 5'-3' direction of a functional nucleic acid molecule. SINE B2 elements can be identified, for example, using a published program such as RepeatMask (Bedell et al., Bioinformatics. 2000 Nov; 16(11):1040-1. MaskerAid: a performance enhancement to RepeatMasker). A sequence can be recognized as a SINE B2 element by returning hits in the Repbase database against the SINE B2 consensus sequence with a Smith-Waterman (SW) score of greater than 225, the default cutoff in the RepeatMasker program. Generally, SINE B2 elements are 20 bp or longer and 400 bp or shorter. Preferably, SINE B2 is derived from a tRNA.

[0020] The term "functionally active fragment of a SINE B2 element" refers to a portion of the sequence of a SINE B2 element that retains its protein translation enhancing ability. The term also includes sequences that have one or more nucleotide mutations relative to the wild-type sequence but retain their protein translation enhancing ability. The term is intended to encompass both forward and reverse orientations of a SINE B2 element relative to the 5'-3' direction of a functional nucleic acid molecule.

[0021] The terms "internal ribosome entry site (IRES) sequence" and "internal ribosome entry site (IRES)-derived sequence" are defined in WO 2019 / 058304. IRES sequences guide 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 of cellular mRNAs encoding stress response genes, thereby stimulating their translation in cis. The term "IRES-derived sequence" is understood to refer to a nucleic acid sequence that has homology to an IRES sequence in order to retain its functional activity, i.e., translation-enhancing activity. In particular, IRES-derived sequences can be obtained from natural IRES sequences by genetic engineering or chemical modification, for example, by isolating a specific sequence of an IRES sequence that retains its function, or by mutating / deleting / introducing one or more nucleotides in the IRES sequence, or by replacing one or more nucleotides in the IRES sequence with structurally modified nucleotides or analogs. More specifically, those skilled in the art know that an IRES-derived sequence is a nucleotide sequence that can promote translation of the second cistron in a bicistronic construct. Typically, a plasmid encoding dual luciferases (firefly luciferase, Renilla luciferase) is used in experimental tests. A major database, IRESite, exists for the annotation of experimentally validated nucleotide sequences as IRESs using dual reporter or bicistronic assays (http: / / iresite.org / IRESite_web.php). Within IRESite, a web-based tool is available to search the database for sequence- and structure-based similarities between a query sequence of interest and the entire set of experimentally validated annotated IRES sequences. The output of the program is a probability score for a nucleotide sequence that can function as an IRES in validation experiments using a bicistronic construct.Additional sequence-based and structure-based web-based browsing tools are available to suggest numerical predictions of the likelihood of IRES activity for any given nucleotide sequence (http: / / rna.informatik.uni-freiburg.de / ; http: / / regrna.mbc.nctu.edu.tw / index1.php).

[0022] The term "mini-SINEUP" intends a nucleic acid molecule comprising (or consisting of) a binding domain (i.e., a sequence complementary to a target mRNA), optionally a spacer sequence, and any SINE or SINE-derived sequence or IRES or IRES-derived sequence as an effector domain (Zucchelli et al., Front Cell Neurosci., 9: 174, 2015).

[0023] The term "microSINEUP" refers to a nucleic acid molecule comprising (or consisting of) a binding domain (i.e., a sequence complementary to a target mRNA), optionally a spacer sequence, and a functionally active fragment of a SINE or SINE-derived sequence or an IRES-derived sequence. For example, the functionally active fragment can be a 77-bp sequence corresponding to nucleotides 44-120 of the 167-bp SINE B2 element within AS Uchl1.

[0024] A polypeptide or polynucleotide sequence is said to be the same as, or "identical to," another polypeptide or polynucleotide sequence if it shares 100% sequence identity over its entire length. Residues in a sequence are numbered from left to right, i.e., from the N-terminus to the C-terminus for polypeptides; from the 5' to the 3' end for polynucleotides.

[0025] For purposes of comparing two closely related polynucleotide sequences, the "% sequence identity" between a first nucleotide sequence and a second nucleotide sequence can be calculated using NCBI BLAST with standard settings for nucleotide sequences (BLASTN). For purposes of comparing two closely related polypeptide sequences, the "% sequence identity" between a first polypeptide sequence and a second polypeptide sequence can be calculated using NCBI BLAST with standard settings for polypeptide sequences (BLASTP). A "difference" between sequences refers to the insertion, deletion, or substitution of a single nucleotide at a position in the second sequence compared to the first sequence. Two sequences can contain one, two, or more such differences. An insertion, deletion, or substitution in a second sequence that is particularly identical (100% sequence identity) to the first sequence reduces the % sequence identity.

[0026] functional nucleic acid molecule The functional nucleic acid molecules of the present invention comprise at least one target binding sequence comprising a sequence reverse complementary to the OPA1 mRNA sequence and at least one regulatory sequence comprising an RNA comprising a SINE B2 element or a functionally active fragment of a SINE B2 element, or an internal ribosome entry site (IRES) sequence or an IRES-derived sequence.

[0027] ( Regulatory sequences ) The regulatory sequence has the ability to enhance protein translation. An improvement in protein translation ability indicates an improvement in ability compared to the absence of the functional nucleic acid molecule of the present invention in the system. In one embodiment, the expression of the protein encoded by the target mRNA is increased by at least 1.5-fold, e.g., at least 2-fold. In a further embodiment, the expression of the protein encoded by the target mRNA is increased by 1.5- to 3-fold, e.g., 1.6- to 2.2-fold. It is anticipated that increasing protein expression within these ranges will enable the treatment of mitochondrial deficiency-related diseases, such as ADOA, without the negative side effects associated with increased OPA1 expression above physiological levels.

[0028] In one embodiment, the regulatory sequence is located 3' to the target binding sequence. The regulatory sequence may be in a forward or reverse orientation relative to the 5'-3' orientation of the functional nucleic acid molecule. Reference to "forward" refers to the situation where the regulatory sequence is embedded (inserted) in the same 5'-3' orientation as the functional nucleic acid molecule. Alternatively, "reverse" refers to the situation where the regulatory sequence is in a 3'-5' orientation relative to the functional nucleic acid molecule.

[0029] Preferably, at least one regulatory sequence comprises a sequence having at least about 80% sequence identity, at least about 85% 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, preferably 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, more preferably 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1-69. In one embodiment, at least one regulatory sequence consists of a sequence having at least about 80% sequence identity, at least about 85% 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, preferably 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, more preferably 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1-69.

[0030] In one embodiment, the regulatory sequence comprises a SINE B2 element or a functionally active fragment of a SINE B2 element. The SINE B2 element is preferably in the reverse orientation relative to the 5'-3' direction of the functional nucleic acid molecule, i.e., an inverted SINE B2 element. As mentioned in the definitions section, inverted SINE B2 elements are disclosed and exemplified in WO 2012 / 133947.

[0031] In one embodiment, the at least one regulatory sequence comprises a sequence having at least about 80% sequence identity, at least about 85% 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, preferably 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, more preferably 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1-51.

[0032] Particularly preferred are SEQ ID NO: 1 (a 167 nucleotide variant of the inverted SINE B2 element in AS Uchl1) and SEQ ID NO: 2 (a 77 nucleotide variant of the inverted SINE B2 element in AS Uchl1 comprising nucleotides 44 to 120), and sequences with percentage identity to these sequences.

[0033] Other inverted SINE B2 elements and functionally active fragments of inverted SINE B2 elements are SEQ ID NOs: 3 to 51. Experimental data demonstrating the protein translation enhancing ability of these sequences are not explicitly presented in the present patent application but have been disclosed in previous patent applications in the name of the same applicant. SEQ ID NOs: 3 to 51 can therefore also be used as regulatory sequences in molecules according to the present invention.

[0034] SEQ ID NOs: 3-6, 8-11, 18, and 43-51 are functionally active fragments of the inverted SINE B2 transposable element from AS Uchl1. The use of functional fragments provides more space for target sequences and / or expression elements, thereby reducing the size of regulatory sequences and being advantageous when used in expression vectors (e.g., viral vectors, which may be size-restricted).

[0035] SEQ ID NO: 7 is the full length 183 nucleotide inverted SINE B2 transposable element from AS Uchl 1. SEQ ID NOs: 12-17, 19, 20, and 39-42 are functionally active mutant fragments of the inverted SINE B2 transposable element from AS Uchl 1.

[0036] SEQ ID NOs: 21 to 25 and 28 to 38 are different SINE B2 transposable elements. SEQ ID NOs: 26 and 27 are sequences in which multiple inverted SINE B2 transposable elements have been inserted.

[0037] Alternatively, the regulatory sequence comprises an IRES sequence or an IRES-derived sequence. Thus, in one embodiment, the regulatory sequence comprises an IRES sequence or an IRES-derived sequence, which sequence enhances translation of the target mRNA sequence.

[0038] Several IRESs with sequences ranging from 48 to 576 nucleotides have been successfully tested, such as the human hepatitis C virus (HCV) IRES (e.g., SEQ ID NOs: 52 and 53), human poliovirus IRES (e.g., SEQ ID NOs: 54 and 55), human encephalomyocarditis (EMCV) virus (e.g., SEQ ID NOs: 56 and 57), human cricket paralysis (CrPV) virus (e.g., SEQ ID NOs: 58 and 59), human Apaf-1 (e.g., SEQ ID NOs: 60 and 61), human ELG-1 (e.g., SEQ ID NOs: 62 and 63), human c-MYC (e.g., SEQ ID NOs: 64-67), and human dystrophin (DMD) (e.g., SEQ ID NOs: 68 and 69).

[0039] Such sequences are disclosed, defined, and exemplified in WO 2019 / 058304. Preferably, such sequences have at least about 80% sequence identity, at least about 85% 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, preferably 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, and more preferably 100% sequence identity to any of SEQ ID NOS: 52-69.

[0040] Target Determining Sequence Human OPA1 is a dynamin-related GTPase protein encoded by the 3q28 gene and localized to the inner mitochondrial membrane (IMM). It is widely expressed, reaching highest levels in the brain, retina, and heart. The gene consists of 30 exons, and the protein itself is translated into eight different isoforms depending on alternative mRNA splicing and processing. In the IMM, cleavage of the mitochondrial targeting sequence (MTS) at the first cleavage site generates a long transmembrane form. Isoforms 4, 6, 7, and 8 also contain a second cleavage site encoded in exon 5b and are further processed to generate shorter forms of the protein. OPA1 plays a key role in mitochondrial homeostasis. Together with the mitofusins ​​MFN1 and MFN2, it promotes mitochondrial fusion, a process associated with improved respiratory efficiency, and contributes to the maintenance of mitochondrial DNA (mtDNA). OPA1 protein polymerization also maintains cristae morphogenesis and promotes the activity of respiratory supercomplexes. Because it is required for the compartmentalization of cytochrome C, it plays a key role in regulating the apoptotic process, and its uncontrolled release leads to cell death. While all eight OPA1 isoforms can support their three essential functions (energy, structure, and mtDNA maintenance), the balance between long and short isoforms appears to be a crucial requirement for fully restoring the mitochondrial network. Therefore, a balance between the long and short forms of at least two isoforms is required to fully rescue the morphology of the mitochondrial network. The data presented herein demonstrate that OPA1-SINEUP is a unique tool for simultaneously targeting all OPA1 transcripts, restoring them to the correct physiological ratio and processing them into the l / s forms in a normal physiological manner. This differs from alternative treatments that favor the expression of a single specific transcript / isoform and potentially disrupt the physiological ratio of OPA1 isoforms and l / s OPA1 protein.

[0041] WO 2012 / 133947 has already shown that a target binding sequence need only have about 60% similarity to the sequence reverse complementary to the target mRNA, and in fact, a target binding sequence can exhibit a significant number of mismatches and retain activity.

[0042] In one embodiment, the target binding sequence comprises a sequence that is reverse complementary to a portion of the OPA1 mRNA sequence that is common to all OPA1 isoforms. By maintaining the relative levels of all OPA1 isoforms, the functional nucleic acid molecule can induce the optimal molecular expression pattern to restore physiological homeostasis. This is not possible with more conventional gene therapy approaches, where only one isoform is ectopically expressed, which can lead to isoform imbalance.

[0043] The target binding sequence comprises a sequence of sufficient length to bind to the OPA1 mRNA transcript. Thus, the target binding sequence can 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, or at least about 18 nucleotides in length. Furthermore, the target binding sequence can 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 about 4 to about 50 nucleotides in length, such as about 18 to about 44 nucleotides in length.

[0044] The target binding sequence can be designed to hybridize to the 5' untranslated region (5'UTR) of the OPA1 mRNA sequence. In one embodiment, the sequence is reverse complementary to 0 to 50 nucleotides of the 5'UTR, e.g., 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, or 0 to 6 nucleotides. Alternatively, or in combination, the target binding sequence can be designed to hybridize to the coding sequence (CDS) of the OPA1 mRNA sequence. In one embodiment, the sequence is reverse complementary to 0 to 40 nucleotides of the CDS, e.g., 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, 0 to 4, or 0 nucleotides.

[0045] The target binding sequence can be designed to hybridize to a region upstream of an AUG site (start codon), such as the start codon, within the CDS of the OPA1 mRNA sequence. In one embodiment, the sequence is reverse complementary to 0 to 80 nucleotides of the AUG site, e.g., 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 nucleotides. Alternatively, or in combination, the target binding sequence can be designed to hybridize to the OPA1 mRNA sequence downstream of the AUG site. In one embodiment, the sequence is reverse complementary to 0 to 40 nucleotides, e.g., 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, 0 to 4, or 0 nucleotides, of the OPA1 mRNA sequence downstream of the AUG site.

[0046] Preferably, the target binding sequence is at least 10 nucleotides in length and is 1) a sequence that is reverse complementary to 0 to 50 nucleotides of the 5'UTR and 0 to 40 nucleotides of the CDS of the OPA1 mRNA sequence; or 2) A sequence that is reverse complementary to 0 to 80 nucleotides of the region upstream of the AUG site (start codon) of OPA1 mRNA and 0 to 40 nucleotides of the OPA1 mRNA sequence downstream of the AUG site. Includes:

[0047] In case 1), the coding sequence begins at the first AUG site (M1) of the mRNA. In case 2), preferred AUG sites are those corresponding to internal start codons, such as methionine 125 (M125) in exon 3. In the context of referring to a sequence reverse-complementary to a region in the 5'UTR and CDS, this is preferably fixed around the AUG site, i.e., the region in the 5'UTR is immediately 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 40 nucleotides in the 5'UTR upstream of the AUG site (-40) and 4 nucleotides in the CDS downstream of the AUG site (+4).

[0048] According to conventional numbering, nucleotides in the 5' UTR sequence are numbered sequentially by decreasing negative numbers (e.g., -3, -2, -1) as they approach the AUG site on the target mRNA. Nucleotides in the CDS sequence are numbered sequentially by increasing positive numbers (e.g., +1, +2, +3) from the AUG site, with the A in the AUG site being numbered +1. Thus, the region bridging the 5' UTR and CDS is numbered -3, -2, -1, +1, +2, +3, with the A in the AUG site being numbered +1.

[0049] More preferably, at least one target binding sequence is at least 14 nucleotides in length and is a sequence reverse complementary to 0 to 40 (preferably 0 to 21, more preferably 0 to 14) nucleotides of the 5'UTR and 0 to 32 (preferably 0 to 4, more preferably 0) nucleotides of the CDS of the OPA1 mRNA sequence; or - a sequence reverse complementary to 0 to 70 (preferably 0 to 40) nucleotides of the region upstream of the AUG site (start codon) of OPA1 mRNA and 0 to 4 (preferably 0) nucleotides of the CDS of the OPA1 mRNA sequence downstream of the AUG site Includes:

[0050] In certain embodiments, the target binding sequence is 18 nucleotides in length and comprises, from 3' to 5', a sequence that is reverse complementary to 14 nucleotides of the 5'UTR and 4 nucleotides of the CDS of the OPA1 mRNA sequence. For example, the target binding sequence can comprise the sequence encoded by the DNA sequence of SEQ ID NO: 82 (i.e., -14 / +4 of M1).

[0051] In a further specific embodiment, the target binding sequence is 15 nucleotides in length and comprises, from 3' to 5', a sequence that is reverse complementary to the 6 nucleotides of the 5'UTR and the 9 nucleotides of the CDS of the OPA1 mRNA sequence. For example, the target binding sequence can comprise the sequence encoded by the DNA sequence of SEQ ID NO: 83 (i.e., -6 / +9 of M1).

[0052] In another specific embodiment, the target binding sequence is 12 nucleotides in length and comprises, from 3' to 5', a sequence that is reverse complementary to 41 nucleotides of the 5'UTR and 30 nucleotides of the CDS of the OPA1 mRNA sequence. For example, the target binding sequence can comprise the sequence encoded by the DNA sequence of SEQ ID NO: 84 (i.e., -41 / +30 of M1).

[0053] In another specific embodiment, the target binding sequence is 12 nucleotides in length and comprises, from 3' to 5', a sequence that is reverse complementary to 41 nucleotides of the 5' UTR and 30 nucleotides of the CDS of the OPA1 mRNA sequence. For example, the target binding sequence can comprise the sequence encoded by the DNA sequence of SEQ ID NO:93 (i.e., -41 / -30 of M1).

[0054] In a further embodiment, the target binding sequence is 14 nucleotides in length and comprises, 3' to 5', a sequence that is reverse complementary to the region between nucleotides 97 and 84 of the 5' UTR of the OPA1 mRNA sequence. For example, the target binding sequence can comprise the sequence encoded by the DNA sequence of SEQ ID NO: 85 (i.e., -97 / -87 of M1).

[0055] In a further embodiment, the target binding sequence is 18 nucleotides in length and comprises, from 3' to 5', the reverse complement of 18 nucleotides of the 5'UTR and 0 nucleotides of the CDS of the OPA1 mRNA sequence. For example, the target binding sequence can comprise the sequence encoded by the DNA sequence of SEQ ID NO: 86 (i.e., -18 / -1 of M1).

[0056] In another embodiment, the target binding sequence is 22 nucleotides in length and comprises, from 3' to 5', the reverse complement of 18 nucleotides of the 5'UTR and 4 nucleotides of the CDS of the OPA1 mRNA sequence. For example, the target binding sequence can comprise the sequence encoded by the DNA sequence of SEQ ID NO: 87 (i.e., -18 / +4 of M1).

[0057] In a further embodiment, the target binding sequence is 14 nucleotides in length and comprises, from 3' to 5', the reverse complement of 14 nucleotides of the 5'UTR and 0 nucleotides of the CDS of the OPA1 mRNA sequence. For example, the target binding sequence can comprise the sequence encoded by the DNA sequence of SEQ ID NO: 88 (i.e., -14 / -1 of M1).

[0058] In one specific embodiment, the target binding sequence is 17 nucleotides in length and includes, from 3' to 5', a sequence that is reverse complementary to 9 nucleotides of the region upstream of the AUG site (start codon) of OPA1 mRNA and 8 nucleotides of the CDS of the OPA1 mRNA sequence downstream of the AUG site. For example, the target binding sequence can include the sequence encoded by the DNA sequence of SEQ ID NO:89 (i.e., -9 / +8 of M125).

[0059] In another embodiment, the target binding sequence is 18 nucleotides in length and includes, from 3' to 5', a sequence that is reverse complementary to 18 nucleotides of the region upstream of the AUG site (start codon) of OPA1 mRNA and 0 nucleotides of the CDS of the OPA1 mRNA sequence downstream of the AUG site. For example, the target binding sequence can include the sequence encoded by the DNA sequence of SEQ ID NO:90 (i.e., -18 / -1 of M125).

[0060] In a further embodiment, the target binding sequence is 22 nucleotides in length and includes, from 3' to 5', a sequence that is reverse complementary to 18 nucleotides of a region upstream of the AUG site (start codon) of OPA1 mRNA and 4 nucleotides of the CDS of the OPA1 mRNA sequence downstream of the AUG site. For example, the target binding sequence can include the sequence encoded by the DNA sequence of SEQ ID NO:91 (i.e., -18 / +4 of M125).

[0061] In a further embodiment, the target binding sequence is 14 nucleotides in length and includes, from 3' to 5', a sequence that is reverse complementary to 14 nucleotides of the region upstream of the AUG site (start codon) of OPA1 mRNA and 0 nucleotides of the CDS of the OPA1 mRNA sequence downstream of the AUG site. For example, the target binding sequence can include the sequence encoded by the DNA sequence of SEQ ID NO:92 (i.e., -14 / -1 of M125).

[0062] In a further embodiment, the target binding sequence is 44 nucleotides in length and includes, from 3' to 5', a sequence that is reverse complementary to 40 nucleotides of the region upstream of the AUG site (start codon) of OPA1 mRNA and 4 nucleotides of the CDS of the OPA1 mRNA sequence downstream of the AUG site. For example, the target binding sequence can include the sequence encoded by the DNA sequence of SEQ ID NO:94 (-40 / +4 of M1).

[0063] In a further embodiment, the target binding sequence is 44 nucleotides in length and includes, from 3' to 5', a sequence that is reverse complementary to 40 nucleotides of the region upstream of the AUG site (start codon) of OPA1 mRNA and 4 nucleotides of the CDS of the OPA1 mRNA sequence downstream of the AUG site. For example, the target binding sequence can include the sequence encoded by the DNA sequence of SEQ ID NO:95 (-40 / +4 of M2).

[0064] Thus, in some embodiments, the target binding sequence comprises a sequence encoded by a DNA sequence having at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, preferably 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, more preferably 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, and even more preferably 100% sequence identity to any of SEQ ID NOs: 82-95, preferably SEQ ID NOs: 82-84 or 89. In a further embodiment, the target binding sequence consists of a sequence encoded by a DNA sequence having at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, preferably at least 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, more preferably 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, and even more preferably 100% sequence identity to any of SEQ ID NOs: 82-95, preferably SEQ ID NOs: 82-84 or 89.

[0065] In one embodiment, the functional nucleic acid molecule comprises a sequence having at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, preferably 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, more preferably 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, and even more preferably 100% sequence identity to any of SEQ ID NOs: 70-79, preferably SEQ ID NOs: 70-74, 78-79. In a further embodiment, the functional nucleic acid molecule consists of a sequence having at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, preferably 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, more preferably 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, and even more preferably 100% sequence identity to any of SEQ ID NOs: 70-79, preferably SEQ ID NOs: 70-74, 78-79.

[0066] More specifically, SEQ ID NOS: 70-74 and 78-79 relate to functional nucleic acid molecules for human OPA1 isoforms, and SEQ ID NOS: 75-77 relate to functional nucleic acid molecules for mouse OPA1 isoforms. Furthermore, SEQ ID NOS: 70-77 comprise a "mini" inverted SINE B2 element (167 nucleotides) within AS Uchl1, SEQ ID NOS: 78 comprise a "micro" inverted SINE B2 element (i.e., nucleotides 44-120 of the inverted SINE B2 transposable element from AS Uchl1) within AS Uchl1, and SEQ ID NOS: 79 comprise a "nano" inverted SINE B2 element (i.e., nucleotides 64-92 of the inverted SINE B2 transposable element from AS Uchl1) within AS Uchl1. Differences between mini-SINEUP sequences are due to targeting binding sequences and / or spacer / linker sequences, as described herein.

[0067] As will be appreciated from the disclosure herein, and for example, from the sequence identity of the human OPA1 isoform to the mouse and rhesus macaque (Macaca mulatta) OPA1 isoforms, the target binding sequences presented herein may have cross-reactivity with other species. For example, human OPA1 mRNA shares 96.19% sequence identity over its entire length with rhesus macaque OPA1 mRNA and 78.36% sequence identity with mouse OPA1 mRNA. Thus, in one embodiment, a target binding sequence comprising or consisting of any of the sequences encoded by the DNA sequences of SEQ ID NOs: 82-95 binds to mouse OPA1 and / or rhesus macaque OPA1, preferably rhesus macaque OPA1.

[0068] In one embodiment, the functional nucleic acid molecules provided herein are chemically modified. The term "modified" or "chemically modified" refers to a structural change in or on the most common naturally occurring ribonucleotides: adenosine, guanosine, cytidine, or uridine ribonucleotides. The chemical modification can be a change in or on the nucleobase (i.e., chemical base modification) or a change in or on the sugar (i.e., chemical sugar modification). The chemical modification can be introduced co-transcriptionally (e.g., by substituting one or more nucleotides with modified nucleotides during synthesis) or post-transcriptionally (e.g., by the action of an enzyme).

[0069] 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 / ). Many modifications occur naturally, such as chemical modifications to natural transfer RNA (tRNA), including 2'-O-methyl (such as 2'-O-methyladenosine, 2'-O-methylguanosine, and 2'-O-methylpseudouridine), 1-methyladenosine, 2-methyladenosine, 1-methylguanosine, 7-methylguanosine, 2-thiocytidine, 5-methylcytidine, 5-formylcytidine, pseudouridine, dihydrouridine, and the like.

[0070] Structural features A functional nucleic acid molecule may contain two or more regulatory sequences, where the same sequence may be repeated two or more times, or different regulatory sequences (i.e., different SINE B2 elements / functionally active fragments of SINE B2 elements / IRES sequences / IRES-derived sequences).

[0071] The at least one target binding sequence and the at least one regulatory sequence are preferably connected by at least one spacer / linker sequence. SEQ ID NO: 80 or 81 are non-limiting examples of spacer / linker sequences that may be used. Fragments of these sequences are also contemplated.

[0072] The functional nucleic acid molecules of the present invention are preferably circular molecules, as this conformation is more difficult to degrade within the cell (exonucleases cannot degrade circular molecules) and therefore results in a much more stable molecule that remains active for a longer period of time.

[0073] In addition, functional nucleic acid molecules may optionally contain a non-coding 3' tail sequence, which contains, for example, restriction sites useful for cloning the molecule into an appropriate plasmid.

[0074] In one embodiment, a 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 a transcript, which confers stability to an RNA molecule and can facilitate translation.

[0075] In one embodiment, a functional nucleic acid molecule comprises a 5'-cap. A "5'-cap" refers to a modified nucleotide at the 5' end of a transcript that provides stability to the molecule, particularly against exonucleolytic degradation, and can facilitate translation.

[0076] It should be noted that functional nucleic acid molecules can enhance the translation of target genes of interest without affecting the amount of target gene mRNA. Therefore, they can be successfully used as molecular tools to verify gene function in cells and to implement pipelines for recombinant protein production.

[0077] DNA molecules and vectors According to a further aspect of the present invention, there is provided a DNA molecule encoding any of the functional nucleic acid molecules disclosed herein. According to a further aspect of the present invention, there is provided an expression vector comprising said DNA molecule.

[0078] Exemplary expression vectors are known in the art and may include, for example, plasmid vectors, viral vectors (e.g., adenoviral, adeno-associated viral, retroviral, or lentiviral vectors), phage vectors, cosmid vectors, etc. The choice of expression vector may depend on the type of host cell used and the intended use. In particular, the following plasmids have been used for efficient expression of functional nucleic acid molecules:

[0079] Mammalian expression plasmids: Plasmid name: pCDNA3.1(-) Expression: CMV promoter BGH Poly(A) Terminator Plasmid name: pDUAL-eGFPΔ (modified from peGFP-C2) Expression: H1 promoter BGH Poly(A) Terminator Viral vectors: Vector name: pAAV Virus: Adeno-associated virus Expression: CAG promoter / CMV enhancer SV40 late poly(A) terminator Vector name: rcLV-TetOne-Puro Virus: Lentivirus (3rd generation) Expression: LTR-TREt (Tre-Tight) promoter (doxycycline-inducible expression) BGH Poly(A) Terminator Vector name: pLPCX-link Viruses: Retroviruses (3rd generation) Expression: CMV It should be noted that any promoter may be used in the vector and function similarly to those described above.

[0080] Compositions and Medical Uses The present invention also relates to compositions comprising the functional nucleic acid molecules, DNA molecules and expression vectors described herein, which may contain components that enable delivery of said functional nucleic acid molecules by viral vectors (e.g., AAV, lentivirus) and non-viral vectors (e.g., nanoparticles, lipid particles).

[0081] The functional nucleic acid molecules of the present invention can also be administered as naked, i.e., unpackaged, RNA. Alternatively, the functional nucleic acid molecules can be administered as part of a composition, such as a composition containing a suitable carrier. In certain embodiments, the carrier is selected based on its ability to facilitate transfection of target cells with one or more functional nucleic acid molecules.

[0082] According to a further aspect of the present invention there is provided a functional nucleic acid molecule, DNA molecule, expression vector or composition as defined herein for use as a medicament.

[0083] It will be appreciated that the functional nucleic acid molecules of the present invention are used to increase the level of OPA1 protein in cells. OPA1 has a major function in mitochondrial homeostasis, and therefore, according to a further aspect of the present invention, there are provided functional nucleic acid molecules, DNA molecules, expression vectors, or compositions for use in treating diseases associated with mitochondrial defects.

[0084] The functional nucleic acid molecules, DNA molecules, and / or compositions described above are preferably used as pharmaceuticals for treating autosomal dominant optic atrophy (ADOA), particularly for promoting recovery from mitochondrial deficiency-related diseases. Retinal ganglion cells (RGCs) expressing mutant 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).

[0085] Therefore, in another embodiment, the mitochondrial deficiency-related disease is ADOA.ADOA is the most common hereditary optic neuropathy, and 75% of cases are caused by heterozygous mutations in OPA1 gene.The main symptom of this disease is bilateral degeneration of retinal ganglion cells (RGC) and optic nerve atrophy, which may be associated with muscle and neurodegenerative symptoms.Various forms of ADOA have been reported, such as ADOA plus, which also shows muscle defects and neurosensory hearing loss, and ADOAC, which also leads to cataracts.In addition, intra- and inter-family variations in the severity of disease have been reported among patients with the same mutation.

[0086] According to a further aspect of the present invention there is provided the use of a functional nucleic acid molecule (or DNA molecule, expression vector or composition) as defined herein for the manufacture of a medicament for treating a mitochondrial defect-associated disease such as ADOA.

[0087] According to another aspect of the present invention there is provided a therapeutically effective amount of a functional nucleic acid molecule, DNA molecule, expression vector, or composition as defined herein for use in the manufacture of a medicament for the treatment of a mitochondrial defect associated disease, such as ADOA.

[0088] In general, OPA1 is one of the major factors controlling mitochondrial fusion, mitochondrial DNA (mtDNA) maintenance, bioenergetics, and cristae integrity. These cellular processes are targets of several diseases that can potentially be rescued by increasing endogenous OPA1 expression. In addition, OPA1 also regulates apoptosis through cristae remodeling and cytochrome c release, independent of mitochondrial fusion (Frezza et al. (2006) Cell 126(1):177-89).

[0089] In addition to ADOA, several reports have proposed that mild increases in 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 improved the phenotype of two mitochondrial disease mouse models with defects in the Ndufs4 or Cox15 genes. In humans, mutations in NDUFS4 are associated with early-onset, fatal Leigh syndrome due to severe complex I (CI) deficiency, while mutations in COX15 have been reported in children with severe isolated cardiomyopathy, encephalopathy, or cardiencephalomyopathies. As another example, Varanita et al. (2015) Cell Metabolism showed that Opa1 tg Mice (models overexpressing OPA1 approximately 1.5-fold—see also Cogliati et al. (2013) Cell 155(1): 160-171) are protected from muscle atrophy, myocardial infarction, are less susceptible to Fas-induced liver injury, and mitochondria are resistant to cristae remodeling and cytochrome C release. In contrast, massive overexpression of OPA1 has been shown to be toxic (Cipolat et al. (2004) PNAS 101(45): 15927-15932). Therefore, the methods provided herein are particularly suitable for treating diseases associated with OPA1 deficiency, as it is only important to increase expression to normal physiological levels. This is expected to avoid undesirable side effects that may be associated with significantly increased OPA1 expression above physiological levels.

[0090] Mitochondrial defect-associated diseases are well known in the art and are described, for example, in Gorman et al. (2016) Nat. Rev. Disease Primers, 2, 16080. They can be characterized by defects in oxidative phosphorylation due to mutations in nuclear DNA or mitochondrial DNA that result in mutated / dysfunctional mitochondrial proteins.

[0091] Mitochondrial defects have been associated with neurological disorders and onset. Thus, in one embodiment, the mitochondrial deficiency-associated disease is a neurological disorder. Caglayan et al. (2020) iScience 23: 101154 described that genetically modified human embryonic stem cells and patient-derived induced pluripotent stem cells with OPA1 haploinsufficiency resulted in abnormal nuclear DNA methylation, significantly altering the transcriptional circuitry of neural progenitor cells (NPCs). In particular, OPA1+ / - NPCs failed to develop into GABAergic interneurons. Alterations to normal OPA1 expression have also been associated with Alzheimer's disease, Huntington's disease, and Parkinson's disease (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; Iannielli et al. (2018) Cell Rep. 22(8): 2066-2079; and Iannielli et al. (2019) Cell Rep. 29(13): 4646-4656). In one embodiment, the neurological disorder is selected from Alzheimer's disease, Huntington's disease, and Parkinson's disease.

[0092] In one embodiment, the mitochondrial defect-associated disease is prion disease. For example, Wu et al. (2019) Cell Death Dis. 10(10): 710 describes that downregulation of OPA1 was observed in vitro and in vivo in a prion disease model, and that this was accompanied by damage and dysfunction of mitochondrial structure, loss of mtDNA, and apoptosis of neuronal cells. These symptoms were alleviated by increasing OPA1 expression.

[0093] (method) According to a further aspect of the present invention, there is provided a method for enhancing protein translation of OPA1 mRNA in a cell, comprising administering to said cell a functional nucleic acid molecule, DNA molecule, expression vector or composition as defined herein. Preferably, the cell is a mammalian cell, such as a human or mouse cell.

[0094] According to a further aspect of the present invention, there is provided a method for increasing the efficiency of protein synthesis of an OPA1 protein in a cell, the method comprising administering to the cell a functional nucleic acid molecule, DNA molecule, expression vector, or composition described herein.

[0095] The methods described herein may include transfecting a cell with a functional nucleic acid molecule, DNA molecule, expression vector or composition defined herein. The functional nucleic acid molecule, DNA molecule, expression vector or composition may be administered to the target cell using methods known in the art, including, for example, microinjection, lipofection, electroporation, the use of calcium phosphate, autoinfection with a vector or viral transduction.

[0096] In one embodiment, the cell is OPA1 haploinsufficient, i.e., the presence of mutant alleles in a heterozygous combination results in the amount of product produced by a single wild-type gene being insufficient for full or normal function. Generally, haploinsufficiency is a condition that occurs when a normal phenotype requires the protein products of both alleles, and a reduction in gene function below 50% results in an abnormal phenotype.

[0097] The methods of the present invention increase intracellular OPA1 protein levels and are therefore useful, for example, in treating diseases associated with OPA1 deficiency (i.e., reduced OPA1 protein levels and / or loss-of-function mutations in the OPA1 gene). The methods of the present invention are particularly useful for diseases caused by a quantitative decrease in a given normal protein level. The methods of the present invention can be performed in vitro, ex vivo, or in vivo.

[0098] According to a further aspect of the present invention there is provided a method for treating a mitochondrial defect associated disease such as ADOA, comprising administering a therapeutically effective amount of a functional nucleic acid molecule, DNA molecule, expression vector or composition as defined herein.

[0099] Gene therapy for diseases such as ADOA is challenging because the bilayer IMM is a relatively impermeable barrier. While several approaches are currently being investigated to engineer the expression of proteins containing specific mitochondrial targeting sequences, the OPA1 functional nucleic acid molecules described herein alleviate the need for such engineering and deliver their effects in the cytoplasm, enhancing the translation of endogenous targets.

[0100] In one embodiment, the therapeutically effective amount is administered to the retina, brain, or heart, particularly the retina.

[0101] It will be understood that the embodiments described herein may be applied to all aspects of the invention, i.e., embodiments described for functional nucleic acid molecules may be equally applied to the claimed methods, etc.

[0102] The invention will now be described with reference to the following non-limiting examples. [Example]

[0103] (Example) Example 1 A synthetic mini-SINEUP was designed to target human OPA1 mRNA. Figure 1A shows a schematic diagram of the SINEUP functional domains. The overlapping domain is the binding domain (BD, gray), which provides SINEUP specificity and is in the antisense orientation relative to the mRNA encoding the sense protein (target mRNA). The inverted SINE B2 (invB2) element of AS Uchl1 is the effector domain (ED), which confers enhanced protein synthesis. The 5' to 3' orientation of the sense and antisense RNA molecules is shown. The structural elements of the target mRNA are shown: the 5' untranslated region (5' UTR, white), the coding sequence (CDS, black), and the 3' untranslated region (3' UTR, white). The diagram is not drawn to scale. (B) Schematic diagram of the human OPA1 gene (5'-UTR, white) and BD (gray) design of the synthetic mini-SINEUP-OPA1, which targets the initiating M1-AUG and the second in-frame M125-AUG. Numbering indicates the position relative to methionine (i.e., -40 / +4, from 40 nucleotides upstream to 4 nucleotides downstream of M1-AUG). All BDs were designed within a region contained in all human OPA1 transcripts. The diagram is not drawn to scale. (C) Schematic diagram of the mouse OPA1 gene (5'-UTR, white) and BD (gray) design of the synthetic mini-SINEUP-OPA1 targeting the initiating M1-AUG and M125-AUG. Numbering indicates the position relative to methionine (i.e., -40 / +4, from 40 nucleotides upstream to 4 nucleotides downstream of M1-AUG). All BDs were designed within a region contained in all mouse OPA1 transcripts. The diagram is not drawn to scale.

[0104] Example 2 This example demonstrates that synthetic mini-SINEUP increases endogenous OPA1 protein levels in human cells in vitro. HEK 293T cells were obtained from ATCC (Cat. No. CRL-11268), transfected with mini-SINEUP lacking BD (ΔBD) and mini-SINEUP-OPA1 mutants encoded by the pCS2+link plasmid, and harvested 48 hours posttransfection. ΔBD served as a negative control.

[0105] Whole cell lysates were analyzed by Western blotting using anti-OPA1 and anti-β-actin antibodies. First, the intensity of the OPA1 bands (L and S forms) was normalized to the relative β-actin band. Then, fold-change values ​​were calculated by normalizing to the control cells (ΔBD). The results are shown in Figure 2A. Mini-SINEUP-OPA1 transfected cells show increased levels of endogenous OPA1 protein. The variation in the expression of both target and mini-SINEUP mRNA between samples was not statistically significant (one-way ANOVA followed by Dunnett's post-hoc test). OPA1 transcripts were quantified using human GAPDH (hGAPDH) expression as an internal control. The OPA1 / hGAPDH ratio of the ΔBD sample was set as the baseline value to which all transcript levels were normalized. This indicates that OPA1 mRNA levels were unchanged, thereby confirming the increase in OPA1 protein synthesis at the post-transcriptional level. Mini-SINEUP transcripts were quantified using hGAPDH expression as an internal control. The ΔBD / hGAPDH ratio of the sample was set as the baseline value to which all transcript levels were normalized.

[0106] Figure 2B shows the average fold change in OPA1 protein levels. All mini-SINEUPs have been shown to increase endogenous OPA1 protein levels.

[0107] Example 3 This example demonstrates that synthetic mini-SINEUP increases endogenous OPA1 protein levels in the mouse Neuro2A cell line in vitro. Neuro2A (N2A) cells were obtained from ATCC (Cat. No. CCL-131), transfected with mini-SINEUP lacking BD (ΔBD) and mini-SINEUP-OPA1 mutants encoded by the pCS2+link plasmid, and harvested 48 hours posttransfection. ΔBD served as a negative control.

[0108] As previously described, whole cell lysates were analyzed by Western blotting using anti-OPA1 antibody (BD Bioscience, catalog no. 612606) and anti-β-actin antibody (Sigma, catalog no. A2066). The intensity of the OPA1 bands (L and S forms) was normalized to the relative β-actin band, and fold-change values ​​were then calculated normalized to the control cells (ΔBD). The results are shown in Figure 3A. Mini-SINEUP-OPA1 transfected cells show increased levels of endogenous OPA1 protein. The variation in the expression of both target and mini-SINEUP mRNA between samples was not statistically significant (one-way ANOVA followed by Dunnett's post-hoc test). OPA1 transcripts were quantified using mouse GAPDH (mGAPDH) expression as an internal control. The OPA1 / mGAPDH ratio of the ΔBD sample was set as the baseline value to which all transcript levels were normalized. This shows that OPA1 mRNA levels were unchanged, thereby confirming increased OPA1 protein synthesis at the post-transcriptional level. Mini-SINEUP transcripts were quantified using mGAPDH expression as an internal control. The ΔBD / mGAPDH ratio of the samples was set as the baseline value to which all transcript levels were normalized.

[0109] Figure 3B shows the average fold change in OPA1 protein levels. All mini-SINEUPs have been shown to increase endogenous OPA1 protein levels.

[0110] Example 4 This example demonstrates that synthetic mini-SINEUP increases endogenous OPA1 protein levels in a mouse astrocyte cell line in vitro. Astrocyte cells were obtained from ATCC (CRL-254), transfected with mini-SINEUP lacking the BD (ΔBD) and the mini-SINEUP-OPA1 mutant encoded by the pCS2+link plasmid, and harvested 48 hours posttransfection. ΔBD served as a negative control.

[0111] As previously described, whole cell lysates were analyzed by Western blotting using anti-OPA1 and anti-β-actin antibodies. The intensities of the OPA1 bands (L and S forms) were normalized to the relative β-actin band, and fold-change values ​​were calculated by normalizing to the control cells (ΔBD). The results are shown in Figure 4A. Mini-SINEUP-OPA1 transfected cells show increased levels of endogenous OPA1 protein. Variation between samples in both target and mini-SINEUP mRNA expression was not statistically significant (one-way ANOVA followed by Dunnett's post-hoc test). OPA1 transcripts were quantified using mGAPDH expression as an internal control. The OPA1 / mGAPDH ratio of the ΔBD sample was set as the baseline value to which all transcript levels were normalized. This indicates that OPA1 mRNA levels were unchanged, thereby confirming increased OPA1 protein synthesis at the post-transcriptional level. Mini-SINEUP transcripts were quantified using mGAPDH expression as an internal control. The ΔBD / mGAPDH ratio of the sample was set as the baseline value to which all transcript levels were normalized.

[0112] Figure 4B shows the average fold change in OPA1 protein levels. All mini-SINEUPs were shown to increase endogenous OPA1 protein levels.

[0113] Example 5 This example describes the optimization of the effector domain (i.e., regulatory sequence). microSINEUP increases endogenous OPA1 protein levels in HEK 293T cells in vitro. HEK 293T cells were transfected with a control vector (ΔBD), miniSINEUP-OPA1 (14 / +4-M1-AUG), and the microSINEUP-OPA1 (14 / +4-M1-AUG) mutant. Cells were harvested 48 hours after transfection. The control vector (ΔBD) and miniSINEUP-OPA1 (14 / +4-M1-AUG) served as negative and positive controls, respectively. microSINEUP-OPA1 exhibits a truncated ED composed of nucleotides 44 to 120 of the invSINEB2 element from AS Uchl1.

[0114] Whole cell lysates were analyzed by Western blotting using anti-OPA1 and anti-β-actin antibodies. As previously described, OPA1 band intensity was normalized to relative β-actin, and fold-change values ​​were calculated normalized to negative control cells (ΔBD). The results are shown in Figure 5. Micro-SINEUP-OPA1 transfected cells show increased levels of endogenous OPA1 protein compared to negative control cells.

[0115] Example 6 This example demonstrates that synthetic nano2SINEUP increases endogenous OPA1 protein levels in human cells in vitro. HEK 293T cells were transfected with mini-SINEUP (ΔBD) lacking the binding domain and nano2SINEUP-OPA1 (-14 / +4-M1-AUG) and harvested 48 hours posttransfection. nano2SINEUP-OPA1 expresses a truncated ED consisting of nucleotides 64-92 of the invSINEB2 element from AS Uchl1.

[0116] Whole cell lysates were analyzed by Western blotting using anti-OPA1 and anti-β-actin antibodies. A clear increase in OPA1 protein levels was demonstrated after transfection with nano2SINEUP, but not with the control SINEUP lacking the BD (Figure 6A). Real-time PCR analysis of OPA1 mRNA and nano2SINEUP RNA expression in transfected cells showed that endogenous OPA1 mRNA was not significantly increased compared to non-transfected (NT) control cells, confirming that the increase in OPA1 protein levels occurs post-transcriptionally (Figure 6B, left panel). Real-time PCR analysis also clearly demonstrated the expression and presence of OPA1-nano2SINEUP in HEK 293T cells (Figure 6B, right panel).

[0117] Example 7 This example demonstrates that mini-SINEUP in two different vector backbones increases luminescence of overexpressed OPA1-nanoluc in vitro. Mouse Neuro2A cells were co-transfected with an expression plasmid containing nanoluciferase-tagged human OPA1 and either mini-SINEUP(ΔBD) lacking the binding domain or mini-SINEUP-OPA1 (with the -14 / +4 binding domain encompassing methionine 1). Two different plasmid backbones, pCS2+ and pDUAL, were used. After 48 hours, cells were subjected to luciferase assays to quantify the amount of luminescence present in each treatment condition. For both plasmid vector backbones, those containing mini-SINEUP-OPA1 showed approximately a 2.5- to 3-fold increase in luminescence over the negative control lacking the binding domain for the target mRNA (Figure 7). pCS2+mini-SINEUP-OPA1 showed a 3.2-fold increase over the control, and pDUAL showed a 2.6-fold increase over the control (n = 4 independent biological experiments, each with three technical replicates).

[0118] Example 8 This example demonstrates that synthetic nano2SINEUP increases endogenous OPA1 protein levels in human cells in vitro when transfected with a plasmid vector (pCS2+) and transfected as naked RNA carrying modified ribonucleotides. The naked RNA molecules were modified with 2'-O-methyladenosine (2'-O-MeA).

[0119] HEK 293T cells were transfected with mini-SINEUP (ΔBD), nano2SINEUP-OPA1(-14 / +4-M1-AUG), nano2SINEUP lacking the binding domain and 2'-O-MeA modified RNA, and nano2SINEUP-OPA1(-14 / +4-M1-AUG). Cells were harvested 48 hours post-transfection. nano2SINEUP-OPA1 displays a truncated ED consisting of nucleotides 64-92 of the invSINEB2 element from AS Uchl1.

[0120] Whole cell lysates were analyzed by Western blotting using anti-OPA1 and anti-β-actin antibodies (Figure 8A). A clear increase in OPA1 protein levels was demonstrated after transfection with nano2SINEUP (both in plasmid and naked RNA form), but not with the control SINEUP lacking the BD (Figures 8A and 8B). Real-time PCR analysis of OPA1 mRNA and nano2SINEUP RNA expression in transfected cells showed that endogenous OPA1 mRNA was not significantly increased compared to control cells, confirming that the increase in OPA1 protein levels occurs posttranscriptionally (Figure 8C). Real-time PCR analysis also clearly demonstrated the expression and presence of OPA1-nano2SINEUP in HEK 293T cells (Figure 8D, right panel).

Claims

1. A functional nucleic acid molecule capable of enhancing translation of OPA1 mRNA, comprising: - at least one target binding sequence comprising a sequence reverse complementary to the OPA1 mRNA sequence; and - at least one regulatory sequence comprising a SINE B2 element or a functionally active fragment of a SINE B2 element, A functional nucleic acid molecule, wherein the at least one target binding sequence comprises a sequence that is reverse complementary to a portion of the OPA1 mRNA sequence that is common to all OPA1 isoforms.

2. 2. The functional nucleic acid molecule of claim 1, wherein the at least one regulatory sequence comprises a sequence having at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 1 to 51.

3. the at least one target binding sequence is at least 10 nucleotides in length and is reverse complementary 3' to 5' to 0-50 nucleotides of the 5' untranslated region (5'UTR) and 0-40 nucleotides of the coding sequence (CDS) of the OPA1 mRNA sequence; or The functional nucleic acid molecule of claim 1 or 2, comprising a sequence that is reverse complementary to 0 to 80 nucleotides of a region upstream of the AUG site (start codon) of OPA1 mRNA and 0 to 40 nucleotides of the CDS of the OPA1 mRNA sequence downstream of the AUG site.

4. the at least one target binding sequence is at least 14 nucleotides in length and is reverse complementary 3' to 5' to 0-40 nucleotides of the 5'UTR and 0-32 nucleotides of the CDS of the OPA1 mRNA sequence; or The functional nucleic acid molecule of claim 3, comprising a sequence that is reverse complementary to 0 to 70 nucleotides of a region upstream of the AUG site (start codon) of OPA1 mRNA and 0 to 4 nucleotides of the CDS of the OPA1 mRNA sequence downstream of the AUG site.

5. The functional nucleic acid molecule of any one of claims 1 to 4, further comprising at least one linker sequence between said at least one target binding sequence and said at least one regulatory sequence.

6. The functional nucleic acid molecule of any one of claims 1 to 5, wherein the molecule is circular.

7. A DNA molecule encoding the functional nucleic acid molecule of any one of claims 1 to 6.

8. An expression vector comprising the functional nucleic acid molecule of any one of claims 1 to 6 or the DNA molecule of claim 7.

9. A composition comprising a functional nucleic acid molecule according to any one of claims 1 to 6, a DNA molecule according to claim 7, or an expression vector according to claim 8.

10. A functional nucleic acid molecule according to any one of claims 1 to 6, a DNA molecule according to claim 7, an expression vector according to claim 8, or a composition according to claim 9, for use as a pharmaceutical.

11. A functional nucleic acid molecule according to any one of claims 1 to 6, a DNA molecule according to claim 7, an expression vector according to claim 8, or a composition according to claim 9, for use in treating a disease associated with a mitochondrial defect.

12. A therapeutically effective amount of the functional nucleic acid molecule of any one of claims 1 to 6, the DNA molecule of claim 7, the expression vector of claim 8, or the composition of claim 9, for use in the manufacture of a medicament for treating a disease associated with mitochondrial defects.

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