CN116568311A - Functional nucleic acid molecules - Google Patents
Functional nucleic acid molecules Download PDFInfo
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- CN116568311A CN116568311A CN202180068542.0A CN202180068542A CN116568311A CN 116568311 A CN116568311 A CN 116568311A CN 202180068542 A CN202180068542 A CN 202180068542A CN 116568311 A CN116568311 A CN 116568311A
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Abstract
The present invention relates to functional nucleic acid molecules for up-regulating OPA1 expression. The functional nucleic acid molecule typically comprises at least one target binding sequence that is reverse complementary to the OPA1mRNA sequence and at least one regulatory sequence comprising a SINE B2 element or an Internal Ribosome Entry Site (IRES) sequence. Methods of treatment using the functional nucleic acids are also described.
Description
Technical Field
The present invention relates to functional nucleic acid molecules for up-regulating OPA1 expression.
Background
Autosomal Dominant Optic Atrophy (ADOA) is the most common hereditary optic neuropathy, with 75% of cases being caused by heterozygous mutations in the OPA1 gene. ADOA is an early-onset autosomal dominant single dose deficient condition with a prevalence of 1:12000 to 1:50000 is a birth population and is characterized by degeneration of retinal ganglion cells, which leads to atrophy and blindness of the optic nerve. Human OPA1 is a ubiquitously expressed dynamin-related gtpase protein that has a critical function in mitochondrial homeostasis, localizes to the Inner Mitochondrial Membrane (IMM), and is expressed at its highest levels in the brain, retina and heart.
The data indicate that both underexpression and overexpression of OPA1 have deleterious consequences, as observed in ADOA patients, and that changes in both OPA1 levels lead to increased apoptosis (Chen et al (2009) cardioasc res.84 (1): 91-9). In vivo, the data is quite complex. Although transgenic mice with mild OPA1 overexpression appear healthy and fertile, and additionally exhibit protective effects against specific tissue, such as liver and brain, prolonged overexpression in SV129 mouse strains was observed to increase the incidence of spontaneous cancer and reduce longevity (Varanita et al (2015) Cell metab.21 (6): 834-44). High expression of OPA1 and other fusion-promoting mitochondrial proteins is 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).
A new class of long non-coding RNAs (lncRNA), termed SINEUPs, is previously described that can selectively enhance translation of their targets. SINEUP activity depends on a combination of two domains: an overlapping domain or Binding Domain (BD), which 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 comprising SINEUP. Another class of lncRNA is described in WO 2019/058304, which uses an effector domain comprising an Internal Ribosome Entry Site (IRES) sequence to provide a trans-acting functional nucleic acid molecule.
It is an object of the present invention to provide a first gene-specific technique targeting OPA1 translation, in particular for the treatment of ADOA.
Disclosure of Invention
According to a first aspect, there is provided a functional nucleic acid molecule comprising:
-at least one target binding sequence comprising a sequence that is reverse complementary to an OPA1 mRNA sequence; and
-at least one regulatory sequence comprising an RNA comprising a sineb 2 element or a functionally active fragment of a sineb 2 element or an Internal Ribosome Entry Site (IRES) sequence or an IRES derivative sequence.
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.
According to a further aspect of the present invention there is provided a composition comprising a functional nucleic acid molecule, a DNA molecule or an expression vector as defined herein.
According to a further aspect of the present invention there is provided a method for increasing the efficiency of protein synthesis of OPA1 in a cell comprising administering to the cell a functional nucleic acid molecule, a DNA molecule, an expression vector or a composition as defined herein.
According to a further aspect of the present invention there is provided a method of treating a mitochondrial deficiency related 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.
According to another aspect of the invention there is provided the use of a therapeutically effective amount of a functional nucleic acid molecule, DNA molecule, expression vector or composition for the manufacture of a medicament for the treatment of a mitochondrial deficiency related disease such as ADOA.
Drawings
Fig. 1: schematic representation of the SINEMP domain and human OPA1 gene, examples of target binding domains of functional nucleic acids according to the invention.
Fig. 2: HEK 293T cells were transfected with the BD-depleted miniSINEMP (. DELTA.BD) and miniSINEMP-OPA 1 variants. (A) Whole cell lysates were analyzed by western blot with anti-OPA 1 and anti- β -actin antibodies. A representative experiment is shown. The figure shows real-time PCR analysis of OPA1 mRNA and miniSINEMP RNA expression in transfected cells. The columns represent the mean.+ -. Standard error of the mean of n.gtoreq.3 independent experiments. (B) average fold change in OPA1 protein level. For each sample, the values are reported as two columns representing the long (left) and short (right) isoforms (isoforms), respectively. Columns represent the mean ± standard error of the mean of n.gtoreq.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 post test).
Fig. 3: mouse Neruo2A cells were transfected with BD-depleted miniSINEUP (Δbd) and miniSINEUP-OPA1 variants. (A) Whole cell lysates were analyzed by western blot with anti-OPA 1 and anti- β -actin antibodies. A representative experiment is shown. The figure shows real-time PCR analysis of OPA1 mRNA and miniSINEMP RNA expression in transfected cells. The columns represent the mean.+ -. Standard error of the mean of n.gtoreq.3 independent experiments. (B) average fold change in OPA1 protein level. For each sample, the values are reported as two columns representing the long (left) and short (right) isoforms, respectively. Columns represent the mean ± standard error of the mean of n.gtoreq.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 post test).
Fig. 4: mouse astrocytes were transfected with the BD-depleted miniSINEUP (Δbd) and miniSINEUP-OPA1 variants. (A) Whole cell lysates were analyzed by western blot with anti-OPA 1 and anti- β -actin antibodies. A representative experiment is shown. The figure shows real-time PCR analysis of OPA1 mRNA and miniSINEMP RNA expression in transfected cells. The columns represent the mean.+ -. Standard error of the mean of n.gtoreq.3 independent experiments. (B) average fold change in OPA1 protein level. For each sample, the values are reported as two columns representing the long (left) and short (right) isoforms, respectively. Columns represent the mean ± standard error of the mean of n.gtoreq.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 post test).
Fig. 5: HEK 293T cells were transfected with the BD-depleted miniSINEMP (. DELTA.BD), miniSINEMP-OPA 1 (-14/+4-M1-AUG), and microSINEMP-OPA 1 (-14/+4-M1-AUG) variants. (A) Whole cell lysates were analyzed by western blot with anti-OPA 1 and anti- β -actin antibodies. A representative experiment is shown. The figure shows real-time PCR analysis of OPA1 mRNA and miniSINEMP RNA expression in transfected cells. The columns represent the mean ± standard error of the mean of n.gtoreq.3 independent experiments. (B) average fold change in OPA1 protein level. For each sample, the values are reported as two columns representing the long (left) and short (right) isoforms, respectively. Columns represent the mean ± standard error of the mean 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 post test).
Fig. 6: HEK 293T cells were transfected with miniSINEP (ΔBD) lacking binding domain and nano2 SINEP-OPA 1 (-14/+4-M1-AUG). (A) Whole cell lysates were analyzed by western blot with anti-OPA 1 and anti- β -actin antibodies. A representative experiment is shown. (B) Real-time PCR analysis of OPA1 mRNA (left panel) and nano2SINEUP RNA expression (right panel). Nt=untransfected.
Fig. 7: miniSINEMP in two different vector backbones showed OPA 1-nanoluminescence in vitro.
Fig. 8: results of synthetic nano2 SINEMP on OPA1 protein and mRNA levels. (A) Whole cell lysates were analyzed by western blot with anti-OPA 1 and anti- β -actin antibodies; (B) average fold change in OPA1 protein level. Real-time PCR analysis of OPA1 mRNA (C) and nano2 SINEMP RNA (D).
Detailed Description
It is an object of the present invention to provide a functional nucleic acid molecule which increases the expression of OPA1 protein without exceeding physiological levels, targets OPA1 expression of all isoforms in a highly gene-specific manner and limits side effects.
The present inventors have developed and targeted OPA1 using SINEUP technology to increase endogenous levels of all OPA1 protein isoforms in human, mouse and patient derived cell lines. Through in vitro screening, OPA 1-specific SINEMP was shown to selectively increase human and murine OPA1 protein. Importantly, SINEUP-OPA1 increased OPA1 protein levels within a 2-fold range when expressed in fibroblasts derived from ADOA patients, showed activity sufficient for functional rescue without causing the negative side effects associated with increasing OPA1 expression above physiological levels. Furthermore, SINEUP-OPA1 does not disrupt the long/short form ratio of OPA1 protein, which is critical for rescuing mitochondrial network morphology.
Definition of the definition
"functional nucleic acid molecule" generally means that the nucleic acid molecule is capable of enhancing translation of a target mRNA of interest, in this particular case OPA1 mRNA.
"OPA1 mRNA sequence" refers to any length of mRNA sequence of at least 10 nucleotides contained in the mRNA of the corresponding OPA1 gene. Alternative splicing of OPA1 transcripts resulted in the production of 8 different isoforms. They share their 5' utr and are ubiquitously expressed. The resulting OPA1 protein also undergoes cleavage to produce 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.
The OPA1 gene encodes OPA1 mitochondrial dynamin-like gtpase (also known as dynamin-like 120KDa protein, mitochondria), referred to herein as "OPA1 protein". OPA1 protein sequences are known in the art, see for example UniProt ID: o60313.
The term "SINE" (short interspersed nuclear element) can be referred to as a non-LTR (long terminal repeat) retrotransposon and is an interspersed repeat sequence that (a) encodes a protein that has neither reverse transcription activity nor endonuclease activity, etc., and (b) whose complete or incomplete copy sequence is found in abundance in the genome of a living organism.
The term "SINE B2 element" is defined in WO 2012/133947, wherein specific examples are also provided (see tables beginning on page 69 of PCT publication). The term is intended to encompass SINE B2 elements that are in a direct orientation and in a reverse orientation relative to the 5 'to 3' orientation of a functional nucleic acid molecule. SINE B2 element can be identified, for example, using a program such as published RepeatMask (Bedell et al, bioinformation.11, 2000; 16 (11): 1040-1, "masquerAid: enhanced performance for RepeatMasker (masquerAid: aperformance enhancement to RepeatMasker)"). By returning hits on the consensus sequence of SINE B2 in the Repbase database, the sequence can be identified as SINE B2 element, with a Smith-Waterman (SW) score exceeding 225, which is the default cutoff in the RepeatMasker program. Typically the SINE B2 element is not less than 20bp and not more than 400bp. Preferably, SINE B2 is derived from tRNA.
The term "functionally active fragment of a SINE B2 element" refers to a portion of the sequence of a SINE B2 element that retains protein translation enhancing efficiency. The term also includes sequences that are mutated in one or more nucleotides relative to the wild-type sequence but retain protein translation enhancing efficiency. The term is intended to encompass SINE B2 elements that are in a direct orientation and in a reverse orientation relative to the 5 'to 3' orientation of a functional nucleic acid molecule.
The terms "Internal Ribosome Entry Site (IRES) sequence" and "Internal Ribosome Entry Site (IRES) derivative sequence" are defined in WO 2019/058304. IRES sequences recruit 40S ribosomal subunits and promote cap-independent translation of protein-encoding mRNA subunits. IRES sequences are typically present in the 5' untranslated region of cellular mRNA encoding stress response genes, thus stimulating their cis-translation. The term "IRES-derived sequence" is understood to mean a nucleic acid sequence having homology to an IRES sequence in order to preserve its functional activity, i.e. translation enhancing activity. In particular, IRES derived sequences may be obtained from naturally occurring IRES sequences by genetic engineering or chemical modification, for example by isolating specific sequences that remain functional in the IRES sequence, or mutating/deleting/introducing one or more nucleotides in the IRES sequence, or replacing one or more nucleotides in the IRES sequence with structurally modified nucleotides or analogues. More particularly, the IRES derived sequence is known to those skilled in the art to be a nucleotide sequence capable of promoting translation of the second cistron in a bicistronic construct. Typically, a double luciferase (firefly luciferase, renilla luciferase) encoding plasmid was used for experimental testing. There is a major database, IRESITE, for annotating nucleotide sequences that have been experimentally verified to be IRES using a dual reporter or bicistronic assay (http:// IRESite org/IRESITE_web. Php). Within IRESITE, web-based tools may be used to search for sequence-based and structure-based similarities between the query sequence of interest and all annotated and experimentally verified IRES sequences within the database. The output of the program is the probability score of any nucleotide sequence capable of acting as an IRES in a validation experiment using a bicistronic construct. Additional sequence-based and structure-based web-based browsing tools can be used to suggest IRES activity potential for any given nucleotide sequence with numerical predictions (http:// rnia. Informatik. Uni-freiburg. De/; http:// regnna. Mbc. Nctu. Edu. Tw/index1. Php).
The term "miniSINEMP" 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 any SINE or SINE-derived sequence or IRES-derived sequence as an effector domain (Zucchelli et al, front Cell neurosci.,9:174, 2015).
The term "microSINEMP" 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 SINE or SINE-derived sequence or IRES-derived sequence. For example, the functionally active fragment may be a 77bp sequence corresponding to nucleotides 44-120 of the 167bp SINE B2 element in AS Uchl 1.
A polypeptide or polynucleotide sequence is considered identical or "identical" to another polypeptide or polynucleotide sequence if it has 100% sequence identity over its entire length. Residues in the sequence are numbered from left to right, i.e., from the N-terminus to the C-terminus of the polypeptide; from the 5 'to the 3' end of the polynucleotide.
To compare two closely related polynucleotide sequences, "% sequence identity" between a first nucleotide sequence and a second nucleotide sequence may be calculated using NCBI BLAST using the standard set-up (BLASTN) of nucleotide sequences. To compare two closely related polypeptide sequences, "% sequence identity" between a first polypeptide sequence and a second polypeptide sequence may be calculated using NCBI BLAST, using the standard set-up (BLASTP) of polypeptide sequences. "difference" between sequences refers to the insertion, deletion or substitution of a single nucleotide at the position of the second sequence as compared to the first sequence. Two sequences may contain one, two or more such differences. In addition, insertions, deletions or substitutions in the second sequence that are identical to the first sequence (100% sequence identity) result in a decrease in% sequence identity.
Functional nucleic acid molecules
The functional nucleic acid molecules of the invention comprise at least one target binding sequence comprising a sequence that is reverse complementary to an OPA1 mRNA sequence and at least one regulatory sequence comprising an RNA comprising a sink 2 element or a functionally active fragment of a sink 2 element or an ribosome entry site (IRES) sequence or an IRES derivative sequence.
Regulatory sequences
Regulatory sequences have protein translation enhancing efficiency. An increase in the efficiency of protein translation indicates an increase in efficiency compared to the absence of the functional nucleic acid molecule according to the 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, such as at least 2-fold. In another embodiment, the expression of the protein encoded by the target mRNA is increased 1.5 to 3-fold, such as 1.6 to 2.2-fold. It is envisaged that increasing protein expression within these ranges will allow the treatment of diseases associated with mitochondrial defects, such as ADOA, without causing the negative side effects associated with increased OPA1 expression beyond physiological levels.
In one embodiment, the regulatory sequence is located 3' to the target binding sequence. The regulatory sequences may be in a direct or reverse orientation relative to the 5 'to 3' orientation of the functional nucleic acid molecule. Reference to "directly" refers to the case where the regulatory sequences are inserted (intercalated) in the same 5 'to 3' direction as the functional nucleic acid molecule. Conversely, "reverse" refers to the case where the regulatory sequences are in the 3 'to 5' orientation relative to the functional nucleic acid molecule.
Preferably, at least one regulatory sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 1-69 has 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. In one embodiment, at least one regulatory sequence consists of a sequence selected from the group consisting of SEQ ID NOs: 1-69 has 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.
In one embodiment, the regulatory sequence comprises a SINE B2 element or functionally active fragment of a SINE B2 element. 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. As mentioned in the definition section, reverse SINE B2 elements are disclosed and exemplified in WO 2012/133947.
In one embodiment, at least one regulatory sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 1-51 has 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.
Particularly preferred are SEQ ID NOs: 1 (167 nucleotide variant of the inverted SINE B2 element in AS Uchl 1) and SEQ ID NO:2 (a 77 nucleotide variant of the inverted SINE B2 element in AS Uchl1, which comprises nucleotides 44 to 120), and sequences having a percent identity to these sequences.
Other reverse SINE B2 elements and functionally active fragments of reverse SINE B2 elements are SEQ ID NO:3-51. Experimental data showing the protein translation enhancing efficiency of these sequences are not explicitly shown in the present patent application but are disclosed in the prior patent application of the same applicant. Thus, SEQ ID NO:3-51 can also be used as regulatory sequences in the molecules according to the invention.
SEQ ID NO:3-6, 8-11, 18 and 43-51 are functionally active fragments of the inverted SINE B2 transposable element derived from AS Uchl 1. The use of a functional fragment reduces the size of the regulatory sequence, which is advantageous when used in an expression vector (e.g. may be a viral vector of limited size), as this provides more room for the target sequence and/or expression element.
SEQ ID NO:7 is a full 183 nucleotide inverted SINE B2 transposable element derived from AS Uchl1 (transposable element). SEQ ID NO:12-17, 19, 20 and 39-42 are mutated functionally active fragments of the inverted SINE B2 transposable element derived from AS Uchl 1.
SEQ ID NO:21-25 and 28-38 are different SINE B2 transposable elements. SEQ ID NO:26 and 27 are sequences into which a plurality of inverted SINE B2 transposable elements are inserted.
Alternatively, the regulatory sequence comprises an IRES sequence or an IRES derivative sequence. Thus, in one embodiment, the regulatory sequence comprises an IRES sequence or an IRES derivative sequence. The sequences enhance translation of the target mRNA sequences.
Several IRES having 48 to 576 nucleotide sequences have been successfully tested, such as human Hepatitis C Virus (HCV) IRES (e.g., SEQ ID NO:52 and 53), human poliovirus IRES (e.g., SEQ ID NO:54 and 55), human encephalomyocarditis virus (EMCV) (e.g., SEQ ID NO:56 and 57), human cricket paralysis virus (CrPV) (e.g., SEQ ID NO:58 and 59), human Apaf-1 (e.g., SEQ ID NO:60 and 61), human ELG-1 (e.g., SEQ ID NO:62 and 63), human c-MYC (e.g., SEQ ID NO: 64-67), and human dystrophin (DMD) (e.g., SEQ ID NO:68 and 69).
Such sequences have been disclosed, defined and exemplified in WO 2019/058304. Preferably, such sequences are identical to SEQ ID NO:52-69 with 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.
Target binding sequences
Human OPA1 is an dynamin-related gtpase protein encoded in locus 3q28, which is located in the Inner Mitochondrial Membrane (IMM) and is ubiquitously expressed, reaching the highest expression levels in brain, retina and heart. The gene consists of 30 exons, and the protein itself is translated into 8 different isoforms according to alternative mRNA splicing and processing. In IMM, a Mitochondrial Targeting Sequence (MTS) is cleaved at a first cleavage site to produce a long transmembrane type. Isoforms 4, 6, 7 and 8 also contain a second cleavage site encoded in exon 5b, which results in their further processing into short forms of the protein. OPA1 has a major function in mitochondrial homeostasis. Together with mitochondrial fusion proteins MFN1 and MFN2, they promote mitochondrial fusion, a process associated with increased respiratory efficiency, and they contribute to mitochondrial DNA (mtDNA) maintenance. OPA1 protein polymerization also maintains ridge morphogenesis, promoting respiratory supercomplex activity. It plays a major role in controlling the apoptotic process, as it is the basis for cytochrome C compartmentalization, and uncontrolled release of cytochrome C will lead to cell death. Any of the eight OPA1 isoforms can support its three basic functions (energetics, structure and mtDNA maintenance), but the balance between long and short isoforms seems to be a critical requirement for complete recovery of the mitochondrial network. Thus, complete rescue of mitochondrial network morphology requires long-short equilibrium of at least two isoforms. The data presented herein indicate that OPA1-SINEUP is a unique tool that targets all OPA1 transcripts simultaneously, restoring the correct physiological ratio and processing to l/s form in a conventional physiological manner. This is different from alternative therapies that might favor single specific transcript/isoform expression and potentially disrupt OPA1 isoform physiological ratios and l/sOPA1 protein ratios.
In WO 2012/133947, it has been shown that the target binding sequence needs to have only about 60% similarity to the sequence reverse complementary to the target mRNA. Indeed, the target binding sequence may even exhibit a large number of mismatches and retain activity.
In one embodiment, the target binding sequence comprises a sequence that is reverse-complementary to a portion of the OPA1mRNA sequence that is common to all OPA1 isoforms. By maintaining the mutual levels of all OPA1 isoforms, the functional nucleic acid molecules are able to induce optimal molecular expression patterns to restore physiological homeostasis. This is not possible with more conventional gene therapy approaches when only one isoform may be ectopic resulting in an isoform imbalance.
The target binding sequence comprises a sequence of sufficient length to bind to an OPA1mRNA transcript. Thus, 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 at least about 18 nucleotides in length. In addition, 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.
The target binding sequence may 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, such as 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 may 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, such as 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.
The target binding sequence can be designed to hybridize to an AUG site (start codon) of the OPA1 mRNA sequence, such as a region upstream of the start codon within the CDS. In one embodiment, the sequence is reverse complementary to 0 to 80 nucleotides of the AUG site, 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 nucleotides. Alternatively, or in combination, the target binding sequence may be designed to hybridize to an OPA1 mRNA sequence downstream of the AUG site. In one embodiment, the sequence is reverse complementary to 0 to 40 nucleotides of the OPA1 mRNA sequence downstream of the AUG site, such as 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.
Preferably, the target binding sequence is at least 10 nucleotides in length and comprises from 3 'to 5':
1) A sequence reverse complementary to 0 to 50 nucleotides of the 5' utr of OPA1 mRNA sequence and 0 to 40 nucleotides of CDS; or (b)
2) A sequence 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 said AUG site.
In case 1), the coding sequence starts at the first AUG site (M1) of the mRNA. In case 2), the preferred AUG site corresponds to an internal start codon, such as methionine 125 (M125) in exon 3. In the context of sequences that are reverse complementary to the 5'UTR and to the region in the CDS, this is preferably anchored 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 of "40/+4" of M1 refers to a target binding sequence that is reverse-complementary to 40 nucleotides (-40) in the 5' UTR upstream of the AUG site and 4 nucleotides (+4) in the CDS downstream of the AUG site.
According to conventional numbering, the nucleotides of the 5' UTR sequence are numbered sequentially using decreasing negative numbers (e.g., -3, -2, -1) approaching the AUG site on the target mRNA. The nucleotides of the CDS sequence are numbered sequentially using positive numbers (e.g., +1, +2, +3) that increase from the AUG site such that the a number of the AUG site is +1. Thus, the region bridging the 5' UTR and CDS will be numbered-3, -2, -1, +1, +2, +3, with A at the AUG site numbered +1.
More preferably, at least one target binding sequence is at least 14 nucleotides in length and comprises from 3 'to 5':
a sequence that is reverse complementary to 0 to 40 (preferably 0 to 21, more preferably 0 to 14) nucleotides of the 5' utr of OPA1 mRNA sequence and 0 to 32 (preferably 0 to 4, more preferably 0) nucleotides of CDS; or (b)
-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 said AUG site.
In a specific embodiment, 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 may comprise a sequence consisting of SEQ ID NO:82 (i.e., -14/+4 of M1).
In another specific embodiment, the target binding sequence is 15 nucleotides in length and comprises, from 3' to 5', a sequence that is reverse complementary to 6 nucleotides of the 5' utr and 9 nucleotides of the CDS of the OPA1 mRNA sequence. For example, the target binding sequence may comprise a sequence consisting of SEQ ID NO:83 (i.e., -6/+9 of M1).
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 of the OPA1 mRNA sequence and 30 nucleotides of the CDS. For example, the target binding sequence may comprise a sequence consisting of SEQ ID NO:84 (i.e., -41/+30 of M1).
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 of the OPA1 mRNA sequence and 30 nucleotides of the CDS. For example, the target binding sequence may comprise a sequence consisting of SEQ ID NO:93 (i.e., -41/-30 of M1).
In another embodiment, the target binding sequence is 14 nucleotides in length and comprises a sequence that is reverse complementary from 3' to 5' to the region between nucleotides 97 and 84 of the 5' utr of the OPA1 mRNA sequence. For example, the target binding sequence may comprise a sequence consisting of SEQ ID NO:85 (i.e., -97/-87 of M1).
In another embodiment, the target binding sequence is 18 nucleotides in length and comprises, from 3' to 5', a sequence that is reverse complementary to 18 nucleotides of the 5' utr of the OPA1 mRNA sequence and 0 nucleotides of the CDS. For example, the target binding sequence may comprise a sequence consisting of SEQ ID NO:86 (i.e., -18/-1 of M1).
In another embodiment, the target binding sequence is 22 nucleotides in length and comprises, from 3' to 5', a sequence that is reverse complementary to 18 nucleotides of the 5' utr and 4 nucleotides of the CDS of the OPA1 mRNA sequence. For example, the target binding sequence may comprise a sequence consisting of SEQ ID NO:87 (i.e., -18/+4 of M1).
In another embodiment, the target binding sequence is 14 nucleotides in length and comprises, from 3' to 5', a sequence that is reverse complementary to 14 nucleotides of the 5' utr and 0 nucleotides of the CDS of the OPA1 mRNA sequence. For example, the target binding sequence may comprise a sequence consisting of SEQ ID NO:88 (i.e., -14/-1 of M1).
In a specific embodiment, the target binding sequence is 17 nucleotides in length and comprises 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 said AUG site. For example, the target binding sequence may comprise a sequence consisting of SEQ ID NO:89 (i.e., -9/+8 of M125).
In another embodiment, the target binding sequence is 18 nucleotides in length and comprises 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 said AUG site. For example, the target binding sequence may comprise a sequence consisting of SEQ ID NO:90 (i.e., -18/-1 of M125).
In another embodiment, the target binding sequence is 22 nucleotides in length and comprises 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 4 nucleotides of the CDS of the OPA1 mRNA sequence downstream of said AUG site. For example, the target binding sequence may comprise a sequence consisting of SEQ ID NO:91 (i.e., -18/+4 of M125).
In another embodiment, the target binding sequence is 14 nucleotides in length and comprises 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 said AUG site. For example, the target binding sequence may comprise a sequence consisting of SEQ ID NO:92 (i.e., -14/-1 of M125).
In another embodiment, the target binding sequence is 44 nucleotides in length and comprises 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 said AUG site. For example, the target binding sequence may comprise a sequence consisting of SEQ ID NO:94 (M1, -40/+4).
In another embodiment, the target binding sequence is 44 nucleotides in length and comprises 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 said AUG site. For example, the target binding sequence may comprise a sequence consisting of SEQ ID NO:95 (M2, -40/+4).
Thus, in some embodiments, the target binding sequence comprises a sequence that hybridizes to SEQ ID NO:82-95, preferably SEQ ID NO:82-84 or 89 has 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, even more preferably 100% sequence identity. In another embodiment, the target binding sequence consists of a sequence that hybridizes to SEQ ID NO:82-95, preferably SEQ ID NO:82-84 or 89 has 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, even more preferably 100% sequence identity.
In one embodiment, the functional nucleic acid molecule comprises a sequence that hybridizes to SEQ ID NO:70-79, preferably SEQ ID NO: any of 70-74, 78-79 has 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, a sequence having at least about 99% sequence identity, even more preferably 100% sequence identity. In another embodiment, the functional nucleic acid molecule consists of a sequence that hybridizes to SEQ ID NO:70-79, preferably SEQ ID NO: any of 70-74, 78-79 has 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, even more preferably 100% sequence identity.
More specifically, SEQ ID NO:70-74, 78-79 relates to a functional nucleic acid molecule directed against the human OPA1 isoform, whereas SEQ ID NO:75-77 to a functional nucleic acid molecule directed against the mouse OPA1 isoform. Furthermore, SEQ ID NO:70-77 comprise a "mini" reverse SINE B2 element (167 nucleotides) in AS Uchl1, SEQ ID NO:78 comprises a "micro" inverted sink B2 element in AS Uchl1 (i.e., nucleotides 44-120 of the inverted sink B2 transposable element derived from AS Uchl 1), and SEQ ID NO:79 comprises a "nano" inverted SINE B2 element in AS Uchl1 (i.e., nucleotides 64-92 of an inverted SINE B2 transposable element derived from AS Uchl 1). Differences between minisinup sequences are derived from the targeting binding sequences and/or spacer/linker sequences described herein.
From the disclosure herein and sequence identity of the human OPA1 isoform to, for example, mice and rhesus monkeys (Macaca mulatta), it is understood that the target binding sequences provided herein may be cross-reactive with other species. For example, human OPA1 mRNA has 96.19% sequence identity over its entire length to rhesus monkey (Macaca mulatta) OPA1 mRNA and 78.36% sequence identity to mouse OPA1 mRNA. Thus, in one embodiment, a polypeptide consisting of SEQ ID NO:82-95 or a target binding sequence consisting of the same, binds to mouse OPA1 and/or rhesus OPA1, preferably rhesus OPA1.
In one embodiment, the functional nucleic acid molecules provided herein are chemically modified. The term "modification" or "chemical modification" refers to a structural change in or on the most common natural ribonucleotides: adenosine, guanosine, cytidine, or uridine ribonucleotides. The chemical modification may be a change in or on a nucleobase (i.e., a chemical base modification) or a change in or on a sugar (i.e., a chemical sugar modification). Chemical modifications can be introduced co-transcribed (e.g., by substituting one or more nucleotides with modified nucleotides during synthesis) or post-transcriptionally (e.g., by the action of an enzyme).
Chemical modifications are known in the art and are described, for example, in the RNA modification database (https:// mods. RNA. Albany. Edu/mods /) provided by RNA research.
Many modifications occur in nature, such as chemical modifications to the natural transfer RNA (tRNA), including, for example: 2 '-O-methyl (such as 2' -O-methyl adenosine, 2 '-O-methyl guanosine and 2' -O-methyl pseudouridine), 1-methyl adenosine, 2-methyl adenosine, 1-methyl guanosine, 7-methyl guanosine, 2-thiocytidine, 5-methyl cytidine, 5-formyl cytidine, pseudouridine, dihydrouridine, and the like.
Structural features
The functional nucleic acid molecule may comprise more than one regulatory sequence, which may be the same sequence repeated more than once or different regulatory sequences (i.e. different SINE B2 elements/functionally active fragments of SINE B2 elements/IRES sequences/IRES derived sequences).
The at least one target binding sequence and the at least one regulatory sequence are preferably linked 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.
The functional nucleic acid molecules of the invention are preferably circular molecules. This conformation results in a more stable molecule that is more difficult to degrade inside the cell (exonucleases cannot degrade cyclic molecules) and thus remains active for a longer period of time.
Furthermore, the functional nucleic acid molecule may optionally comprise a non-coding 3' tail sequence, which for example comprises restriction sites that can be used to clone the molecule into an appropriate plasmid.
In one embodiment, the functional nucleic acid molecule comprises a 3' -polyadenylation (polyA) tail. "3'-polyA tail" refers to a long chain of adenine nucleotides added to the 3' -end of transcription, which provides stability to the RNA molecule and can facilitate translation.
In one embodiment, the functional nucleic acid molecule comprises a 5' -cap. "5 '-cap" refers to an altered nucleotide at the 5' -end of a transcript that provides stability to the molecule, particularly from degradation by exonucleases, and can facilitate translation.
It should be noted that functional nucleic acid molecules can enhance translation of a target gene of interest without affecting the amount of mRNA of the target gene. Thus, they can be successfully used as molecular tools to verify gene function in cells and as a conduit for implementing recombinant protein production.
DNA molecules and vectors
According to another aspect of the present invention there is provided a DNA molecule encoding any of the functional nucleic acid molecules disclosed herein. According to another aspect of the present 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 (e.g., adenovirus, adeno-associated virus, retrovirus or lentivirus vectors), phage vectors, cosmid vectors, and the like. The choice of expression vector may depend on the type of host cell used and the purpose of use. In particular, the following plasmids have been used to efficiently express functional nucleic acid molecules:
Mammalian expression plasmid:
plasmid name: pCDNA3.1 (-)
Expression: CMV promoter
BGH poly (A) terminator
Plasmid name: pDOAL-eGFP delta (modified from pepGFP-C2)
Expression: h1 promoter
BGH poly (A) terminator
Viral vectors:
carrier name: PAAV (PAAV)
Virus: adeno-associated virus
Expression: CAG promoter/CMV enhancer
SV40 late poly (A) terminator
Carrier name: rcLV-TetOne-Puro
Virus: lentivirus (3 rd generation)
Expression: LTR-TREt (Tre-light) promoter (doxycycline inducible expression)
BGH poly (A) terminator
Carrier name: pLPCX-link
Virus: retrovirus (3 rd generation)
Expression: CMV (CMV)
It should be noted that any promoter may be used in the vector and functions as the promoter described above.
Composition and medical use
The invention also relates to compositions comprising the functional nucleic acid molecules, DNA molecules and expression vectors described herein. The composition may comprise components capable of delivering the functional nucleic acid molecule via viral vectors (AAV, lentivirus, etc.) and non-viral vectors (nanoparticles, lipid particles, etc.).
The functional nucleic acid molecules of the invention may also be administered as naked or unpackaged RNA. Alternatively, the functional nucleic acid molecule may be administered as part of a composition, such as a composition comprising a suitable carrier. In certain embodiments, the vectors are selected based on their ability to facilitate transfection of the target cells with one or more functional nucleic acid molecules.
According to a further aspect of the present invention there is provided the use of a functional nucleic acid molecule, DNA molecule, expression vector or composition as defined herein as a medicament.
It will be appreciated that the functional nucleic acid molecules of the invention may be used to increase the level of OPA1 protein in a cell. Thus OPA1 has a major function in mitochondrial homeostasis. According to another aspect of the invention there is provided the use of a functional nucleic acid molecule, a DNA molecule, an expression vector or a composition for the treatment of a disease associated with mitochondrial defects.
The above-described functional nucleic acid molecules, DNA molecules and/or compositions are useful as medicaments, preferably for the treatment of Autosomal Dominant Optic Atrophy (ADOA), in particular for promoting recovery of disease-related mitochondrial defects. Retinal Ganglion Cells (RGCs) from mice deficient in OPA1 and RGC-specific OPA1 expressing mutations have been shown to play a role in autophagy in the pathogenesis of ADOA (Zaninello et al (2020) Nat.Comm.11 (1): 4029).
Thus, in another embodiment, the mitochondrial defect related disease is ADOA. ADOA is the most common hereditary optic neuropathy, caused in 75% of cases by heterozygous mutations in the OPA1 gene. The main symptoms of the disease are bilateral degeneration of Retinal Ganglion Cells (RGCs) and optic atrophy, possibly accompanied by associated muscle and neurodegenerative symptoms. Various forms of ADOA have been reported, such as adoa+ (ADOA plus) (which also shows muscle defects and sensorineural deafness) and ADOAC (which also causes cataracts). Furthermore, patients with the same mutations have been reported to have intra-and inter-family changes in terms of disease severity.
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 the treatment of a mitochondrial deficiency related disease such as ADOA.
According to a further aspect of the present invention there is provided the use of a therapeutically effective amount of a functional nucleic acid molecule, DNA molecule, expression vector or composition as defined herein for the manufacture of a medicament for the treatment of a mitochondrial deficiency related disease such as ADOA.
In general, OPA1 is one of the major factors controlling mitochondrial fusion, mitochondrial DNA (mtDNA) maintenance, bioenergy, and cristae integrity. These cellular processes are targets for several diseases that can potentially be rescued by increasing the endogenous expression of OPA 1. Furthermore, OPA1 also controls apoptosis independently of mitochondrial fusion through cristae remodeling and cytochrome c release (Frezza et al, (2006) Cell 126 (1): 177-89).
In addition to ADOA, some reports suggest that a slight increase in OPA1 protein expression may be useful in the treatment of other diseases. For example, civiletto et al (2015) Cell Metab.21 (6): 845-854 shows that moderate OPA1 overexpression improved the phenotype of two mouse models of mitochondrial disease with defects in the Ndefs 4 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 cardiomyopathy. As another example, varanita et al (2015) Cell Metabolism shows OPA1 tg Mice (a model that overexpresses OPA1 approximately 1.5-see also cog et al (2013) Cell 155 (1): 160-171) were protected from muscle atrophy, myocardial infarction, less sensitivity to Fas-induced liver injury, mitochondria were resistant to cristae remodeling and cytochrome C release. In contrast, large amounts of OPA1 overexpression has proven to be toxic (Cipolat et al (2004) PNAS 101 (45): 15977-15932), and therefore the methods provided herein are particularly useful for treating OPA1 deficiency-related diseases, as it is critical to increase expression only to normal physiological levels. It is envisaged that this will avoid the possible correlation with a substantial increase in OPA1 expression above physiological levelsIs an undesirable side effect of (a).
Mitochondrial defect related diseases are well known in the art, for example, as described by Gorman et al (2016) nat. Rev. Disease Primers,2,16080. They may be characterized as defective oxidative phosphorylation due to mutations in nuclear or mitochondrial DNA that result in mutated/dysfunctional mitochondrial proteins.
Mitochondrial defects are associated with neurological diseases and development. Thus, in one embodiment, the mitochondrial defect related disease is a neurological disease. Castayan et al (2020) iScience 23:101154 describe genetically modified human embryonic and patient-derived induced pluripotent stem cells with single dose starvation of OPA1, which lead to aberrant nuclear DNA methylation and significantly alter transcriptional loops in Neural Progenitor Cells (NPCs). In particular, OPA1+/-NPC is unable to develop into gamma-aminobutyric acid capable interneurons. Changes in normal OPA1 expression are also associated with 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; itanielli et al (2018) Cell Rep.22 (8): 2066-2079; and Itanielli 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 prion disease. For example, wu et al (2019) Cell Death Dis.10 (10): 710 describes that downregulation of OPA1 was observed in prion disease models in vitro and in vivo, and this occurs with mitochondrial structural damage and dysfunction, mtDNA loss, and neuronal apoptosis. These symptoms are alleviated by increasing OPA1 expression.
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 the 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.
According to a further aspect of the present invention there is provided a method for increasing the efficiency of protein synthesis of OPA1 protein in a cell comprising administering to the cell a functional nucleic acid molecule, DNA molecule, expression vector or composition as defined herein.
The methods described herein may comprise transfecting a functional nucleic acid molecule, DNA molecule, expression vector or composition as defined herein into a cell. The functional nucleic acid molecule, DNA molecule, expression vector or composition can be administered to the target cell using methods known in the art including, for example, microinjection, lipofection, electroporation, use of calcium phosphate, self-infection by a vector or transduction by a virus.
In one embodiment, the cells are in a single dose deficient of OPA1, i.e. wherein the presence of variant alleles in the heterozygous combination results in insufficient amounts of product produced by a single wild-type gene to fulfill complete or normal function. In general, a single dose deficiency is a condition that occurs when a normal phenotype requires a protein product of both alleles, and a decrease in gene function to 50% or less results in an abnormal phenotype.
The methods of the invention result in increased levels of OPA1 protein in the cells and are therefore useful, for example, in methods of treating diseases associated with OPA1 defects (i.e., reduced levels of OPA1 protein and/or loss of function mutations in the OPA1 gene). The method of the invention is particularly useful for diseases caused by a quantitative decrease in the level of a predetermined normal protein. The method of the invention may be performed in vitro, ex vivo or in vivo.
According to a further aspect of the present invention there is provided a method of treating a mitochondrial deficiency related 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.
Gene therapy is challenging in diseases such as ADOA, because bilayer IMM is a relatively impermeable barrier. Although the method of designing proteins to be expressed to contain allotropic (allotropic) specific mitochondrial targeting sequences is currently being investigated, OPA1 functional nucleic acid molecules described herein reduce the need for such designs, causing their role in the cytoplasm to enhance translation of endogenous targets.
In one embodiment, the therapeutically effective amount is administered in the retina, brain or heart (particularly the retina).
It is to be understood that the embodiments described herein are applicable to all aspects of the invention, i.e. the embodiments described for functional nucleic acid molecules are equally applicable to the claimed methods and the like.
The invention will now be illustrated with reference to the following non-limiting examples.
Examples
Example 1
Synthetic miniSINEMP was designed to target human OPA1 mRNA. FIG. 1A shows a schematic representation of the SINEMP domain. The overlap is a binding domain (BD, grey) providing SINEUP specificity and is in antisense orientation with the mRNA encoding the sense protein (target mRNA). The inverted SINE B2 (invB 2) element from AS Uchl1 is the Effector Domain (ED) and enhances protein synthesis. Indicating the 5 'to 3' orientation of the sense and antisense RNA molecules. The structural elements of the target mRNA are shown as: a 5 'untranslated region (5' UTR, white), a coding sequence (CDS, black), and a 3 'untranslated region (3' UTR, white). The schemes are not drawn to scale. (B) Scheme design of human OPA1 gene (5' -UTR, white) and BD (grey) targeting synthesis of miniSINEMP-OPA 1 starting M1-AUG and second M125-AUG in frame. Numbering references are based on the position of methionine (i.e., -40/+4, from 40 nucleotides upstream to 4 nucleotides downstream of M1-AUG). All BDs were designed in the region included in all human OPA1 transcripts. The schemes are not drawn to scale. (C) Scheme of mouse OPA1 gene (5' -UTR, white) and BD (grey) design targeting the synthesis of miniSINEMP-OPA 1 starting with M1-AUG and M125-AUG. Numbering references are based on the position of methionine (i.e., -40/+4, from 40 nucleotides upstream to 4 nucleotides downstream of M1-AUG). All BDs were designed in the region included in all mouse OPA1 transcripts. The schemes are not drawn to scale.
Example 2
This example shows that synthesis of miniSINEMP increases endogenous OPA1 protein levels in human cells in vitro. HEK 293T cells were obtained from ATCC (cat# CRL-11268) transfected with the BD-deleted miniSINEMP (. DELTA.BD) and miniSINEMP-OPA 1 variants encoded on the pCS2+ ligation plasmid and harvested 48 hours after transfection. Δbd served as a negative control.
Whole cell lysates were analyzed by western blot with anti-OPA 1 and anti- β -actin antibodies. First, OPA1 band (L-and S-type) intensities were normalized to the relative β -actin band. Then, fold change values (Δbd) normalized to control cells were calculated. The results are shown in FIG. 2A.
Cells transfected with miniSINEMP-OPA 1 showed increased endogenous OPA1 protein levels. The changes in target and miniSINEMP mRNA expression in the samples were not statistically significant (one-way ANOVA followed by a post Dunnett test). Human GAPDH (hGAPDH) expression was used as an internal control to quantify OPA1 transcripts. The OPA1/hGAPDH ratio of the Δbd samples was set to baseline values, and all transcript levels were normalized to baseline values. OPA1 mRNA levels were shown to be unchanged, confirming that OPA1 increased protein synthesis at post-transcriptional levels. miniSINEMP transcripts were quantified using hGAPDH expression as an internal control. The Δbd/hGAPDH ratio samples were set to baseline values, and all transcript levels were normalized to baseline values.
Figure 2B shows the mean fold change in OPA1 protein levels. All minisinup showed an increase in endogenous OPA1 protein levels.
Example 3
This example shows that synthesis of miniSINEMP increases endogenous OPA1 protein levels in a mouse Neuro2A cell line in vitro. Neuro2A (N2A) cells were obtained from ATCC (cat# CCL-131), transfected with BD-depleted miniSINEMP (. DELTA.BD) and miniSINEMP-OPA 1 variants encoded on pCS2+ ligation plasmids, and harvested 48 hours after transfection. Δbd served as a negative control.
Western blot analysis was performed as described above with anti-OPA 1 (BD Bioscience, cat# 612606) and anti-beta-actin antibody (Sigma, cat# A2066). OPA1 band (L-and S-type) intensities were normalized to the relative β -actin band, and then fold change values (Δbd) normalized to control cells were calculated. The results are shown in FIG. 3A. Cells transfected with miniSINEMP-OPA 1 showed increased endogenous OPA1 protein levels. The changes in target and miniSINEMP mRNA expression in the samples were not statistically significant (one-way ANOVA followed by Dunnett post-test). Mouse GAPDH (mGAPDH) expression was used as an internal control to quantify OPA1 transcripts. The OPA1/mGAPDH ratio of the Δbd samples was set to baseline values, and all transcript levels were normalized to baseline values. OPA1 mRNA levels were shown to be unchanged, confirming that OPA1 increased protein synthesis at post-transcriptional levels. mGAPDH expression was used as an internal control to quantify miniSINEUP transcripts. The Δbd/mGAPDH ratio samples were set to baseline values, and all transcript levels were normalized to baseline values.
Figure 3B shows the mean fold change in OPA1 protein levels. All minisinup showed an increase in endogenous OPA1 protein levels.
Example 4
This example shows that synthesis of miniSINEMP increases endogenous OPA1 protein levels in a mouse astrocyte cell line in vitro. Astrocytes were obtained from ATCC (CRL-254), transfected with BD-depleted miniSINEMP (. DELTA.BD) and miniSINEMP-OPA 1 variants encoded on pCS2+ ligation plasmids, and harvested 48 hours after transfection. Δbd served as a negative control.
Whole cell lysates were analyzed by western blot using anti-OPA 1 and anti- β -actin antibodies as described above. OPA1 band (L-and S-type) intensities were normalized to the relative β -actin band and calculated normalized to fold change value (Δbd) of control cells. The results are shown in FIG. 4A. Cells transfected with miniSINEMP-OPA 1 showed increased endogenous OPA1 protein levels. The changes in target and miniSINEMP mRNA expression in the samples were not statistically significant (one-way ANOVA followed by Dunnett post-test). OPA1 transcripts were quantified using mGAPDH expression as an internal control. The OPA1/mGAPDH ratio of the Δbd samples was set to baseline values, and all transcript levels were normalized to baseline values. OPA1 mRNA levels were shown to be unchanged, confirming that OPA1 increased protein synthesis at post-transcriptional levels. mGAPDH expression was used as an internal control to quantify miniSINEUP transcripts. The Δbd/mGAPDH ratio samples were set to baseline values, and all transcript levels were normalized to baseline values.
Figure 4B shows the mean fold change in OPA1 protein levels. All minisinup showed an increase in endogenous OPA1 protein levels.
Example 5
This example describes effector domain (i.e., regulatory sequence) optimization. microSINEMP increases endogenous OPA1 protein levels in HEK 293T cells in vitro. HEK 293T cells were transfected with control vector (. DELTA.BD), miniSINEMP-OPA 1 (-14/+4-M1-AUG) and microSINEMP-OPA 1 (-14/+4-M1-AUG) variants. Cells were harvested 48 hours after transfection. Control vector (. DELTA.BD) and miniSINEMP-OPA 1 (-14/+4-M1-AUG) served as negative and positive controls, respectively. microSINEMP-OPA 1 presents a truncated ED consisting of nucleotides 44-120 of the invSINE B2 element of AS Uchl 1.
Whole cell lysates were analyzed by western blot with anti-OPA 1 and anti- β -actin antibodies. OPA1 band intensities were normalized to relative β actin and fold change values normalized to negative control cells (Δbd) were calculated as described above. The results are shown in FIG. 5. MicroSINEMP-OPA 1 transfected cells showed increased endogenous OPA1 protein levels compared to negative control cells.
Example 6
This example shows that synthesizing nano2 SINEMP increases endogenous OPA1 protein levels in human cells in vitro. HEK 293T cells were transfected with miniSINEP (ΔBD) lacking binding domain and nano2 SINEP-OPA 1 (-14/+4-M1-AUG) and harvested 48 hours after transfection. Nano2 SINEMP-OPA 1 presents a truncated ED consisting of nucleotides 64-92 of the invSINE B2 element from AS Uchl 1.
Whole cell lysates were analyzed by western blot with anti-OPA 1 and anti- β -actin antibodies. OPA1 protein levels increased significantly after transfection with nano2SINEUP, but not with control SINEUP lacking BD (fig. 6A). Real-time PCR analysis of OPA1 mRNA and nano2SINEUP RNA expression in transfected cells showed no significant increase in endogenous OPA1 mRNA compared to untransfected (NT) control cells, confirming increased post-transcriptional OPA1 protein levels (fig. 6B, left panel). Real-time PCR analysis also clearly demonstrated that OPA1-nano2SINEUP was expressed and present in HEK 293T cells (fig. 6B, right panel).
Example 7
This example shows that miniSINEMP in two different vector backbones increases over-expressed OPA1-nanoluc luminescence in vitro. Mouse Neuro2A cells were co-transfected with expression plasmids containing nano-luciferase-tagged human OPA1 plus miniSINEUP (Δbd) lacking the binding domain or miniSINEUP-OPA1 (with-14/+4 binding domain comprising methionine 1). Two different plasmid backbones were used, pcs2+ and pDUAL. After 48 hours, cells were subjected to a luciferase assay to quantify the amount of luminescence present under each treatment condition. For both plasmid vector backbones, those containing miniSINEMP-OPA 1 showed about 2.5 to 3 fold increased luminescence over the negative control lacking the target mRNA binding domain (FIG. 7). pcs2+minisineup-OPA1 showed a 3.2-fold increase over control and pDUAL 2.6-fold increase over control (n=4 independent biological experiments, each with three technical replicates).
Example 8
This example shows that synthetic nano2SINEUP increases endogenous OPA1 protein levels in human cells in vitro when transfected and endogenously transcribed in a plasmid vector (pcs2+) and transfected as naked RNA carrying modified ribonucleotides. The naked RNA molecule was modified with 2 '-O-methyladenosine (2' -O-MeA).
HEK 293T cells were transfected with miniSINEP (ΔBD) lacking the binding domain, nano2 SINEP-OPA 1 (-14/+4-M1-AUG), nano2 SINEP 2'-O-MeA modified RNA lacking the binding domain, and nano2 SINEP-OPA 1 (-14/+4-M1-AUG) 2' -O-MeA modified RNA. Cells were harvested 48 hours after transfection. Nano2 SINEMP-OPA 1 presents a truncated ED consisting of nucleotides 64-92 of the invSINE B2 element from AS Uchl 1.
Whole cell lysates were analyzed by western blot with anti-OPA 1 and anti- β -actin antibodies (fig. 8A). Following transfection with nano2 SINEMP (plasmid and naked RNA forms), OPA1 protein levels were significantly increased, but not for BD-deficient control SINEMP (FIGS. 8A and 8B). Real-time PCR analysis of OPA1mRNA and nano2SINEUP RNA expression in transfected cells showed no significant increase in endogenous OPA1mRNA compared to control cells, confirming increased post-transcriptional OPA1 protein levels (fig. 8C). Real-time PCR analysis also clearly demonstrated that OPA1-nano2SINEUP was expressed and present in HEK 293T cells (fig. 8D, right panel).
Sequence listing
<110> International higher institute (Scuola Internazionale Superiore Di Studi Avanzati-SISSA)
Telansaien theraphy Co., ltd (Transine Therapeutics Limited)
<120> functional nucleic acid molecules
<130> P123587PCT
<150> GB2015997.6
<151> 2020-10-08
<150> GB2019325.6
<151> 2020-12-08
<160> 96
<170> patent in version 3.5
<210> 1
<211> 167
<212> RNA
<213> mice (Mus musculus)
<400> 1
cagugcuaga ggaggucaga agagggcauu ggauccccca gaacuggagu uauacgguaa 60
ccucguggug guugugaacc accaugugga uggauauuga guuccaaaca cugguccugu 120
gcaagagcau ccagugcucu uaagugcuga gccaucucuu uagcucc 167
<210> 2
<211> 77
<212> RNA
<213> mice (Mus musculus)
<400> 2
gaacuggagu uauacgguaa ccucguggug guugugaacc accaugugga uggauauuga 60
guuccaaaca cuggucc 77
<210> 3
<211> 29
<212> RNA
<213> mice (Mus musculus)
<400> 3
ccucguggug guugugaacc accaugugg 29
<210> 4
<211> 61
<212> RNA
<213> mice (Mus musculus)
<400> 4
guuauacggu aaccucgugg ugguugugaa ccaccaugug gauggauauu gaguuccaaa 60
c 61
<210> 5
<211> 98
<212> RNA
<213> mice (Mus musculus)
<400> 5
aucccccaga acuggaguua uacgguaacc ucgugguggu ugugaaccac cauguggaug 60
gauauugagu uccaaacacu gguccugugc aagagcau 98
<210> 6
<211> 129
<212> RNA
<213> mice (Mus musculus)
<400> 6
gaagagggca uuggaucccc cagaacugga guuauacggu aaccucgugg ugguugugaa 60
ccaccaugug gauggauauu gaguuccaaa cacugguccu gugcaagagc auccagugcu 120
cuuaagugc 129
<210> 7
<211> 183
<212> RNA
<213> mice (Mus musculus)
<400> 7
gggcagugcu agaggagguc agaagagggc auuggauccc ccagaacugg aguuauacgg 60
uaaccucgug gugguuguga accaccaugu ggauggauau ugaguuccaa acacuggucc 120
ugugcaagag cauccagugc ucuuaagugc ugagccaucu cuuuagcucc agucucuuaa 180
gcu 183
<210> 8
<211> 67
<212> RNA
<213> mice (Mus musculus)
<400> 8
gaacuggagu uauacgguaa ccucguggug guugugaacc accaugugga uggauauuga 60
guuccaa 67
<210> 9
<211> 107
<212> RNA
<213> mice (Mus musculus)
<400> 9
ggauccccca gaacuggagu uauacgguaa ccucguggug guugugaacc accaugugga 60
uggauauuga guuccaaaca cugguccugu gcaagagcau ccagugc 107
<210> 10
<211> 127
<212> RNA
<213> mice (Mus musculus)
<400> 10
agagggcauu ggauccccca gaacuggagu uauacgguaa ccucguggug guugugaacc 60
accaugugga uggauauuga guuccaaaca cugguccugu gcaagagcau ccagugcucu 120
uaagugc 127
<210> 11
<211> 38
<212> RNA
<213> mice (Mus musculus)
<400> 11
gguaaccucg uggugguugu gaaccaccau guggaugg 38
<210> 12
<211> 180
<212> RNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 12
cagugcuaga ggaggucaga agagggcauu ggauccccca gaacuggagu uauacgauaa 60
ccucguggug guugugaacc accaugugga uggauauuga guuccaaaca cugguccugu 120
gcaagagcau ccagugcucu uaagugcuga gccaucucuu uagcuccagu cucuuaagcu 180
<210> 13
<211> 180
<212> RNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 13
cagugcuaga ggaggucaga agagggcauu ggauccccca gaacuggagu uauacgcuaa 60
ccucguggug guugugaacc accaugugga uggauauuga guuccaaaca cugguccugu 120
gcaagagcau ccagugcucu uaagugcuga gccaucucuu uagcuccagu cucuuaagcu 180
<210> 14
<211> 180
<212> RNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 14
cagugcuaga ggaggucaga agagggcauu ggauccccca gaacuggcgu uauacgguaa 60
ccucguggug guugugaacc accaugugga uggauauuga guuccaaaca cugguccugu 120
gcaagagcau ccagugcucu uaagugcuga gccaucucuu uagcuccagu cucuuaagcu 180
<210> 15
<211> 180
<212> RNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 15
cagugcuaga ggaggucaga agagggcauu ggauccccca gaaguggagu uauacgguaa 60
ccucguggug guugugaacc accaugugga uggauauuga guuccaaaca cugcuccugu 120
gcaagagcau ccagugcucu uaagugcuga gccaucucuu uagcuccagu cucuuaagcu 180
<210> 16
<211> 180
<212> RNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 16
cagugcuaga ggaggucaga agagggcauu ggauccccca gauggugagu uauacgguaa 60
ccucguggug guugugaacc accaugugga uggauauuga guuccaaaca cgucaccugu 120
gcaagagcau ccagugcucu uaagugcuga gccaucucuu uagcuccagu cucuuaagcu 180
<210> 17
<211> 180
<212> RNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 17
cagugcuaga ggaggucaga agagggcauu ggauccccca gaacugcacu auacgguaac 60
cucguggugg uugugaacca ccauguggau ggauauugag uuccaaauga gugguccugu 120
gcaagagcau ccagugcucu uaagugcuga gccaucucuu uagcuccagu cucuuaagcu 180
<210> 18
<211> 116
<212> RNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 18
gggcauugga ucccccagaa cuggaguuau acgguaaccu cguggugguu gugaaccacc 60
auguggaugg auauugaguu ccaaacacug guccugugca agagcaucca gugcuc 116
<210> 19
<211> 77
<212> RNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 19
ggacuggagu uauacgguaa ccucguggug guugugaacc accaugugga uggauauuga 60
guuccaaaca cuggucc 77
<210> 20
<211> 77
<212> RNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 20
gaacuggcgu uauacgguaa ccucguggug guugugaacc accaugugga uggauauuga 60
guuccaaaca cuggucc 77
<210> 21
<211> 107
<212> RNA
<213> mice (Mus musculus)
<400> 21
gaugccuuag aaguggaguu aagaguugug agcugccguu uuuugguucu gggacucgaa 60
cucguuuccu cugauacuau caaccaccaa gccaucucuu cagcccc 107
<210> 22
<211> 131
<212> RNA
<213> mice (Mus musculus)
<400> 22
gccagaagaa guugugggau ucccuggaac uggagcaacc aacaguuugu gugcaccaug 60
uggguaaugg gaaucgaacc uggguccucu auaagacugg ccagugcucu uaacuacuga 120
ggugcauuuc u 131
<210> 23
<211> 187
<212> RNA
<213> mice (Mus musculus)
<400> 23
uuauuuuaaa uauaugagua uuucaccugc auaggcgcac aguacccaca gagacuagaa 60
gaggguggca gaucuccuga gacuggaguu aaugcuugug agcugccaug uggaugcugg 120
aaaucaaacc cagguccuuu ggaaggcagg caggugcucu uaaucaugga agcaucucuu 180
cagcucc 187
<210> 24
<211> 131
<212> RNA
<213> mice (Mus musculus)
<400> 24
cagcgacauc agaagaggau auuggauccc auuacagaug guugaaggcc accaugucgu 60
ugcugggaau gaacucaaga ccucuggaag agcagucagu gcucuuaacc ucugagccau 120
cucuccagcc c 131
<210> 25
<211> 114
<212> RNA
<213> mice (Mus musculus)
<400> 25
auccccucca aagcucaaga ugguuguaag ccacccugug auugcuggga uuugaacuca 60
agaccuccgg aagagcaauu agugcucuua accgcugagc aaucucucca gccc 114
<210> 26
<211> 357
<212> RNA
<213> mice (Mus musculus)
<400> 26
gugcagugcu agaggagguc agaagagggc auuggauccc ccagaacugg aguuauacgg 60
uaaccucgug gugguuguga accaccaugu ggauggauau ugaguuccaa acacuggucc 120
ugugcaagag cauccagugc ucuuaagugc ugagccaucu cuuuagcucc uuauuuuaaa 180
uauaugagua uuucaccugc auaggcgcac aguacccaca gagacuagaa gaggguggca 240
gaucuccuga gacuggaguu aaugcuugug agcugccaug uggaugcugg aaaucaaacc 300
cagguccuuu ggaaggcagg caggugcucu uaaucaugga agcaucucuu cagcucc 357
<210> 27
<211> 532
<212> RNA
<213> mice (Mus musculus)
<400> 27
gugcagugcu agaggagguc agaagagggc auuggauccc ccagaacugg aguuauacgg 60
uaaccucgug gugguuguga accaccaugu ggauggauau ugaguuccaa acacuggucc 120
ugugcaagag cauccagugc ucuuaagugc ugagccaucu cuuuagcucc gugcgaauuc 180
ggugcagugc uagaggaggu cagaagaggg cauuggaucc cccagaacug gaguuauacg 240
guaaccucgu ggugguugug aaccaccaug uggauggaua uugaguucca aacacugguc 300
cugugcaaga gcauccagug cucuuaagug cugagccauc ucuuuagcuc cgugcgaauu 360
cggugcagug cuagaggagg ucagaagagg gcauuggauc ccccagaacu ggaguuauac 420
gguaaccucg uggugguugu gaaccaccau guggauggau auugaguucc aaacacuggu 480
ccugugcaag agcauccagu gcucuuaagu gcugagccau cucuuuagcu cc 532
<210> 28
<211> 228
<212> RNA
<213> mice (Mus musculus)
<400> 28
uuuuuuuaaa aauuuauuuu uauuuuaugu guaugagugu uuugccugca uguaugucug 60
uguaccacgu gcgugccugg ugcccgcgga ggccagaaga gggcgucgga uccccuggaa 120
cuggaguuac agaugguugu gagccgccau gugggugcug ggaaucgaac ccggguccuc 180
uggaagagca gccagugcuc uuaaccgcug agccaucucu ccagcccc 228
<210> 29
<211> 214
<212> RNA
<213> mice (Mus musculus)
<400> 29
uuuuuuuuac uuguauaggu guuuugccug cauguguauc uaucuaugua ccgaauaugu 60
uccugguauc cacagagacc aaaaguggau guuguaucuc cugaaauugg agucauagac 120
aguuaugagc ugccauuuga gugcuuggaa uagaacccag guccucuuaa agagcaucca 180
gugcucuuaa aaacugagac aucucuguag ccuc 214
<210> 30
<211> 200
<212> RNA
<213> mice (Mus musculus)
<400> 30
uuuauuuugc uuuauguguc ugaguguuug cuugaaugua ugucugugua ccacgccugu 60
accuugugcc uucagaguug agaggagggc auaggaucuc cuggaacugg aauugcaggu 120
gguugugagc cacccugugg guccugggga ccauacucca gcaagaacau caugugcucu 180
uaauuccuga gucuccaacc 200
<210> 31
<211> 214
<212> RNA
<213> mice (Mus musculus)
<400> 31
uuuauuuacu uaucuuuaug uguaugagug uguugucaga cuguuauguc ugugugucac 60
augcaugccu gcuguucaug gaguccagaa gagggcaucg gauccccugg aacuggaguu 120
acagaugagu ggccauguga auguuaagaa ccaaaccugg guccucugaa agagcagaca 180
augcucuuaa cuacugagcu gucucuccag cccc 214
<210> 32
<211> 205
<212> RNA
<213> mice (Mus musculus)
<400> 32
uuauuuuauu cguguaagug uuuugccagc aucuaugucu ucgcacuaug ugcaggucug 60
gugccugagg gguccagacg agagcacugg gucuccggga acuggaguua cagaucauug 120
ugagccacca ugugggugca gggaaucgaa ccugggaccu cuggaggagc agccacugcu 180
cuuaaccacu acacuauuuc uccag 205
<210> 33
<211> 121
<212> RNA
<213> mice (Mus musculus)
<400> 33
ucuguggacc acuguguaca gaagccugag aaggcuagca gauccccaga acuggaacug 60
ugagacgcug ugcuauggag gugcuaggaa cugaaaaugg auggguccuc ugcaagagca 120
g 121
<210> 34
<211> 191
<212> RNA
<213> mice (Mus musculus)
<400> 34
uuguuuuaau ugaauggcua uaggguguuu cuucuguaug uauaucuaug uuugguaccu 60
acagaggcau cagauccucu ggaacuguag uugcugacag uugugagcug ucauggggau 120
gcuggaauug aaccuggauc cuaugaaaga acagccagug uucuuaaccg cugagcuauc 180
ucuccaggcc c 191
<210> 35
<211> 205
<212> RNA
<213> mice (Mus musculus)
<400> 35
uuuuuuuuuu aauuuuaaaa aaaaagauuu uauuuauuua uuuuauauau gaugaguaca 60
cugucacucu uuucagacac ccuagaaaag gggggcauca gaucccauua cagaugguug 120
ugagccacau gguugcuggg aauugaccuc aggaccucug aaagagcagu cagugcucuc 180
aaccuuugag ucaucucucc agccc 205
<210> 36
<211> 190
<212> RNA
<213> mice (Mus musculus)
<400> 36
auguauaucu guaaugggac auacucacau acaugggcac gugaguauaa aaggccagaa 60
gagagcacug gacccucugg aguugagauu cuaagcaguu gugaaccauc ugauguaggu 120
gcugggaacu gaacuugggu ccuuugcuag agaaguaugu cucuuaacca cugagccgua 180
ucuccauccc 190
<210> 37
<211> 169
<212> RNA
<213> mice (Mus musculus)
<400> 37
uaaagauuua uucauuaagu acacuguagc uaucuucaga cgcaucagaa gagggcguca 60
gaucucuuua caggugguug ugagccacca ugugguugcu ggaauuugaa cucaggaccu 120
ucaaaagagc agucaguguu cuuaaccgcu gagccaucuc uccaacccc 169
<210> 38
<211> 159
<212> RNA
<213> mice (Mus musculus)
<400> 38
uuauuuauua uaaguacacu guagcugucu ucagacacaa caaaagaggg cgucagaucu 60
cauuacaggu gguugagcca ccaugugguu gcugggauuu gaacucagga ccuucagaac 120
agucagugcu cuuacccacu gagccagcga gccagcccc 159
<210> 39
<211> 77
<212> RNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 39
gaacuggagu uauacgguaa ccucguggug guugggaacc accaugugga uggauauuga 60
guuccaaaca cuggucc 77
<210> 40
<211> 77
<212> RNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 40
gaacuggagu uauacgguaa ccucguggug guucccaacc accaugugga uggauauuga 60
guuccaaaca cuggucc 77
<210> 41
<211> 77
<212> RNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 41
ggaccggagu uauacgguaa ccgcguggug guugugaacc accacgcgga uggauauuga 60
guuccaaaca ccggucc 77
<210> 42
<211> 77
<212> RNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 42
gaacuagagu uauacgguaa ccacauggug guugugaacc accaugugga uggauauuga 60
guuccaaaca cuaguuc 77
<210> 43
<211> 206
<212> RNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 43
uuauuuuaaa uauaugagua uuucaccugc auaggcgcac aguacccaca gagacuagaa 60
gaggguagua gauccccuag aacuggaguu auacgguaac cucguggugg uugugagcua 120
ccauguggau ggauacuggg aaucaaaccc agguccugug gaaggcaggc aggugcucuc 180
aagcacugag ccaucucuuc agcucc 206
<210> 44
<211> 206
<212> RNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 44
uuauuuuaaa uauaugagua uuucaccugc auaggcgcac agugcucaag gagaucagaa 60
gagggcauca gaucuccuga gacuggaguu auacgguaac cucgugaugg uugugaacua 120
ccauguggau ggauauugag uuccaaacac agguccugug caagagcagc aggugcucuu 180
aagcacggaa ccaucucuuu agcucc 206
<210> 45
<211> 156
<212> RNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 45
gaggcuagaa gaggguauca gauccccuga gacuggaguu auacgguaac cucguggugg 60
uugugagcca ccauguggau ggauacugag aaccaaaccc ugguccugug caagagcauc 120
aggugcucuu aagcacggaa ccaucucuuc agcucc 156
<210> 46
<211> 68
<212> RNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 46
guccugugca agagcaucga acucggugcu cuuaagcaca gaagccacca agccaucucu 60
ucagcccc 68
<210> 47
<211> 110
<212> RNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 47
cagugcuaga ggaggucaga agagggcauc ccccagccuc guggugguug ugaaccacca 60
uguggcugug caagagcaug cucuuaagug cugagccauc ucuuuagcuc 110
<210> 48
<211> 126
<212> RNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 48
gagggcauug gaucccccag aacuggaguu auacgguaac cucguggugg uugugaacca 60
ccauguggau ggauauugag uuccaaacac ugguccugug caagagcauc cagugcucuu 120
aagugc 126
<210> 49
<211> 89
<212> RNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 49
ggauccccca gaacuggagu uauacgguaa ccucguggug guugugaacc accaugugga 60
uggauauuga guuccaaaca cugguccug 89
<210> 50
<211> 72
<212> RNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 50
ugcuagagga ggucagaaga gggcauugga ugcaaaucca gugcucuuaa gugcugagcc 60
aucucuuuag cu 72
<210> 51
<211> 94
<212> RNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 51
gagggcauug gaucccccag aacuggaguu auacgguaac gauggauauu gaguuccaaa 60
cacugguccu gugcaagagc auccagugcu cuua 94
<210> 52
<211> 383
<212> RNA
<213> hepatitis C Virus
<400> 52
gccagccccc ugaugggggc gacacuccac caugaaucac uccccuguga ggaacuacug 60
ucuucacgca gaaagcgucu agccauggcg uuaguaugag ugucgugcag ccuccaggac 120
ccccccuccc gggagagcca uaguggucug cggaaccggu gaguacaccg gaauugccag 180
gacgaccggg uccuuucuug gauaaacccg cucaaugccu ggagauuugg gcgugccccc 240
gcaagacugc uagccgagua guguuggguc gcgaaaggcc uugugguacu gccugauagg 300
gugcuugcga gugccccggg aggucucgua gaccgugcac caugagcacg aauccuaaac 360
cucaaagaaa aaccaaacgu aac 383
<210> 53
<211> 383
<212> RNA
<213> hepatitis C Virus
<400> 53
guuacguuug guuuuucuuu gagguuuagg auucgugcuc auggugcacg gucuacgaga 60
ccucccgggg cacucgcaag cacccuauca ggcaguacca caaggccuuu cgcgacccaa 120
cacuacucgg cuagcagucu ugcgggggca cgcccaaauc uccaggcauu gagcggguuu 180
auccaagaaa ggacccgguc guccuggcaa uuccggugua cucaccgguu ccgcagacca 240
cuauggcucu cccgggaggg gggguccugg aggcugcacg acacucauac uaacgccaug 300
gcuagacgcu uucugcguga agacaguagu uccucacagg ggagugauuc augguggagu 360
gucgccccca ucagggggcu ggc 383
<210> 54
<211> 312
<212> RNA
<213> human poliovirus
<400> 54
augagucugg acaucccuca ccggugacgg ugguccaggc ugcguuggcg gccuaccuau 60
ggcuaacgcc augggacgcu aguugugaac aaggugugaa gagccuauug agcuacauaa 120
gaauccuccg gccccugaau gcggcuaauc ccaaccucgg agcagguggu cacaaaccag 180
ugauuggccu gucguaacgc gcaaguccgu ggcggaaccg acuacuuugg guguccgugu 240
uuccuuuuau uuuauugugg cugcuuaugg ugacaaucac agauuguuau cauaaagcga 300
auuggauugg cc 312
<210> 55
<211> 312
<212> RNA
<213> human poliovirus
<400> 55
ggccaaucca auucgcuuua ugauaacaau cugugauugu caccauaagc agccacaaua 60
aaauaaaagg aaacacggac acccaaagua gucgguuccg ccacggacuu gcgcguuacg 120
acaggccaau cacugguuug ugaccaccug cuccgagguu gggauuagcc gcauucaggg 180
gccggaggau ucuuauguag cucaauaggc ucuucacacc uuguucacaa cuagcguccc 240
auggcguuag ccauagguag gccgccaacg cagccuggac caccgucacc ggugagggau 300
guccagacuc au 312
<210> 56
<211> 576
<212> RNA
<213> encephalomyocarditis Virus
<400> 56
cccccccucu cccucccccc ccccuaacgu uacuggccga agccgcuugg aauaaggccg 60
gugugcguuu gucuauaugu uauuuuccac cauauugccg ucuuuuggca augugagggc 120
ccggaaaccu ggcccugucu ucuugacgag cauuccuagg ggucuuuccc cucucgccaa 180
aggaaugcaa ggucuguuga augucgugaa ggaagcaguu ccucuggaag cuucuugaag 240
acaaacaacg ucuguagcga cccuuugcag gcagcggaac cccccaccug gcgacaggug 300
ccucugcggc caaaagccac guguauaaga uacaccugca aaggcggcac aaccccagug 360
ccacguugug aguuggauag uuguggaaag agucaaaugg cucuccucaa gcguauucaa 420
caaggggcug aaggaugccc agaagguacc ccauuguaug ggaucugauc uggggccucg 480
gugcacaugc uuuacaugug uuuagucgag guuaaaaaac gucuaggccc cccgaaccac 540
ggggacgugg uuuuccuuug aaaaacacga ugauaa 576
<210> 57
<211> 576
<212> RNA
<213> encephalomyocarditis Virus
<400> 57
uuaucaucgu guuuuucaaa ggaaaaccac guccccgugg uucggggggc cuagacguuu 60
uuuaaccucg acuaaacaca uguaaagcau gugcaccgag gccccagauc agaucccaua 120
caauggggua ccuucugggc auccuucagc cccuuguuga auacgcuuga ggagagccau 180
uugacucuuu ccacaacuau ccaacucaca acguggcacu gggguugugc cgccuuugca 240
gguguaucuu auacacgugg cuuuuggccg cagaggcacc ugucgccagg ugggggguuc 300
cgcugccugc aaagggucgc uacagacguu guuugucuuc aagaagcuuc cagaggaacu 360
gcuuccuuca cgacauucaa cagaccuugc auuccuuugg cgagagggga aagaccccua 420
ggaaugcucg ucaagaagac agggccaggu uuccgggccc ucacauugcc aaaagacggc 480
aauauggugg aaaauaacau auagacaaac gcacaccggc cuuauuccaa gcggcuucgg 540
ccaguaacgu uagggggggg ggagggagag gggggg 576
<210> 58
<211> 192
<212> RNA
<213> cricket paralysis virus
<400> 58
aaagcaaaaa ugugaucuug cuuguaaaua caauuuugag agguuaauaa auuacaagua 60
gugcuauuuu uguauuuagg uuagcuauuu agcuuuacgu uccaggaugc cuaguggcag 120
ccccacaaua uccaggaagc ccucucugcg guuuuucaga uuagguaguc gaaaaaccua 180
agaaauuuac cu 192
<210> 59
<211> 192
<212> RNA
<213> cricket paralysis virus
<400> 59
agguaaauuu cuuagguuuu ucgacuaccu aaucugaaaa accgcagaga gggcuuccug 60
gauauugugg ggcugccacu aggcauccug gaacguaaag cuaaauagcu aaccuaaaua 120
caaaaauagc acuacuugua auuuauuaac cucucaaaau uguauuuaca agcaagauca 180
cauuuuugcu uu 192
<210> 60
<211> 231
<212> RNA
<213> human (homosapiens)
<400> 60
cagagaucca ggggaggcgc cugugaggcc cggaccugcc ccggggcgaa ggguaugugg 60
cgagacagag cccugcaccc cuaauucccg guggaaaacu ccuguugccg uuucccucca 120
ccggccugga gucucccagu cuugucccgg cagugccgcc cuccccacua agaccuaggc 180
gcaaaggcuu ggcucauggu ugacagcuca gagagagaaa gaucugaggg a 231
<210> 61
<211> 231
<212> RNA
<213> human (homosapiens)
<400> 61
ucccucagau cuuucucucu cugagcuguc aaccaugagc caagccuuug cgccuagguc 60
uuagugggga gggcggcacu gccgggacaa gacugggaga cuccaggccg guggagggaa 120
acggcaacag gaguuuucca ccgggaauua ggggugcagg gcucugucuc gccacauacc 180
cuucgccccg gggcaggucc gggccucaca ggcgccuccc cuggaucucu g 231
<210> 62
<211> 460
<212> RNA
<213> human (homosapiens)
<400> 62
acuuuuggug ggcauuuaaa aaugugugug uauguguaua uauguaugug uauguaugug 60
uauauaugua uauguaugua uguaucgcgu guaugugugu auguaugcau guguauguau 120
guauaugcau guauguguau guguauauau guaugugugu guauguauau guguguguau 180
guguaugugu guguguaugu guguguguau guauguaugu auguauaugu auuauacaca 240
uauacacaua uugguuuuuu uaaucauuug agaguuaguu gaagauaaaa acccaucacc 300
ccuaaaugua uuccaaagaa uaagaacauu guuuuauaca uagcacacuu aacaaaauca 360
agaaauuuaa cauuaauaca guacuguuac cuaauccgua gucgauuuuc aaauuuuguc 420
aguuguucca auaauguccu uuauauauuc cccgcccagc 460
<210> 63
<211> 460
<212> RNA
<213> human (homosapiens)
<400> 63
gcugggcggg gaauauauaa aggacauuau uggaacaacu gacaaaauuu gaaaaucgac 60
uacggauuag guaacaguac uguauuaaug uuaaauuucu ugauuuuguu aagugugcua 120
uguauaaaac aauguucuua uucuuuggaa uacauuuagg ggugaugggu uuuuaucuuc 180
aacuaacucu caaaugauua aaaaaaccaa uauguguaua uguguauaau acauauacau 240
acauacauac auacacacac acauacacac acacauacac auacacacac auauacauac 300
acacacauac auauauacac auacacauac augcauauac auacauacac augcauacau 360
acacacauac acgcgauaca uacauacaua uacauauaua cacauacaua cacauacaua 420
uauacacaua cacacacauu uuuaaaugcc caccaaaagu 460
<210> 64
<211> 395
<212> RNA
<213> human (homosapiens)
<400> 64
aauuccagcg agaggcagag ggagcgagcg ggcggccggc uaggguggaa gagccgggcg 60
agcagagcug cgcugcgggc guccugggaa gggagauccg gagcgaauag ggggcuucgc 120
cucuggccca gcccucccgc uugauccccc aggccagcgg uccgcaaccc uugccgcauc 180
cacgaaacuu ugcccauagc agcgggcggg cacuuugcac uggaacuuac aacacccgag 240
caaggacgcg acucucccga cgcggggagg cuauucugcc cauuugggga cacuuccccg 300
ccgcugccag gacccgcuuc ucugaaaggc ucuccuugca gcugcuuaga cgcuggauuu 360
uuuucgggua guggaaaacc agcagccucc cgcga 395
<210> 65
<211> 395
<212> RNA
<213> human (homosapiens)
<400> 65
ucgcgggagg cugcugguuu uccacuaccc gaaaaaaauc cagcgucuaa gcagcugcaa 60
ggagagccuu ucagagaagc ggguccuggc agcggcgggg aagugucccc aaaugggcag 120
aauagccucc ccgcgucggg agagucgcgu ccuugcucgg guguuguaag uuccagugca 180
aagugcccgc ccgcugcuau gggcaaaguu ucguggaugc ggcaaggguu gcggaccgcu 240
ggccuggggg aucaagcggg agggcugggc cagaggcgaa gcccccuauu cgcuccggau 300
cucccuuccc aggacgcccg cagcgcagcu cugcucgccc ggcucuucca cccuagccgg 360
ccgcccgcuc gcucccucug ccucucgcug gaauu 395
<210> 66
<211> 48
<212> RNA
<213> human (homosapiens)
<400> 66
gggcacuuug cacuggaacu uacaacaccc gagcaaggac gcgacucu 48
<210> 67
<211> 48
<212> RNA
<213> human (homosapiens)
<400> 67
agagucgcgu ccuugcucgg guguuguaag uuccagugca aagugccc 48
<210> 68
<211> 71
<212> RNA
<213> human (homosapiens)
<400> 68
guacugacau cguagaugga aaucauaaac ugacucuugg uuugauuugg aauauaaucc 60
uccacuggca g 71
<210> 69
<211> 71
<212> RNA
<213> human (homosapiens)
<400> 69
cugccagugg aggauuauau uccaaaucaa accaagaguc aguuuaugau uuccaucuac 60
gaugucagua c 71
<210> 70
<211> 232
<212> RNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 70
acaucccgcc ggcggggagg ucacgcaggc gccgagacgg ccacaucugc agaauuccag 60
ugcuagagga ggucagaaga gggcauugga ucccccagaa cuggaguuau acgguaaccu 120
cguggugguu gugaaccacc auguggaugg auauugaguu ccaaacacug guccugugca 180
agagcaucca gugcucuuaa gugcugagcc aucucuuuag cuccagucuc uu 232
<210> 71
<211> 206
<212> RNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 71
acaucccgcc ggcggggaau cugcagaauu ccagugcuag aggaggucag aagagggcau 60
uggauccccc agaacuggag uuauacggua accucguggu gguugugaac caccaugugg 120
auggauauug aguuccaaac acugguccug ugcaagagca uccagugcuc uuaagugcug 180
agccaucucu uuagcuccag ucucuu 206
<210> 72
<211> 199
<212> RNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 72
acaucccgcc ggcggggaga auuccagugc uagaggaggu cagaagaggg cauuggaucc 60
cccagaacug gaguuauacg guaaccucgu ggugguugug aaccaccaug uggauggaua 120
uugaguucca aacacugguc cugugcaaga gcauccagug cucuuaagug cugagccauc 180
ucuuuagcuc cagucucuu 199
<210> 73
<211> 232
<212> RNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 73
ucauaucuuu ccacugauca aaagucuuuu uggcugugua gccaaucugc agaauuccag 60
ugcuagagga ggucagaaga gggcauugga ucccccagaa cuggaguuau acgguaaccu 120
cguggugguu gugaaccacc auguggaugg auauugaguu ccaaacacug guccugugca 180
agagcaucca gugcucuuaa gugcugagcc aucucuuuag cuccagucuc uu 232
<210> 74
<211> 206
<212> RNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 74
ucauaucuuu ccacugauau cugcagaauu ccagugcuag aggaggucag aagagggcau 60
uggauccccc agaacuggag uuauacggua accucguggu gguugugaac caccaugugg 120
auggauauug aguuccaaac acugguccug ugcaagagca uccagugcuc uuaagugcug 180
agccaucucu uuagcuccag ucucuu 206
<210> 75
<211> 232
<212> RNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 75
acaucccgcc agagcagacc acacacaggc gcugagacgg ccacaucugc agaauuccag 60
ugcuagagga ggucagaaga gggcauugga ucccccagaa cuggaguuau acgguaaccu 120
cguggugguu gugaaccacc auguggaugg auauugaguu ccaaacacug guccugugca 180
agagcaucca gugcucuuaa gugcugagcc aucucuuuag cuccagucuc uu 232
<210> 76
<211> 206
<212> RNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 76
acaucccgcc agagcagaau cugcagaauu ccagugcuag aggaggucag aagagggcau 60
uggauccccc agaacuggag uuauacggua accucguggu gguugugaac caccaugugg 120
auggauauug aguuccaaac acugguccug ugcaagagca uccagugcuc uuaagugcug 180
agccaucucu uuagcuccag ucucuu 206
<210> 77
<211> 233
<212> RNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 77
ucauaucuuu ccauucaucg aagguuuuuu uggcuguaua gccacaucug cagaauucca 60
gugcuagagg aggucagaag agggcauugg aucccccaga acuggaguua uacgguaacc 120
ucgugguggu ugugaaccac cauguggaug gauauugagu uccaaacacu gguccugugc 180
aagagcaucc agugcucuua agugcugagc caucucuuua gcuccagucu cuu 233
<210> 78
<211> 114
<212> RNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 78
acaucccgcc ggcggggaau cugcagaauu cgcccuugaa cuggaguuau acgguaaccu 60
cguggugguu gugaaccacc auguggaugg auauugaguu ccaaacacug gucc 114
<210> 79
<211> 66
<212> RNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 79
acaucccgcc ggcggggaau cugcagaauu cgcccuuccu cguggugguu gugaaccacc 60
augugg 66
<210> 80
<211> 13
<212> RNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 80
aucugcagaa uuc 13
<210> 81
<211> 6
<212> RNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 81
gaauuc 6
<210> 82
<211> 18
<212> DNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 82
acatcccgcc ggcgggga 18
<210> 83
<211> 15
<212> DNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 83
tcgccacatc ccgcc 15
<210> 84
<211> 12
<212> DNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 84
gagacggcca cg 12
<210> 85
<211> 14
<212> DNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 85
aatgacccag gaag 14
<210> 86
<211> 18
<212> DNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 86
cccgccggcg gggaggtc 18
<210> 87
<211> 22
<212> DNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 87
acatcccgcc ggcggggagg tc 22
<210> 88
<211> 14
<212> DNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 88
cccgccggcg ggga 14
<210> 89
<211> 17
<212> DNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 89
ggtatcatat ctttcca 17
<210> 90
<211> 18
<212> DNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 90
atctttccac tgatcaaa 18
<210> 91
<211> 22
<212> DNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 91
tcatatcttt ccactgatca aa 22
<210> 92
<211> 14
<212> DNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 92
atctttccac tgat 14
<210> 93
<211> 12
<212> DNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 93
gagacggcca cg 12
<210> 94
<211> 44
<212> DNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 94
acatcccgcc ggcggggagg tcacgcaggc gccgagacgg ccac 44
<210> 95
<211> 44
<212> DNA
<213> artificial sequence
<220>
<223> Artificial sequence
<400> 95
tcatatcttt ccactgatca aaagtctttt tggctgtgta gcca 44
<210> 96
<211> 18
<212> DNA
<213> artificial sequence
<220>
<223> M125 -14/+4
<400> 96
tcatatcttt ccactgat 18
Claims (20)
1. A functional nucleic acid molecule comprising:
-at least one target binding sequence comprising a sequence that is reverse complementary to an OPA1 mRNA sequence; and
-at least one regulatory sequence comprising an RNA comprising a sineb 2 element or a functionally active fragment of a sineb 2 element or an Internal Ribosome Entry Site (IRES) sequence or an IRES derivative sequence.
2. The functional nucleic acid molecule of claim 1, wherein the at least one regulatory sequence comprises a sequence that hybridizes to a sequence selected from the group consisting of SEQ ID NOs: 1-69 has a sequence having at least 75% sequence identity.
3. The functional nucleic acid molecule of claim 2, wherein the at least one regulatory sequence comprises a sequence that hybridizes to a sequence selected from the group consisting of SEQ ID NOs: 1-69 has a sequence having at least 90% sequence identity.
4. The functional nucleic acid molecule of any one of claims 1 to 3, wherein the at least one target binding sequence comprises a sequence that is reverse-complementary to a portion of an OPA1 mRNA sequence that is common to all OPA1 isoforms.
5. The functional nucleic acid molecule of any one of claims 1 to 4, wherein the at least one target binding sequence is at least 10 nucleotides in length and comprises from 3 'to 5':
-a sequence reverse complementary to 0 to 50 nucleotides of the 5 'untranslated region (5' utr) of said OPA1 mRNA sequence and 0 to 40 nucleotides of the coding sequence (CDS); or (b)
-a sequence reverse complementary to 0 to 80 nucleotides of the region upstream of the AUG site (start codon) of said OPA1 mRNA and 0 to 40 nucleotides of the CDS of said OPA1 mRNA sequence downstream of said AUG site.
6. The functional nucleic acid molecule of claim 5, wherein the at least one target binding sequence is at least 14 nucleotides in length and comprises from 3 'to 5':
-a sequence reverse complementary to 0 to 40 nucleotides of the 5' utr of said OPA1 mRNA sequence and 0 to 32 nucleotides of CDS; or (b)
-a sequence reverse complementary to 0 to 70 nucleotides of the region upstream of the AUG site (start codon) of the OPA1 mRNA and 0 to 4 nucleotides of the CDS of the OPA1 mRNA sequence downstream of the AUG site.
7. The functional nucleic acid molecule of any one of claims 1 to 6, further comprising at least one linker sequence located between the at least one target binding sequence and the at least one regulatory sequence.
8. The functional nucleic acid molecule of any one of claims 1 to 7, wherein the molecule is circular.
9. A DNA molecule encoding the functional nucleic acid molecule according to any one of claims 1 to 8.
10. An expression vector comprising the functional nucleic acid molecule according to any one of claims 1 to 8 or the DNA molecule according to claim 9.
11. A composition comprising the functional nucleic acid molecule of any one of claims 1 to 8, the DNA molecule of claim 9, or the expression vector of claim 10.
12. A method for increasing the protein synthesis efficiency of OPA1 in a cell, comprising administering to the cell the functional nucleic acid molecule of any one of claims 1 to 8, the DNA molecule of claim 9, the expression vector of claim 10 or the composition of claim 11.
13. The method of claim 12, wherein the functional nucleic acid molecule is administered as naked RNA.
14. The method of claim 12 or claim 13, wherein the cells are OPA1 single dose deficient.
15. Use of the functional nucleic acid molecule according to any one of claims 1 to 8, the DNA molecule according to claim 9, the expression vector according to claim 10 or the composition according to claim 11 as a medicament.
16. Use of a functional nucleic acid molecule according to any one of claims 1 to 8, a DNA molecule according to claim 9, an expression vector according to claim 10 or a composition according to claim 11 for the treatment of a mitochondrial defect related disease.
17. A method of treating a disease associated with mitochondrial defects comprising administering a therapeutically effective amount of the functional nucleic acid molecule of any one of claims 1 to 8, the DNA molecule of claim 9, the expression vector of claim 10, or the composition of claim 11.
18. The method of claim 17, wherein the therapeutically effective amount is administered in the retina, brain, or heart.
19. Use of a therapeutically effective amount of a functional nucleic acid molecule according to any one of claims 1 to 8, a DNA molecule according to claim 9, an expression vector according to claim 10 or a composition according to claim 11 for the manufacture of a medicament for the treatment of a disease associated with mitochondrial defects.
20. The functional nucleic acid molecule, DNA molecule, expression vector or composition for use according to claim 16, the method according to claim 17 or claim 18 or the therapeutically effective amount according to claim 19, wherein the mitochondrial defect related disease is ADOA.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB2015997.6 | 2020-10-08 | ||
| GB2019325.6 | 2020-12-08 | ||
| GBGB2019325.6A GB202019325D0 (en) | 2020-12-08 | 2020-12-08 | Functional nucleic acid molecules |
| PCT/GB2021/052607 WO2022074396A1 (en) | 2020-10-08 | 2021-10-08 | Functional nucleic acid molecules |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116568311A true CN116568311A (en) | 2023-08-08 |
Family
ID=74165790
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202180068542.0A Pending CN116568311A (en) | 2020-10-08 | 2021-10-08 | Functional nucleic acid molecules |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN116568311A (en) |
| GB (1) | GB202019325D0 (en) |
-
2020
- 2020-12-08 GB GBGB2019325.6A patent/GB202019325D0/en not_active Ceased
-
2021
- 2021-10-08 CN CN202180068542.0A patent/CN116568311A/en active Pending
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| Publication number | Publication date |
|---|---|
| GB202019325D0 (en) | 2021-01-20 |
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