EP4532715A1 - Functional nucleic acid molecule - Google Patents

Abstract

The present invention relates to circular functional nucleic acid molecules comprising one or more target binding sequences and a regulatory sequence comprising an internal ribosome entry site (IRES). The invention also encompasses linear functional nucleic acid molecules comprising one or more target binding sequences and a regulatory sequence comprising an IRES. Also included are methods of enhancing protein translation efficiency, and methods of treating gene defects using the circular functional nucleic acid molecules and linear nucleic acid molecules of the invention.

Description

FUNCTIONAL NUCLEIC ACID MOLECULE

FIELD OF THE INVENTION

The present invention relates to a circularfunctional nucleic acid molecule comprising one or more target binding sequences and a regulatory sequence comprising an internal ribosome entry site (IRES). The invention also encompasses a linear functional nucleic acid molecule comprising one or more target binding sequences and a regulatory sequence comprising an IRES. Also included are methods of enhancing protein translation efficiency, and methods of treating gene defects using the circular and linear functional nucleic acid molecules of the invention.

BACKGROUND OF THE INVENTION

SINEUPs are antisense long non-coding RNAs (IncRNAs) that increase translation of overlapping mRNAs through the activity of two domains: a SINEB2 sequence which up- regulates translation (Effector Domain, ED) and an antisense region providing target specificity (Binding Domain, BD).

Natural SINEUPs are a functional class of antisense IncRNAs that promote translation of their sense protein encoding genes. They are generated from genomic loci with partially overlapping sense/antisense transcript pairs organized in a head-to-head conformation.

SINEUP activity depends on the combination of two RNA domains: an overlapping antisense region, defined as the Binding Domain (BD), which drives SINEUP specificity, and a SINEB2 element, which is often in inverted orientation (invSINEB2), and acts as an Effector Domain (ED), determining the up-regulation of target m RNA translation. A representative member of natural SINEUPs is AS Uchl1 , a IncRNA antisense to the mouse orthologue of the human ubiquitin C-terminal hydrolase L1 (Uchl1) gene. When overexpressed, AS Uchl1 increases UchL1 protein expression without affecting its m RNA levels. This activity requires the concomitant presence of ED and BD RNA sequences. Upon stress, AS Uchl1 promotes the association of Uchl1 mRNA to heavy polysomes and consequent increased UCHL1 protein expression.

An artificial SINEUP can be synthesized by designing BD sequences antisense to an mRNA of interest in order to redirect AS Uchl1 activity to target ectopically expressed transcripts or endogenous mRNAs. The target site (TS) is usually located within the 5’ untranslated region (5’UTR) of the m RNA and can include the AUG translation initiation site.

SINEUPs can increase expression of the protein encoded by a target mRNA by around 1.5 to 3 fold, making SINEUPs an ideal tool to increase target protein levels in vivo within a normal physiological range.

The molecular mechanism of natural and synthetic SINEUP activity remains somewhat unclear. However, it is clear that SINEUPs allow rapid and efficient changes in protein levels, such as for global repression of protein synthesis during cellular stress and activation of stress-responsive protein translation.

IRESs were first discovered as complex structures in the 5’UTRs of picornavirus transcripts and were later found to occur in other viral and cellular mRNAs. IRESs can interact with translation initiation factors or directly with the small ribosomal subunit leading to ribosomal positioning at or near the initiation codon and promoting translation initiation. Their activity is regulated by IRES trans-acting factors (ITAFs) that are RNA-binding proteins (RBPs). IRESs have also been found as short sequence elements that can form base pairs with ribosomal RNA (rRNA), similarto Shine-Dalgamo sequences in bacterial translation initiation.

The inventors have previously shown that the invSINEB2 sequence from the natural SINEUP AS Uchl1 RNA exhibits the functions of an Internal Ribosomal Entry Site (IRES), and that viral and cellular IRES sequences can act as EDs in synthetic SINEUPs, promoting protein expression in trans.

The present invention seeks to optimise the role of IRES containing functional nucleic acids.

SUMMARY OF THE INVENTION

Recently, IRES sequences have been found in circular RNAs (circRNAs). CircRNAs are single-stranded, covalently closed RNA molecules produced from pre-m RNAs through a process called back-splicing. They were first observed in several viroid genomes and then visualized in the cytoplasm of HeLa cells with electron microscopy. With the advent of nextgeneration sequencing, thousands of circRNAs have been identified. They are regulated separately from their linear counterparts and are mostly conserved. CircRNAs can regulate protein expression through several mechanisms, such as binding to RBPs or by miRNA sponging. Without wishing to be bound by theory, it is envisaged that circularfunctional nucleic acids may be more resistant to degradation than their linear counterparts.

The inventors provide herein a circular functional nucleic acid molecule in which the molecular functionality of IRES sequences is retained when utilised as a trans- acting effector domain (ED), such that targeted translational upregulation of specific proteins is achieved when utilising IRES sequence EDs in conjunction with antisense target binding domains.

Further provided herein is a functional nucleic acid molecule wherein the nucleic acid molecule is linear and may act as a precursorto the circularfunctional nucleic acids.

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

(a) one or more target binding sequences comprising one or more sequences reverse complementary to one or more target mRNA sequences for which protein translation is to be enhanced; and

(b) a regulatory sequence comprising an internal ribosome entry site (IRES), wherein the functional nucleic acid molecule is circular.

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

(a) one or more target binding sequences comprising one or more sequences reverse complementary to one or more target mRNA sequences for which protein translation is to be enhanced; and

(b) a regulatory sequence comprising an internal ribosome entry site (IRES), wherein the functional nucleic acid molecule is linear and wherein the one or more target binding sequences and the regulatory sequence are positioned between two complementary intronic repeats.

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

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

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

According to a further aspect of the invention, there is provided use of the functional nucleic acid molecule, the expression vector or the composition, as defined herein, for enhancing translation of one or more target mR NA sequences.

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

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

According to a further aspect of the invention, there is provided a method of treating a disease associated with gene defects comprising administering the functional nucleic acid molecule, the DNA molecule, the expression vector, the composition or the pharmaceutical composition, as defined herein, to a subject.

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

According to a further aspect of the invention, there is provided an in vitro method for enhancing protein translation, comprising administering to a cell the functional nucleic acid molecule, the DNA molecule, the expression vector, the composition or the pharmaceutical composition, as defined herein, to a subject. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 - Circularised SINEUP RNA maintains its activity in trans

(a) Schematic representation of circularized SINEUP(IRES) RNA (CircUP). SyntheticCircUP(c-myc IRES)-GFP was generated using pcDNA3.1 (+)ZKSCAN MCS Exon circularizing vector. A synthetic SINEUP(c-myc IRES)-GFP comprising a BD (green) targeting GFP mRNA (-40/+4, green) and a c-myc IRES element as ED (light grey) was cloned into pcDNA3.1 (+)ZKSCAN MCS Exon vector in order to be circularized, (b) CircUP(c- myc IRES)-GFP activity. HEK293T cells were co-transfected with circRNA-expressing vector and pEGFP (to express GFP protein) in a 6:1 ratio. At 48 h after transfection, GFP protein fluorescence was measured and total RNA extracted. GFP fluorescence intensities were normalized on NucBlue signal. Empty pCDNA3.1 (+) was used as negative control (Ctrl=1 , dotted line) and linear miniSINEUP-GFP and miniSINEUP(c-myc IRES)-GFP were positive controls. CircUP(c-myc IRES)-GFP increased GFP protein levels more than the linear positive controls. Dot plot reports GFP fold induction mean ± SD; it represents 3 independent biological replicates; *p <0.05 was calculated with one-sample t-test. (c) GFP mRNA levels. RNAs were extracted, reverse transcribed and analyzed with real time PCR. GAPDH was used to normalize RNA expression. Empty pCDNA3.1 (-) was utilised as the negative control (Ctrl=1 , dotted line). GFP mRNA relative expression was quantified and plotted. No significant variations were observed, (d) SINEUP and circUP RNA. Expression of all transfected constructs was confirmed with real time PCR. All graphs indicate mean ± SD, and represent n=3 independent replicates.

Figure 2 - Circ5533 is an IRES containing circRNA transcribed from the c-myc locus and it affects cellular proteome upon overexpression.

(a) Schematic representation of circ5533. Circ5533 comprises an IRES sequence (light grey) as ED and an upstream region potentially acting as mono- or multi BD (yellow) to one or more endogenous target mRNAs. HEK293T cells were transfected with pcDNA3.1 (+)- ZKSCAN-circ5533 (N=6). Empty pcDNA3.1(+)-ZKSCAN was used as negative control. 48 h after transfection, cells were harvested; cell pellets were divided into two tubes in order to extract both proteins and RNA from the same sample, (b) Circ5533 acts as a circUP increasing target protein levels. Protein extract was analyzed by mass-spectrometry; resulting data were normalized assuming that the number of proteins that did not exhibit a change in level was larger than the number of proteins that did (MLR method). Foldinductions were calculated compared to the negative control. Volcano plot reports detected protein levels following circ5533 overexpression. The Iog2 fold change (FC), on the x-axis, indicates the mean expression level for each protein; the — Iog10 p-values are plotted on the y-axis. Dots on the left, with respect to log2FC=0 on the x-axis, represent downregulated proteins when circ5533 is expressed. Dots on the right are proteins whose levels increased. Red dots represent proteins whose fold inductions (FC>1.5) were validated through western blot, p <0.05 was considered significant, (c) RNA-seq volcano plot. Mean expression level for each RNA, detected with RNA-seq, was plotted on the x-axis as the log2FC. The — Iog10 p-values are plotted on the y-axis. Concerning log2FC=0, dots on the left represent downregulated RNAs, dots on the right represent upregulated RNAs. Red dots represent mRNA coding for proteins whose fold inductions (FC>1.5) were validated with western blot. mRNA levels of interest were validated with real time PCR. p <0.05 was considered significant.

Figure 3 - Circ5533 increases target protein levels without altering their mRNA levels, validation results

HEK293T cells were transfected with pcDNA3.1 (+) ZKSCAN1 MCS Exon circ5533 (n=6). Control cells were transfected with empty pcDNA3.1 (+) ZKSCAN1 MCS Exon Vector (as described in figure 2). (a) Mass spectrometry results validation. Total protein extract was analyzed with western blot, p-actin and GAPDH were used to normalize; fold-inductions were calculated relative to the empty pcDNA3.1 (+) ZKSCAN1 MCS Exon Vector. PXK, MFN, CHK1 , GUCY1 B1 Abs were used to detect levels of proteins of interest. circ5533 overexpression led to upregulation of all proteins of interest, (b) circ5533 target mRNA levels. Extracted RNAs were reverse-transcribed and analyzed with real time PCR. GAPDH was used to normalize. Empty pcDNA3.1 (+) ZKSCAN1 MCS Exon Vector was the negative control (Ctrl=1 , dotted line). Target mRNA relative expressions were quantified and reported on the graph. No significant variations were observed, (c) circ5533 RNA expression.

Overexpression of circ5533 was observed with qRT-PCR using specific divergent primers, normalized on GAPDH mRNA levels and compared to the empty control. The graph indicates mean ± standard deviation (n=6).

Figure 4 - Circ5533 induces PXK protein translation

Circ5533 increases natural PXK protein levels. HEK293T cells were transfected with pcDNA3.1 (+)-Laccase2-circ5533 (N=9). Empty pcDNA3.1 (+)-Laccase2 vector was the negative control. 48 h after transfection, cells were harvested and RNA and proteins extracted, (a) Western blot analysis was performed using anti-PXK Ab to detect PXK protein. PXK fold induction was calculated compared to the empty control (Ctrl=1 , dotted line), p-actin was used to normalize. The ability of circ5533 to increase PXK was thus confirmed. Circ5533 activity was plotted as mean fold induction values ± SD. **p <0.01 was considered significant (one-sample t-test). (b) Pxk mRNA expression. Total RNA was reverse transcribed and analyzed with real time PCR. GAPDH was used to normalize.

Empty pcDNA3.1 (+)-Laccase2 MCS Exon Vector was the negative control (Ctrl=1 , dotted line). Pxk mRNA relative expression was quantified and reported on the graph. No significant variations were observed, (c) Circ5533, from pcDNA3.1 (+)-Laccase 2 vector, RNA expression. Over-expression of circ5533 was observed with qRT-PCR using specific divergent primers, normalized on GAPDH mRNA levels and compared to empty pcDNA3.1 (+)-Laccase2 MCS Exon Vector. The graph indicates mean ± standard deviation (n=6).

Figure 5 - Circ5533 mutants circ5533 mutants were generated by deleting both BD1 and BD2 (circ5533 ABD1-2) or inverting their sequence orientations (circ5533 invBDI -2). HEK293T cells were transfected with circ5533 mutants (n=3). Empty pcDNA3.1 (+)-Laccase2 vector was the negative control while pcDNA3.1(+)-Laccase2-circ5533was the positive control. 48 h after transfection, cells were harvested and proteins and RNAs were extracted, (a) Western blot analysis was performed using anti-PXK Ab to detect PXK protein. PXK fold induction was calculated compared to the empty control (Ctrl= 1 , dotted line), p-actin was used to normalize. Neither circ5533 ABD1-2 nor circ5533 invBDI -2 induced PXK expression. Circ5533 mutant activities were plotted as mean fold-induction values ± SD. *p <0.05 was considered significant. One- sample t-test or one-way ANOVA were used to calculate significance, (b) Total RNA was reverse-transcribed and analyzed with real time PCR. GAPDH was used to normalize.

Empty pcDNA3.1 (+)-Laccase2 MCS Exon Vector was the negative control (Ctrl=1 , dotted line). Pxk mRNA relative expressions were quantified and reported on the graph. No significant variations were observed, (c) CircRNA expression. Overexpression of circRNAs were observed with qRT-PCR using specific divergent primers, normalized on GAPDH mRNA levels and compared to empty pcDNA3.1 (+)-Laccase2 MCS Exon Vector. Both circ5533 mutants were expressed at lower levels than WT circ5533. The graph indicates mean ± standard deviation (n=3). Figure 6 - Circ 5533 increases PXK protein expression through an IRES activity in trans

(a) Schematic design of putative Binding Domains (BDs) in circ5533 RNA. Putative BD1 (pink) and BD2 (blue) are located on the region upstream of the IRES sequence of circ5533 (dark yellow). BDs were identified by looking for the longest sequence pairing, in antisense orientation, between circ5533 RNA and Pxk mRNA; a minimal perfect match of 8nts was considered. BD1 can potentially target 3 different regions on the 5’UTR of Pxk mRNA, identified as target sites (TSs) 1 , 2 and 3 (pink). BD2 could pair with TS4 and/or TS5 (blue) on the 3’UTR of Pxk mRNA. Pxk CDS is highlighted in green.

(b) Circ5533 activity on PXK-FI_AG mutant fold inductions. Identified TSs were individually deleted from Pxk-flag RNA; each derived construct was co-transfected with circ5533 in HEK293T cells (1 :12). After 48 h, cells were harvested and proteins and RNA extracted. Empty pcDNA3.1 (+)-Laccase2 vector, co-transfected with different PXK-mutants, was used as negative control; PXK-FI-AG WT + circ5533was the positive control. Deletion of TS1 from Pxk 5’UTR or of TS4 from Pxk 3’UTR inhibited the circ5533-mediated increase in PXK- FI-AG protein. Anti-FI_AG Ab was used to detect ectopic PXK-FLAG. Fold inductions were calculated compared to each relative negative control (Ctrl=1 , dotted line), p-actin was used to normalize. Plots report PXK-FLAG fold induction mean ± SD; they represent at least 5 independent biological replicates; *p <0.05, **p <0.01 and ****p <0.0001 were analyzed with one-sample t-test.

(c) Representative western blot image and plots of circ5533 activity on PXK-FLAG mutants with only TS1 and/or TS4 at their 5’ and 3’UTRs respectively. UTR regions were completely removed from Pxk-flag mRNA. Four new Pxk-flag mutated RNAs were assembled: TS1 PXK-FLAG with only TS1 at the 5’UTR; PXK-FLAG TS4 with only TS4 at the 3’UTR; TS1 PXK-FLAG TS4 with TS1 at the 5’ and TS4 at the 3’UTRs; TS4 PXK-FLAG TS1 with TS4 at the 5’ and TS1 at the 3’UTRs. Empty pcDNA3.1 (+)-Laccase2 vector, co-transfected with different PXK-mutants, was used as negative control; PXK-FLAG WT co-transfected with circ5533 was the positive control. TS1 on Pxk 5’UTR was sufficient to allow circ5533- mediated upregulation of PXK-FLAG protein. TS4 at the 3’UTR restored the protein fold induction at lower levels than the positive control. Anti-FLAG Ab was used to detect PXK- FLAG. Fold inductions were calculated compared to each relative negative control (Ctrl=1 , dotted line), p-actin was used to normalize. Plots report PXK-FLAG fold induction mean ± SD; they represent 4 biological replicates; *p <0.05 and **p <0.01 were calculated with one- sample t-test. (d) ATSs Pxk mutant mRNA and TSs Pxk mutant mRNA. RNA was extracted, reverse- transcribed and analyzed with qRT-PCR. GAPDH was used to normalize. Empty pcDNA3.1 (+)-Laccase2 co-transfected with each Pxk mutant was the negative control (Ctrl=1 , dotted line). Pxk mRNA relative expressions were quantified and reported on the graph. No significant variations were observed, (e) CircRNA expression. Overexpression of circRNAs was observed with qRT-PCR using specific divergent primers, normalized on GAPDH mRNA levels and com pared to empty pcDNA3.1 (+)-Laccase2 MCS Exon Vector. Graph indicates mean ± standard deviation (n=3).

DETAILED DESCRIPTION OF THE INVENTION

It is an object of the present invention to provide a circularfunctional nucleic acid molecule comprising one or more target binding sequences and a regulatory sequence comprising an internal ribosome entry site (IRES). It is envisaged that the functional nucleic acid molecule will act post-transcriptionally to increase target protein levels. A functional nucleic acid molecule of the invention may be circular or linear. The linear functional nucleic acid molecule provided herein may constitute a precursor of the circularfunctional nucleic acid molecule of the invention.

The results provided herein demonstrate that IRESs can act as trans- acting translational up- regulators of protein expression within a circular functional nucleic acid. Utilising one or more target Binding Domains (BD), the functional nucleic acid molecule provided may be utilised for the targeted upregulation of proteins of interest without affecting mRNA levels. The functional nucleic acid molecule described herein may be used to enhance translation of a target mRNA sequence, such as a therapeutic target mRNA sequence which encodes a therapeutic target protein, without inducing negative side-effects associated with increasing expression of the target above physiological levels.

Functional Nucleic Acid Molecule

The “functional nucleic acid molecule” of the invention is a molecule as described herein. In particular, the term “functional nucleic acid molecule” describes a nucleic acid molecule (e.g. DNA or RNA) that is capable of enhancing translation of a target mRNA of interest.

The functional nucleic acid molecule of the invention is preferably an RNA molecule. The term “functional RNA molecule” refers to instances wherein the functional nucleic acid molecule is formed of RNA. The functional RNA molecule is generally capable of enhancing the translation of a target m RNA.

A “circular nucleic acid molecule”, such as a circular RNA (circRNA), is a nucleic acid molecule that adopts a ring structure by virtue of the covalent linkage of the 5’ and 3’ termini within the same molecule. Natural circRNAs are formed through the process of back-splicing of linear precursor (pre-) mRNAs. While synthetic circularfunctional nucleic acid molecules may be generated by back-splicing, their synthesis is not limited thereby. For exam pie, they may be constructed by routine molecular biology methods.

In some embodiments, the functional nucleic acid molecule of the present invention comprises one or more target binding sequences comprising one or more sequences reverse complementary to one or more target mRNA sequences; and a regulator sequence comprising an internal ribosome entry site (IRES), wherein the functional nucleic acid molecule is circular.

A circular functional nucleic acid molecule according to the invention may be more stable than a linear nucleic acid molecule as it may be less susceptible to degradation, e.g., since exonucleases cannot degrade circular molecules as they lack ‘free ends’. A circular functional nucleic acid molecule of the invention may therefore remain active for a longer time than a corresponding linear functional nucleic acid molecule. Thus, a circular functional nucleic acid molecule may exhibit prolonged activity due to a prolonged lifetime and therefore be advantageous over a conventional non-circular (i.e. linear) nucleic acid molecule which does not or cannot circularise. For example, it has been shown that circular RNAs have an average half-life that is more than 2.5 time longer than that of their linear counterparts (Enuka Y, et al. Circular RNAs are long-lived and display only minimal early alterations in response to a growth factor. Nucleic Acids Res. 2016;44(3): 1370-1383. doi:10.1093/nar/gkv1367). Enhanced stability of circular nucleic acid molecules over noncircular nucleic acid molecules may afford advantages in terms of lifetime, e.g., product lifetime, prior to any use, or increased lifetime in cellulo. Enhanced stability may allow a functional nucleic acid to remain active even when present in very small amounts. This can allow the administration of smaller amounts of functional nucleic acid, which may be particularly advantageous in a therapeutic setting.

In some embodiments a functional nucleic acid molecule of the present invention comprises one or more target binding sequences comprising one or more sequences reverse complementary to one or more target mRNA sequences; and a regulator sequence comprising an internal ribosome entry site (IRES), wherein the functional nucleic acid molecule is linear and wherein the one or more target binding sequences and the regulatory sequence are positioned between two complementary intronic repeats.

As used herein, the term “linear functional nucleic acid molecule” refers to a nucleic acid molecule that possesses two termini: a 5’- and 3’ -terminus. Such nucleic acids may generally be considered to adopt a linear structure.

The linear functional nucleic acid molecule described herein may be circularised to form a circularfunctional nucleic acid molecule of the invention.

The linear functional nucleic acid molecule of the invention comprises two complementary intronic repeats, within which the regulatory sequences (binding domain(s) and effector domain) are positioned. Intronic repeats are known to promote circRNA formation in cis, and are further regulated (in trans) by RNA binding proteins, such as splicing factors. Hence, the intronic repeats disclosed herein may promote the formation of the circularfunctional nucleic acid molecule of the invention.

Intronic repeats may be DNA or RNA sequences derived from a suitable source. For example, intronic repeats may com prise or consist of DNA or RNA sequences derived from one or more of circ5533; or the circularizing vectors pcDNA3.1 (+) ZKSCAN, or pcDNA3.1 (+) Laccase2 MCS Exon Vector.

The RNA ‘circ5533’ is endogenously expressed in eukaryotic cells. Complementary intronic repeats, such as those disclosed herein, are used for the over-expression of exogenous circ5533 through circularizing plasmids such as pcDNA3.1 (+)ZKSCAN or pcDNA3.1 (+) Laccase2 MCS Exon Vectors.

In one embodiment, the intronic repeats are derived from the pCDNA3.1 (+) ZKSCAN1 MCS Exon Vector and comprise or consist of SEQ ID NO: 21 (‘upstream 5533 insert’), or an RNA sequence encoded thereby.

In one embodiment, the intronic repeats are derived from the pCDNA3.1 (+) ZKSCAN1 MCS Exon Vector and comprise orconsist of SEQ ID NO: 22 (‘downstream 5533 insert’) or an RNA sequence encoded thereby. In one embodiment, the intronic repeats are derived from the pcDNA3.1 (+) Laccase2 MCS Exon Vector and comprise orconsist of SEQ ID NO: 23 (‘upstream 5533 insert’), or an RNA sequence encoded thereby.

In one embodiment, the intronic repeats are derived from the pcDNA3.1 (+) Laccase2 MCS Exon Vector and comprise or consist of SEQ ID NO: 24 (‘downstream 5533 insert’), or an RNA sequence encoded thereby.

In one embodiment the intronic repeats are selected from the group consisting of SEQ ID NOs: 21 - 24.

In one embodiment, the intronic repeats comprise or consist of RNA sequences encoded by the DNA sequences of any one or more of SEQ ID NOs: 21 - 24.

The linear functional nucleic acid molecule of the invention may constitute a precursor of the circularfunctional nucleic acid molecule of the invention. The linear functional nucleic acid molecule of the invention may circularise or be circularised, e.g., in vitro prior to use; in vitro within a cell, or in vivo (e.g., within a cell); by a natural process, such as endogenous back- splicing; or by synthetic methods, such as in vitro ligation.

It is to be understood that the terms “linear” and “circular” nucleic acid molecule, as described herein, refer to a nucleic acid molecule having a generally or overall linear (i.e., possessing 5’ and 3’ termini) or circular (i.e., covalently ‘closed’) configuration. These terms do not preclude, for example, the existence of regions within said functional nucleic acid molecule that possess some structural characteristics themselves, e.g., RNA secondary structure.

The functional nucleic acid molecule of the present invention is preferably an RNA molecule or modified RNA molecule as described herein.

The functional nucleic acid molecule of the invention is preferably a circRNA or linear precursor thereof.

In some embodiments, the functional nucleic acid molecule of the invention comprises or consists of a sequence from the circRNAs identified in Table 1. The functional nucleic acid molecule provided herein is trans- acting such that it functionally modulates sequences present on another RNA molecule.

In one embodiment, the functional nucleic acid molecule further comprises at least one spacer sequence between the target determinant sequence and the regulatory sequence.

SEQ ID NO: 1 is a non-limiting example of a spacer/linker sequence which may be used in the functional nucleic acid molecule of the present invention.

In one embodiment the spacer/linker sequence may comprise SEQ ID NO: 1 .

In another embodiment the spacer/linker sequence may consist of SEQ ID NO: 1.

In one embodiment the functional nucleic acid molecule is single stranded.

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

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

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

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

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

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

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

The term “modification” or "chemical modification" refers to a structural change in, or on, the most com mon, natural ribonucleotides: adenosine, guanosine, cytidine, thymidine, or uridine ribonucleotides. In particular, the chemical modifications described herein may be changes in or on a nucleobase (i.e. a chemical base modification), or in or on a sugar (i.e. a chemical sugar modification). The chemical modifications may be introduced co-transcriptionally (e.g. by substitution of one or more nucleotides with a modified nucleotide during synthesis), or post-transcriptionally (e.g. by the action of an enzyme). Chemical modifications are known in the art, for exam pie as described in The RNA

Modification Database provided by The RNA Institute (htps://mods.rna.albany.edu/mods/).

Examples of chemical modifications which may be useful in the present invention are described in PCT/GB2021/052607, which is incorporated herein by reference in its entirety.

In one embodiment, the chemical modification is a chemical base modification. The chemical base modification may be selected from a modification of an adenine, cytosine, thymine and/or uracil base.

In one embodiment, the chemical base modification is selected from methylation and/or isomerisation.

In a further embodiment, the chemical base modification is selected from the group consisting of: Pseudouridine (ψ), N1 -Methylpseudouridine (N1mψ), 5-Methylcytidine (m5C) and N6-Methyladenosine (m6A). In a further embodiment, the chemical base modification is selected from the group consisting of: Pseudouridine, N1 -Methylpseudouridine and N6- Methyladenosine.

In one embodiment, the chemical modification is a chemical sugar modification.

In one embodiment, the chemical sugar modification is methylation.

In one embodiment, the chemical sugar modification is a 2’ modification, such as a 2'-O- Methyl modification.

In a further embodiment, the chemical sugar modification is 2'-O-Methyladenosine (Am).

In one embodiment, the functional nucleic acid molecule comprises a 3’ -polyadenylation (polyA) tail. A “3’-polyA tail” refers to a long chain of adenine nucleotides added to the 3’-end of the nucleic acid which provides stability to the RNA molecule and can promote translation.

In one embodiment the functional nucleic acid molecule comprises a 5’ -cap. A “5’-cap” refers to an altered nucleotide at the 5’ -end of the transcript which provides stability to the molecule, particularly from degradation from exonucleases, and can promote translation. Most commonly, the 5’-cap may be a 7-m ethylguanylate cap (m7G), i.e. a guanine nucleotide connected to the RNA via a 5' to 5' triphosphate linkage and methylated on the 7 position.

It would be understood that such 5’-caps and 3’-polyA tails would not apply to the circular functional nucleic acid molecule in so far as it does not possess 5’ and 3’ termini.

It is to be understood that the term “functional nucleic acid” as it applies to the linear functional nucleic acid molecule disclosed herein may refer to functionality of the linear functional nucleic acid and/or functionality, e.g., translational regulatory activity, of the circular functional nucleic acids for which the linear nucleic acid molecule serves as a precursor. Thus the linear functional nucleic acid molecule, when acting as a precursor, may not necessarily exhibit the functionality of the circular functional nucleic acid molecule, which it generates.

Herein the term “SINEUP” may be used to encompass both traditional SINEUPs containing a SINE element as well as corresponding functional nucleic acids containing an IRES as the effector domain.

Target determinant sequences

The target determinant sequence (also referred to as the target binding sequence) is the portion of the functional nucleic acid molecule that binds to the target m RNA.

In one aspect, the at least one target determinant sequence comprises a sequence reverse complementary to a target m RNA sequence for which protein translation is to be enhanced.

As used herein, “target” or “target m RNA sequence” refers to an endogenous or exogenous mRNA within a cell in vitro, an endogenous or exogenous mRNA /n vivo, e.g., within a cell; or any mRNA/n vitro, with which the functional nucleic acid molecule of the invention is reverse complementary, such that their translation is enhanced when in the presence of the functional nucleic acid molecule of the invention.

It will be understood that by “enhancing translation of one or more target mRNA sequences” it is meant that translation of the entire protein-coding region of the target mRNA will be enhanced such that synthesis of the protein encoded thereby is increased. In one embodiment, the at least one target determinant sequence comprises a sequence reverse complementary to a therapeutic target mRNA sequence for which protein translation is to be enhanced.

As used herein, “therapeutic target” or “therapeutic target mRNA sequence” refers to a target which may be used to treat a disease or condition in said subject when its translation is enhanced, such as enhanced by using a functional nucleic acid molecule according to the present invention.

For example when expressed in a subject (such as in a cell of a subject), a therapeutic target may: restore or otherwise increase levels of a protein that are deficient or abnormal in a cell; augment an existing pathway in a cell; and/or provide a novel function or activity in a cell; thereby treating a disease or condition of said subject.

In one embodiment, the therapeutic target comprises at least one gene defect that results in abnormal levels of a protein of interest.

In one embodiment the gene defect may be haploinsufficiency.

In WO 2012/133947, which is incorporated herein by reference in its entirety, it was shown that a target binding sequence needs to have only about 60% similarity with a sequence reverse complementary to the target mRNA in order to increase protein translation. In fact, the target binding sequence can even display a large number of mismatches and retain activity.

The target binding sequences of the functional nucleic acid molecule of the invention may each display about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% similarity with a sequence reverse complementary to the target mRNA.

Herein, polypeptide or polynucleotide sequences are said to be the same as or “identical” to other polypeptide or polynucleotide sequences, if they share 100% sequence identity over their entire length. Residues in sequences are numbered from left to right, i.e. from N- to C- terminus for polypeptides; from 5’ to 3’ terminus for polynucleotides. If closely related sequences are not identical they may be similar, i.e., they may possess a certain, quantifiable, degree of sequence identity, e.g., a sequence may have 50%, 60%, 70%, 80%, 90%, 95% or 99% sequence identity to another sequence. Unless a specific reference range is given, e.g., with respect to the nucleotide positions, any quoted sequence identity will be understood as being calculated across the residue range over which the two sequences are aligned. The aligned residue range may represent the entirety of one or more of the input sequences or a contiguous section of sequence of one or more of the input sequences, and is typically determined by standard tools known in the art, e.g., NCBI BLAST.

For the purposes of comparing two closely-related polynucleotide sequences, the “% sequence identity” between a first nucleotide sequence and a second nucleotide sequence may be calculated using NCBI BLAST, using standard settings for nucleotide sequences (BLASTN). For the purposes of comparing two closely-related polypeptide sequences, the “% sequence identity” between a first polypeptide sequence and a second polypeptide sequence may be calculated using NCBI BLAST, using standard settings for polypeptide sequences (BLASTP). A “difference” between sequences refers to an insertion, deletion or substitution of a single nucleotide in a position of the second sequence, compared to the first sequence. Insertions, deletions or substitutions in a second sequence which is otherwise identical (100% sequence identity) to a first sequence result in reduced % sequence identity.

“Complementarity” relates to the Watson-Crick base pairing principle that ‘A nucleotides will hydrogen bond with T (or ‘U’) nucleotides, and ‘G’ nucleotides with ‘C’ nucleotides to form double stranded structures that associate via said “complementary” nucleotides. Herein, a “complementary” sequence is a sequence closely-related to another sequence such that such base pairing can occur. Complementary sequences may be 100% complementary such that they may base pair across their entire length, or they may be e.g., 99%, 90%, 80%, 70%, or 60% complementary etc., such that they base pair across portions of their sequence. Here, as is common in the art, a complementary sequence may also be called a “reverse complementary” sequence.

The target binding sequence comprises a sequence which is sufficient in length to bind to the target mR NA transcript. Therefore, the target binding sequence may be at least about 10 nucleotides long, such as at least about 14 nucleotides long, such as at least about 15 nucleotides long, such as at least about 16 nucleotides long, such as at least about 17 nucleotides long, such as least 18 nucleotides long. Furthermore, the target binding sequence may be less than about 250 nucleotides long, preferably less than about 200 nucleotides long, less than about 150 nucleotides long, less than about 140 nucleotides long, less than about 130 nucleotides long, less than about 120 nucleotides long, less than about 110 nucleotides long, less than about 100 nucleotides long, less than about 90 nucleotides long, less than about 80 nucleotides long, less than about 70 nucleotides long, less than about 60 nucleotides long or less than about 50 nucleotides long. In one embodiment, the target binding sequence is between about 4 and about 50 nucleotides in length, such as between about 18 and about 44 nucleotides in length.

The target binding sequence may be designed to hybridise with the 5’-untranslated region (5’ UTR) of the target mRNA sequence. In one embodiment, the sequence is reverse complementary to 0 to 50 nucleotides, such as 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 of the 5’ UTR.

Alternatively, or in combination, the target binding sequence may be designed to hybridise to the coding sequence (CDS) of the target mRNA sequence. In one embodiment, the sequence is reverse complementary to 0 to 40 nucleotides, 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, or 0 to 4 nucleotides of the CDS.

The target binding sequence may be designed to hybridise to a region upstream of an AUG site (start codon), such as a start codon within the CDS, of the target mRNA sequence. In one embodiment, the sequence is reverse complementary to 0 to 80 nucleotides, 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 upstream of the AUG site.

Alternatively, or in combination, the target binding sequence may be designed to hybridise to the target mRNA sequence downstream of said AUG site. In one embodiment, the sequence is reverse complementary to 0 to 40 nucleotides, 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, or 0 to 4 nucleotides of the target mRNA sequence downstream of said AUG site. In one embodiment, the target determinant sequence is at least 10 nucleotides long and comprises, from 3’ to 5’:

- a sequence reverse complementary to 0 to 50 nucleotides of the 5’ untranslated region (5’ UTR) and 0 to 40 nucleotides of the coding sequence (CDS) of the target mRNA sequence; or

- a sequence reverse complementary to 0 to 80 nucleotides of the region upstream of an AUG site (start codon) of the target mRNA and 0 to 40 nucleotides of the CDS of the target mRNA sequence downstream of said AUG site.

In one embodiment, the target determinant sequence is at least 14 nucleotides long and comprises, from 3’ to 5’:

- a sequence reverse complementary to 0 to 40 nucleotides of the 5’ UTR and 0 to 32 nucleotides of the CDS of the target mRNA sequence; or

- a sequence reverse complementary to 0 to 70 nucleotides of the region upstream of an AUG site (start codon) of the target mRNA and 0 to 4 nucleotides of the CDS of the target mRNA sequence downstream of said AUG site.

In one embodiment, the coding sequence starts on the first AUG site (M1) of the mRNA.

In one embodiment, the preferred AUG site is that corresponding to an internal start codon (e.g. M2).

In the context of referencing a sequence reverse complementary to a region in the 5’ UTR and the CDS, this is preferably anchored around the AUG site, i.e. the region in the 5’ UTR is directly upstream of the AUG site of the target mRNA. For example, reference to a target binding sequence that is “-40/+4 of M1” refers to a target binding sequence that is reverse complementary to the 40 nucleotides within the 5’ UTR upstream of the AUG site (-40) and the 4 nucleotides within the CDS downstream of the AUG site (+4).

In accordance with conventional numbering, the nucleotides of the 5’UTR sequence are numbered sequentially using decreasing negative numbers approaching the AUG site on the target mRNA (e.g. -3, -2, -1). The nucleotides of the CDS sequence are numbered sequentially using increasing positive numbers (e.g. +1 , +2, +3) from the AUG site, such that the A of the AUG site is numbered +1. The region bridging the 5’UTR and the CDS will therefore be numbered -3, -2, -1 , +1 , +2, +3, with the A of the AUG site numbered +1 . Exemplary targets

The functional nucleic acid of the invention may be designed to target any suitable mRNA in order to increase protein translation of said mRNA. Particularly suitable targets are mRNAs for which their natural abundance is reduced e.g., due to a haploinsufficiency or microdeletion.

Suitable targets may include, but are not limited to, OPA1 , FXN, GRN, GBA, PRPF31 , GDNF, BDNF, NGF, TRKA, TRKB, and/or RET.

The functional nucleic acid molecule of the invention may be designed to target a given mRNA using the principles disclosed in the foregoing section, “Target determinant sequences”.

PXK

PX domain-containing protein kinase-like protein (PXK) is a protein that is encoded by the PX gene. PXK is implicated in the regulation of electrical excitability, synaptic transmission, and the internalization and degradation of epidermal growth factors. Variation in PXK expression has been linked to susceptibility to systemic lupus erythematosus.

In one embodiment, there is provided a functional nucleic acid molecule according to the invention, wherein the one or more target binding sequence comprises a sequence reverse complementary to a PXK mRNA sequence.

In some embodiments, the one or more target binding sequence comprises a sequence reverse complementary to a target mRNA sequence selected from the group consisting of: human PXK and mouse PXK.

In one embodiment, the one or more target binding sequence comprises a sequence reverse complementary to a sequence within SEQ ID NO: 25.

In one embodiment, the one or more target binding sequence consists of a sequence reverse complementary to a sequence within SEQ ID NO: 25.

In one embodiment, the target binding sequence comprises a sequence reverse complementary to a sequence with about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a sequence within SEQ ID NO: 25.

In one embodiment, the target binding sequence consists of a sequence reverse complementary to a sequence with about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a sequence within SEQ ID NO: 25.

In one embodiment, the target sequence comprises a sequence encoded by SEQ ID NO: 25.

In one embodiment, the target sequence consists of a sequence encoded by SEQ ID NO: 25.

In one embodiment, the target sequence comprises a sequence with about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a sequence encoded by SEQ ID NO: 25.

In one embodiment, the target sequence consists of a sequence with about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a sequence encoded by SEQ ID NO: 25.

SEQ ID NO: 25 is a PXK transcript with the accession numbers ENST00000356151 (ensemble) and NM_017771.5 (NCBI).

In a further embodiment, the one or more target binding sequence comprises or consists of a sequence as selected from SEQ ID NOs: 26 and SEQ ID NO: 27.

In a further embodiment, the one or more target binding sequence comprises or consists of a sequence reverse complementary to a sequence selected from SEQ ID NOs: 28 and SEQ ID NO: 29. In a further embodiment, the target sequence comprises or consists of a sequence selected from SEQ ID NOs: 28 and SEQ ID NO: 29.

In one embodiment, the target sequence comprises a sequence with about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a sequence encoded by SEQ ID NOs: 28 and/or 29.

CHK1

Checkpoint kinase 1 (CHK1 or Chk1), is a serine/threonine protein kinase that is encoded by the CHEK1 gene. CHK1 haploinsufficiency is associated with anaemia and defective erythropoiesis. Since CHK1 overexpression is implicated in deleterious processes such as tumorigenesis, the use of the functional nucleic acid of the present invention to restore protein levels (i.e. , without exceeding wild-type physiological levels) is advantageous in avoiding such unwanted effects.

In one embodiment, there is provided a functional nucleic acid molecule according to the invention, wherein the one or more target binding sequence comprises a sequence reverse complementary to a CHK1 mRNA sequence.

In some embodiments, the one or more target binding sequence comprises a sequence reverse complementary to a target mRNA sequence selected from the group consisting of: human CHK1 and mouse CHK1.

In one embodiment, the one or more target binding sequence comprises a sequence reverse complementary to a sequence within SEQ ID NO: 30.

In one embodiment, the one or more target binding sequence consists of a sequence reverse complementary to a sequence within SEQ ID NO: 30.

In one embodiment, the target binding sequence comprises a sequence reverse complementary to a sequence with about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a sequence within SEQ ID NO: 30. In one embodiment, the target binding sequence consists of a sequence reverse complementary to a sequence with about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a sequence within SEQ ID NO: 30.

In one embodiment, the target sequence comprises a sequence encoded by SEQ ID NO: 30.

In one embodiment, the target sequence consists ofa sequence encoded by SEQ ID NO: 30.

In one embodiment, the target sequence comprises a sequence with about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a sequence encoded by SEQ ID NO: 30.

In one embodiment, the target sequence consists of a sequence with about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a sequence encoded by SEQ ID NO: 30.

SEQ ID NO: 30 is a CHK1 transcript with the accession numbers ENST00000438015 (ensemble) and NM_001114122.3 (NCBI).

MFN2

Mitofusin-2 is a mitochondrial outer membrane GTPase that is encoded by the MFN2 gene. Herein, Mitofusin-2 protein may be referred to as MFN2, as may the corresponding mRNA that encodes it. Mutations in MFN2 are associated with Charcot-Marie-Tooth (CMT) disease-2A, which is a neurological disorder that presents neuropathy-related features and systemic impairment of the central nervous system. Up-regulation of MFN2 triggers apoptotic cell death of vascular smooth muscle cells and cardiomyocytes, indeed MFN2 protein levels are associated with several heart diseases. Down-regulation of MFN2 leads to vascular proliferative disorders and cardiac dysfunction. In one embodiment, there is provided a functional nucleic acid molecule according to the invention, wherein the one or more target binding sequence comprises a sequence reverse complementary to a MFN2 mR NA sequence.

In some embodiments, the one or more target binding sequence comprises a sequence reverse complementary to a target mRNA sequence selected from the group consisting of: human MFN2 and mouse MFN2.

In one embodiment, the one or more target binding sequence comprises a sequence reverse complementary to a sequence within SEQ ID NO: 31 .

In one embodiment, the one or more target binding sequence consists of a sequence reverse complementary to a sequence within SEQ ID NO: 31 .

In one embodiment, the target binding sequence comprises a sequence reverse complementary to a sequence with about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a sequence within SEQ ID NO: 31.

In one embodiment, the target binding sequence consists of a sequence reverse complementary to a sequence with about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a sequence within SEQ ID NO: 31.

In one embodiment, the target sequence comprises a sequence encoded by SEQ ID NO: 31.

In one embodiment, the target sequence consists of a sequence encoded by SEQ ID NO: 31.

In one embodiment, the target sequence comprises a sequence with about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a sequence encoded by SEQ ID NO: 31. In one embodiment, the target sequence consists of a sequence with about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a sequence encoded by SEQ ID NO: 31.

SEQ ID NO: 31 is a MFN2 transcript with the accession numbers ENST00000235329 (ensemble) and NM_014874.4 (NCBI).

GUCY1 B1

Guanylate cyclase soluble subunit beta-1 is a protein encoded by the GYCY1B1 gene that forms part of a soluble guanylate cyclase which mediates responses to nitric oxide by catalysing the synthesis of cGMP. GUCY1 B1 is associated with hypertensive disease and the GUCYB1 gene was found to be among 97 common genes that are downregulated in both Alzheimer’s and Parkinson’s diseases.

In one embodiment, there is provided a functional nucleic acid molecule according to the invention, wherein the one or more target binding sequence comprises a sequence reverse complementary to a GUCY1 B1 mRNA sequence.

In some embodiments, the one or more target binding sequence comprises a sequence reverse complementary to a target mRNA sequence selected from the group consisting of: human GUCY1 B1 and mouse GUCY1 B1.

In one embodiment, the one or more target binding sequence comprises a sequence reverse complementary to a sequence within SEQ ID NO: 32.

In one embodiment, the one or more target binding sequence consists of a sequence reverse complementary to a sequence within SEQ ID NO: 32.

In one embodiment, the target binding sequence comprises a sequence reverse complementary to a sequence with about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a sequence within SEQ ID NO: 32. In one embodiment, the target binding sequence consists of a sequence reverse complementary to a sequence with about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a sequence within SEQ ID NO: 32.

In one embodiment, the target sequence comprises a sequence encoded by SEQ ID NO: 32.

In one embodiment, the target sequence consists of a sequence encoded by SEQ ID NO: 32.

In one embodiment, the target sequence comprises a sequence with about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a sequence encoded by SEQ ID NO: 32.

In one embodiment, the target sequence consists of a sequence with about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a sequence encoded by SEQ ID NO: 32.

SEQ ID NO: 32 is a GUCY1 B1 transcript with the accession numbers ENST00000264424 (ensemble) and NM_000857.5 (NCBI).

Regulatory Sequences

The functional nucleic acid molecule of the invention comprises a regulatory sequence comprising an internal ribosome entry site (IRES).

The terms “internal ribosome entry site (IRES) sequence” is defined in WO 2019/058304, which is incorporated herein by reference in its entirety. IRES sequences recruit the 40S ribosomal subunit and promote cap-independent translation of a subset of protein coding mRNAs. IRES sequences are generally found in the 5’ untranslated region (5’UTR) of cellular mRNAs coding for stress-response genes, thus stimulating their translation in cis. The person skilled in the art would know that an IRES sequence is a nucleotide sequence capable of promoting translation of a second cistron in a bicistronic construct. Typically, a dual luciferase (Firefly luciferase [Flue], Renilla Luciferase [Rluc]) encoding plasmid is used for experimental tests. Said test may be considered “The Standard Bicistronic Plasmid Test for Cellular mRNA IRESs” used to test putative IRES sequences. The foregoing is a functional test wherein the putative IRES sequence is inserted between RLuc and FLuc, e.g., as described in Jasckson, Cold Spring Harb Perspect Biol 2013; 5:a011569, wherein the translational function of the putative IRES sequence is determined by the Fluc/RLuc value, thus measuring c/s-acting activity.

A major database exists, namely IRESite, for the annotation of nucleotide sequences that have been experimentally validated as IRES, using dual reporter or bicistronic assays (http://iresite.org/IRESite_web.php).

Within the IRESite, a web-based tool is available to search for sequence-based and structure-based similarities between a query sequence of interest and the entirety of annotated and experimentally validated IRES sequences within the database. The output of the program is a probability score for any nucleotide sequence to be able to act as IRES in a validation experiment with bicistronic constructs. Additional sequence-based and structurebased web-based browsing tools are available to suggest, with a numerical predicting value, the IRES activity potentials of any given nucleotide sequence (http://rna.informatik.uni- freiburg.de/; http://regrna.mbc.nctu.edu.tw/index1.php).

The regulatory sequence has protein translation enhancing activity.

The regulatory sequence increases or enhances translation of the target m RNA sequence.

Increased or enhanced protein translation activity indicates that the efficiency or activity of translation is increased as compared to a case where the functional nucleic acid molecule according to the present invention is not present in a system.

In one embodiment, expression of the protein encoded by the target mRNA is increased by at least 1 .2 fold, such as at least 1 .5 fold, in particular at least 2 fold.

In a further embodiment, expression of the protein encoded by the target mRNA is increased between 1 .5 to 3 fold, such as between 1 .6 and 2.2 fold. These increases in protein expression are within physiological ranges. It is envisaged that increasing protein expression within these ranges will allow the treatment of diseases associated with one or more gene defects, such as cancer or neurodegenerative diseases, without leading to negative side effects associated with increasing expression of the target above non-disease state or ‘wildtype’ physiological levels.

In one embodiment the expression of the protein encoded by the target mRNA is increased by at least about 1.1 fold, at least about 1 .2 fold, at least about 1 .3 fold, at least about 1 .4 fold, at least about 1 .5 fold, at least about 1 .6 fold, at least about 1 .7 fold, at least about 1 .8 fold, at least about 1 .9 fold, at least about 2.0 fold, at least about 2.1 fold, at least about 2.2 fold, at least about 2.3 fold, at least about 2.4 fold, at least about 2.5 fold, at least about 2.6 fold, at least about 2.7 fold, at least about 2.8 fold, at least about 2.9 fold, or at least about

3.0 fold.

In one embodiment the expression of the protein encoded by the target mRNA is increased about 1.1 fold, about 1.2 fold, about 1.3 fold, about 1.4 fold, about 1.5 fold, about 1.6 fold, about 1.7 fold, about 1.8 fold, about 1.9 fold, about 2.0 fold, about 2.1 fold, about 2.2 fold, about 2.3 fold, about 2.4 fold, about 2.5 fold, about 2.6 fold, about 2.7 fold, about 2.8 fold, about 2.9 fold, or about 3.0 fold.

In one embodiment the expression of the protein encoded by the target mRNA is increased by less than about 1 .2 fold, less than about 1 .3 fold, less than about 1 .4 fold, less than about

1 .5 fold, less than about 1 .6 fold, less than about 1 .7 fold, less than about 1 .8 fold, less than about 1 .9 fold, less than about 2.0 fold, less than about 2.1 fold, less than about 2.2 fold, less than about 2.3 fold, less than about 2.4 fold, less than about 2.5 fold, less than about

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

In one embodiment, the regulatory sequence is in a direct orientation relative to the 5’ to 3’ orientation of the functional nucleic acid molecule, i.e., the regulatory sequence is embedded (inserted) with the same 5’ to 3’ orientation as the functional nucleic acid molecule.

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

The regulatory sequence com prises an IRES sequence, or a fragment thereof. Said sequence enhances translation of the target mRNA sequence. Several IRESs having sequences ranging from 48 to 576 nucleotides have been tested with success, e.g. human Hepatitis C Virus (HCV) IRESs (e.g. SEQ ID NO: 2 and 3), human poliovirus IRESs (e.g. SEQ ID NO: 4 and 5), human encephalomyocarditis (EMCV) virus (e.g. SEQ ID NO: 6 and 7), human cricket paralysis (CrPV) virus (e.g. SEQ ID NO: 8 and 9), human Apaf-1 (e.g. SEQ ID NO: 10 and 11), human ELG-1 (e.g. SEQ ID NO: W and 13), human c-MYC (e.g. SEQ ID NO: 14-17) and human dystrophin (DMD) (e.g. SEQ ID NO: 18 and 19).

In some embodiments the regulatory sequence comprises a sequence selected from the group consisting of SEQ ID NOs 2 - 20, or a fragment thereof.

In some embodiments the regulatory sequence consists of a sequence selected from the group consisting of SEQ ID NOs 2 - 20, or a fragment thereof.

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

In one embodiment the regulatory element has at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 86% sequence identity, at least about 87% sequence identity, at least about 88% sequence identity, at least about 89% sequence identity, at least about 90% sequence identity, at least about 91 % sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or 100% sequence identity to any one of SEQ ID NOs 2 - 20.

In one embodiment, the at least one regulatory sequence consists of a sequence with at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 86% sequence identity, at least about 87% sequence identity, at least about 88% sequence identity, at least about 89% sequence identity, at least about 90% sequence identity, at least about 91 % sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs 2 - 20.

In some embodiments, the regulatory sequence is a sequence comprising or consisting of a sequence selected from the circRNAs identified in Table 1.

In some embodiments the regulatory sequence comprises or consists of a fragment of any one of SEQ ID NOs 2 - 20, wherein the fragment is about is about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about

120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about

200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about

280, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about

360, about 370 or more nucleotides in length.

In some embodiments the fragment is a functionally active fragment that retains IRES activity within the definition provided above.

In some embodiments the fragment is a functionally active fragment that retains protein translation enhancing activity.

It will be understood that, owing to the functional nature of The Standard Bicistronic Plasmid Test for Cellular mRNA IRESs, a “functionally active fragment” of an IRES might also be considered an IRES perse. Herein, “functionally active fragment” of an IRES is utilised to delineate IRES sequences that are shorter in length as compared with ‘parental’ IRES sequences from which they are designed or derived.

DNA molecules and vectors

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

According to a further aspect of the invention, there is provided an expression vector comprising said DNA molecule.

Exemplary expression vectors are known in the art and may include, for exam pie, plasmid vectors, viral vectors (for example adenovirus, adeno-associated virus, retrovirus or lentivirus vectors), phage vectors, cosmid vectors and the like. The choice of expression vector may be dependent upon the type of host cell to be used and the purpose of use. In particular, and without limitation, the following plasmids have been used for expression of the functional nucleic acid molecule:

Mammalian expression plasmids:

- pCDNA3.1 (-)

- pDUAL-eGFPΔ (modified from peGFP-C2)

- PCDNA3.1 (+) ZKSCAN1 MCS Exon Vector

- pcDNA3.1 (+) Laccase2 MCS Exon Vector

Viral vectors:

- pAAV (an Adeno-Associated Virus vector)

- rcLV -TetOne-Puro (a 3^rd generation Lentivirus vector)

- pLPCX-link (a 3^rd generation Retrovirus vector)

In one embodiment the mammalian expression plasmid is a pCDNA3.1 (+) ZKSCAN1 MCS Exon Vector.

In another embodiment the mammalian expression plasmid is a pcDNA3.1 (+) Laccase2 MCS Exon Vector

Plasmids of the invention may comprise any one of more features selected from the list comprising: a CMV promoter, a H1 promoter, and/or a BGH poly(A) terminator.

In one embodiment the viral vector is pAAV.

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

In one embodiment the viral vector is pLPCX-link.

Vectors of the invention may comprise any one of more features selected from the list comprising: a CAG promoter, a CMV enhancer, SV40 late poly(A) terminator, a LTR-TREt (Tre-Tight) promoter, and/or a BGH poly(A) terminator.

It should be noted that any promoter may be used in the vector. Since the activity of the functional nucleic acids of the invention is independent of the promoter it is envisaged that these will work just as well as those exemplified above. Compositions and methods

The present invention also relates to com positions com prising the functional nucleic acid molecule, the DNA molecule or the expression vector described herein.

The composition may comprise components which enable delivery of said functional nucleic acid molecule by viral vectors (AAV, lentivirus and the like) and non-viral vectors (nanoparticles, lipid particles and the like). Alternatively, the functional nucleic acid molecule of the invention may be administered as naked or unpackaged RNA

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

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

In one embodiment, there is provided a pharmaceutical composition comprising at least one functional nucleic acid molecule, at least one DNA molecule, or at least one expression vector according to the present invention.

Suitably, a pharmaceutical composition may com prise at least one functional nucleic acid molecule, at least one DNA molecule, or at least one expression vector according to the present invention with a suitable pharmaceutical excipient, diluent or carrier.

The suitable pharmaceutical excipient, diluent or carrier may depend on the intended route of administration and standard pharmaceutical practice.

A suitable carrier may include any of the standard pharmaceutical carriers, vehicles, diluents or excipients known in the art and which are generally intended for use in facilitating the delivery of nucleic acids, such as RNA. Liposomes, exosomes, lipidic particles or nanoparticles are examples of suitable carriers that may be used for the delivery of RNA. In a preferred embodiment, the carrier or vehicle delivers its contents to the target cell such that the functional nucleic acid molecule is delivered to the appropriate subcellular compartment, such as the cytoplasm. Methods, methods of treatment and medical uses

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

According to a further aspect of the invention, there is provided an in vitro method for increasing the protein synthesis of a target in a cell or cell-free system comprising administering the functional nucleic acid molecule, DNA molecule, expression vector or the composition described herein, to the cell or cell-free system.

According to a further aspect of the invention, there is provided a method for increasing the protein synthesis of a target in a cell comprising administering the functional nucleic acid molecule, DNA molecule, expression vector or the composition described herein, to the cell. Preferably the cell is a mammalian cell, such as a human or a mouse cell.

According to a further aspect of the invention, there is provided a method for increasing the protein synthesis efficiency of a target in a cell comprising administering the functional nucleic acid molecule, DNA molecule, expression vector or the composition described herein, to the cell. Preferably the cell is a mammalian cell, such as a human or a mouse cell.

Methods of the invention result in increased levels of target protein in a cell and therefore find use, for example, in methods of treatment of diseases which are associated with gene defects (e.g. one or more gene defects which result in reduced protein levels and/or loss-of- function mutations of the encoding gene). Methods of the invention find particular use in diseases caused by a quantitative decrease in the predetermined, normal protein level, such as haploinsufficiency.

Methods of the invention can be performed in vitro, ex vivo or in vivo.

The methods described herein may com prise transfecting into a cell the functional nucleic acid molecule, DNA molecule, expression vector or composition as defined herein. The functional nucleic acid molecule, DNA molecule, expression vector or composition may be administered to target cells using methods known in the art and include, for exam pie, microinjection, lipofection, electroporation, using calcium phosphate, self-infection by the vector or transduction of a virus.

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

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

It will be understood that the functional nucleic acid molecule of the invention find use in increasing the level of a target protein, such as a therapeutic target within a cell.

Thus the functional nucleic acid molecule, DNA molecule, expression vector or composition, such as a pharmaceutical composition may be administered to a subject having an existing disease or condition in order to lessen, reduce or improve at least one symptom associated with the disease and/or to slow down, reduce or block the progression of the disease.

In one aspect there is provided the functional nucleic acid molecule, DNA molecule, expression vector or composition, such as pharmaceutical composition, for use in the treatment of a disease-associated with one or more gene defects.

As used herein, “gene defect” or “gene defects”, refer to one or more abnormalities in a gene which results in reduced protein levels and/or loss-of-function mutations of the encoding gene. For exam pie, a gene defect may be caused by a mutation in a single gene, mutations in multiple genes, chromosomal abnormality, or mutation(s) in mitochondrial DNA or in nuclear genes.

For exam pie, a disease associated with one or more gene defects may be a cancer or a neurodegenerative disease.

In one embodiment there is provided the functional nucleic acid molecule, DNA molecule, expression vector or composition, such as pharmaceutical composition, for use in the treatment of cancer. In one embodiment there is provided the functional nucleic acid molecule, DNA molecule, expression vector or composition, such as pharmaceutical composition, for use in the treatment of a neurodegenerative disease.

In one aspect, there is provided a method of treating a disease associated with one or more gene defects comprising administering a therapeutically effective amount of the functional nucleic acid molecule, the DNA molecule, the expression vector, or the composition, such as pharmaceutical composition, as defined herein to a subject in need thereof.

In one aspect, there is provided a method of treating a disease associated with one or more gene defects comprising administering a therapeutically effective amount of the functional nucleic acid molecule, the DNA molecule, the expression vector, or the composition, such as the pharmaceutical composition, as defined herein to a subject in need thereof, wherein the disease is a cancer or a neurodegenerative disease.

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

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

EXAMPLES

Example 1 - Circularized SINEUP(IRES) RN A maintains its activities in trans

Building on observations that natural AS Uchl1 SINEUP RNA can function through an IRES sequence and that viral and cellular IRES RNAs can promote translation in trans, we aimed to identify new potential natural non-coding RNAs with an embedded IRES sequence that exhibited SINEUP-like activity. In contrast to the invSINEB2 in natural SINEUPs, IRES- containing RNAs mostly correspond to protein-coding genes. Furthermore, target mRNA sequences cannot be identified for their localization within a sense/antisense pair in the genome. Therefore, we decided to focus on a specific class of RNAs, circular RNAs (circRNAs), which arise from protein-coding genes and have been reported to include IRES RNA sequences. CircRNAs can be both non-coding and coding for proteins. Here, we investigated non-coding circRNAs with an embedded IRES sequence.

As a proof-of-principle that circularized SINEUP(IRES) RNAs are post-transcriptionally active like their linear counterparts, we generated synthetic circularized SINEUP RNAs (named circUP since they are “circular RNAs UP-regulating protein translation”) combining c-myc IRES element as Effector Domain (ED) and a previously established GFP targeting sequence as Binding Domain (BD) in the circularizing pcDNA3.1 (+) ZKSCAN1 MCS Exon Vector (Figure 1a). The sequence was inserted between two highly complementary intronic repeats (derived from the zkscan gene; SEQ ID NO: 21 & SEQ ID NO: 22) that facilitate circularization of the inserted sequence.

An empty pcDNA3.1 (+) ZKSCAN1 MCS Exon Vector was used as negative control. This constructwas named circUP(c-myc IRES)-GFP. Linear miniSINEUP-GFP (Zucchelli 2015) and miniSINEUP(c-myc IRES)-GFP were used as positive controls (Figure 1).

When these constructs were co-transfected with a vector expressing GFP (pEGFP) in HEK293T cells, circUP(c-myc IRES)-GFP induced an increase in GFP protein expression similar to canonical linear SINEUPs (Figure 1b). The presence of circularized transcripts was proved with the use of divergent primers. As expected, no variation in GFP mRNA levels was observed, confirming that both linear and circularized SINEUPs act post- transcriptionally (Figure 1c). These results demonstrated that synthetically designed circUPs can sustain SINEUP-like activity.

Example 2 - Identification of circ5533 as a representative member of circUPs

To investigate the existence of natural circUPs, we took advantage of the circBase database and a list of oligonucleotide sequences present in cellular and viral RNAs that supported IRES activity in bicistronic assays.

In total, 140,790 unique IDs from circBase of putative spliced circRNAs were mapped on the human genome, while 570 sequences with IRES activity were retrieved from https://bitbucket.org/alexeyg-com/52. The 570 trimmed IRES sequences, of a length of 174 nts, were aligned using BLAST against the 140,790 circR NA sequences. Considering only alignments with 100% IRES coverage and mismatches </=1 nt without gaps, 411 unique circRNAs containing sequences with potential IRES activity were identified (Data not shown). We then filtered out circRNAs with ORF sequences longer than 100 aa to look for potential non-coding circRNAs.

The Virtual Ribosome tool (Dna2pep v1 .1) was used to predict the longest complete ORF for each circRNA by searching across all positive reading frames with methionine as start codon and a canonical stop codon. circRNAs from 32 genomic loci presented a predicted ORF of 100 or fewer aa in length (Table 1 , below). Among them, only hsa_circ_0085533 showed complete absence of ORF according to the used constraints. To experimentally validate a potential mechanism of IRES activity in trans, we focused on this non-coding circRNA (circ5533, hsa_circ_0085533). Circ5533 is transcribed from the c-myc locus, is 555-nts-long, and contains an IRES sequence of a length of 380 nts, usually included in the 5’UTR of c- myc mRNA, with well-established evidence of cis activity. In addition, as reported above, c- myc IRES acts in trans as ED in both linear synthetic SINEUP and in circlIP (Figure 1 ). Notably, circ5533 also presents additional 175 nts of unknown function that could contain BD sequences (Figure 2a).

To investigate circ5533’s ability to function as a circular SINEUP and identify its potential to target mRNAs, we overexpressed circ5533 RNA in HEK 293T cells by taking advantage of the circularizing vector previously used to express circUP(c-myc IRES)-GFP. The effect of circ5533 overexpression was analyzed with untargeted proteomics and RNA-seq experiments to identify proteins with increased expression and no change in the respective mRNA level. Experiments were carried out in six biological replicates. Based on the proteomics results, 117 proteins were significantly upregulated in circ5533-transfected cells com pared to controls (Figure 2b) while no mRNAs were differentially expressed (Figure 2c). Notably, the absence of significant changes in mRNA levels strongly suggests there was no potential sponge effect on miRNAs activity in our experimental settings. For validation, we chose six representative examples from the most reproducibly increased proteins that could be efficiently detected with commercially available antibodies.

Western blot experiments (Figure 3a) confirmed statistically significant protein synthesis induction by circ5533 in four of the six proteins tested (PXK, MFN, CHK1 , GUCY1 B1). RT- qPCR (Figure 3b-c) analysis of the corresponding transcripts confirmed no change in mRNA level.

Table 1 - List of circRNAs containing an IRES sequence with a predicted ORF of 100 aa or fewer in length

List of putative non-coding circRNAs with an embedded IRES element identified as per Example 2. To investigate the existence of natural circRNAs with SINEUP-like activity (circUPs) we took advantage of the circBase database and a list of sequences present in cellular and viral RNAs whose IRES activity was experimentally validated (Gritsenko, A. A. et al. Sequence features of viral and human Internal Ribosome Entry Sites predictive of their activity. PLoS Comput. Biol. 13, (2017)). IRES sequences were aligned against the circRNA sequences and coding circRNAs were filtered out. In this way, we identified a group of 49 circRNAs, with an embedded IRES element and a predicted ORF sequence shorter than 100 aa (conventional threshold for non-coding RNAs). Among putative non-coding circRNAs, only circ5533 has no ORF regions. All reported circRNAs could potentially show SINEUP- like activity where the IRES sequences represent the effector domain (ED) while the sequences, upstream the ED, function as single- or multi-target binding domain (BD). The table shows identified circRNAs by reporting their chromosome coordinates (Chromosome, Chrom Start and ChromEnd), their ID reference numbers in circBase (circBase ID), the gene strand on which they are encoded (Strand), the number of exons contained in the circRNA (Exon number), their predicted ORF length (ORF length), the best transcript RefSeq ID (Best Transcript) and the gene name.

Example 3 - ci rc 5533 positively regulates PXK protein levels through two defined BDs in trans We focused on Pxk as a representative mRNA target of the circlIP activity of circ5533. To this end, we cloned circ5533 annotated sequence (NM_002467) into a second circularizing vector named pcDNA3.1 (+) Laccase2 MCS Exon Vector, demonstrating that circ5533 activity was not dependent on the type of circularizing plasmid. We thus confirmed circ5533’s ability to increase endogenous PXK protein levels with no change in mRNA levels (Figure 4a-c).

Furthermore, we carried out co-transfection analysis with a full-length cDNAfor Pxk fused to an in-frame FLAG tag at the C-terminus of the protein. Detecting PXK-FLAG protein with an anti-FLAG antibody in western blot analysis, we demonstrated that circ5533 increased protein levels with no change in mRNA quantity (Figure 6b-d).

We then carried out a bioinformatic analysis looking for the longest pairing sequence in antisense orientation that would allow binding between Pxk mRNA and circ5533 RNA Matches with a minimal length of 8 nts between Pxk and the non-IRES sequence of circ5533 were found. As described in Figure 6a and Table 2, three distinct potential target sites (TS1 , TS2, TS3) in the 5’UTR of Pxk mRNA could pair to the same antisense region of circ5533 from nucleotide 23 to position 48 (named BD1 ). In addition, two potential TSs (TS4 and TS5) were identified in the 3’UTR of Pxk mRNA, that could pair with a second antisense region (BD2) in the non-IRES sequence of circ5533, which spans position 104 to 112.

We first deleted or inverted the orientation of the sequences of both potential BD sequences in circ5533 to assess their influence on activity. Unfortunately, both mutations strongly diminished expression levels of circ5533, constraining this type of analysis (Figure 5). We therefore proceeded by individually mutating all five TS sites on the PXK-FLAG construct and verified the maintenance of circ5533 activity. As shown in Figure 6, the mutations on TS2, TS3, and TS5 did not influence circ5533’s effect on PXK-FLAG protein expression, as detected with the anti-FLAG antibody. However, deletions of TS1 and TS4 inhibited the circ5533-mediated increase of PXK-FLAG protein, demonstrating that TS1 and TS4 are essential for circ5533 activity and suggesting that both BD1 and BD2 are functionally active.

We then investigated whether TS1 and TS4 were sufficient for circ5533 activity by assembling three PXK-FLAG mutants: (i) only TS1 at the 5’UTR; (ii) only TS4 at the 3’UTR; (iii) both TS1 and TS4 respectively at 5’ and 3’UTRs; and (iv) both TS4 and TS1 in inverted positions respectively at 5’ and 3’UTRs . Interestingly, the presence of TS1 alone was sufficient to sustain full circ5533 activity, while the exclusive presence of TS4 increased protein expression at lower levels. The combination of TS1 and TS4 restored full circ5533 activity while inverting the two transcript sites reduced the activity of circ5533 (Figure 6c). Circularized transcripts were evidenced with the use of divergent primers. No change in mRNA levels was observed in any of the experiments, demonstrating that the observed protein upregulation effects were post-transcriptional (Figure 6d-e).

Table 2 - Identification of potential BDs and TSs between circ5533 and Pxk mRNA

Identification of putative target sites (TSs) on Pxk mRNA pairing in antisense orientation with circ5533 RNA. Here, we report 18 alignments from the blast analysis between Pxk mRNA (subject sequence) and circ5533 RNA (query sequence) with a percentage of identity > 80% (% of identity) on a minimal match length of 8 nts (match length). S-start and S-end represent pairing coordinates on Pxk mRNA. Start and end match region coordinates on circ5533 sequence are reported in Q-start and Q-end respectively. Numbers of mismatches and/or gaps are also reported. BTOP value is the maximal perfect match before the first mismatch or gap. For the bioinformatics analysis, query sequence (circ5533) was divided in two main regions identified as non-IRES and IRES sequences, and then aligned against Pxk 5’UTR, CDS and 3’UTR, respectively. For each region, overlapping matches are indicated with 1 (Yes) and no matches with 0. SEQUENCES

Claims

1. A functional nucleic acid molecule comprising:

- one or more target binding sequences comprising one or more sequences reverse complementary to one or more target mRNA sequences for which protein translation is to be enhanced; and

- a regulatory sequence comprising an internal ribosome entry site (IRES), wherein the functional nucleic acid molecule is circular.

2. The functional nucleic acid molecule of claim 1 , wherein the functional nucleic acid molecule is a trans-acting functional nucleic acid molecule.

3. A functional nucleic acid molecule com prising:

- one or more target binding sequences comprising one or more sequences reverse complementary to one or more target mRNA sequences for which protein translation is to be enhanced; and

- a regulatory sequence comprising an internal ribosome entry site (IRES), wherein the functional nucleic acid molecule is linear and wherein the one or more target binding sequences and the regulatory sequence are positioned between two complementary intronic repeats.

4. The functional nucleic acid molecule of any one of claims 1 to 3, wherein the IRES is orientated, within the functional nucleic acid molecule, in the direct orientation relative to the 5’ to 3’ orientation of the functional nucleic acid molecule.

5. The functional nucleic acid molecule of any one of claims 1 to 4, wherein the regulatory sequence is located 3’ of the one or more target binding sequences.

6. The functional nucleic acid molecule of any one of claims 1 to 5, wherein the IRES comprises a sequence which has at least about 75% identity to a sequence selected from the group consisting of SEQ ID NOs 2 - 20, or a fragment thereof.

7. The functional nucleic acid molecule of any one of claims 1 to 6, wherein the IRES comprises a sequence which has at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a sequence selected from the group consisting of SEQ ID NOs 2 - 20, or a fragment thereof.

8. The functional nucleic acid molecule of any one of claims 1 to 7, wherein the IRES has 100% identity to a sequence selected from the group consisting of SEQ ID NOs 2 - 20, or a fragment thereof.

9. The functional nucleic acid molecule of any one of claims 6 to 8, wherein identity is defined across the length of overlap between the IRES sequence and the sequence selected from the group consisting of SEQ ID NOs 2 - 20.

10. The functional nucleic acid molecule of any one of claims 1 to 9, wherein the IRES comprises a sequence selected from the group consisting of SEQ ID NOs 2 - 20, or a fragment thereof.

11. The functional nucleic acid molecule of any one of claims 1 to 10, wherein the IRES consists of a sequence selected from the group consisting of SEQ ID NOs 2 - 20, or a fragment thereof.

12. The functional nucleic acid molecule of any one of claims 1 to 11 , wherein the fragment is about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370 or more nucleotides in length.

13. The functional nucleic acid molecule of any one of claims 1 to 12, wherein the functional nucleic acid molecule com prises R NA nucleotides or modified RNA nucleotides.

14. The functional nucleic acid molecule of claim 13, wherein the functional nucleic acid molecule consists of RNA nucleotides or modified RNA nucleotides.

15. The functional nucleic acid molecule of any one of claims 1 to 14, wherein the functional nucleic acid molecule is single stranded.

16. The functional nucleic acid molecule of any one of claims 1 to 15, comprising a spacer between the one or more target binding sequences and the regulatory sequence.

17. A DNA molecule encoding the functional nucleic acid molecule of any one of claims 1

18. An expression vector comprising the DNA molecule of claim 17.

19. A composition comprising the functional nucleic acid molecule of any one of claims 1 to 16, the DNA molecule of claim 17 or the expression vector of claim 18.

20. A pharmaceutical composition comprising the functional nucleic acid molecule of any one of claims 1 to 16, the DNA molecule of claim 17 or the expression vector of claim 18, and a pharmaceutically acceptable excipient.

21. Use of a functional nucleic acid molecule of any one of claims 1 to 16, the DNA molecule of claim 17, the expression vector of claim 18, the composition of claim 19, or the pharmaceutical composition of claim 20, for enhancing translation of one or more target mR NA sequences.

22. The functional nucleic acid molecule of any one of claims 1 to 16, the DNA molecule of claim 17, the expression vector of claim 18, the composition of claim 19 or the pharmaceutical composition of claim 20 for use in therapy.

23. The functional nucleic acid molecule of any one of claims 1 to 16, the DNA molecule of claim 17, the expression vector of claim 18, the composition of claim 19 or the pharmaceutical composition of claim 20 for use in a method of treating a disease associated with gene defects.

24. A method of treating a disease associated with gene defects comprising administering the functional nucleic acid molecule of any one of claims 1 to 16, the DNA molecule of claim 17, the expression vector of claim 18, the composition of claim 19 or the pharmaceutical composition of claim 20 to a subject.

25. The functional nucleic acid molecule of any one of claims 1 to 16, the DNA molecule of claim 17, the expression vector of claim 18, the composition of claim 19 or the pharmaceutical composition of claim 20 for use in the manufacture of a medicament for treating a gene defect.

26. An in vitro method for enhancing protein translation comprising administering to a cell the functional nucleic acid molecule of any one of claims 1 to 16, the DNA molecule of claim 17, the expression vector of claim 18, the composition of claim 19, or the pharmaceutical composition of claim 20.

27. The method of claim 26, wherein one or more of the target mR NA sequences is a therapeutic target mRNA sequence.