FUNCTIONAL NUCLEIC ACID MOLECULES UPREGULATING THE TRANSLATION OF A FRATAXIN MRNA

Abstract

There are disclosed functional nucleic acid molecules comprising at least one target binding sequence comprising a sequence reverse complementary to a frataxin mRNA sequence; and a regulatory sequence comprising a SINE B2 element or a functionally active fragment of a SINE B2 element or an internal ribosome entry site (IRES) sequence or an IRES derived sequence.

Claims

1. A functional nucleic acid molecule comprising: at least one target binding sequence comprising a sequence reverse complementary to a frataxin mRNA sequence; and at least one regulatory sequence comprising an RNA comprising a SINE B2 element or a functionally active fragment of a SINE B2 element or an internal ribosome entry site (IRES) sequence or an IRES derived sequence.

2. The functional nucleic acid molecule according to claim 1, wherein the at least one regulatory sequence comprises a sequence with at least 75% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 51 and SEQ ID NO: 53 to SEQ ID NO: 82.

3. The functional nucleic acid molecule according to claim 2, wherein the at least one regulatory sequence comprises a sequence with at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 51 and SEQ ID NO: 53 to SEQ ID NO: 82.

4. The functional nucleic acid molecule according to claim 1, wherein the at least one target binding 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 frataxin mRNA sequence; or a sequence reverse complementary to 0 to 80 nucleotides of the region upstream of an AUG site (start codon) of the frataxin mRNA and 0 to 40 nucleotides of the CDS of the frataxin mRNA sequence downstream of said AUG site.

5. The functional nucleic acid molecule according to claim 4, wherein the at least one target binding 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 frataxin mRNA sequence; or a sequence reverse complementary to 0 to 70 nucleotides of the region upstream of an AUG site (start codon) of the frataxin mRNA and 0 to 4 nucleotides of the CDS of the frataxin mRNA sequence downstream of said AUG site.

6. The functional nucleic acid molecule according to claim 1, further comprising at least one linker sequence between the at least one target binding sequence and the at least one regulatory sequence.

7. The functional nucleic acid molecule according to claim 1, wherein the molecule is circular.

8. A DNA molecule encoding the functional nucleic acid molecule according to claim 1.

9. An expression vector comprising the functional nucleic acid molecule according to claim 1.

10. A composition comprising the functional nucleic acid molecule according to claim 1, or comprising an expression vector that comprises the functional nucleic acid molecule.

11. A method for increasing protein synthesis efficiency of frataxin in a target cell comprising administering the functional nucleic acid molecule according to claim 1, or administering an expression vector that comprises the functional nucleic acid molecule, to the target cell.

12. The method according to claim 11, wherein the target cell exhibits a level of frataxin that is lower than the level of frataxin in a normal cell.

13-14. (canceled)

15. A method of treating Friedreich's ataxia (FRDA) comprising administering, to a patient with FRDA, a therapeutically effective amount of the functional nucleic acid molecule according to claim 1, or administering to the patient a therapeutically amount of an expression vector that comprises the functional nucleic acid molecule.

16. The method according to claim 12, wherein the cell is a mammalian cell.

17. The method according to claim 16, wherein the method is performed in vivo.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 shows a schematic representation of SINEUP functional domains and of the human FXN gene with examples of the target binding domains of the functional nucleic acid according to the invention.

[0023] FIG. 2 shows that synthetic SINEUPs increase endogenous frataxin protein level in human cells in vitro.

[0024] FIG. 3 shows SINEUP effect on FXN knockdown HEK293T/17 cells.

[0025] FIG. 4 shows that miniSINEUPs increase endogenous frataxin protein level in human cells in vitro.

[0026] FIG. 5 shows that miniSINEUPs increase endogenous frataxin protein level in SH-SY5Y cells in vitro.

[0027] FIG. 6 shows that the binding domain is specific and that frataxin protein expression in vitro increases selectively.

[0028] FIG. 7 shows effector domain optimization.

[0029] FIG. 8 shows miniSINEUPs lentiviral transduction optimization.

[0030] FIG. 9 shows lentiviral infection of HEK 293T/17 cells.

[0031] FIG. 10 shows increased endogenous FXN protein expression in FRDA-derived fibroblasts.

[0032] FIG. 11 shows that AAV9-miniSINEUPs increase endogenous frataxin protein level in HEK 293T/17 cells in vitro.

[0033] FIG. 12 shows protein rescue of FRDA-derived lymphoblasts.

[0034] FIG. 13 shows the phenotypic rescue of FRDA-derived lymphoblasts.

[0035] FIG. 14 shows the miniSINEUP-FXN effect on off-target protein expression.

DETAILED DESCRIPTION OF THE INVENTION

[0036] A functional nucleic acid molecule of the present invention comprises at least one target binding sequence comprising a sequence reverse complementary to a frataxin mRNA sequence and at least one regulatory sequence comprising an RNA comprising a SINE B2 element or a functionally active fragment of a SINE B2 element or an internal ribosome entry site (IRES) sequence or an IRES derived sequence.

[0037] The functional nucleic acid molecules of the invention are able to modulate protein translation of the mRNA target however, compared to other methods, the modulation is not the result of modifying the target gene and therefore does not include the risks associated with genome editing. Furthermore, the functional nucleic acid molecules are highly specific to the target, reducing any off-target side effects.

[0038] Regulatory Sequences

[0039] In one embodiment, the regulatory sequence has protein translation enhancing efficiency. The increase of the protein translation efficiency indicates that the efficiency 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 1.5 fold, such as at least 2 fold. In a further embodiment, expression of the protein encoded by the target mRNA is increased between 1.2 to 3 fold, such as between 1.5 and 2.2 fold.

[0040] In one embodiment, the regulatory sequence is located 3′ of the target binding sequence. The regulatory sequence may be in a direct or inverted orientation relative to the 5′ to 3′ orientation of the functional nucleic acid molecule. Reference to “direct” refers to the situation in which the regulatory sequence is embedded (inserted) with the same 5′ to 3′ orientation as the functional nucleic acid molecule. Instead, “inverted” refers to the situation in which the regulatory sequence is 3′ to 5′ oriented relative to the functional nucleic acid molecule.

[0041] Preferably, the at least one regulatory sequence comprises a sequence with at least 75% sequence identity, preferably at least 90% sequence identity, more preferably at least 95% sequence identity, even more preferably 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 1-82. In one embodiment, the at least one regulatory sequence consists of a sequence with at least 75% sequence identity, preferably at least 90% sequence identity, more preferably at least 95% sequence identity, even more preferably 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 1-82.

[0042] In one embodiment, the regulatory sequence comprises a SINE B2 element or a functionally active fragment of a SINE B2 element. The SINE B2 element is preferably in an inverted orientation relative to the 5′ to 3′ orientation of the functional nucleic acid molecule, i.e. an inverted SINE B2 element. As mentioned in the definitions section, inverted SINE B2 elements are disclosed and exemplified in WO 2012/133947.

[0043] Preferably, the at least one regulatory sequence comprises a sequence with at least 75% sequence identity, preferably 90% sequence identity, more preferably 95% sequence identity, even more preferably 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:51.

[0044] SEQ ID NO:1 (the inverted SINE B2 element in AS Uchl1) and SEQ ID NO:2 (the 77 nucleotide variant of the inverted SINE B2 element in AS Uchl1 that includes nucleotides 44 to 120) are particularly preferred.

[0045] Other inverted SINE B2 elements and functionally active fragments of inverted SINE B2 elements are SEQ ID NO:3 to SEQ ID NO:51. Experimental data showing the protein translation enhancing efficiency of these sequences is not explicitly shown in the present patent application, but is disclosed in a previous patent application in the name of the same applicant. SEQ ID NO:3 to SEQ ID NO:51 can therefore be used as regulatory sequences in molecules according to the present invention.

[0046] SEQ ID NO:3 to SEQ ID NO:6, SEQ ID NO:8 to SEQ ID NO:11, SEQ ID NO:18, SEQ ID NO:43 to SEQ ID NO:51 are functionally active fragments of inverted SINE B2 transposable element derived from AS Uchl1. The use of functional fragments reduces the size of the regulatory sequence which is advantageous if used in an expression vector (e.g. viral vectors which may be size-limited) because this provides more space for the target sequence and/or expression elements.

[0047] SEQ ID NO:7 is a full length 183 nt inverted SINE B2 transposable element derived from AS Uchl1. SEQ ID NO:12 to SEQ ID NO:17, SEQ ID NO:19 and SEQ ID NO:20, SEQ ID NO:39 to SEQ ID NO:42 are mutated functionally active fragments of inverted SINE B2 transposable element derived from AS Uchl1.

[0048] SEQ ID NO:21 to SEQ ID NO:25 and SEQ ID NO:28 to SEQ ID NO:38 are different SINE B2 transposable elements. SEQ ID NO:26 and SEQ ID NO:27 are sequences in which multiple inverted SINE B2 transposable element have been inserted.

[0049] Alternatively, the regulatory sequence comprises an IRES sequence or an IRES derived sequence. Therefore, in one embodiment, the regulatory sequence comprises an IRES sequence or an IRES derived sequence. Said sequence enhances translation of the target mRNA sequence.

[0050] 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: 53 and 54), human poliovirus IRESs (e.g. SEQ ID NO: 55 and 56), human encephalomyocarditis (EMCV) virus (e.g. SEQ ID NO: 57 and 58), human cricket paralysis (CrPV) virus (e.g. SEQ ID NO: 59 and 60), human Apaf-1 (e.g. SEQ ID NO: 61 and 62), human ELG-1 (e.g. SEQ ID NO: 63 and 64), human c-MYC (e.g. SEQ ID NO: 65-68), human dystrophin (DMD) (e.g. SEQ ID NO: 69 and 70).

[0051] Such sequences have been disclosed, defined and exemplified in WO 2019/058304. Preferably, such sequences have 75% sequence identity, preferably 90% sequence identity, more preferably 95% sequence identity, even more preferably 100% sequence identity to any of SEQ ID NO:53 to SEQ ID NO:82. More preferably, such sequences have 75% sequence identity, preferably 90% sequence identity, more preferably 95% sequence identity, even more preferably 100% sequence identity to any of SEQ ID NO:53 to SEQ ID NO:70.

[0052] Target Binding Sequences

[0053] In WO 2012/133947 it was already shown that the target binding sequence needs to have only about 60% similarity with a sequence reverse complementary to the target mRNA. As a matter of fact, the target binding sequence can even display a large number of mismatches and retain activity.

[0054] The target binding sequence comprises a sequence which is sufficient in length to bind to the frataxin mRNA transcript. Therefore, the target binding sequence may be at least 10 nucleotides long, such as at least 14 nucleotides long, such as least 18 nucleotides long. Furthermore, the target binding sequence may be less than 250 nucleotides long, preferably less than 200 nucleotides long, less than 150 nucleotides long, less than 100 nucleotides long, less than 80 nucleotides long, less than 60 nucleotides long or less than 50 nucleotides long. In one embodiment, the target binding sequence is between 4 and 50 nucleotides in length, such as between 18 and 44 nucleotides long.

[0055] The target binding sequence may be designed to hybridise with the 5′-untranslated region (5′ UTR) of the frataxin mRNA sequence. In one embodiment, the sequence is reverse complementary to 0 to 50 nucleotides, such as 0 to 40, 0 to 21 or 0 to 14 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 frataxin mRNA sequence. In one embodiment, the sequence is reverse complementary to 0 to 40 nucleotides, such as 0 to 32, 0 to 18 or 0 to 4 nucleotides of the CDS.

[0056] 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 frataxin mRNA sequence. In one embodiment, the sequence is reverse complementary to 0 to 80 nucleotides, such as 0 to 70 or 0 to 40 nucleotides of the AUG site. Alternatively, or in combination, the target binding sequence may be designed to hybridise to the frataxin mRNA sequence downstream of said AUG site. In one embodiment, the sequence is reverse complementary to 0 to 40 nucleotides, such as 0 to 4 nucleotides of the frataxin mRNA sequence downstream of said AUG site.

[0057] Preferably, the at least one target binding sequence is at least 10 nucleotides long and comprises, from 3′ to 5′: [0058] 1) a sequence reverse complementary to 0 to 50 nucleotides of the 5′ UTR and 0 to 40 nucleotides of the CDS of the frataxin mRNA sequence; or [0059] 2) a sequence reverse complementary to 0 to 80 nucleotides of the region upstream of an AUG site (start codon) of the frataxin mRNA and 0 to 40 nucleotides, preferably 0 to 10 nucleotides, of the CDS of the frataxin mRNA sequence downstream of said AUG site.

[0060] Of course in case 1) the coding sequence starts on the first AUG site (M1) of the mRNA. In case 2), the preferred AUG site is that corresponding to methionine 76 (M76) in exon 2.

[0061] More preferably, the at least one target binding sequence is at least 14 nucleotides long and comprises, from 3′ to 5′: [0062] a sequence reverse complementary to 0 to 40 (preferably 0 to 21, more preferably 0 to 14) nucleotides of the 5′ UTR and 0 to 32 (preferably 0 to 4, more preferably 0) nucleotides of the CDS of the frataxin mRNA sequence; or [0063] a sequence reverse complementary to 0 to 70 (preferably 0 to 40) nucleotides of the region upstream of an AUG site (start codon) of the frataxin mRNA and 0 to 4 (preferably 0) nucleotides of the CDS of the frataxin mRNA sequence downstream of said AUG site.

[0064] In one embodiment, the functional nucleic acid molecule comprises a sequence with at least 75% sequence identity, preferably at least 90% sequence identity, more preferably at least 95% sequence identity, even more preferably 100% sequence identity to any of SEQ ID NO: 83-98. In a further embodiment, the functional nucleic acid molecule consists of a sequence with at least 75% sequence identity, preferably at least 90% sequence identity, more preferably at least 95% sequence identity, even more preferably 100% sequence identity to any of SEQ ID NO: 83-98.

[0065] Structural Features

[0066] The functional nucleic acid molecule preferably comprises more than one regulatory sequence, which can be the same sequence repeated more than once, or a different regulatory sequence (i.e. a different SINE B2 element/functionally active fragment of a SINE B2 element/an IRES sequence/an IRES derived sequence).

[0067] The at least one target binding sequence and the at least one regulatory sequence are preferably connected by at least one spacer/linker sequence. In case of multiple sequences, several spacer/linker sequences can be inserted in-between the sequences. SEQ ID NO:52 is a non-limiting example of the spacer/linker sequence.

[0068] The functional nucleic acid molecule of the present invention is preferably a circular molecule. This conformation leads to a much more stable molecule that is degraded with greater difficulty within the cell (exonucleases cannot degrade circular molecules) and therefore remains active for a longer time.

[0069] Furthermore, the functional nucleic acid molecule may optionally comprise a non-coding 3′ tail sequence, which e.g. includes restriction sites useful for cloning the molecule in appropriate plasmids.

[0070] It should be noted that the functional nucleic acid molecules can enhance translation of the target gene of interest with no effects on mRNA quantities of the target gene. Therefore they can successfully be used as molecular tools to validate gene function in cells as well as to implement the pipelines of recombinant protein production.

[0071] DNA Molecules and Vectors

[0072] According to a further aspect of the invention, there is provided a DNA molecule encoding any of the above disclosed functional nucleic acid molecules. According to a further aspect of the invention, there is provided an expression vector comprising the above said DNA molecule.

[0073] Exemplary expression vectors are known in the art and may include, for example, plasmid vectors, viral vectors (for example adenovirus, adeno-associated virus, retrovirus or lentivirus vectors), phage vectors, cosmid vectors and the like. The choice of expression vector may be dependent upon the type of host cell to be used and the purpose of use. In particular the following plasmids have been used for efficient expression of functional nucleic acid molecules.

[0074] Mammalian Expression Plasmids: [0075] Plasmid Name: pCDNA3.1 (−) [0076] Expression: CMV promoter [0077] BGH poly(A) terminator [0078] Plasmid Name: pDUAL-eGFPΔ (modified from peGFP-C2) [0079] Expression: H1 promoter [0080] BGH poly(A) terminator

[0081] Viral Vectors: [0082] Vector Name: pAAV [0083] Virus: Adeno-Associated Virus [0084] Expression: CAG promoter/CMV enhancer [0085] SV40 late poly(A) terminator [0086] Vector Name: rcLV-TetOne-Puro [0087] Virus: Lentivirus (3rd generation) [0088] Expression: LTR-TREt (Tre-Tight) promoter (doxycycline-inducible expression) [0089] BGH poly(A) terminator [0090] Vector Name: pLPCX-link [0091] Virus: Retrovirus (3rd generation) [0092] Expression: CMV

[0093] It should be noted that any promoter may be used in the vector and will work just as well as those mentioned above.

[0094] Compositions

[0095] The present invention also relates to compositions comprising the above said functional nucleic acid molecules or the above said DNA molecules. Any compositions are included allowing to deliver the above said functional nucleic acid molecules by viral vectors (AAV, lentivirus and the like) and non-viral vectors (nanoparticles, lipid particles and the like).

[0096] According to a further aspect of the invention, there is provided the functional nucleic acid molecule, DNA molecule, expression vector or the composition as defined herein for use as a medicament.

[0097] It will be understood that the functional nucleic acid molecules of the invention find use in increasing the level of frataxin protein within a cell. Therefore, the above said functional nucleic acid molecules, DNA molecules and/or compositions may be used as medicaments, preferably for treating Friedreich's ataxia and in particular promoting the recovery of disease-associated mitochondrial defects.

[0098] Friedreich's ataxia is a rare genetic disorder caused by an insufficient quantity of frataxin protein. The main root of the pathology is the impaired transcription of the FXN gene as a result of GAA repeat expansion. As shown in the Examples provided herein, the functional nucleic acid molecules were able to rescue the physiological translation of frataxin even in patient cells with a mRNA deficit (e.g. see FIG. 11A showing Western blot comparison between healthy, patient lymphoblasts and patient lymphoblasts stably expressing miniSINEUP-FXN). Use of the functional nucleic acid molecules to treat Friedreich's ataxia has several advantages including inducing target gene expression within the range of 1.5 to 2.5 fold thus limiting side effects due to exaggerated overexpression and enabling exclusively in situ translation enhancement avoiding ectopic protein synthesis in the absence of the target mRNA. They also do not trigger any hereditable genome editing.

[0099] According to a further aspect of the invention, there is provided the use of the functional nucleic acid molecule (or DNA molecule, expression vector or composition) as defined herein for the manufacture of a medicament for the treatment of Friedreich's ataxia.

[0100] Methods

[0101] According to a further aspect of the invention, there is provided a method for enhancing protein translation of FXN 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.

[0102] According to a further aspect of the invention, there is provided a method for increasing the protein synthesis efficiency of frataxin in a cell comprising administering the functional nucleic acid molecule, DNA molecule, expression vector or composition as defined herein to the cell.

[0103] The methods described herein may comprise transfecting into a cell the functional nucleic acid molecule, DNA molecule or expression vector as defined herein. The functional nucleic acid molecule, DNA molecule or expression vector may be administered to target cells using methods known in the art and include, for example, microinjection, lipofection, electroporation, using calcium phosphate, self-infection by the vector or transduction of a virus.

[0104] The target cell to be treated may comprise a reduced amount of frataxin. In one embodiment, the level of frataxin in the cell is lower than the level of frataxin in a normal cell (i.e. a cell comprising a normal phenotype with functional copies of the FXN gene). For example, the level of frataxin in the cell may be less than 70% of the level of frataxin in a normal cell, such as less than 60% or less than 50% of the level of frataxin in a normal cell. In a further embodiment, the level of frataxin in the cell is about 50% of the level of frataxin in a normal cell.

[0105] In a further embodiment, the cell is FXN haploinsufficient, i.e. wherein the presence of a variant allele in a heterozygous combination results in the amount of product generated by the single wild-type gene is not sufficient for complete or normal function. Generally, haploinsufficiency is a condition that arises when the normal phenotype requires the protein product of both alleles, and reduction to 50% or less of gene function results in an abnormal phenotype.

[0106] Methods of the invention result in increased levels of frataxin in a cell and therefore find use, for example, in methods of treatment for diseases which are associated with FXN defects (i.e. reduced frataxin levels and/or loss-of-function mutations of the FXN gene). Methods of the invention find particular use in diseases caused by a quantitative decrease in the predetermined, normal protein level. Methods of the invention can be performed in vitro, ex vivo or in vivo.

[0107] According to a further aspect of the invention, there is provided a method of treating Friedreich's ataxia comprising administering a therapeutically effective amount of the functional nucleic acid molecule, DNA molecule, expression vector or composition as defined herein.

[0108] It will be understood that the embodiments described herein may be applied to all aspects of the invention, i.e. the embodiment described for the functional nucleic acid molecules may equally apply to the claimed methods and so forth.

[0109] The invention will now be illustrated with reference to the following non-limiting examples.

EXAMPLES

Example 1

[0110] This example shows how the regulatory sequences and the target binding sequences of the functional nucleic acid according to the invention have been designed.

[0111] In particular, FIG. 1A shows a schematic representation of SINEUPs functional domains. The binding domain (BD, grey) provides SINEUP specificity and is in antisense orientation to the sense protein-coding mRNA (Target mRNA). The inverted SINEB2 element (invB2) is the effector domain (ED) and confers enhancement of protein synthesis. 5′ to 3′ orientation of sense and antisense RNA molecules is indicated. Structural elements of target mRNA are shown: 5′ untranslated region (5′UTR, white), coding sequence (CDS, black) and 3′ untranslated region (3′UTR, white). Scheme is not drawn in scale. FIG. 1B shows a scheme of human FXN gene (5′-end, white) and BDs (grey) design of synthetic SINEUP-FXN targeting the initiating M1-AUG and the M76-AUG downstream GAA expansions. The numbering refers to the position according to the methionine (i.e. −40/+32, from 40 nucleotides upstream and to 32 nucleotides downstream the M1-AUG). Scheme is not drawn in scale.

[0112] The sequences used in the examples are as follows.

TABLE-US-00001 SEQ ID NO. Definition Features 83 SINEUP-FXN in antisense BD = −40/+32 relative to M1-AUG (72 bp) orientation to FXN mRNA ED = inverted SINEB2 repeat (167 bp) isoform 1 Partial Alu element (45 bp) Extra sequence (13 bp) Backbone = Δ5'ASUchl1 (1105 bp) 84 SINEUP-FXN in antisense BD = −40/+4 relative to M1-AUG (44 bp) orientation to FXN mRNA ED = inverted SINEB2 repeat (167 bp) isoform 1 Partial Alu element (45 bp) Extra sequence (13 bp) Backbone = Δ5'ASUchl1 (1105 bp) 85 SINEUP-FXN in antisense BD = −21/+32 relative to M1-AUG (53 bp) orientation to FXN mRNA ED = inverted SINEB2 repeat (167 bp) isoform 1 Partial Alu element (45 bp) Extra sequence (13 bp) Backbone = Δ5'ASUchl1 (1105 bp) 86 SINEUP-FXN in antisense BD = −21/+4 relative to M1-AUG (25 bp) orientation to FXN mRNA ED = inverted SINEB2 repeat (167 bp) isoform 1 Partial Alu element (45 bp) Extra sequence (13 bp) Backbone = Δ5'ASUchl1 (1105 bp) 87 SINEUP-FXN in antisense BD = −40/+0 relative to M1-AUG (40 bp) orientation to FXN mRNA ED = inverted SINEB2 repeat (167 bp) isoform 1 Partial Alu element (45 bp) Extra sequence (13 bp) Backbone = Δ5'ASUchl1 (1105 bp) 88 SINEUP-FXN in antisense BD = −14/+0 relative to M1-AUG (14 bp) orientation to FXN mRNA ED = inverted SINEB2 repeat (167 bp) isoform 1 Partial Alu element (45 bp) Extra sequence (13 bp) Backbone = Δ5'ASUchl1 (1105 bp) 89 SINEUP-FXN in antisense BD = −14/+4 relative to M1-AUG (18 bp) orientation to FXN mRNA ED = inverted SINEB2 repeat (167 bp) isoform 1 Partial Alu element (45 bp) Extra sequence (13 bp) Backbone = Δ5'ASUchl1 (1105 bp) 90 SINEUP-FXN in antisense BD = −40/+4 relative to M76-AUG (44 bp) orientation to FXN mRNA ED = inverted SINEB2 repeat (167 bp) isoform 1 Partial Alu element (45 bp) Extra sequence (13 bp) Backbone = Δ5'ASUchl1 (1105 bp) 91 SINEUP-FXN in antisense BD = −40/+4 relative to M76-AUG (40 bp) orientation to FXN mRNA ED = inverted SINEB2 repeat (167 bp) isoform 1 Partial Alu element (45 bp) Extra sequence (13 bp) Backbone = Δ5'ASUchl1 (1105 bp) 92 SINEUP-FXN in antisense BD = (−10/−60/+ 0 relative to orientation to FXN mRNA M76-AUG (70 bp) isoform 1 ED = inverted SINEB2 repeat (167 bp) Partial Alu element (45 bp) Extra sequence (13 bp) Backbone = Δ5'ASUchl1 (1105 bp) 93 miniSINEUP-FXN in BD = −40/+0 relative to M1-AUG (40 bp) antisense orientation to FXN ED = inverted SINEB2 repeat (167 bp) mRNA isoform 1 Extra sequence (13 bp) 94 miniSINEUP-FXN in BD = −14/+0 relative to M1-AUG (14 bp) antisense orientation to FXN ED = inverted SINEB2 repeat (167 bp) mRNA isoform 1 Extra sequence (13 bp) 95 miniSINEUP-FXN in BD = −14/+4 relative to M1-AUG (18 bp) antisense orientation to FXN ED = inverted SINEB2 repeat (167 bp) mRNA isoform 1 Extra sequence (13 bp) 96 miniSINEUP-FXN in BD = −40/+4 relative to M76-AUG (44 bp) antisense orientation to FXN ED = inverted SINEB2 repeat (167 bp) mRNA isoform 1 Extra sequence (13 bp) 97 microSINEUP-FXN in BD = −40/+0 relative to M1-AUG (40 bp) antisense orientation to FXN ED = nt 44-120 of inverted SINEB2 mRNA isoform 1 element (77 bp) Extra sequence (19 bp) 98 microSINEUP-FXN in BD = −14/+0 relative to M1-AUG (14 bp) antisense orientation to FXN ED = nt 44−120 of inverted SINEB2 mRNA isoform 1 element (77 bp) Extra sequence (19 bp) BD: Binding domain; ED: Effector domain

Example 2

[0113] This example shows that synthetic SINEUPs increase endogenous frataxin protein level in human cells in vitro. In particular, HEK 293T/17 cells (ATCC Cat. No. CRL-11268) were transfected with empty vector (ctrl) and SINEUP-FXN variants and harvested 48 hours post transfection. Empty vector (ctrl) and miniSINEUP-FXN −40/+4 M1-AUG were taken as negative and positive controls respectively. HEK 293T/17 cells were used to screen the activity of SINEUP-FXN because they endogenously express frataxin.

[0114] FIG. 2A, left panel shows a Western blot with anti-FXN and anti-β-actin antibodies of whole cell lysates. One representative experiment is shown. First, FXN band intensity was normalized to the relative β-actin band. Then, fold change values were calculated normalizing to control cells (ctrl). SINEUP-FXN-transfected cells show increased levels of endogenous FXN protein. FIG. 2A, right panel shows real-time PCR analysis of FXN mRNA and SINEUP RNA expression in transfected cells. In particular, BDs with minimal (−40/+4 M1-AUG) or no (−40/+0 M1-AUG) overlap to the CDS induced up-regulation of mature frataxin in the range of 1.4-fold. When the overlapping region corresponded exactly to the 5′UTR, as in the case for −14/+0 and −14/+4 M1-AUG configurations, SINEUPs reached the highest potency (1.5- to 2-fold increase). Columns represent mean±S.E.M. of n≥4 independent experiments. Variation in both target and SINEUP mRNA expression among samples are not statistically significant (one-way ANOVA followed by Dunnett's post-test). FXN transcripts were quantified, using human GAPDH (hGAPDH) expression as internal control. The FXN/hGAPDH ratio for ctrl sample was set as a baseline value to which all transcripts levels were normalized. Unchanged FXN mRNA levels are shown, thereby confirming FXN increased protein synthesis at post-transcriptional level. SINEUP transcripts were quantified, using hGAPDH expression as internal control. The SINEUP/hGAPDH ratio for −40/+4 M1-AUG sample was set as a baseline value to which all transcripts levels were normalized. FIG. 2B shows average fold change of FXN protein levels. Columns represent mean±S.E.M. of n≥4 independent experiments; ns, p>0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 (one-way ANOVA followed by Dunnett's post-test).

Example 3

[0115] This example shows SINEUP effect on FXN knockdown HEK293T/17 cells. Silencing of FXN by shFXN (sh, Short Hairpin) in HEK 293T/17 cells. Cells were co-transfected with shCTRUSINEUP ctrl (empty vectors), shFXN/SINEUP ctrl and shFXN/SINEUP-FXN −40/+0 M1-AUG. shCTRL/SINEUP and shFXN/SINEUP were taken as negative and positive silencing controls respectively. 48 hours post transfection, whole cell lysates were analysed by Western blotting with anti-FXN and anti-β-actin antibodies (FIG. 3). First, FXN band intensity was normalized to the relative f3-actin. Then, fold change values were calculated normalizing to shCTRL/SINEUP ctrl sample. SINEUP-FXN −40/+0 M1-AUG drove a 30% increase in FXN protein level (n=1).

Example 4

[0116] This example shows that miniSINEUPs increase endogenous frataxin protein level in human cells in vitro.

[0117] In particular FIG. 4A is a scheme of human FXN gene (5′-end) and binding domains anatomy of tested synthetic miniSINEUP-FXN targeting the initiating M1-AUG and the M76-AUG downstream GAA expansions. In FIGS. 4B and 4C HEK 293T/17 cells were transfected with empty vector (ctrl) and miniSINEUP-FXN variants (−40/+0; −14/+0 and −14/+4 M1-AUG or −40/+4 M76-AUG) and harvested 48 hours post transfection. Empty vector (ctrl) and miniSINEUP-FXN −40/+0 M1-AUG were taken as negative control and positive controls respectively. In FIG. 4B, left panel: 48 hours post transfection, whole cell lysates were analysed by Western blotting with anti-FXN and anti-β-actin antibodies. One representative experiment is shown. First, FXN band intensity was normalized to the relative β-actin band. Then, fold change values were calculated normalizing to control cells (ctrl). miniSINEUP-FXN-transfected cells show increased levels of endogenous FXN protein. miniSINEUPs-FXN promoted a protein induction consistently in the range of 1.4- to 1.7-fold, proving they retain the same efficacy of their full-length counterpart with the advantage of being shorter. In FIG. 4B, right panel: real-time PCR analysis of FXN mRNA and miniSINEUP RNA expression in transfected cells. Columns represent mean±S.E.M. of n=3 independent experiments. Variation in both target and SINEUP mRNA expression among samples are not statistically significant (one-way ANOVA followed by Dunnett's post-test). FXN transcripts were quantified, using human GAPDH (hGAPDH) expression as internal control. The FXN/hGAPDH ratio for ctrl sample was set as a baseline value to which all transcripts levels were normalized. Unchanged FXN mRNA levels are shown, thereby confirming FXN increased protein synthesis at post-transcriptional level. miniSINEUP transcripts were quantified, using hGAPDH expression as internal control. The miniSINEUP/hGAPDH ratio for −40/+0 M1-AUG sample was set as a baseline value to which all transcripts levels were normalized. FIG. 4C shows average fold change of FXN protein levels. Columns represent mean±S.E.M. of n=3 independent experiments; ns, p>0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 (one-way ANOVA followed by Dunnett's post-test).

Example 5

[0118] This example shows that miniSINEUPs increase endogenous frataxin protein level in SH-SY5Y cells in vitro.

[0119] In FIGS. 5A and 5B, SH-SY5Y cells (ATCC Cat. No. CRL-2266) were transfected with empty vector (ctrl) and miniSINEUP-FXN variants (−40/+0; −14/+0 and −14/+4 M1-AUG or −40/+4 M76-AUG) and harvested 48 hours post transfection. Empty vector (ctrl) and miniSINEUP-FXN −40/+0 M1-AUG were taken as negative control and positive controls respectively. In FIG. 5A, left panel: whole cell lysates were analysed by Western blotting with anti-FXN and anti-β-actin antibodies. One representative experiment is shown. First, FXN band intensity was normalized to the relative β-actin band. Then, fold change values were calculated normalizing to control cells (ctrl). miniSINEUP-FXN-transfected cells show increased levels of endogenous FXN protein. However, the up-regulation is not statistically significant. In FIG. 5A, right panel: real-time PCR analysis of FXN mRNA and miniSINEUP RNA expression in transfected cells. Columns represent mean±S.E.M. of n=3 independent experiments. Variation in both target and miniSINEUP mRNA expression among samples are not statistically significant (one-way ANOVA followed by Dunnett's post-test). FXN transcripts were quantified, using human GAPDH (hGAPDH) expression as internal control. The FXN/hGAPDH ratio for ctrl sample was set as a baseline value to which all transcripts levels were normalized. Unchanged FXN mRNA levels are shown, thereby confirming FXN increased protein synthesis at post-transcriptional level (top). minSINEUP transcripts were quantified, using hGAPDH expression as internal control. The miniSINEUP/hGAPDH ratio for −40/+0 M1-AUG sample was set as a baseline value to which all transcripts levels were normalized (bottom). FIG. 5B shows average fold change of FXN protein levels. Columns represent mean±S.E.M. of n=3 independent experiments; ns, p>0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 (one-way ANOVA followed by Dunnett's post-test).

Example 6

[0120] This example shows the binding domain's specificity: a selective increase of frataxin protein expression in vitro is demonstrated. In FIGS. 6A and 6B, HEK 293T/17 cells were transfected with empty vector (ctrl), deltaBD (ΔBD, construct lacking the overlapping region to FXN mRNA), miniSINEUP-FXN −40/+0 M1-AUG and harvested 48 hours post transfection. Empty vector (ctrl) and miniSINEUP-FXN −40/+0 M1-AUG were taken as negative control and positive controls respectively. In FIG. 6A, whole cell lysates were analysed by Western blotting with anti-FXN and anti-β-actin antibodies. One representative experiment is shown. First, FXN band intensity was normalized to the relative β-actin band. Then, fold change values were calculated normalizing to control cells (ctrl). miniSINEUP-FXN-transfected cells show increased levels of endogenous FXN protein, while deltaBD-transfected cells showed unchanged protein levels. FIG. 6B shows average fold change of FXN protein levels. Columns represent mean±S.E.M. of n≥4 independent experiments; ns, p>0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 (one-way ANOVA followed by Dunnett's post-test).

Example 7

[0121] This example shows effector domain optimization. microSINEUPs increase endogenous frataxin protein level in HEK 293T/17 cells in vitro. HEK 293T/17 cells were transfected with empty vector (ctrl), miniSINEUP-FXN −40/+0 M1-AUG and microSINEUP-FXN variants (−40/+0; −14/+0 M1-AUG). Cells were harvested 48 hours post transfection. Empty vector (ctrl) and miniSINEUP-FXN −40/+0 M1-AUG were taken as negative control and positive controls respectively. In FIG. 7A, whole cell lysates were analysed by Western blotting with anti-FXN and anti-β-actin antibodies. One representative experiment is shown. First, FXN band intensity was normalized to the relative β-actin. Then, fold change values were calculated normalizing to control cells (ctrl). microSINEUP-FXN-transfected cells show increased levels of endogenous FXN protein. FIG. 7B shows average fold change of FXN protein levels. Columns represent mean±S.E.M. of n=3 independent experiments; ns, p>0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 (one-way ANOVA followed by Dunnett's post-test).

Example 8

[0122] This example shows miniSINEUPs lentiviral transduction optimization. In particular, FIGS. 8A to 8C show infection of human neuroblastoma cells (SH-SY5Y) with inducible lentiviral vectors driving the expression of empty virus (ctrl) and LVminiSINEUP-FXN variants (−14/+0 M1-AUG or −40/+4 M76-AUG). FIG. 8A shows doxycycline treatment timelines. Single induction timing (top). 48 hrs after infection (time 0), cells were subjected to doxycycline treatment and harvested 96 hrs after infection. Double induction timing (bottom). Cells were treated twice with doxycycline (time 48 and 96 hrs) and harvested 144 hrs after infection. In FIG. 8B SH-SY5Y cells were infected following both protocols timing. Whole cell lysates were analysed by western blotting with anti-FXN and anti-β-actin antibodies. One representative experiment is shown. First, FXN band intensity was normalized to the relative β-actin. Then, fold change values were calculated normalizing to control cells (ctrl). LVminiSINEUP-FXN-infected cells show increased levels of endogenous FXN protein only with double doxycycline induction, while no up-regulation was observed after a single treatment. FIG. 8C shows average fold change of FXN protein levels. Columns represent mean±S.E.M. of n≥3 independent experiments; ns, p>0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 (one-way ANOVA followed by Dunnett's post-test).

Example 9

[0123] This example shows lentiviral infection of HEK293T/17 cells. In FIGS. 9A and 9B, HEK 293T/17 cells were infected with inducible lentiviral vectors driving the expression of empty virus (ctrl) and LVminiSINEUP-FXN variants (−14/+0 M1-AUG or −40/+4 M76-AUG), induced 48- and 96-hours post infection, and harvested 6 days post infection. Empty vector (ctrl) and LVminiSINEUP-FXN −40/+0 M1-AUG were taken as negative control and positive controls respectively. In FIG. 9A, whole cell lysates were analysed by western blotting with anti-FXN and anti-β-actin antibodies. One representative experiment is shown. First, FXN band intensity was normalized to the relative β-actin. Then, fold change values were calculated normalizing to control cells (ctrl). LVminiSINEUP-FXN-infected cells show increased levels of endogenous FXN protein. FIG. 9B shows average fold change of FXN protein levels. Columns represent mean±S.E.M. of n=3 independent experiments; ns, p>0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 (one-way ANOVA followed by Dunnett's post-test).

Example 10

[0124] This example shows increased endogenous FXN protein expression in FRDA-derived fibroblasts. GM04078 cells (patients' primary fibroblasts) showed an intermediate phenotype carrying a hyper-expansion of about 541 repeats on one allele and 420 repeats on the other one. In FIGS. 10A and 10B GM04078 cells were infected with inducible lentiviral vectors driving the expression of empty virus (ctrl) and LVminiSINEUP-FXN variants (−40/+0; −14/+0 and −14/+4 M1-AUG or −40/+4 M76-AUG), induced 48 and 96 hours post infection, and harvested 6 days post infection. Empty vector (ctrl) and LVminiSINEUP-FXN −40/+0 M1-AUG were taken as negative control and positive controls respectively.

[0125] In FIG. 10A, left panel: whole cell lysates were analysed by Western blotting with anti-FXN and anti-β-actin antibodies. One representative experiment is shown. First, FXN band intensity was normalized to the relative β-actin. Then, fold change values were calculated normalizing to control cells (ctrl). LVminiSINEUP-FXN-infected cells show increased levels of endogenous FXN protein. All LV miniSINEUPs led to an increase in frataxin quantities in the range of 1.6- to 2.1 fold (FIG. 10A). Importantly, the position of SINEUP BD relative to the GAA expansion and the presence of the pathological expansion itself did not interfere with the observed protein increase in patients' cells. Considering that GM04078 cells show reduced levels of frataxin, averaging around 40% when compared to age- or sex-matched healthy-derived cells (Gomez-Sebastian et al. (2007) Mol. Ther., 15: 248-254), SINEUP activity rescued physiological protein quantities in this FRDA cellular model.

[0126] In FIG. 10A, right panel: real-time PCR analysis of FXN mRNA and miniSINEUP RNA expression in transfected cells. Columns represent mean±S.E.M. of n≥4 independent experiments. Variation in both target and miniSINEUP mRNA expression among samples are not statistically significant (one-way ANOVA followed by Dunnett's post-test). FXN transcripts were quantified, using human GAPDH (hGAPDH) expression as internal control. The FXN/hGAPDH ratio for ctrl sample was set as a baseline value to which all transcripts levels were normalized. Unchanged FXN mRNA levels are shown, thereby confirming FXN increased protein synthesis at post-transcriptional level (top). miniSINEUP transcripts were quantified, using hGAPDH expression as internal control. The miniSINEUP/hGAPDH ratio for −40/+0 M1-AUG sample was set as a baseline value to which all transcripts levels were normalized (bottom). FIG. 10B shows average fold change of FXN protein levels. Columns represent mean±S.E.M. of n≥4 independent experiments; ns, p>0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 (one-way ANOVA followed by Dunnett's post-test).

Example 11

[0127] This example shows that AAV9-miniSINEUPs increase endogenous frataxin protein level in HEK 293T/17 cells in vitro. In FIGS. 11A and 11B, HEK 293T/17 cells were transfected with adeno-associated serotype 9 (AAV9) empty vector (ctrl) and AAV9miniSINEUP-FXN variants (−40/+0; −14/+0 and −14/+4 M1-AUG or −40/+4 M76-AUG) and harvested 48 hours post transfection. Empty vector (ctrl) and miniSINEUP-FXN −40/+0 M1-AUG were taken as negative control and positive controls respectively. In FIG. 11A, whole cell lysates were analysed by Western blotting with anti-FXN and anti-β-actin antibodies. MiniSINEUP-FXN-transfected cells show increased levels of endogenous FXN protein. One representative experiment is shown. First, FXN band intensity was normalized to the relative β-actin. Then, fold change values were calculated normalizing to control cells (ctrl). miniSINEUP-FXN-transfected cells show increased levels of endogenous FXN protein. However, the up-regulation is not statistically significant. FIG. 11B shows average fold change of FXN protein levels. Columns represent mean±S.E.M. of n=3 independent experiments; ns, p>0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 (one-way ANOVA followed by Dunnett's post-test).

Example 12

[0128] This example shows protein rescue of FRDA-derived lymphoblasts which carry the pathogenic expansion of GAA repeats and show reduced levels of the protein when compared to controls. GM16214 cells (patients' primary lymphoblasts) were stably transfected with empty vector (ctrl) and miniSINEUP-FXN variants. Ctrl, −40/+0 M1-AUG and −40/+4 M76-AUG stable clones were obtained from at least 15 days of G418 selection. Untransfected GM16214 cells and ctrl clone were taken as negative controls, while GM16215 cells (primary lymphoblasts derived from the healthy heterozygous patient's mother) were taken as positive control. In FIG. 12A, left panel: whole cell lysates were analysed by Western blotting with anti-FXN and anti-β-actin antibodies. Two representative experiments are shown. First, FXN band intensity was normalized to the relative β-actin. Then, fold change values were calculated normalizing to control cells (ctrl). GM16214 cells expressing miniSINEUP-FXN show increased levels of endogenous FXN protein. In FIG. 12A, right panel: real-time PCR analysis of FXN mRNA and miniSINEUP RNA expression in transfected cells. Columns represent mean±S.E.M. of n≥4 independent experiments. Variation in both target and miniSINEUP mRNA expression among samples are not statistically significant (one-way ANOVA followed by Dunnett's post-test). FXN transcripts were quantified, using human GAPDH (hGAPDH) expression as internal control. The FXN/hGAPDH ratio for ctrl sample was set as a baseline value to which all transcripts levels were normalized. Unchanged FXN mRNA levels are shown, thereby confirming FXN increased protein synthesis at post-transcriptional level (top). miniSINEUP transcripts were quantified, using hGAPDH expression as internal control. The miniSINEUP/hGAPDH ratio for −40/+0 M1-AUG sample was set as a baseline value to which all transcripts levels were normalized (bottom).

[0129] FIG. 12B shows average fold change of FXN protein levels. Extracts from FRDA cells showed a significant deficit of FXN protein expression averaging ˜2.3-fold when compared to control lymphoblasts derived from the healthy heterozygous patient's mother (FIG. 12B). Analysis of independent miniSINEUP clones revealed a strong rescue of frataxin levels while negative control transfectants (ctrl) showed no significative change. In particular, an up-regulation ranging from 1.6- to 2.9-fold is observed when compared to negative controls (FIG. 12B). Columns represent mean±S.E.M. of n≥4 independent experiments; ns, p>0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 (one-way ANOVA followed by Dunnett's post-test).

Example 13

[0130] This example shows the phenotypic rescue of FRDA-derived lymphoblasts. Frataxin-deficient cells are primarily affected by defective iron-sulfur cluster biosynthesis. Accordingly, insufficient frataxin levels trigger a typical loss in the activity of aconitases, two different ISC-dependent enzymes located in mitochondrial and cytosolic compartments. To assess the functional impact of SINEUPs, aconitase activity was chosen as a functional readout of restoring frataxin physiological levels for FRDA stable transfectants. Citrate synthase assay is used as an internal control.

[0131] The effect of miniSINEUP-FXN expression in vitro on aconitase activity was measured on whole cells lysates. Untransfected GM16214 cells and ctrl clones were taken as negative controls, while GM16215 cells (primary lymphoblasts derived from the healthy heterozygous patient's mother) were taken as positive control, as previously shown for other experiments. GM16214 cells expressing miniSINEUP-FXN show restored activity of endogenous aconitase as compared to GM16215 positive control. Activity of citrate synthase, the Krebs cycle enzyme catalysing the preceding step respect to aconitase, but lacking ISC, did not show significant fluctuations in assayed extracts. Aconitase (FIG. 13A) and citrate synthase (FIG. 13B) activities are expressed as mU/mg ratio. Columns represent mean±S.E.M. (n≥4) of mU/mg values; ns, p>0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 (one-way ANOVA followed by Dunnett's post-test).

Example 14

[0132] This example shows analysis of off-target effects. To predict putative off-targets, the Basic Local Alignment Search Tool (BLAST) of Ensembl genome browser was used to align the binding domain sequences to the human mRNA dataset. Results were filtered for match orientation while non-functional genes (pseudogenes and patch chromosomes) were removed. Potential off-target mRNAs were identified for their 100% identity to SINEUP-FXNs within the 5′UTRs of STX1B, FAM49A and CBX3 genes with length ranging from 13 to 20 nucleotides. All complementary sequences were distant from the translation initiation site of the target mRNA. Unconventional positions of target binding sites were also identified for TUBGCP5 (CDS) and for SH3GLB2, EIF4E and DISC1 (3′UTRs).

[0133] HEK 293T/17 cells were transfected with empty vector (ctrl) and miniSINEUP-FXN variants (−40/+0; −14/+0 and −14/+4 M1-AUG or −40/+4 M76-AUG) and harvested 48 hours post transfection. Empty vector (ctrl) was taken as negative control. Whole cell lysates were analysed by western blotting. Average fold changes of both target and off-targets for each binding domain are shown. First, band intensity was normalized to the relative β-actin band. Then, fold change values were calculated normalizing to control cells (ctrl). The mean FXN fold changes are plotted as the mean±S.E.M. (n≥4); ns, P>0.05; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 (one-way ANOVA followed by Dunnett's post-test). Off-targets fold changes are plotted as the mean±SEM (n=3) and p values are calculated by unpaired t-test with Welch's correction. ns, P>0.05. As shown in FIG. 14, in the very same lysates where miniSINEUP-FXNs were active, no effects on these additional targets were observed highlighting the specificity of the designed SINEUP RNAs.

[0134] The above disclosed examples show that a number of antisense long non-coding SINEUP RNAs targeting human FXN mRNA are capable to up-regulate frataxin protein to physiological amounts acting at post-transcriptional level. FXN-specific SINEUPs promote the recovery of disease-associated mitochondrial defects in FRDA-derived cells. SINEUPs are feasibly the first gene-specific therapeutic approach to activate FXN translation in FRDA.

[0135] In detail: [0136] 1) binding domains efficient to target FXN mRNA have been identified and optimised. [0137] 2) Effector domains efficient to upregulate frataxin protein levels including those indicated as miniSINEUPs and microSINEUPs and their modifications have been identified and optimised. [0138] 3) Experimental proof has been obtained of SINEUP ability to increase frataxin protein levels within physiological range, even when endogenous frataxin levels are very low as in FRDA patients. [0139] 4) The ability of SINEUP-FRXs to function when delivered with different types of plasmid and viral vectors has been shown. [0140] 5) The ability of SINEUP-FRXs to function within different types of cells, including those of FRDA patients has been shown. [0141] 6) The ability of SINEUP-FRXs to restore mitochondrial activity within physiological range in patients' cells has been shown.

[0142] The present invention therefore provides functional nucleic acids that are able to increase endogenous frataxin protein levels within physiological range and restore mitochondrial activity in FRDA patients' cells representing a new therapeutic strategy for this untreatable disease. By restoring physiological levels of frataxin, the molecules according to the invention limit potential side effects due to exaggerated overexpression of frataxin proteins by more conventional gene therapy approaches. In addition, by taking advantage of RNA base pairing between the binding domain and FRX mRNA, the molecules according to the invention limit potential side effects present when a small molecule approach is used to treat FRDA patients.