Functional nucleic acid molecule and use thereof

Abstract

There is disclosed a trans-acting functional nucleic acid molecule comprising a eukaryotic target binding sequence comprising a sequence reverse complementary to a target mRNA sequence for which protein translation is to be enhanced, and a regulatory sequence comprising an internal ribosome entry site (IRES) sequence or an internal ribosome entry site (IRES) derived sequence and enhancing translation of the target mRNA sequence, wherein the regulatory sequence is located 3′ of the target binding sequence.

Claims

1. A trans-acting functional nucleic acid molecule comprising: a target binding sequence comprising a sequence reverse complementary to a eukaryotic target mRNA sequence for which protein translation is to be enhanced; and a regulatory sequence comprising an internal ribosome entry site (IRES) sequence or an internal ribosome entry site (IRES) derived sequence and enhancing translation of the target mRNA sequence, wherein the regulatory sequence is located 3′ of the target binding sequence.

2. The trans-acting functional nucleic acid molecule according to claim 1, wherein the target binding sequence consists, from 3′ to 5′, of a sequence reverse complementary to 1 to 50 nucleotides of the 5′ untranslated region (5′ UTR) and 1 to 40 nucleotides of the coding sequence (CDS) of the target mRNA sequence.

3. The trans-acting functional nucleic acid molecule according to claim 2, wherein the target binding sequence consists, from 3′ to 5′, of a sequence reverse complementary to 10 to 45 nucleotides of the 5′ untranslated region (5′ UTR) and 2 to 6 nucleotides of the coding sequence (CDS) of the target mRNA sequence.

4. The trans-acting functional nucleic acid molecule according to claim 1, wherein the IRES sequence or IRES derived sequence is oriented, in the trans-acting functional nucleic acid molecule, in direct orientation relative to the 5′ to 3′ orientation of the functional nucleic acid molecule.

5. The trans-acting functional nucleic acid molecule according to claim 1, wherein the IRES sequence or IRES derived sequence is a sequence with 75% homology to a sequence selected from the group consisting of SEQ ID NO:36 to SEQ ID NO:65.

6. The trans-acting functional nucleic acid molecule according to claim 5, wherein the IRES sequence or IRES derived sequence is a sequence with 90% homology to a sequence selected from the group consisting of SEQ ID NO:36 to SEQ ID NO:65.

7. The trans-acting functional nucleic acid molecule according to claim 6, wherein the IRES sequence or IRES derived sequence is a sequence selected from the group consisting of SEQ ID NO:36 to SEQ ID NO:65.

8. The trans-acting functional nucleic acid molecule according to claim 1, wherein the trans-acting functional nucleic acid molecule is an RNA molecule or a modified RNA molecule.

9. The trans-acting functional nucleic acid molecule according to claim 1, further comprising a spacer sequence between the target binding sequence and the regulatory sequence.

10. A DNA molecule encoding the trans-acting functional nucleic acid molecule according to claim 1.

11. An expression vector comprising the DNA molecule according to claim 10.

12. A method for enhancing protein translation, the method comprising transfecting into a cell (a) the trans-acting functional nucleic acid molecule according to claim 1, (b) a DNA molecule encoding the trans-acting functional nucleic acid molecule according to claim 1, or (c) an expression vector comprising a DNA molecule encoding the trans-acting functional nucleic acid molecule according to claim 1.

13. A composition comprising (a) the trans-acting functional nucleic acid molecule according to claim 1, (b) a DNA molecule encoding the trans-acting functional nucleic acid molecule according to claim 1, or (c) an expression vector comprising a DNA molecule encoding the trans-acting functional nucleic acid molecule according to claim 1.

14. A method for enhancing translation of a target mRNA sequence, the method comprising hybridizing the target mRNA sequence to (a) the trans-acting functional nucleic acid molecule according to claim 1, (b) a DNA molecule encoding the trans-acting functional nucleic acid molecule according to claim 1, or (c) an expression vector comprising a DNA molecule encoding the trans-acting functional nucleic acid molecule according to claim 1.

15. A method for treating a genetic disease caused by down-regulation of a protein-coding mRNA, the method comprising administering (a) the trans-acting functional nucleic acid molecule according to claim 1, (b) a DNA molecule encoding the trans-acting functional nucleic acid molecule according to claim 1, or (c) an expression vector comprising a DNA molecule encoding the trans-acting functional nucleic acid molecule according to claim 1.

16. A method for treating a genetic or sporadic disease where reduced gene dosage is detrimental, the method comprising administering (a) the trans-acting functional nucleic acid molecule according to claim 1, (b) a DNA molecule encoding the trans-acting functional nucleic acid molecule according to claim 1, or (c) an expression vector comprising a DNA molecule encoding the trans-acting functional nucleic acid molecule according to claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a schematic diagram of the trans-acting functional nucleic acid molecule according to the present invention.

(2) FIG. 2A shows a schematic diagram of a functional nucleic acid molecule according to the prior art (SINEUP).

(3) FIG. 2B shows the results of a Western blot carried out on lysates of human embryonic kidney 293T/17 cells (hereinafter also referred to as HEK 293T/17 cells) transfected respectively with empty control plasmid (−), full-length SINEUP-DJ-1 (FL) and its deletion mutants (ΔED=mutant with deleted effector domain, ΔBD=mutant with deleted binding domain).

(4) FIG. 2C shows the results of a qRT-PCR to quantify expression of endogenous DJ-1 mRNA (top panel) and SINEUP RNA (bottom panel) carried out on samples as in FIG. 2B.

(5) FIG. 2D shows a graphical representation of full-length SINEUP-DJ-1, ΔED and ΔBD translation enhancement activity on endogenous DJ-1 mRNA in HEK 293T/17 cells (N=5). p<0.05

(6) FIG. 3 shows a schematic diagram of a generic trans-acting functional nucleic acid molecule according to the present invention (IRUP) targeting DJ-1 mRNA.

(7) FIG. 4 shows a schematic diagram of the experimental procedure for testing translation up-regulation activity of IRES-containing functional antisense nucleic acid molecules.

(8) FIG. 5A shows a schematic diagram of a functional nucleic acid molecule according to the present invention (IRUP) targeting the DJ-1 gene and including an HCV IRES.

(9) FIG. 5B shows the results of a Western blot carried out on lysates of HEK 293T/17 cells transfected respectively with empty control plasmid, SINEUP-DJ-1, and an IRUP including an HCV IRES in direct (HCV(d)) and inverted orientation (HCV(i)).

(10) FIG. 5C shows the results of a qRT-PCR to quantify expression of endogenous DJ-1 mRNA (top panel) and IRUP RNA (bottom panel) carried out on samples as in FIG. 5B.

(11) FIG. 6A shows a schematic diagram of a functional nucleic acid molecule according to the present invention (IRUP) targeting the DJ-1 gene and including a Poliovirus IRES.

(12) FIG. 6B shows the results of a Western blot carried out on lysates of HEK 293T/17 cells transfected respectively with empty control plasmid, SINEUP-DJ-1, and an IRUP including a Poliovirus IRES in direct (Polio (d)) and inverted orientation (Polio(i)).

(13) FIG. 6C shows the results of a qRT-PCR to quantify expression of endogenous DJ-1 mRNA (top panel) and IRUP RNA (bottom panel) carried out on samples as in FIG. 6B.

(14) FIG. 7A shows a schematic diagram of a functional nucleic acid molecule according to the present invention (IRUP) targeting the DJ-1 gene and including an encephalomyocarditis virus (EMCV) IRES and a cricket paralysis virus (CrPV) IRES.

(15) FIG. 7B shows the results of a Western blot carried out on lysates of HEK 293T/17 cells transfected respectively with empty control plasmid, SINEUP-DJ-1, and an IRUP including an EMCV IRES in direct (EMCV(d)) and inverted orientation (EMCV(i)), and a CrPV IRES in direct (CrPV(d)) and inverted orientation (CrPV(i)).

(16) FIG. 7C shows the results of a qRT-PCR to quantify expression of endogenous DJ-1 mRNA (top panel) and IRUP RNA (bottom panel) carried out on samples as in FIG. 7B.

(17) FIGS. 8A and 8B show graphs summarising the increase in quantities of DJ-1 protein in HEK 293T/17 cells transfected with HCV(d) and HCV(i) IRUPs, Polio(d) and Polio(i) IRUPs and EMCV(d), EMCV(i), CrPV(d) and CrPV(i) IRUPs of the present invention relative to empty vector transfected 293T/17 cells.

(18) FIG. 9A shows a schematic diagram of a functional nucleic acid molecule according to the present invention (IRUP) targeting the DJ-1 gene and including a human Apoptotic Peptidase Activating Factor 1 (Apaf-1) mRNA IRES.

(19) FIG. 9B shows the results of a Western blot carried out on lysates of HEK 293T/17 cells transfected respectively with empty control plasmid, SINEUP-DJ-1, and an IRUP including an Apaf-1 IRES in direct (Apaf-1(d)) and inverted orientation (Apaf-1(i)).

(20) FIG. 9C shows the results of a qRT-PCR to quantify expression of endogenous DJ-1 mRNA (top panel) and IRUP RNA (bottom panel) carried out on samples as in FIG. 9B.

(21) FIG. 10A shows a schematic diagram of a functional nucleic acid molecule according to the present invention (IRUP) targeting the DJ-1 gene and including a human Enhanced Level of Genomic instability 1 (ELG-1) mRNA IRES.

(22) FIG. 10B shows the results of a Western blot carried out on lysates of HEK 293T/17 cells transfected respectively with empty control plasmid, SINEUP-DJ-1, and an IRUP including an ELG-1 IRES in direct (ELG-1(d)) and inverted orientation (ELG-1(i)).

(23) FIG. 10C shows the results of a qRT-PCR to quantify expression of endogenous DJ-1 mRNA (top panel) and IRUP RNA (bottom panel) carried out on samples as in FIG. 10B.

(24) FIG. 11A shows a schematic diagram of a functional nucleic acid molecule according to the present invention (IRUP) targeting the DJ-1 gene and including a human V-Myc Avian Myelocytomatosis Viral Oncogene Homolog (cMYC) mRNA IRES.

(25) FIG. 11B shows the results of a Western blot carried out on lysates of HEK 293T/17 cells transfected respectively with empty control plasmid, SINEUP-DJ-1, and an IRUP including an cMYC IRES in direct (cMYC(d)) and inverted orientation (cMYC(i)).

(26) FIG. 11C shows the results of a qRT-PCR to quantify expression of endogenous DJ-1 mRNA (top panel) and IRUP RNA (bottom panel) carried out on samples as in FIG. 11B.

(27) FIG. 12A shows a schematic diagram of a functional nucleic acid molecule according to the present invention (IRUP) targeting the DJ-1 gene and including a shorter version of the human V-Myc Avian Myelocytomatosis Viral Oncogene Homolog (cMYC) mRNA IRES of FIGS. 11A to 11C.

(28) FIG. 12B shows the results of a Western blot carried out on lysates of HEK 293T/17 cells transfected respectively with empty control plasmid, SINEUP-DJ-1, and an IRUP including an cMYC IRES (short variant) in direct (cMYC(d)) and inverted orientation (cMYC(i)).

(29) FIG. 12C shows the results of a qRT-PCR to quantify expression of endogenous DJ-1 mRNA (top panel) and IRUP RNA (bottom panel) carried out on samples as in FIG. 12B.

(30) FIG. 13A shows a schematic diagram of a functional nucleic acid molecule according to the present invention (IRUP) targeting the DJ-1 gene and including a human Dystrophin (DMD) mRNA IRES.

(31) FIG. 13B shows the results of a Western blot carried out on lysates of HEK 293T/17 cells transfected respectively with empty control plasmid, SINEUP-DJ-1, and an IRUP including an DMD IRES in direct (DMD(d)) and inverted orientation (DMD(i)).

(32) FIG. 13C shows the results of a qRT-PCR to quantify expression of endogenous DJ-1 mRNA (top panel) and IRUP RNA (bottom panel) carried out on samples as in FIG. 13B.

(33) FIGS. 14A and 14B show graphs summarising the increase in quantities of DJ-1 protein in HEK 293T/17 cells transfected with Apaf-1(d) and Apaf-1(i) IRUPs, ELG-1(d) and ELG-1(i) IRUPs, cMYC(d) short version and cMYC(i) short version IRUPs, DMD(d) and DMD(i) IRUPs, and cMYC(d) long version and cMYC(i) long version IRUPs of the present invention relative to empty vector transfected 293T/17 cells.

(34) FIG. 15A-15D show the results of Western blots carried out on lysates of human hepatocellular carcinoma (HepG2) cells transfected respectively with empty control plasmid, SINEUP-DJ-1, an IRUP including an HCV IRES sequence in direct (d) or inverted (i) orientation, a Polio and cMYC IRES sequence in direct (d) or inverted (i) orientation, an Apaf-1 and ELG-1 IRES sequence in direct (d) or inverted (i) orientation, and a DMD IRES sequence in direct (d) or inverted (i) orientation.

(35) FIGS. 16A and 16B show graphs summarising the increase in quantities of DJ-1 protein in HepG2 cells transfected with HCV(d) and HCV(i) IRUPs, Polio(d) and Polio(i) IRUPs, cMYC(d) short version and cMYC(i) short version IRUPs, Apaf-1(d) and Apaf-1(i) IRUPs, ELG-1(d) and ELG-1(i) IRUPs, and DMD(d) and DMD(i) IRUPs of the present invention relative to empty vector transfected HepG2 cells.

(36) FIG. 17A shows a schematic diagram of a miniaturised version of functional nucleic acid molecules according to the present invention (miniIRUP) targeting the DJ-1 gene.

(37) FIG. 17B shows the results of a Western blot carried out on lysates of HEK 293T/17 cells transfected respectively with empty control plasmid, SINEUP-DJ-1 and miniIRUPs including an HCV IRES in direct orientation (HCV(d)), a Poliovirus IRES in direct (Polio(d)) and inverted orientation (Polio(i)) and a cMYC short version IRES in direct orientation (cMYC short(d)).

(38) FIG. 17C shows the results of a qRT-PCR to quantify expression of endogenous DJ-1 mRNA (top panel) and IRUP RNA (bottom panel) carried out on samples as in FIG. 17B.

(39) FIG. 17D shows a graphical representation of SINEUP-DJ-1 and mini-IRUP translation enhancement activity on endogenous DJ-1 mRNA in HEK 293T/17 cells (N=6).

(40) FIG. 18A shows a schematic diagram of a miniaturised version of functional nucleic acid molecules according to the present invention (miniIRUP) targeting the DJ-1 gene. With respect to the miniIRUPs of FIGS. 17A-17D, the IRES-containing functional nucleic acid sequences are cloned under the control of a different promoter.

(41) FIG. 18B shows the results of a Western blot carried out on lysates of HEK 293T/17 cells transfected respectively with empty control plasmid, SINEUP-DJ-1 and miniIRUPs including an HCV IRES in direct orientation (HCV(d)), a Poliovirus IRES in direct (Polio(d)) and inverted orientation (Polio(i)) and a cMYC short version IRES in direct orientation.

(42) FIG. 18C shows the results of a qRT-PCR to quantify expression of endogenous DJ-1 mRNA (top panel) and IRUP RNA (bottom panel) carried out on samples as in FIG. 18B.

(43) FIG. 18D shows a graphical representation of SINEUP-DJ-1 and mini-IRUP translation enhancement activity on endogenous DJ-1 mRNA in HEK 293T/17 cells (N=6).

(44) FIG. 19A shows a schematic diagram of pDUAL-GFP plasmids for the concomitant expression of functional nucleic acid molecule and genes of interest in mammalian cells.

(45) FIG. 19B shows the results of a Western blot carried out on lysates of HEK 293T/17 cells transfected respectively with empty control plasmid, SINE-containing (SINE) or IRES-containing (IRES) mini-functional nucleic acid molecules shown in FIG. 19A.

(46) FIG. 19C shows the results of a qRT-PCR to quantify expression of overexpressed GFP mRNA in HEK 293T/17 cells transfected with the constructs of FIG. 19A.

(47) FIG. 19D shows the results of a qRT-PCR to quantify expression of functional nucleic acid SINE or IRES RNA.

(48) FIG. 20A shows a schematic diagram of pDUAL-GFP plasmids with GFP targeting functional nucleic acid molecules in which the Effector Domain is represented by the SINE or HCV IRES sequence.

(49) FIG. 20B shows the results of a Western blot carried out on lysates of human neuroblastoma Neuro2a cells transfected respectively with empty control plasmid, SINE-containing (SINE) or IRES-containing (IRES) mini-functional nucleic acid molecules shown in FIG. 20A.

(50) FIG. 21 shows a schematic representation of HCV IRES RNA secondary structure.

(51) FIG. 22A shows the results of a Western blot carried out on lysates of HEK 293T/17 cells transfected respectively with empty control plasmid, SINEUP-DJ-1 and IRUPs including a WT HCV IRES and the M2 and M5 mutated HCV IRESs.

(52) FIG. 22B shows the results of a qRT-PCR to quantify expression of endogenous DJ-1 mRNA (top panel) and IRUP RNA (bottom panel) carried out on samples as in FIG. 22A.

(53) FIG. 22C shows a graphical representation of SINEUP-DJ-1 and IRUP translation enhancement activity on endogenous DJ-1 mRNA in HEK 293T/17 cells (N=7).

(54) FIG. 23A shows a schematic representation of how the sequence at the 5′ of an IRES sequence and within an IRES-containing cellular mRNA can be considered as Binding Domain.

(55) FIG. 23B shows functional nucleic acid sequence elements within cMYC mRNA reference sequence (NM_002467). The Binding Domain (black), IRES sequence (dark grey), the coding sequence or CDS (light grey) and 3′ untranslated region (white) are indicated.

(56) FIG. 23C shows the results of the bioinformatics analysis (BLAST) using the c-MYC mRNA as query sequence to identify partially overlapping, in antisense orientation, target mRNA protein-coding sequences.

(57) FIG. 24A shows a schematic representation of a mammalian expression plasmid encoding for full-length mRNA of MYC (cMYC-FL).

(58) FIG. 24B shows Western blots carried out on lysates of mammalian SAGS cells with anti-JAG2, anti-DYRK2, anti-LYS, anti-UBE3A, anti-NRF1 antibodies.

(59) FIGS. 24C and 24D show results of qRT-PCR to quantify expression of endogenous JAG2, DYRK2, LIS1, UBE3A, NRF1 and cMYC mRNAs.

(60) FIG. 24E shows average cMyc full-length mRNA levels.

(61) FIG. 25A shows a schematic representation of a mammalian expression plasmid encoding for full-length mRNA of MYC (cMYC-FL) or variants that lack cMYC DNA binding domain (deltaC) and are comprised of the 5′UTR only (5′UTR) or of the IRES only (IRES) domains.

(62) FIG. 25B shows Western blots carried out on lysates of mammalian SAGS cells with anti-JAG2, anti-DYRK2, anti-LYS, anti-UBE3A, anti-NRF1 antibodies.

(63) FIG. 25C shows results of qRT-PCR to quantify expression of endogenous JAG2, DYRK2, LIS1, UBE3A, NRF1 and cMYC mRNAs.

DEFINITIONS

(64) Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although many methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, preferred methods and materials are described below. Unless mentioned otherwise, the techniques described herein for use with the invention are standard methodologies well known to persons of ordinary skill in the art.

(65) By the term “internal ribosome entry site (IRES) derived sequence” there is intended a sequence of nucleic acid with a homology to an internal ribosome entry site (IRES) sequence such as to retain the functional activity thereof, i.e. a translation enhancing activity. In particular, the internal ribosome entry site (IRES) derived sequence can be obtained from a naturally occurring IRES sequence by genetic engineering or chemical modification, e.g. by isolating a specific sequence of the IRES sequence which remains functional, or mutating/deleting/introducing one or more nucleotides in the IRES sequence, or replacing one or more nucleotides in the IRES sequence with structurally modified nucleotides or analogs. More in particular, the skilled in the art would know that an internal ribosome entry site (IRES) derived sequence is a nucleotide sequence capable of promoting translation of a second cistron in a bicistronic construct. Typically, a dual luciferase (Firefly luciferase, Renilla Luciferase) encoding plasmid is used for experimental tests. A large-scale screening based on a dual reporter or bicistronic plasmid has been recently employed to survey sequences from the human genome for their ability to act as IRES (Weingarten-Gabbay S, et al., Science. 2016, 351:6270). 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 (http://iresite.org./IRESite_web.php?page=search). 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 structure-based 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/;

(66) http://regrna.mbc.nctu.edu.tw/index1.php).

DETAILED DESCRIPTION OF THE INVENTION

(67) With reference to FIG. 1, the trans-acting functional nucleic acid molecule of the invention (also referred to in the following as “IRUP”) comprises a target binding sequence (also referred to as “binding domain”) and a regulatory sequence (also referred to as “effector domain”).

(68) The target binding sequence comprises a sequence reverse complementary to a eukaryotic target mRNA sequence for which protein translation is to be enhanced.

(69) The eukaryotic target mRNA sequence is preferably an animal or human target mRNA sequence, more preferably a human target mRNA sequence.

(70) The regulatory sequence comprises an internal ribosome entry site (IRES) sequence or an internal ribosome entry site (IRES) derived sequence and enhances translation of the target mRNA sequence.

(71) The regulatory sequence is located 3′ of the target binding sequence.

(72) The trans-acting functional nucleic acid molecule hybridises to the target mRNA sequence through the target binding sequence and the IRES or IRES derived sequence enhances the translation of the target mRNA sequence.

(73) The functional nucleic acid molecule of the invention allows to exploit IRES sequences as trans-regulatory elements for gene-specific increase of translation of virtually any cellular endogenous or overexpressed protein-coding mRNA.

(74) Preferably, the target binding sequence consists, from 3′ to 5′, of a sequence reverse complementary to 1 to 50 nucleotides of the 5′ untranslated region (5′ UTR) and 1 to 40 nucleotides of the coding sequence (CDS) of the target mRNA sequence. Specific non-limiting examples include target binding sequences consisting of: a sequence reverse complementary to 40 nucleotides of the 5′ untranslated region (5′ UTR) and 4 nucleotides of the coding sequence (CDS) of the target mRNA sequence (referred to the initiation methionine codon or to internal in-frame methionine codons); a sequence reverse complementary to 40 nucleotides of the 5′ untranslated region (5′ UTR) and 32 nucleotides of the coding sequence (CDS) of the target mRNA sequence; a sequence reverse complementary to 14 nucleotides of the 5′ untranslated region (5′ UTR) and 4 nucleotides of the coding sequence (CDS) of the target mRNA sequence (referred to the initiation methionine codon).

(75) The regulatory sequence comprises an internal ribosome entry site (IRES) sequence preferably derived from human viruses or human protein-coding genes. Several IRESs having sequences ranging from 48 to 576 nucleotides have been tested with success, e.g. human Hepatitis C Virus (HCV) IRESs (SEQ ID NO:36 and SEQ ID NO:37), human poliovirus IRESs (SEQ ID NO:38 and SEQ ID NO:39), human encephalomyocarditis (EMCV) virus (SEQ ID NO:40 and SEQ ID NO:41), human cricket paralysis (CrPV) virus (SEQ ID NO:42 and SEQ ID NO:43), human Apaf-1 (SEQ ID NO:44 and SEQ ID NO:45), human ELG-1 (SEQ ID NO:46 and SEQ ID NO:47), human c-MYC (SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO: 50, and SEQ ID NO:51), human dystrophin (DMD) (SEQ ID NO:52 and SEQ ID NO:53). More detail can be found in the example section. Sequences coding for structural elements of the IRESs, fundamental for the translation enhancing activity, have been identified, isolated and used as regulatory sequences of the trans-acting functional nucleic acid molecule.

(76) As already mentioned in the definitions, internal ribosome entry site (IRES) derived sequences can include genetically engineered or chemically modified IRESs.

(77) Chemical modifications include, but are not limited to, the following:

(78) Base modifications: pseudouridine; 5′-Bromo-uridine; 5′-methylcytidine.

(79) Sugar modifications (2′ modifications): 2′-O-methyl-(2′-O-Me); 2′-O-methoxyethyl (2′-MOE); locked nucleic acid (LNA).

(80) Backbone modifications (phosphate backbone modifications): Phosphorothioate (PS); phosphotriester.

(81) Others (cell-type specific targeting domains): GalNAc linkage (hepatocytes).

(82) Although the IRES sequence or IRES derived sequence is functional whether inserted—in the trans-acting functional nucleic acid molecule—in direct or inverted orientation relative to the 5′ to 3′ orientation of the functional nucleic acid molecule, it is preferably oriented in direct orientation. In other words, by “direct” there is intended the situation in which the IRES sequence is embedded (inserted) with the same 5′ to 3′ orientation as the functional nucleic acid molecule. Instead, by “inverted” there is intended the situation in which a reverse complement of the IRES sequence is inserted in the nucleic acid molecule (the IRES sequence is 3′ to 5′ oriented relative to the functional nucleic acid molecule).

(83) Preferably, the IRES sequence or IRES derived sequence is a sequence with 75% homology to a sequence selected from the group consisting of SEQ ID NO:36 to SEQ ID NO:65, more preferably a sequence with 90% homology to a sequence selected from the group consisting of SEQ ID NO:36 to SEQ ID NO:65, even more preferably a sequence selected from the group consisting of SEQ ID NO:36 to SEQ ID NO:65.

(84) The trans-acting functional nucleic acid molecule is preferably an RNA molecule or a modified RNA molecule. Examples of modifications are:

(85) Base modifications: pseudouridine; 5′-Bromo-uridine; 5′-methylcytidine.

(86) Sugar modifications (2′ modifications): 2′-O-methyl-(2′-O-Me); 2′-O-methoxyethyl (2′-MOE); locked nucleic acid (LNA).

(87) Backbone modifications (phosphate backbone modifications): Phosphorothioate (PS); phosphotriester.

(88) Others (cell-type specific targeting domains): GalNAc linkage (hepatocytes).

(89) The trans-acting functional nucleic acid molecule preferably further comprises a spacer sequence between the target binding sequence and the regulatory sequence.

(90) In addition, the trans-acting functional nucleic acid molecule optionally comprises a non-coding 3′ tail sequence, which e.g. includes restriction sites useful for cloning the molecule in appropriate plasmids.

(91) Several trans-acting functional nucleic acid molecule have been generated according to the invention.

(92) The features of some of these molecules are summarised in the following. (BD=Binding Domain; numbering in parenthesis is with respect to AUG triplet A=+1)

(93) SEQ ID NO:1

(94) Definition: IRUP Functional Nucleic Acid Molecule

(95) IRES: viral IRES, Human Hepatitis C Virus, HCV, 383 nucleotides, direct orientation

(96) Features: BD=DJ-1s (−40/+4)

(97) ED=IRES HCV (383 ntds) (SEQ ID NO:36)

(98) Backbone=Delta5′ASUchl1

(99) IRES orientation: direct

(100) SEQ ID NO: 2

(101) Definition: IRUP Functional Nucleic Acid Molecule

(102) IRES: viral IRES, Human Hepatitis C Virus, HCV, 383 nucleotides, inverted orientation

(103) Features: BD=DJ-1s (−40/+4)

(104) ED=IRES HCV (383 ntds) (SEQ ID NO:37)

(105) Backbone=Delta5′ASUchl1

(106) IRES orientation: inverted (reverse complement)

(107) SEQ ID NO: 3

(108) Definition: IRUP Functional Nucleic Acid Molecule

(109) IRES: viral IRES, Human Polio Virus, 312 nucleotides, direct orientation

(110) Features: BD=DJ-1s (−40/+4)

(111) ED=IRES poliovirus (312 ntds) (SEQ ID NO:38)

(112) Backbone=Delta5′ASUchl1

(113) IRES orientation: direct

(114) SEQ ID NO: 4

(115) Definition: IRUP Functional Nucleic Acid Molecule

(116) IRES: viral IRES, Human Polio Virus, 312 nucleotides, inverted orientation

(117) Features: BD=DJ-1s (−40/+4)

(118) ED=IRES poliovirus (312 ntds) (SEQ ID NO:39)

(119) Backbone=Delta5′ASUchl1

(120) IRES orientation: inverted (reverse complement)

(121) SEQ ID NO: 5

(122) Definition: IRUP Functional Nucleic Acid Molecule

(123) IRES: viral IRES, Human Encephalomyocarditis virus, EMCV-R, 576 nucleotides, direct orientation

(124) Features: BD=DJ-1s (−40/+4)

(125) ED=IRES EMCV-R (576 ntds) (SEQ ID NO:40)

(126) Backbone=Delta5′ASUchl1

(127) IRES orientation: direct

(128) SEQ ID NO: 6

(129) Definition: IRUP Functional Nucleic Acid Molecule

(130) IRES: viral IRES, Human Encephalomyocarditis virus, EMCV-R, 576 nucleotides, inverted orientation

(131) Features: BD=DJ-1s (−40/+4)

(132) ED=IRES EMCV-R (576 ntds) (SEQ ID NO:41)

(133) Backbone=Delta5′ASUchl1

(134) IRES orientation: inverted (reverse complement)

(135) SEQ ID NO: 7

(136) Definition: IRUP Functional Nucleic Acid Molecule

(137) IRES: viral IRES, Human Cricket Paralysis Virus, CrPV, 192 nucleotides, direct orientation

(138) Features: BD=DJ-1s (−40/+4)

(139) ED=IRES CrPV (192 ntds) (SEQ ID NO:42)

(140) Backbone=Delta5′ASUchl1

(141) IRES orientation: direct

(142) SEQ ID NO: 8

(143) Definition: IRUP Functional Nucleic Acid Molecule

(144) IRES: viral IRES, Human Cricket Paralysis Virus, CrPV, 192 nucleotides, inverted orientation

(145) Features: BD=DJ-1s (−40/+4)

(146) ED=IRES CrPV (192 ntds) (SEQ ID NO:43)

(147) Backbone=Delta5′ASUchl1

(148) IRES orientation: inverted (reverse complement)

(149) SEQ ID NO: 9

(150) Definition: IRUP Functional Nucleic Acid Molecule

(151) IRES: cellular IRES, Human Apaf-1, 231 nucleotides, direct orientation

(152) (Ensembl: ENSG00000120868; MIM:602233)

(153) Features: BD=DJ-1s (−40/+4)

(154) ED=IRES Apaf-1 (231 ntds) (SEQ ID NO:44)

(155) Backbone=Delta5′ASUchl1

(156) IRES orientation: direct

(157) SEQ ID NO: 10

(158) Definition: IRUP Functional Nucleic Acid Molecule

(159) IRES: cellular IRES, Human Apaf-1, 231 nucleotides, inverted orientation

(160) (Ensembl: ENSG00000120868; MIM:602233)

(161) Features: BD=DJ-1s (−40/+4)

(162) ED=IRES Apaf-1 (231 ntds) (SEQ ID NO:45)

(163) Backbone=Delta5′ASUchl1

(164) IRES orientation: inverted (reverse complement)

(165) SEQ ID NO: 11

(166) Definition: IRUP Functional Nucleic Acid Molecule

(167) IRES: cellular IRES, Human ELG-1, 460 nucleotides, direct orientation

(168) (Ensembl: ENSG00000176208; MIM:609534)

(169) Features: BD=DJ-1s (−40/+4)

(170) ED=IRES ELG-1 (460 ntds) (SEQ ID NO:46)

(171) Backbone=Delta5′ASUchl1

(172) IRES orientation: direct

(173) SEQ ID No: 12

(174) Definition: IRUP Functional Nucleic Acid Molecule

(175) IRES: cellular IRES, Human ELG-1, 460 nucleotides, inverted orientation

(176) (Ensembl: ENSG00000176208; MIM:609534)

(177) Features: BD=DJ-1s (−40/+4)

(178) ED=IRES ELG-1 (460 ntds) (SEQ ID NO:47)

(179) Backbone=Delta5′ASUchl1

(180) IRES orientation: inverted (reverse complement)

(181) SEQ ID NO: 13

(182) Definition: IRUP Functional Nucleic Acid Molecule

(183) IRES: cellular IRES, Human c-MYC, 395 nucleotides, direct orientation

(184) (Ensembl: ENSG00000136997; MIM:190080)

(185) Features: BD=DJ-1s (−40/+4)

(186) ED=IRES c-MYC full-length (395 ntds) (SEQ ID NO:48)

(187) Backbone=Delta5′ASUchl1

(188) IRES orientation: direct

(189) Others: includes the 48 nt minimal sequence (SEQ ID NO:50) included in SEQ ID NO:15.

(190) SEQ ID NO: 14

(191) Definition: IRUP Functional Nucleic Acid Molecule

(192) IRES: cellular IRES, Human c-MYC, 395 nucleotides, inverted orientation

(193) (Ensembl: ENSG00000136997; MIM:190080)

(194) Features: BD=DJ-1s (−40/+4)

(195) ED=IRES c-MYC full-length (395 ntds) (SEQ ID NO:49)

(196) Backbone=Delta5′ASUchl1

(197) IRES orientation: inverted (reverse complement)

(198) Others: includes the 48nt minimal sequence (SEQ ID NO:51) included in SEQ ID NO:16.

(199) SEQ ID NO: 15

(200) Definition: IRUP Functional Nucleic Acid Molecule

(201) IRES: cellular IRES, Human c-MYC, 48 nucleotides, direct orientation

(202) (Ensembl: ENSG00000136997; MIM:190080)

(203) Features: BD=DJ-1s (−40/+4)

(204) ED=IRES c-MYC (48 ntds) (SEQ ID NO:50)

(205) Backbone=Delta5′ASUchl1

(206) IRES orientation: direct

(207) SEQ ID NO: 16

(208) Definition: IRUP Functional Nucleic Acid Molecule

(209) IRES: cellular IRES, Human c-MYC, 48 nucleotides, inverted orientation

(210) (Ensembl: ENSG00000136997; MIM:190080)

(211) Features: BD=DJ-1s (−40/+4)

(212) ED=IRES c-MYC (48 ntds) (SEQ ID NO:51)

(213) Backbone=Delta5′ASUchl1

(214) IRES orientation: inverted (reverse complement)

(215) SEQ ID NO: 17

(216) Definition: IRUP Functional Nucleic Acid Molecule

(217) IRES: cellular IRES, Human dystrophin (DMD), 71 nucleotides, direct orientation

(218) (Ensembl: ENSG00000198947; MIM:300377)

(219) Features: BD=DJ-1s (−40/+4)

(220) ED=IRES DMD (71 ntds) (SEQ ID NO:52)

(221) Backbone=Delta5′ASUchl1

(222) IRES orientation: direct

(223) SEQ ID NO: 18

(224) Definition: IRUP Functional Nucleic Acid Molecule

(225) IRES: cellular IRES, Human dystrophin (DMD), 71 nucleotides, inverted orientation

(226) (Ensembl: ENSG00000198947; MIM:300377)

(227) Features: BD=DJ-1s (−40/+4)

(228) ED=IRES DMD (71 ntds) (SEQ ID NO:53)

(229) Backbone=Delta5′ASUchl1

(230) IRES orientation: inverted (reverse complement)

(231) SEQ ID NO: 19

(232) Definition: IRUP Functional Nucleic Acid Molecule

(233) IRES: viral IRES, Human Hepatitis C Virus, HCV mutant #1, 303 nucleotides, delta II (40-119), interaction with ribosomal proteins

(234) Features: BD=DJ-1s (−40/+4)

(235) ED=HCV IRES direct, deltaII (SEQ ID NO:54)

(236) Backbone=Delta5′ASUchl1

(237) Mutant #1: delta II (40-119), interaction with ribosomal proteins

(238) SEQ ID NO: 20

(239) Definition: IRUP Functional Nucleic Acid Molecule

(240) IRES: viral IRES, Human Hepatitis C Virus, HCV mutant #2, 367 nucleotides, delta IIIa (156-171), eIF3 binding site

(241) Features: BD=DJ-1s (−40/+4)

(242) ED=HCV IRES direct, deltaIIIa (SEQ ID NO:55)

(243) Backbone=Delta5′ASUchl1

(244) Mutant #2: delta IIIa (156-171), eIF3 binding site

(245) SEQ ID NO: 21

(246) Definition: IRUP Functional Nucleic Acid Molecule

(247) IRES: viral IRES, Human Hepatitis C Virus, HCV mutant #3, 356 nucleotides, delta IIId (253-279), 18S rRNA binding region

(248) Features: BD=DJ-1s (−40/+4)

(249) ED=HCV IRES direct, deltaIIId (SEQ ID NO:56)

(250) Backbone=Delta5′ASUchl1

(251) Mutant #3: delta IIId (253-279), 18S rRNA binding region

(252) SEQ ID NO: 22

(253) Definition: IRUP Functional Nucleic Acid Molecule

(254) IRES: viral IRES, Human Hepatitis C Virus, HCV mutant #4, 330 nucleotides, delta IV (331-383), AUG-containing terminal sequence

(255) Features: BD=DJ-1s (−40/+4)

(256) ED=HCV IRES direct, deltaIV (SEQ ID NO:57)

(257) Backbone=Delta5′ASUchl1

(258) Mutant #4: delta IV (331-383), AUG-containing terminal sequence

(259) SEQ ID NO: 23

(260) Definition: IRUP Functional Nucleic Acid Molecule

(261) IRES: viral IRES, Human Hepatitis C Virus, HCV mutant #5, 383 nucleotides, G266.fwdarw.C; single point mutation, contact with 18S rRNA

(262) Features: BD=DJ-1s (−40/+4)

(263) ED=HCV IRES direct, G266.fwdarw.C (SEQ ID NO:58)

(264) Backbone=Delta5′ASUchl1

(265) Mutant #5: G266.fwdarw.C; single point mutation, contact with 18S rRNA

(266) SEQ ID NO: 24

(267) Definition: IRUP Functional Nucleic Acid Molecule

(268) IRES: viral IRES, Human Hepatitis C Virus, HCV mutant #6, 383 nucleotides, U228.fwdarw.C; control single point mutation in another site of HCV IRES, decreases IRES activity without disrupting formation of pre-initiation complex

(269) Features: BD=DJ-1s (−40/+4)

(270) ED=HCV IRES direct, U228.fwdarw.C (SEQ ID NO:59)

(271) Backbone=Delta5′ASUchl1

(272) Mutant #6: U228.fwdarw.C; control single point mutation in another site of HCV IRES, decreases IRES activity without disrupting formation of pre-initiation complex. Mutated version has reduced affinity for eIF3.

(273) SEQ ID NO: 25

(274) Definition: IRUP Functional Nucleic Acid Molecule

(275) IRES: viral IRES, Human Hepatitis C Virus, HCV mutant #7, 383 nucleotides, G267.fwdarw.C; IIId loop, single point mutation, contact with 18S rRNA

(276) Features: BD=DJ-1s (−40/+4)

(277) ED=HCV IRES direct, G267.fwdarw.C (SEQ ID NO:60)

(278) Backbone=Delta5′ASUchl1

(279) Mutant #7: G267.fwdarw.C; IIId loop, single point mutation, contact with 18S rRNA

(280) SEQ ID NO: 26

(281) Definition: IRUP Functional Nucleic Acid Molecule

(282) IRES: viral IRES, Human Hepatitis C Virus, HCV mutant #8, 383

(283) nucleotides, G268.fwdarw.C; IIId loop, single point mutation, contact with 18S rRNA

(284) Features: BD=DJ-1s (−40/+4)

(285) ED=HCV IRES direct, G268.fwdarw.C (SEQ ID NO:61)

(286) Backbone=Delta5′ASUchl1

(287) Mutant #8: G268.fwdarw.C; IIId loop, single point mutation, contact with 18S rRNA

(288) SEQ ID NO: 27

(289) Definition: IRUP Functional Nucleic Acid Molecule

(290) IRES: viral IRES, Human Hepatitis C Virus, HCV mutant #9, 383 nucleotides, G.sub.266G.sub.267G.sub.268.fwdarw.C.sub.266C.sub.267C.sub.268; IIId loop, triple point mutation, contact with 18S rRNA

(291) Features: BD=DJ-1s (−40/+4)

(292) ED=HCV IRES direct, G.sub.266G.sub.267G.sub.268.fwdarw.C.sub.266C.sub.267C.sub.268 (SEQ ID NO:62)

(293) Backbone=Delta5′ASUchl1

(294) Mutant #9: G.sub.266G.sub.267G.sub.268.fwdarw.C.sub.266C.sub.267C.sub.268; IIId loop, triple point mutation, contact with 18S rRNA

(295) SEQ ID NO: 28

(296) Definition: IRUP Functional Nucleic Acid Molecule

(297) IRES: viral IRES, Human Hepatitis C Virus, HCV mutant #10, 383 nucleotides, G266.fwdarw.A/G268.fwdarw.T; double point mutant; HCV 5a isolate with poor infectivity

(298) Features: BD=DJ-1s (−40/+4)

(299) ED=HCV IRES direct, G266.fwdarw.A/G268.fwdarw.T (SEQ ID NO:63)

(300) Backbone=Delta5′ASUchl1

(301) Mutant #10: G266.fwdarw.A/G268.fwdarw.T; double point mutant; HCV 5a isolate with poor infectivity

(302) SEQ ID NO: 29

(303) Definition: IRUP Functional Nucleic Acid Molecule

(304) IRES: viral IRES, Human Hepatitis C Virus, HCV mutant #11, 383 nucleotides, IIIa.fwdarw.IIIa-comp; AGTA.fwdarw.TCAT

(305) Features: BD=DJ-1s (−40/+4)

(306) ED=HCV IRES direct, IIIa.fwdarw.IIIa-comp; AGTA.fwdarw.TCAT (SEQ ID NO:64)

(307) Backbone=Delta5′ASUchl1

(308) Mutant #11: HCV IRES direct, IIIa.fwdarw.IIIa-comp; AGTA.fwdarw.TCAT)

(309) SEQ ID NO: 30

(310) Definition: IRUP Functional Nucleic Acid Molecule

(311) IRES: viral IRES, Human Hepatitis C Virus, HCV mutant #12, 383 nucleotides, IIe.fwdarw.IIIe-comp; TGATAG.fwdarw.ACTATC

(312) Features: BD=DJ-1s (−40/+4)

(313) ED=HCV IRES direct, IIIe.fwdarw.IIIe-comp; TGATAG.fwdarw.ACTATC (SEQ ID NO:65)

(314) Backbone=Delta5′ASUchl1

(315) Mutant #12: HCV IRES direct, IIIe.fwdarw.IIIe-comp; TGATAG.fwdarw.ACTATC

(316) SEQ ID NO: 31

(317) Definition: miniIRUP Functional Nucleic Acid Molecule

(318) IRES: viral IRES, Human Hepatitis C Virus, HCV, 383 nucleotides, direct orientation

(319) Features: BD=DJ-1s (−40/+4)

(320) ED=IRES HCV (383 ntds) (SEQ ID NO:36)

(321) IRES orientation: direct

(322) SEQ ID NO: 32

(323) Definition: miniIRUP Functional Nucleic Acid Molecule

(324) IRES: viral IRES, Human Polio Virus, 312 nucleotides, direct orientation

(325) Features: BD=DJ-1s (−40/+4)

(326) ED=IRES poliovirus (312 ntds) (SEQ ID NO:38)

(327) IRES orientation: direct

(328) SEQ ID NO: 33

(329) Definition: miniIRUP Functional Nucleic Acid Molecule

(330) IRES: viral IRES, Human Polio Virus, 312 nucleotides, inverted orientation

(331) Features: BD=DJ-1s (−40/+4)

(332) ED=IRES poliovirus (312 ntds) (SEQ ID NO:39)

(333) IRES orientation: inverted (reverse complement)

(334) SEQ ID NO: 34

(335) Definition: miniIRUP Functional Nucleic Acid Molecule

(336) IRES: cellular IRES, Human c-MYC, 48 nucleotides, direct orientation) (Ensembl: ENSG00000136997; MIM:190080)

(337) Features: BD=DJ-1s (−40/+4)

(338) ED=IRES c-MYC (48 ntds) (SEQ ID NO:50)

(339) IRES orientation: direct

(340) SEQ ID NO: 35

(341) Definition: miniIRUP Functional Nucleic Acid Molecule

(342) IRES: viral IRES, Human Hepatitis C Virus, HCV, 383 nucleotides, direct orientation

(343) Features: BD=GFP (−40/+32)

(344) ED=IRES HCV (383 ntds) (SEQ ID NO:36)

(345) IRES orientation: direct

(346) A DNA molecule according to the present invention encodes any of the above disclosed trans-acting functional nucleic acid molecules.

(347) An expression vector according to the present invention comprises the above said DNA molecule. In particular, the following plasmids have been used for efficient expression of functional nucleic acid molecules.

(348) Mammalian Expression Plasmids:

(349) Plasmid Name: pCS2+

(350) Expression: CMVie92 promoter

(351) SV40polyA terminator

(352) Plasmid Name: pCDN3.1 (−)

(353) Expression: CMV promoter

(354) BGH terminator

(355) Plasmid Name: pDUAL-eGFPΔ (modified from peGFP-C1)

(356) Expression: H1 promoter; CMV promoter

(357) BGH terminator; SV40 terminator

(358) Viral Vectors:

(359) Vector Name: pAAV

(360) Virus: Adeno-Associated Virus

(361) Expression: CAG promoter/CMV enhancer

(362) SV40polyA terminator

(363) Vector Name: pLVX-TetOne-Puro

(364) Virus: Lentivirus

(365) Expression: TRE3G promoter (inducible expression)

(366) SV40polyA terminator

(367) It should be noted that the experiments carried out have highlighted that the function of the trans-acting functional nucleic acid molecule is not influenced by the plasmid used.

(368) A method for enhancing protein translation according to the invention comprises transfecting into a cell the above disclosed trans-acting functional nucleic acid molecule or DNA molecule or expression vector.

(369) A composition according to the present invention comprises the above disclosed trans-acting functional nucleic acid molecule or DNA molecule or expression vector. The trans-acting functional nucleic acid molecule can be delivered as naked RNA, the RNA optionally including modifications adapted to increase RNA stability. As an alternative the trans-acting functional nucleic acid molecule can be an in vitro transcribed RNA encapsulated in an array of lipid-based nanoparticles or an in vitro transcribed RNA encapsulated in exosome-based particles.

(370) The above disclosed trans-acting functional nucleic acid molecule or DNA molecule or expression vector can be used for enhancing translation of a target mRNA sequence. The examples show enhancement of translation of two different target mRNA sequences, PARK7/DJ-1 (also referred to as DJ-1) and GFP, but any other mRNA sequence could be targeted with success without influencing mRNA levels.

(371) It should be noted that IRUPs can enhance translation of the gene of interest with no effects on its own mRNA quantities.

(372) The above disclosed trans-acting functional nucleic acid molecule or DNA molecule or expression vector can therefore be successfully used as molecular tools to validate gene function in cells as well as to implement the pipelines of recombinant protein production.

(373) The above disclosed trans-acting functional nucleic acid molecule or DNA molecule or expression vector can be used for treating a genetic disease caused by down-regulation of a protein-coding mRNA or a sporadic disease where reduced levels of a certain protein-coding mRNA is detrimental. The following are examples of such diseases. 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. This is the cause of a wide spectrum of diseases including specific types of cancers, ataxias and those due to failures of developmental programs. A large number of Rare Diseases are caused by mutations or microdeletions that lead to reduced gene dosage. Transcription factors, synaptic proteins and chromatin remodeling enzymes seem to be particularly sensitive to gene dosage. Reduced gene expression can be also observed during aging.

EXAMPLES

Example 1

(374) FIG. 2 shows that the embedded Effector Domain (ED) is required for translation up-regulation function of an antisense long non-coding RNA (lncRNA) targeting endogenous human DJ-1 mRNA.

(375) FIG. 2A shows a schematic diagram of a functional antisense lncRNA molecule that up-regulates translation of target-specific mRNAs. The original molecule that uses an embedded murine transposable element of the SINE B2 family is indicated (SINEUP). SINEUP functional domains are highlighted: SINEUP Binding Domain (BD): SINEUP sequence that overlaps, in antisense orientation, to the sense protein-coding mRNA; SINEUP Effector Domain (ED): the inverted SINEB2 element (SINE) in the non-overlapping portion of SINEUPs, that confers activation of protein synthesis. 5′ to 3′ orientation of sense and antisense RNA molecules is indicated. Target-specific mRNA includes 5′ untranslated region (5′UTR), coding sequence (CDS) and 3′ untranslated region (3′UTR). Translation initiating AUG codon is also shown.

(376) A synthetic SINEUP was designed to target endogenous human DJ-1 mRNA (SINEUP-DJ-1). SINEUP-DJ-1 mutants were generated lacking the Binding Domain (ΔBD) or the Effector Domain (ΔED). Human embryonic kidney (HEK) 293T/17 cells were transfected with plasmids encoding for SINEUP-DJ-1 full length (FL) or its deletion mutants (ΔED=mutant with deleted effector domain, ΔBD=mutant with deleted binding domain). Control cells were transfected with an empty control plasmid (−). 48 hours after transfection, cells were lysed and processed for protein quantities. Western blot (FIG. 2B) was performed with anti-DJ-1 antibody. β-actin was used as loading control. Fold-induction was calculated on Western blot images normalized to β-actin and relative to empty control samples.

(377) RNA was purified from transfected cells. Expression of endogenous DJ-1 mRNA and SINEUP RNA was monitored by qRT-PCR using specific primers (FIG. 2C). Data indicate mean±st. dev. Data are representative of N=5 independent replicas.

(378) FIG. 2D shows a graphical representation of SINEUP-DJ-1 FL, ΔBD and ΔED translation enhancement activity on endogenous DJ-1 mRNA in HEK 293T/17 cells (N=5). p<0.05

Example 2

(379) Synthetic IRUPs were designed as follows to target endogenous human DJ-1 mRNA. As shows in FIG. 3, IRES-containing functional nucleic acid molecules were generated by swapping the original SINE B2 sequence with IRES sequences derived from human viruses or human mRNAs having IRES activity.

(380) Table 1 includes the list of some of the IRES sequences used in the trans-acting functional nucleic acid molecule according to the present invention.

(381) IRES name, IRES origin, cloning orientation and IRES length are indicated.

(382) TABLE-US-00001 TABLE 1 IRES Origin Orientation Length (nt) HCV Human Virus Direct 383 HCV Human Virus Inverted 383 Polio Human Virus Direct 312 Polio Human Virus Inverted 312 EMCV Human Virus Direct 576 EMCV Human Virus Inverted 576 CrPV Human Virus Direct 192 CrPV Human Virus Inverted 192 Apaf-1 Human mRNA Direct 231 Apaf-1 Human mRNA Inverted 231 ELG-1 Human mRNA Direct 460 ELG-1 Human mRNA Inverted 460 cMYC (long) Human mRNA Direct 395 cMYC (long) Human mRNA Inverted 395 cMYC (short) Human mRNA Direct  48 cMYC (short) Human mRNA Inverted  48 DMD Human mRNA Direct  71 DMD Human mRNA Inverted  71

(383) FIG. 4 shows a schematic diagram of the experimental procedure for testing translation up-regulation activity of IRES-containing functional antisense nucleic acid molecules. Antisense functional nucleic acid molecules for translation activation were generated with human DJ-1 overlapping sequence (Binding Domain) and IRES sequences (Effector Domain). IRES-containing functional nucleic acid molecules were cloned into mammalian expression vectors for expression in mammalian cells in culture in vitro. Cells that express endogenous quantities of human DJ-1 mRNA were used. Timing for cell seeding, cell transfection and harvesting are shown. Cells were harvested to purify RNA (for quantitative real-time PCR, qRT-PCR) and proteins (for Western blot, WB).

(384) FIG. 5A shows a schematic diagram of a trans-acting functional nucleic acid molecule in which the effector domain is an IRES sequence from human Hepatitis C virus (HCV). IRES-containing functional nucleic acid molecules were generated with the HCV IRES sequence cloned in direct (HCV(d)—SEQ ID NO:1) or inverted (HCV(i)—SEQ ID NO:2) orientation relative to the 5′ to 3′ orientation of the functional nucleic acid molecule.

(385) HEK 293T/17 cells were transfected with plasmids encoding for IRES-containing functional nucleic acid molecules with HCV IRES sequence in direct (d) or inverted (i) orientation, as indicated. Control cells were transfected with an empty control plasmid (−). Cells transfected with SINEUP-DJ-1 were used as reference for testing the potency of IRES-containing molecules. 48 hours after transfection, cells were lysed and processed for protein quantities. Western blot (FIG. 5B) was performed with anti-DJ-1 antibody. β-actin was used as loading control. Fold-induction was calculated on Western blot images normalized to β-actin and relative to empty control samples. Potency of IRES-containing functional nucleic acid molecules was higher or similar with respect to SINE-containing functional nucleic acid molecules.

(386) RNA was purified from transfected cells. Expression of endogenous DJ-1 mRNA and IRUP RNA was monitored by qRT-PCR using specific primers (FIG. 5C). To compare RNA quantities across SINE- and IRES-containing functional nucleic acid molecules, primers were positioned at the 3′ end of the Effector Domain. Data indicate mean±st. dev. Data are representative of N>5 independent replicas.

Example 3

(387) FIG. 6A shows a schematic diagram of a trans-acting functional nucleic acid molecule in which the effector domain is an IRES sequence from human Poliovirus. IRES-containing functional nucleic acid molecules were generated with the Polio IRES sequence cloned in direct (Polio(d)—SEQ ID NO:3) or inverted (Polio(i)—SEQ ID NO:4) orientation relative to the 5′ to 3′ orientation of the functional nucleic acid molecule.

(388) HEK 293T/17 cells were transfected with plasmids encoding for IRES-containing functional nucleic acid molecules with Polio IRES sequence in direct (d) or inverted (i) orientation, as indicated. Control cells were transfected with an empty control plasmid (−). Cells transfected with SINEUP-DJ-1 were used as reference for testing the potency of IRES-containing molecules. 48 hours after transfection, cells were lysed and processed for protein quantities. Western blot (FIG. 6B) was performed with anti-DJ-1 antibody. β-actin was used as loading control. Fold-induction was calculated on Western blot images normalized to β-actin and relative to empty control samples. Potency of IRES-containing functional nucleic acid molecules was higher than SINE-containing functional nucleic acid molecules.

(389) RNA was purified from transfected cells. Expression of endogenous DJ-1 mRNA and IRUP RNA was monitored by qRT-PCR using specific primers (FIG. 6C). To compare RNA quantities across SINE- and IRES-containing functional nucleic acid molecules, primers were positioned at the 3′ end of the Effector Domain. Data indicate mean±st. dev. Data are representative of N>5 independent replicas.

Example 4

(390) FIG. 7A shows a schematic diagram of a trans-acting functional nucleic acid molecules in which the effector domain is respectively an IRES sequence from human Encephalomyocarditis virus (EMCV) and an IRES sequence from Cricket Paralysis virus (CrPV). IRES-containing functional nucleic acid molecules were generated with the EMCV IRES sequence cloned in direct (EMCV(d)—SEQ ID NO:5) or inverted (EMCV(i)—SEQ ID NO:6) orientation relative to the 5′ to 3′ orientation of the functional nucleic acid molecule and with the CrPV IRES sequence cloned in direct (CrPV(d)—SEQ ID NO:7) or inverted (CrPV(i)—SEQ ID NO:8) orientation relative to the 5′ to 3′ orientation of the functional nucleic acid molecule.

(391) HEK 293T/17 cells were transfected with plasmids encoding for IRES-containing functional nucleic acid molecules with EMCV and CrPV IRES sequence in direct (d) or inverted (i) orientation, as indicated. Control cells were transfected with an empty control plasmid (−). Cells transfected with SINEUP-DJ-1 were used as reference for testing the potency of IRES-containing molecules. 48 hours after transfection, cells were lysed and processed for protein quantities. Western blot (FIG. 7B) was performed with anti-DJ-1 antibody. β-actin was used as loading control. Fold-induction was calculated on Western blot images normalized to β-actin and relative to empty control samples. Potency of IRES-containing functional nucleic acid molecules was higher than SINE-containing functional nucleic acid molecules.

(392) RNA was purified from transfected cells. Expression of endogenous DJ-1 mRNA and IRUP RNA was monitored by qRT-PCR using specific primers (FIG. 7C). To compare RNA quantities across SINE- and IRES-containing functional nucleic acid molecules, primers were positioned at the 3′ end of the Effector Domain. Data indicate mean±st. dev. Data are representative of N>5 independent replicas.

Example 5

(393) The increase in quantities of endogenous DJ-1 protein in HEK 293T/17 cells transfected with HCV(d) and HCV(i) IRUPs of example 2, Polio(d) and Polio(i) IRUPs of example 3 and EMCV(d), EMCV(i), CrPV(d) and CrPV(i) IRUPs of example 4 relative to empty vector transfected HEK 293T/17 cells was measured by Western blot.

(394) The results are summarised in FIGS. 8A and 8B. As evident, IRES-containing functional nucleic acid molecules activate translation in trans and are more active than SINE-containing molecules. Data represents average and stdev of N>5 biological replicas. Single asterisks (*) indicate IRES sequences that have statistically significant translation enhancement activity in trans relative to empty control cells; double asterisks indicate IRES sequences that show statistically significant increased potency relative to SINE-containing functional nucleic acid molecules.

Example 6

(395) FIG. 9A shows a schematic diagram of a trans-acting functional nucleic acid molecule in which the effector domain is an IRES sequence from human Apoptotic Peptidase Activating Factor 1 (Apaf-1) mRNA. IRES-containing functional nucleic acid molecules were generated with the Apaf-1 IRES sequence cloned in direct (Apaf-1(d)—SEQ ID NO:9) or inverted (Apaf-1(i)—SEQ ID NO:10) orientation relative to the 5′ to 3′ orientation of the functional nucleic acid molecule.

(396) HEK 293T/17 cells were transfected with plasmids encoding for IRES-containing functional nucleic acid molecules with Apaf-1 IRES sequence in direct (d) or inverted (i) orientation, as indicated. Control cells were transfected with an empty control plasmid (−). Cells transfected with SINEUP-DJ-1 were used as reference for testing the potency of IRES-containing molecules. 48 hours after transfection, cells were lysed and processed for protein quantities. Western blot (FIG. 9B) was performed with anti-DJ-1 antibody. β-actin was used as loading control. Fold-induction was calculated on Western blot images normalized to β-actin and relative to empty control samples. Potency of IRES-containing functional nucleic acid molecules was higher or similar with respect to SINE-containing functional nucleic acid molecules.

(397) RNA was purified from transfected cells. Expression of endogenous DJ-1 mRNA and IRUP RNA was monitored by qRT-PCR using specific primers (FIG. 9C). To compare RNA quantities across SINE- and IRES-containing functional nucleic acid molecules, primers were positioned at the 3′ end of the Effector Domain. Data indicate mean±st. dev. Data are representative of N>5 independent replicas.

Example 7

(398) FIG. 10A shows a schematic diagram of a trans-acting functional nucleic acid molecule in which the effector domain is an IRES sequence from human Enhanced Level of Genomic instability 1 (ELG-1) mRNA. IRES-containing functional nucleic acid molecules were generated with the ELG-1 IRES sequence cloned in direct (ELG-1(d)—SEQ ID NO:11) or inverted (ELG-1(i)—SEQ ID NO:12) orientation relative to the 5′ to 3′ orientation of the functional nucleic acid molecule.

(399) HEK 293T/17 cells were transfected with plasmids encoding for IRES-containing functional nucleic acid molecules with ELG-1 IRES sequence in direct (d) or inverted (i) orientation, as indicated. Control cells were transfected with an empty control plasmid (−). Cells transfected with SINEUP-DJ-1 were used as reference for testing the potency of IRES-containing molecules. 48 hours after transfection, cells were lysed and processed for protein quantities. Western blot (FIG. 10B) was performed with anti-DJ-1 antibody. β-actin was used as loading control. Fold-induction was calculated on Western blot images normalized to β-actin and relative to empty control samples. Potency of IRES-containing functional nucleic acid molecules was higher with respect to SINE-containing functional nucleic acid molecules.

(400) RNA was purified from transfected cells. Expression of endogenous DJ-1 mRNA and IRUP RNA was monitored by qRT-PCR using specific primers (FIG. 10C). To compare RNA quantities across SINE- and IRES-containing functional nucleic acid molecules, primers were positioned at the 3′ end of the Effector Domain. Data indicate mean±st. dev. Data are representative of N>5 independent replicas.

Example 8

(401) FIG. 11A shows a schematic diagram of a trans-acting functional nucleic acid molecule in which the effector domain is an IRES sequence from human V-Myc Avian Myelocytomatosis Viral Oncogene Homolog (cMYC) mRNA. IRES-containing functional nucleic acid molecules were generated with the cMYC IRES sequence (long variant) cloned in direct (cMYC full length(d)—SEQ ID NO:13) or inverted (cMYC full length(i)—SEQ ID NO:14) orientation relative to the 5′ to 3′ orientation of the functional nucleic acid molecule.

(402) HEK 293T/17 cells were transfected with plasmids encoding for IRES-containing functional nucleic acid molecules with cMYC IRES sequence in direct (d) or inverted (i) orientation, as indicated. Control cells were transfected with an empty control plasmid (−). Cells transfected with SINEUP-DJ-1 were used as reference for testing the potency of IRES-containing molecules. 48 hours after transfection, cells were lysed and processed for protein quantities. Western blot (FIG. 11B) was performed with anti-DJ-1 antibody. β-actin was used as loading control. Fold-induction was calculated on Western blot images normalized to β-actin and relative to empty control samples. Potency of IRES-containing functional nucleic acid molecules was higher or similar with respect to SINE-containing functional nucleic acid molecules.

(403) RNA was purified from transfected cells. Expression of endogenous DJ-1 mRNA and IRUP RNA was monitored by qRT-PCR using specific primers (FIG. 11C). To compare RNA quantities across SINE- and IRES-containing functional nucleic acid molecules, primers were positioned at the 3′ end of the Effector Domain. Data indicate mean±st. dev. Data are representative of N>5 independent replicas.

Example 9

(404) FIG. 12A shows a schematic diagram of a trans-acting functional nucleic acid molecule in which the effector domain is a shorter version of the IRES sequence from human V-Myc Avian Myelocytomatosis Viral Oncogene Homolog (cMYC) mRNA of Example 8. IRES-containing functional nucleic acid molecules were generated with the cMYC IRES sequence (short variant) cloned in direct (cMYC short variant(d)—SEQ ID NO:15) or inverted (cMYC short variant(i)—SEQ ID NO:16) orientation relative to the 5′ to 3′ orientation of the functional nucleic acid molecule.

(405) HEK 293T/17 cells were transfected with plasmids encoding for IRES-containing functional nucleic acid molecules with cMYC IRES sequence in direct (d) or inverted (i) orientation, as indicated. Control cells were transfected with an empty control plasmid (−). Cells transfected with SINEUP-DJ-1 were used as reference for testing the potency of IRES-containing molecules. 48 hours after transfection, cells were lysed and processed for protein quantities. Western blot (FIG. 11B) was performed with anti-DJ-1 antibody. β-actin was used as loading control. Fold-induction was calculated on Western blot images normalized to β-actin and relative to empty control samples. Potency of IRES-containing functional nucleic acid molecules was higher or similar with respect to SINE-containing functional nucleic acid molecules.

(406) RNA was purified from transfected cells. Expression of endogenous DJ-1 mRNA and IRUP RNA was monitored by qRT-PCR using specific primers (FIG. 12C). To compare RNA quantities across SINE- and IRES-containing functional nucleic acid molecules, primers were positioned at the 3′ end of the Effector Domain. Data indicate mean±st. dev. Data are representative of N>5 independent replicas.

Example 10

(407) FIG. 13A shows a schematic diagram of a trans-acting functional nucleic acid molecule in which the effector domain is an IRES sequence from human Dystrophin (DMD) mRNA. IRES-containing functional nucleic acid molecules were generated with the DMD IRES sequence cloned in direct (DMD(d)—SEQ ID NO:17) or inverted (DMD(i)—SEQ ID NO:18) orientation relative to the 5′ to 3′ orientation of the functional nucleic acid molecule.

(408) HEK 293T/17 cells were transfected with plasmids encoding for IRES-containing functional nucleic acid molecules with DMD IRES sequence in direct (d) or inverted (i) orientation, as indicated. Control cells were transfected with an empty control plasmid (−). Cells transfected with SINEUP-DJ-1 were used as reference for testing the potency of IRES-containing molecules. 48 hours after transfection, cells were lysed and processed for protein quantities. Western blot (FIG. 13B) was performed with anti-DJ-1 antibody. β-actin was used as loading control. Fold-induction was calculated on Western blot images normalized to β-actin and relative to empty control samples. Potency of IRES-containing functional nucleic acid molecules was higher with respect to SINE-containing functional nucleic acid molecules.

(409) RNA was purified from transfected cells. Expression of endogenous DJ-1 mRNA and IRUP RNA was monitored by qRT-PCR using specific primers (FIG. 13C). To compare RNA quantities across SINE- and IRES-containing functional nucleic acid molecules, primers were positioned at the 3′ end of the Effector Domain. Data indicate mean±st. dev. Data are representative of N>5 independent replicas.

Example 11

(410) The increase in quantities of endogenous DJ-1 protein in HEK 293T/17 cells transfected with Apaf-1(d) and Apaf-1(i) IRUPs of Example 6, ELG-1(d) and ELG-1(i) IRUPs of Example 7, cMYC full length(d) and cMYC full length(i) IRUPs of Example 8, cMYC short variant(d) and cMYC short variant(i) IRUPs of Example 9, and DMD(d) and DMD(i) IRUPs of Example 10 relative to empty vector transfected HEK 293T/17 cells was measured by Western blot.

(411) The results are summarised in FIGS. 14A and 14B. As evident, IRES-containing functional nucleic acid molecules activate translation in trans and are more active than SINE-containing molecules. Data represents average and stdev of N>5 biological replicas. Single asterisks (*) indicate IRES sequences that have statistically significant translation enhancement activity in trans relative to empty control cells; double asterisks indicate IRES sequences that show statistically significant increased potency relative to SINE-containing functional nucleic acid molecules.

Example 12

(412) Human hepatocellular carcinoma (HepG2) cells were transfected with plasmids encoding for IRES-containing functional nucleic acid molecules with HCV (FIG. 15A) IRES sequence in direct (d) or inverted (i) orientation (SEQ ID NO:1 or SEQ ID NO:2), with Polio and cMYC (FIG. 15B) IRES sequence in direct (d) or inverted (i) orientation (SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:13 or SEQ ID NO:14), with Apaf-1 and ELG-1 (FIG. 15C) IRES sequence in direct (d) or inverted (i) orientation (SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12), and with DMD (FIG. 15D) IRES sequence in direct (d) or inverted (i) orientation (SEQ ID NO:17 or SEQ ID NO:18), as indicated. Control cells were transfected with an empty control plasmid (−). Cells transfected with SINEUP-DJ-1 were used as reference for testing the potency of IRES-containing molecules. 48 hours after transfection, cells were lysed and processed for protein quantities. Western blot was performed with anti-DJ-1 antibody. β-actin was used as loading control. Fold-induction was calculated on Western blot images normalized to β-actin and relative to empty control samples. Potency of IRES-containing functional nucleic acid molecules was almost always higher than SINE-containing functional nucleic acid molecules.

Example 13

(413) The increase in quantities of endogenous DJ-1 protein in HepG2 cells transfected with HCV(d) and HCV(i) IRUPs, Polio(d) and Polio(i) IRUPs and cMYC short variant(d) and cMYC short variant(i) IRUPs, Apaf-1(d) and Apaf-1(i) IRUPs, ELG-1(d) and ELG-1(i) IRUPs, DMD (d) and DMD (i) IRUPs relative to empty vector transfected HepG2 cells was measured by Western blot.

(414) The results are summarised in FIGS. 16A-16B. IRES-containing functional nucleic acid molecules activate translation in trans and are more active than SINE-containing molecules. Data represents average and stdev of N>2 biological replicas.

Example 14

(415) FIG. 17A shows a schematic diagram of a miniaturised version of a trans-acting functional nucleic acid molecule (miniIRUP) in which the effector domain is an IRES sequence from HCV, Poliovirus or cMYC short version. IRES-containing functional nucleic acid molecules were generated with the HCV IRES sequence cloned in direct (HCV(d)—SEQ ID NO:31) orientation, with the Polio IRES sequence cloned in direct (Polio(d)—SEQ ID NO:32) and inverted (Polio(i)—SEQ ID NO:33) orientation and with the cMYC short IRES sequence cloned in direct (cMYC short variant(d)—SEQ ID NO:34) orientation, under the control of an SV40-based promoter for expression by RNA polymerase II in mammalian cells.

(416) HEK 293T/17 cells were transfected with plasmids encoding the above said mini IRES-containing functional nucleic acid molecules, as indicated. Control cells were transfected with an empty control plasmid (−). Cells transfected with SINEUP-DJ-1 were used as reference for testing the potency of IRES-containing molecules. 48 hours after transfection, cells were lysed and processed for protein quantities. Western blot (FIG. 17B) was performed with anti-DJ-1 antibody. β-actin was used as loading control. Fold-induction was calculated on Western blot images normalized to β-actin and relative to empty control samples. Potency of mini IRES-containing functional nucleic acid molecules was higher than SINE-containing functional nucleic acid molecules.

(417) RNA was purified from transfected cells. Expression of endogenous DJ-1 mRNA and mini-IRUP RNA was monitored by qRT-PCR using specific primers (FIG. 17C).

(418) FIG. 17D shows a graphical representation of SINEUP-DJ-1 and mini-IRUP translation enhancement activity on endogenous DJ-1 mRNA in HEK 293T/17 cells (N=6).

Example 15

(419) FIG. 18A shows a schematic diagram of a miniaturised version of a trans-acting functional nucleic acid molecule (miniIRUP) in which the effector domain is an IRES sequence from HCV, Poliovirus or cMYC short version. IRES-containing functional nucleic acid molecules were generated with the HCV IRES sequence cloned in direct (HCV(d)—SEQ ID NO:31) orientation, with the Polio IRES sequence cloned in direct (Polio(d)—SEQ ID NO:32) and inverted (Polio(i)—SEQ ID NO:32) orientation and with the cMYC short IRES sequence cloned in direct (cMYC short version(d)—SEQ ID NO:34) orientation, under the control of an H1-based promoter for expression by RNA polymerase III in mammalian cells.

(420) HEK 293T/17 cells were transfected with plasmids encoding the above said mini IRES-containing functional nucleic acid molecules, as indicated. Control cells were transfected with an empty control plasmid (−). Cells transfected with SINEUP-DJ-1 were used as reference for testing the potency of IRES-containing molecules. 48 hours after transfection, cells were lysed and processed for protein quantities. Western blot (FIG. 18B) was performed with anti-DJ-1 antibody. β-actin was used as loading control. Fold-induction was calculated on Western blot images normalized to β-actin and relative to empty control samples. Potency of mini IRES-containing functional nucleic acid molecules was higher (similar in one case) with respect to SINE-containing functional nucleic acid molecules.

(421) RNA was purified from transfected cells. Expression of endogenous DJ-1 mRNA and mini-IRUP RNA was monitored by qRT-PCR using specific primers (FIG. 18C).

(422) FIG. 18D shows a graphical representation of SINEUP-DJ-1 and mini-IRUP translation enhancement activity on endogenous DJ-1 mRNA in HEK 293T/17 cells (N=6).

Example 16

(423) FIG. 19A shows a schematic diagram of pDUAL-GFP plasmids for the concomitant expression of two genes of interest in mammalian cells. pDUAL-GFP plasmids contain a CMV promoter element for the expression of GFP mRNA by RNA Polymerase II and an H1 promoter (in opposite orientation) for the expression of GFP-targeting miniaturized version of functional nucleic acid molecules for GFP translation enhancement. A control plasmid is produced that lacks the translation enhancer functional nucleic acid molecule and expressed basal levels of GFP protein. pDUAL-GFP/miniGFP plasmids were created in which the effector domain is represented by a SINE B2 sequence or by the HCV IRES, as indicated.

(424) HEK 293T/17 cells were transfected with pDUAL plasmids encoding for GFP alone or GFP in combination with SINE-containing (SINE) or IRES-containing (IRES) mini-functional nucleic acid molecules. Cells transfected with pDUAL-GFP alone were used as reference for testing the potency of IRES-containing molecules. 48 hours after transfection, cells were lysed and processed for protein quantities. Western blot (FIG. 19B) was performed with anti-GFP antibody. β-actin was used as loading control.

(425) RNA was purified from transfected cells. Expression of overexpressed GFP mRNA (FIG. 19C) and functional nucleic acid SINE or IRES RNA (FIG. 19D) was monitored by qRT-PCR using specific primers.

(426) The results show that IRES-containing functional nucleic acid molecules bearing a Binding Domain antisense to GFP mRNA enhance translation of GFP mRNA when GFP is overexpressed in HEK 293T/17 cells.

Example 17

(427) FIG. 20A shows a schematic diagram of pDUAL-GFP plasmids with GFP targeting functional nucleic acid molecules in which the Effector Domain is represented by the SINE or HCV IRES sequence.

(428) Human neuroblastoma Neuro2a cells were transfected with the pDUAL-GFP plasmids shown in FIG. 20A. 48 hours after transfection, cells were lysed and processed for protein quantities. Western blot (FIG. 20B) was performed with anti-GFP antibody. β-actin was used as loading control. Fold-induction was calculated on Western blot images normalized to β-actin and relative to empty control samples. Potency of mini IRES-containing functional nucleic acid molecules was higher than SINE-containing functional nucleic acid molecules.

Example 18

(429) Functional nucleic acid molecules containing DJ-1 targeting Binding Domain and HCV IRES Effector Domain harboring specific mutations in structural regions important for HCV IRES activity in cis were designed.

(430) FIG. 21 shows a schematic representation of HCV IRES RNA secondary structure. HCV IRES structural domains (IIa, IIb, IIIa, IIIb, IIIc, IIId, IIIe and IIIf) are indicated in the different squares. The functionality of each structural domain for the Internal Ribosome Entry activity of the sequence is also shown, with contacts to ribosomal RNA and/or ribosomal proteins. A functional nucleic acid molecule in which the Effector Domain is represented by the IRES sequence derived from HCV and containing DJ-1 targeting Binding Domain was used as template for mutagenesis. An M2 mutant was produced by deletion of the IIIa stem loop (aminoacids 156-171 of HCV IRES sequence−IRES=SEQ ID NO:55; IRUP=SEQ ID NO:20), involved in contacts with the eukaryotic translation initiation factor eIF3 and the ribosomal protein eS27. An M5 mutant was produced by site-directed nucleotide substitution of nucleotide G266 (G266.fwdarw.C−IRES=SEQ ID NO:58; IRUP=SEQ ID NO:23), fundamental for base-pairing to 18S rRNA and for HCV IRES activity in cis.

Example 19

(431) HEK 293T/17 cells were transfected with a mammalian expression plasmid encoding for IRES-containing functional nucleic acid molecule with HCV IRES sequence in direct orientation (WT) or with HCV IRES DIIIa (M2) or G266.fwdarw.C (M5) mutants, as indicated. Control cells were transfected with an empty control plasmid (−). Cells transfected with SINEUP-DJ-1 were used as reference for testing the potency of IRES-containing molecules. 48 hours after transfection, cells were lysed and processed for protein quantities. Western blot (FIG. 22A) was performed with anti-DJ-1 antibody. β-actin was used as loading control. Fold-induction was calculated on Western blot images normalized to β-actin and relative to empty control samples. Potency of WT and mutated IRES-containing functional nucleic acid molecule was higher or similar with respect to SINE-containing functional nucleic acid molecule.

(432) RNA was purified from transfected cells. Expression of endogenous DJ-1 mRNA and functional nucleic acid RNA was monitored by qRT-PCR using specific primers (FIG. 22B). To compare RNA quantities across SINE- and IRES-containing functional nucleic acid molecules, primers were positioned at the 3′ end of the Effector Domain. Data indicate mean±st. dev.

(433) FIG. 22C shows a graphical representation of translation enhancement activity of functional nucleic acid molecules with SINE- or HCV IRES-embedded sequences on endogenous DJ-1 mRNA in HEK 293T/17 cells (N=7). Double asterisks (**) indicate mutant IRES sequences that show statistically significant reduction in translation enhancement relative to WT HCV IRES-containing functional nucleic acid molecule.

(434) This example shows that structural elements required for HCV IRES activity in cis contribute to the increased translation enhancement activity of HCV IRES in trans as embedded Effector Domain (ED) in a functional nucleic acid molecule containing DJ-1 targeting Binding Domain.

Example 20

(435) This example shows that any nucleic acid sequence in the target mRNA can be recognised by the Binding Domain of an IRES-derived sequence containing a functional nucleic acid molecule.

(436) FIG. 23A shows a schematic representation of how the sequence at the 5′ of an IRES sequence and within an IRES-containing cellular mRNA can be considered as Binding Domain and FIG. 23B shows functional nucleic acid sequence elements within cMYC mRNA reference sequence (NM 002467).

(437) FIG. 23C shows the results of the bioinformatics analysis (BLAST) using the c-MYC mRNA as query sequence to identify partially overlapping, in antisense orientation, target mRNA protein-coding sequences. In the table, annotated gene ENSEMBL nomenclature, Gene Name and Gene pairing region are indicated. The IRES-derived Binding Domain can overlap, in antisense orientation, to target mRNA in the 5′ UTR, first exon, internal exon and with different pairing length, ranging from 18 to 198.

(438) Mammalian SAGS cells were transfected with a mammalian expression plasmid encoding for full-length mRNA of MYC (cMYC-FL) (FIG. 24A). Control cells were transfected with an empty vector plasmid (control). 48 hours after transfection, cells were lysed and processed for protein quantities. Western blot (FIG. 24B) was performed with anti-JAG2, anti-DYRK2, anti-LYS, anti-UBE3A, anti-NRF1 antibodies as indicated. β-actin was used as loading control. Anti-cMYC antibody was used as additional control to verify expression of cMYC protein after transfection. Fold-induction was calculated on Western blot images normalized to β-actin and relative to empty control samples (C). Potency of cMYC IRES-containing functional nucleic acid molecules was evident for all tested target mRNAs, with binding domains ranging from 144 (JAG2) to 20 (UBE3A, NRF1) nucleotides in length.

(439) RNA was purified from transfected cells. Expression of endogenous JAG2, DYRK2, LIS1, UBE3A, NRF1 and cMYC mRNAs was monitored by qRT-PCR using specific primers (FIG. 24C and FIG. 24D).

(440) This example shows that the pairing region between the Binding Domain of an IRES-containing functional nucleic acid molecule and the target protein-coding mRNA can vary in position and in length, retaining its full translation enhancement activity.

Example 21

(441) This example shows that the protein-coding CDS portion and the DNA-binding domain of cMYC are not required for the IRES-containing functional nucleic acid molecule to increase translation of partially-overlapping protein-coding mRNAs.

(442) Mammalian SAGS cells were transfected with a mammalian expression plasmid encoding for full-length mRNA of MYC (cMYC-FL) or variants that lack cMYC DNA binding domain (deltaC) and are comprised of the 5′UTR only (5′UTR) or of the IRES only (IRES) domains. (FIG. 25A). Control cells were transfected with an empty vector plasmid (control). 48 hours after transfection, cells were lysed and processed for protein quantities. Western blot (FIG. 25B) was performed with anti-JAG2, anti-DYRK2, anti-LYS, anti-UBE3A, anti-NRF1 antibodies as indicated. β-actin was used as loading control. Anti-cMYC antibody was used as additional control to verify expression of cMYC protein after transfection. Fold-induction was calculated on Western blot images normalized to β-actin and relative to empty control samples. Fold-changes of protein quantities relative to control cells are indicated at the bottom of the western blot images.

(443) RNA was purified from transfected cells. Expression of endogenous JAG2, DYRK2, LIS1, UBE3A, NRF1 and cMYC mRNAs was monitored by qRT-PCR using specific primers (FIG. 25C), proving the post-transcriptional mechanism of the IRES-containing functional nucleic acid molecule.

Advantages

(444) The trans-acting functional nucleic acid molecule of the present invention allows to enhance the translation of virtually any target mRNA sequence without affecting target mRNA levels.

(445) With respect to the functional nucleic acid molecules disclosed in EP 2691522, those of the present invention avoid the risk of retrotransposition due to mouse SINE sequences and promote higher levels of enhancement of protein translation, as shown in Examples 2 to 11 in HEK 293T/17 and in Example 12 in HepG2 cells. In particular, examples of the more potent IRES sequences are given in FIG. 5 (HCV IRES, direct), in FIG. 6 (Polio IRES, direct and inverted), FIG. 7 (ECMV IRES, direct and inverted; CrPV IRES, direct and inverted), FIG. 9 (Apaf1 IRES, direct), FIG. 10 (ELG-1 IRES, direct and inverted), FIG. 11 (cMYC IRES, direct), FIG. 12 (short cMYC IRES, direct), FIG. 12 (DMD IRES, direct and inverted) in HEK cells. In addition, examples of more potent IRES sequences are given also in HepG2 cells (FIG. 15).

(446) Some IRES sequences are as short as 40 to 50 nucleotides. This allows the engineering of very short trans-acting functional nucleic acid molecules.

(447) In addition, the trans-acting functional nucleic acid molecules of the invention can include a particularly short target binding sequence, in particular as compared to functional nucleic acid molecules disclosed in EP2691522.

(448) Both the limited length of the regulatory sequence and the target binding sequence contribute to keeping the length of the molecule short while allowing an optimal targeting and protein synthesis enhancement. One of the main advantages of having a short molecule, is to overcome the difficulty in synthesizing RNAs longer than 100 nucleotides.

(449) Another advantage of the trans-acting functional nucleic acid molecules of the invention is they have a modular structure, i.e. have an independent target binding domain and an independent effector domain.