WO2025011876A2

WO2025011876A2 - Antisense long noncoding rnas for the modification of gene expression and for therapeutic applications

Info

Publication number: WO2025011876A2
Application number: PCT/EP2024/066684
Authority: WO
Inventors: Heike KREBBER; Holger BASTIANS
Original assignee: Georg August Universitaet Goettingen; Universitaetsmedizin Goettingen Georg August Universitaet
Current assignee: Georg August Universitaet Goettingen; Universitaetsmedizin Goettingen Georg August Universitaet
Priority date: 2023-07-08
Filing date: 2024-06-14
Publication date: 2025-01-16
Anticipated expiration: 2026-01-08
Also published as: WO2025011876A3

Abstract

The present invention relates to an antisense long non-encoding RNA (aslncRNA) that may form a partial double stranded (ds) complex with a corresponding cellular mRNA in eukaryotic cells and to a biological complex comprising the antisense lncRNA and a target mRNA that may together form a partial double stranded complex in vitro. The dsRNA complex is preferentially exported from the nucleus and translated in the cytoplasm and thereby gene expression is boosted. In addition to boosting aslnc-RNAs inhibitiory aslnc RNAs are disclosed. An expression vector comprising a nucleic acid sequence of the aslncRNA is also disclosed. The present invention can be used to increase or decrease export of a target mRNA from the nucleus and thus, to increase or decrease gene expression without interfering with genomic DNA or with mRNA levels. Methods to increase or decrease the export and/or expression of a target mRNA, cell transfections using plasmids expressing the antisense lncRNA or using conjugates comprising the aslncRNA, and a pharmaceutical composition comprising the as-lncRNA are also disclosed. The aslncRNA, the complex, the expression vector, and the conjugate are useful for enhancing the production of recombinant proteins that are used in biotechnological applications and for medical purposes. The aslncRNA is also useful for increasing antigen presentation upon mRNA vaccination.

Description

ANTISENSE LONG NONCODING RNAS FOR THE MODIFICATION OF GENE EXPRESSION AND FOR THERAPEUTIC APPLICATIONS

BACKGROUND OF THE INVENTION

[0001] A dogma in gene expression is that mRNAs, which are generated in the nucleus, are subsequently transported into the cytoplasm where they are translated into protein. High throughput technologies have identified a large number of non-coding RNAs (ncRNAs), some of which represent antisense strands that are complementary to parts of the mRNAs. Their function, however, remained mostly elusive, but it seemed that they have important roles in cells because several reports showed that their dysregulation is associated with diseases such as cancer or neurodegenerative diseases. Such antisense (as)RNAs exist in all organisms.

[0002] Typically, ncRNAs are divided into small (below 200 nucleotides) and long ncRNAs (exceeding 200 nucleotides) (IncRNAs). IncRNAs are represented globally in living organisms and are perhaps the least well-understood type of transcripts. IncRNAs share many similar characteristics to mRNA, for instance, they are transcribed by RNA polymerase II with similar chromatin states, undergo 5'-capping, splicing and 3'-polyadenlation. Although it has been described that IncRNAs contain a small open reading frame (smORF) from which functional small peptides are generated, the majority of IncRNAs lack an ORF and therefore the coding potential of mRNAs.

[0003] While reports exist that speculate on potential functions for IncRNAs in regulating transcription, for example, in response to environmental changes, a general function for many IncRNAs is currently unknown. In fact, significant amounts of antisense IncRNAs travel into the cytoplasm and are eliminated through the cytoplasmic exonuclease known as Xrnl, similar to the so- called XUTs identified in yeast. Xrnl mediated degradation follows recognition through the nonsense mediated decay (NMD) system, which detects a lack of correct ORFs and has been suggested to eliminate these cytoplasmic IncRNAs. Exporting and translating such antisense (as)RNAs appears to involve undue energy consumption by the cell at first sight, since no general cytoplasmic function for these IncRNAs has yet been discovered in eukaryotes.

SUMMARY OF THE INVENTION

[0004] In one aspect, the present invention relates to an antisense long non-coding RNA (abbreviated herein as antisense IncRNA, asIncRNA, or IncRNA) comprising a first region and a second region, wherein the first region is complementary to a portion of a target mRNA, and wherein the second region is not complementary to the target mRNA. In an embodiment, the first region is complementary to the 5' end of the target mRNA. In another embodiment, the first region is complementary to the 5' UTR of the target mRNA. In another embodiment, the second region is 3' of the first region. In another embodiment, the antisense IncRNA is capable of forming a partial double strand complex with the mRNA thereby promoting or inhibiting the nuclear export of the mRNA. In an embodiment, the overlap of the first region with the 5' end of the target mRNA has a length of between 1 and thousands of nucleotides. In an embodiment, the complementary region has a length of between 1 and thousands of nucleotides. In another embodiment, the complementary region has a length of between 20 and 3000. In another embodiment, the complementary region has a length of between 20 and 1000. In another embodiment, the complementary region has a length of between 50 and 500. In another embodiment, the antisense IncRNA is capable of forming, with the target mRNA, a complex that is optimized for binding double-strand formation mediating proteins and/or binding export mediating proteins, preferably to Dbp2, Yral, Mex67, Mtr2, or its human homologues, DDX5, ALY/REV/NXF2, TAP, pl5. Other or additional helicases may be comprised in the complex.

[0005] In an embodiment, the second region of the antisense IncRNA has a length of between 1 and thousands of nucleotides. In another embodiment, the second region has a length of between 1 and 5000. In another embodiment, the second region has a length of between 1 and 2000. In another embodiment, the second region has a length of between 1 and 800. In an embodiment, the antisense IncRNA consists of a first region and a second region. In an embodiment, the antisense IncRNA consists of a first and a second region, wherein the second region has a length of between 1 and thousands of nucleotides. In an embodiment, the antisense IncRNA consists of a first and a second region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 1 and thousands of nucleotides, and wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and thousands of nucleotides. In an embodiment, the antisense IncRNA consists of a first and a second region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 20 and 3000 nucleotides, and wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and 5000.

[0006] In another embodiment, the antisense IncRNA further comprises a third region that is 5' of the first region and which is not complementary to the target mRNA. In an embodiment, the third region comprises between 1 and thousands of nucleotides. In an embodiment, the antisense IncRNA comprises a first region, a second region, and a third region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 1 and thousands of nucleotides, wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and thousands of nucleotides, and wherein the third region is 5' of the first region, wherein the third region is not complementary to the mRNA and wherein the third region has a length of between 1 and thousands of nucleotides. In another embodiment, the second region has a length of between 1 and 5000. In another embodiment, the second region has a length of between 1 and 2000. In another embodiment, the second region has a length of between 1 and 800. In an embodiment, the third region has a length of between 1 and 5000. In another embodiment, the third region has a length of between 1 and 2000. In another embodiment, the third region has a length of between 1 and 800. In an embodiment, the second region and the third region have a length of between 1 and 5000. In an embodiment, the antisense IncRNA comprises a first region, a second region, and a third region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 20 and 3000 nucleotides, wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and 5000 nucleotides, and wherein the third region is 5' of the first region, wherein the third region is not complementary to the mRNA and wherein the third region has a length of between 1 and 5000 nucleotides.

[0007] The present invention also relates to a biological complex comprising an antisense IncRNA as described herein and the target mRNA, wherein the IncRNA and mRNA form a partial RNA double strand complex.

[0008] In another embodiment, the present invention relates to a biological complex comprising an antisense IncRNA as described herein and the target mRNA, wherein the IncRNA and mRNA form a partial double strand complex, wherein the IncRNA consists of a first and a second region, wherein the second region of the antisense IncRNA is between 1 and thousands of nucleotides in length. In another embodiment, the second region has a length of between 1 and 5000. In another embodiment, the second region has a length of between 1 and 2000. In another embodiment, the second region has a length of between 1 and 800. In an embodiment, the antisense IncRNA comprises a first region and a second region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 20 and 3000 nucleotides, and wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and 5000 nucleotides.

[0009] In other embodiments, the biological complex comprises an antisense IncRNA as described herein and the target mRNA, wherein the IncRNA and mRNA form a partial double strand complex, wherein the IncRNA comprises a first region, a second region, and a third region. In other embodiments, the biological complex comprises an antisense IncRNA as described herein and the target mRNA, wherein the IncRNA and mRNA form a partial double strand complex, wherein the IncRNA comprises a first region, a second region, and a third region, wherein the first, second and third regions comprises between 1 and thousands of nucleotides. In other embodiments, the biological complex comprises an antisense IncRNA as described herein and the target mRNA, wherein the IncRNA and mRNA form a partial double strand complex, wherein the IncRNA comprises a first region, a second region, and a third region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 1 and thousands of nucleotides, wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and thousands of nucleotides, and wherein the third region is 5' of the first region, wherein the third region is not complementary to the mRNA and wherein the third region has a length of between 1 and thousands of nucleotides. In another embodiment, the second region has a length of between 1 and 5000. In another embodiment, the second region has a length of between 1 and 2000. In another embodiment, the second region has a length of between 1 and 800. In an embodiment, the third region has a length of between 1 and 5000. In another embodiment, the third region has a length of between 1 and 2000. In another embodiment, the third region has a length of between 1 and 800. In an embodiment, the second region and the third region have a length of between 1 and 5000. In an embodiment, the antisense IncRNA comprises a first region, a second region, and a third region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 20 and 3000 nucleotides, wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and 5000 nucleotides, and wherein the third region is 5' of the first region, wherein the third region is not complementary to the mRNA and wherein the third region has a length of between 1 and 5000 nucleotides.

[00010] In an embodiment, the antisense IncRNA disclosed herein comprises a poly(A)-tail at the 3'-end of the second region.

[00011] In another aspect, the present invention relates to an expression vector comprising a nucleic acid sequence encoding an antisense IncRNA as described herein. In an embodiment, the expression vector further comprises a nucleic acid sequence encoding a target mRNA of the IncRNA. [00012] In other embodiments, the present invention relates to an expression vector comprising a nucleic acid sequence encoding an antisense IncRNA as described herein, wherein the antisense IncRNA consists of a first region and a second region. In an embodiment, the present invention relates to an expression vector comprising a nucleic acid sequence encoding an antisense IncRNA as described herein, wherein the antisense IncRNA consists of a first and a second region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 1 and thousands of nucleotides, and wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and thousands of nucleotides. In an embodiment, the expression vector further comprises a nucleic acid sequence encoding a target mRNA of the IncRNA. In another embodiment, the second region has a length of between 1 and 5000. In another embodiment, the second region has a length of between 1 and 2000. In another embodiment, the second region has a length of between 1 and 800. In an embodiment, the antisense IncRNA comprises a first region and a second region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 20 and 3000 nucleotides, and wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and 5000 nucleotides.

[00013] In further embodiments, the present invention relates to an expression vector comprising a nucleic acid sequence encoding an antisense IncRNA as described herein, wherein the IncRNA comprises a first region, a second region, and a third region. In other embodiments, the present invention relates to an expression vector comprising a nucleic acid sequence encoding an antisense IncRNA as described herein, wherein the IncRNA comprises a first region, a second region, and a third region, wherein the first, second and third regions comprises between 1 and thousands of nucleotides. In other embodiments, the present invention relates to an expression vector comprising a nucleic acid sequence encoding an antisense IncRNA as described herein, wherein the IncRNA comprises a first region, a second region, and a third region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 1 and thousands of nucleotides, wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and thousands of nucleotides, and wherein the third region is 5' of the first region, wherein the third region is not complementary to the mRNA and wherein the third region has a length of between 1 and thousands of nucleotides. In another embodiment, the second region has a length of between 1 and 5000. In another embodiment, the second region has a length of between 1 and 2000. In another embodiment, the second region has a length of between 1 and 800. In an embodiment, the third region has a length of between 1 and 5000. In another embodiment, the third region has a length of between 1 and 2000. In another embodiment, the third region has a length of between 1 and 800. In an embodiment, the second region and the third region have a length of between 1 and 5000. In an embodiment, the antisense IncRNA comprises a first region, a second region, and a third region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 20 and 3000 nucleotides, wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and 5000 nucleotides, and wherein the third region is 5' of the first region, wherein the third region is not complementary to the mRNA and wherein the third region has a length of between 1 and 5000 nucleotides. [00014] In another aspect, the present invention relates to the use of the antisense IncRNA, or the biological complex, or the expression vector as described herein, for increasing export of the target mRNA from the nucleus of a eukaryotic cell via a partial double strand complex with the IncRNA. In an embodiment, the present invention relates to the use of the antisense IncRNA, or the biological complex, or the expression vector as described herein, for increasing export of the target mRNA from the nucleus of a eukaryotic cell via a partial double strand complex with the IncRNA, wherein the antisense IncRNA consists of a first region and a second region. In an embodiment, the present invention relates to the use of the antisense IncRNA, or the biological complex, or the expression vector as described herein, for increasing export of the target mRNA from the nucleus of a eukaryotic cell via a partial double strand complex with the IncRNA, wherein the antisense IncRNA consists of a first and a second region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 1 and thousands of nucleotides, and wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and thousands of nucleotides. In an embodiment, the expression vector further comprises a nucleic acid sequence encoding a target mRNA of the IncRNA.

[00015] In another aspect, the present invention relates to the use of the antisense IncRNA, or the biological complex, or the expression vector as described herein, for increasing expression of the target mRNA in a eukaryotic cell by increasing its nuclear export via a partial double strand complex with the IncRNA. In an embodiment, the present invention relates to the use of the antisense IncRNA, or the biological complex, or the expression vector as described herein, for increasing expression of the target mRNA in a eukaryotic cell by increasing its nuclear export via a partial double strand complex with the IncRNA, wherein the antisense IncRNA consists of a first region and a second region. In an embodiment, the present invention relates to the use of the antisense IncRNA, or the biological complex, or the expression vector as described herein, for increasing expression of the target mRNA in a eukaryotic cell by increasing its nuclear export via a partial double strand complex with the IncRNA, wherein the antisense IncRNA consists of a first and a second region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 1 and thousands of nucleotides, and wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and thousands of nucleotides . In an embodiment, the expression vector further comprises a nucleic acid sequence encoding a target mRNA of the IncRNA.

[00016] In other embodiments, the present invention relates to the use of the antisense IncRNA, or the biological complex, or the expression vector as described herein, for decreasing export of the target mRNA from the nucleus of a eukaryotic cell via a partial double strand complex with the IncRNA, wherein the IncRNA comprises a first region, a second region, and a third region. In other embodiments, the present invention relates to the use of the antisense IncRNA, or the biological complex, or the expression vector as described herein, for decreasing export of the target mRNA from the nucleus of a eukaryotic cell via a partial double strand complex with the IncRNA, wherein the IncRNA comprises a first region, a second region, and a third region, wherein the first, second and third regions comprises between 1 and thousands of nucleotides. In other embodiments, the present invention relates to the use of the antisense IncRNA, or the biological complex, or the expression vector as described herein, for decreasing export of the target mRNA from the nucleus of a eukaryotic cell via a partial double strand complex with the IncRNA, wherein the IncRNA comprises a first region, a second region, and a third region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 1 and thousands of nucleotides, wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and thousands of nucleotides, and wherein the third region is 5' of the first region, wherein the third region is not complementary to the mRNA and wherein the third region has a length of between 1 and thousands of nucleotides.

[00017] In yet further embodiments, the present invention relates to the use of the antisense IncRNA, or the biological complex, or the expression vector as described herein, for decreasing expression of the target mRNA in a eukaryotic cell by decreasing its nuclear export via a partial double strand complex with the IncRNA, wherein the IncRNA comprises a first region, a second region, and a third region. In other embodiments, the present invention relates to the use of the antisense IncRNA, or the biological complex, or the expression vector as described herein, for decreasing expression of the target mRNA in a eukaryotic cell by decreasing its nuclear export via a partial double strand complex with the IncRNA, wherein the IncRNA comprises a first region, a second region, and a third region, wherein the first, second and third regions comprises between 1 and thousands of nucleotides. In other embodiments, the present invention relates to the use of the antisense IncRNA, or the biological complex, or the expression vector as described herein, for decreasing expression of the target mRNA in a eukaryotic cell by decreasing its nuclear export via a partial double strand complex with the IncRNA, wherein the IncRNA comprises a first region, a second region, and a third region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 1 and thousands of nucleotides, wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and thousands of nucleotides, and wherein the third region is 5' of the first region, wherein the third region is not complementary to the mRNA and wherein the third region has a length of between 1 and thousands of nucleotides. [00018] In another aspect, the present invention relates to a method of increasing export of a target mRNA from the nucleus of a eukaryotic cell, the method comprising transfecting a cell with the antisense IncRNA, or the biological complex, or the expression vector as described herein. In an embodiment, the present invention relates to a method of increasing export of a target mRNA from the nucleus of a eukaryotic cell, the method comprising transfecting a cell with the antisense IncRNA, or the biological complex, or the expression vector as described herein, wherein the antisense IncRNA or the antisense IncRNA of the biological complex or expression vector consists of a first region and a second region. In an embodiment, the present invention relates to a method of increasing export of a target mRNA from the nucleus of a eukaryotic cell, the method comprising transfecting a cell with the antisense IncRNA, or the biological complex, or the expression vector as described herein, wherein the antisense IncRNA consists of a first and a second region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 1 and thousands of nucleotides, and wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and thousands of nucleotides In an embodiment, the expression vector further comprises a nucleic acid sequence encoding a target mRNA of the IncRNA. In another embodiment, the second region has a length of between 1 and 5000. In another embodiment, the second region has a length of between 1 and 2000. In another embodiment, the second region has a length of between 1 and 800. In an embodiment, the antisense IncRNA comprises a first region and a second region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 20 and 3000 nucleotides, and wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and 5000 nucleotides.

[00019] In another aspect, the present invention relates to a method of increasing expression of a target mRNA in a eukaryotic cell, the method comprising transfecting a cell with the antisense IncRNA, or the biological complex, or the expression vector as described herein. In an embodiment, the present invention relates to a method of increasing expression of a target mRNA in a eukaryotic cell, the method comprising transfecting a cell with the antisense IncRNA, or the biological complex, or the expression vector as described herein, wherein the antisense IncRNA or the antisense IncRNA of the biological complex or expression vector consists of a first region and a second region. In an embodiment, the present invention relates to a method of increasing expression of a target mRNA in a eukaryotic cell, the method comprising transfecting a cell with the antisense IncRNA, or the biological complex, or the expression vector as described herein, wherein the antisense IncRNA consists of a first and a second region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 1 and thousands of nucleotides, and wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and thousands of nucleotides. In another embodiment, the second region has a length of between 1 and 5000. In another embodiment, the second region has a length of between 1 and 2000. In another embodiment, the second region has a length of between 1 and 800. In an embodiment, the antisense IncRNA comprises a first region and a second region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 20 and 3000 nucleotides, and wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and 5000 nucleotides. In an embodiment, the expression vector further comprises a nucleic acid sequence encoding a target mRNA of the IncRNA.

[00020] In another aspect, the present invention relates to a method of decreasing export of a target mRNA from the nucleus of a eukaryotic cell, the method comprising transfecting a cell with the antisense IncRNA, or the biological complex, or the expression vector as described herein, wherein the IncRNA comprises a first region, a second region, and a third region. In other embodiments, the present invention relates to a method of decreasing export of a target mRNA from the nucleus of a eukaryotic cell, the method comprising transfecting a cell with the antisense IncRNA, or the biological complex, or the expression vector as described herein, wherein the IncRNA comprises a first region, a second region, and a third region, wherein the first, second and third regions comprises between 1 and thousands of nucleotides. In other embodiments, the present invention relates to a method of decreasing export of a target mRNA from the nucleus of a eukaryotic cell, the method comprising transfecting a cell with the antisense IncRNA, or the biological complex, or the expression vector as described herein, wherein the IncRNA comprises a first region, a second region, and a third region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 1 and thousands of nucleotides, wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and thousands of nucleotides, and wherein the third region is 5' of the first region, wherein the third region is not complementary to the mRNA and wherein the third region has a length of between 1 and thousands of nucleotides. In another embodiment, the second region has a length of between 1 and 5000. In another embodiment, the second region has a length of between 1 and 2000. In another embodiment, the second region has a length of between 1 and 800. In an embodiment, the third region has a length of between 1 and 5000. In another embodiment, the third region has a length of between 1 and 2000. In another embodiment, the third region has a length of between 1 and 800. In an embodiment, the second region and the third region have a length of between 1 and 5000. In an embodiment, the antisense IncRNA comprises a first region, a second region, and a third region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 20 and 3000 nucleotides, wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and 5000 nucleotides, and wherein the third region is 5' of the first region, wherein the third region is not complementary to the mRNA and wherein the third region has a length of between 1 and 5000 nucleotides.

[00021] In another aspect, the present invention relates to a method of decreasing expression of a target mRNA in a eukaryotic cell, the method comprising transfecting a cell with the antisense IncRNA, or the biological complex, or the expression vector as described herein, wherein the IncRNA comprises a first region, a second region, and a third region. In other embodiments, the present invention relates to a method of decreasing expression of a target mRNA in a eukaryotic cell, the method comprising transfecting a cell with the antisense IncRNA, or the biological complex, or the expression vector as described herein, wherein the IncRNA comprises a first region, a second region, and a third region, wherein the first, second and third regions comprises between 1 and thousands of nucleotides. In other embodiments, the present invention relates to a method of decreasing expression of a target mRNA in a eukaryotic cell, the method comprising transfecting a cell with the antisense IncRNA, or the biological complex, or the expression vector as described herein, wherein the IncRNA comprises a first region, a second region, and a third region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 1 and thousands of nucleotides, wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and thousands of nucleotides, and wherein the third region is 5' of the first region, wherein the third region is not complementary to the mRNA and wherein the third region has a length of between 1 and thousands of nucleotides. In another embodiment, the second region has a length of between 1 and 5000. In another embodiment, the second region has a length of between 1 and 2000. In another embodiment, the second region has a length of between 1 and 800. In an embodiment, the third region has a length of between 1 and 5000. In another embodiment, the third region has a length of between 1 and 2000. In another embodiment, the third region has a length of between 1 and 800. In an embodiment, the second region and the third region have a length of between 1 and 5000. In an embodiment, the antisense IncRNA comprises a first region, a second region, and a third region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 20 and 3000 nucleotides, wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and 5000 nucleotides, and wherein the third region is 5' of the first region, wherein the third region is not complementary to the mRNAand wherein the third region has a length of between 1 and 5000 nucleotides. [00022] In another aspect, the present invention relates to a cell transfected with the antisense IncRNA, or the biological complex, or the expression vector as described herein. In an embodiment, the expression vector further comprises a nucleic acid sequence encoding a target mRNA of the IncRNA.

[00023] In another aspect, the present invention relates to a cell transfected with the antisense IncRNA, or the biological complex, or the expression vector as described herein, wherein the antisense IncRNA or the antisense IncRNA of the biological complex or expression vector consists of a first region and a second region. In an embodiment, the present invention relates to a cell transfected with the antisense IncRNA, or the biological complex, or the expression vector as described herein, wherein the antisense IncRNA consists of a first and a second region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 1 and thousands of nucleotides, and wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and thousands of nucleotides. In another embodiment, the second region has a length of between 1 and 5000. In another embodiment, the second region has a length of between 1 and 2000. In another embodiment, the second region has a length of between 1 and 800. In an embodiment, the antisense IncRNA comprises a first region and a second region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 20 and 3000 nucleotides, and wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and 5000 nucleotides. In an embodiment, the expression vector further comprises a nucleic acid sequence encoding a target mRNA of the IncRNA.

[00024] In another aspect, the present invention relates to a cell transfected with the antisense IncRNA, or the biological complex, or the expression vector as described herein, wherein the IncRNA comprises a first region, a second region, and a third region. In other embodiments, the present invention relates to a cell transfected with the antisense IncRNA, or the biological complex, or the expression vector as described herein, wherein the IncRNA comprises a first region, a second region, and a third region, wherein the first, second and third regions comprises between 1 and thousands of nucleotides. In other embodiments, the present invention relates to a cell transfected with the antisense IncRNA, or the biological complex, or the expression vector as described herein, wherein the IncRNA comprises a first region, a second region, and a third region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 1 and thousands of nucleotides, wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and thousands of nucleotides, and wherein the third region is 5' of the first region, wherein the third region is not complementary to the mRNA and wherein the third region has a length of between 1 and thousands of nucleotides. In another embodiment, the second region has a length of between 1 and 5000. In another embodiment, the second region has a length of between 1 and 2000. In another embodiment, the second region has a length of between 1 and 800. In an embodiment, the third region has a length of between 1 and 5000. In another embodiment, the third region has a length of between 1 and 2000. In another embodiment, the third region has a length of between 1 and 800. In an embodiment, the second region and the third region have a length of between 1 and 5000. In an embodiment, the antisense IncRNA comprises a first region, a second region, and a third region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 20 and 3000 nucleotides, wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and 5000 nucleotides, and wherein the third region is 5' of the first region, wherein the third region is not complementary to the mRNA and wherein the third region has a length of between 1 and 5000 nucleotides.

[00025] In another aspect, the present invention relates to a conjugate comprising the antisense IncRNA, or the biological complex, or the expression vector as described herein, and a cell penetrating peptide. In an embodiment, the expression vector further comprises a nucleic acid sequence encoding a target mRNA of the IncRNA.

[00026] In another aspect, the present invention relates to a conjugate comprising the antisense IncRNA, or the biological complex, or the expression vector as described herein, wherein the antisense IncRNA or the antisense IncRNA of the biological complex or expression vector consists of a first region and a second region. In an embodiment, the present invention relates to a conjugate comprising the antisense IncRNA, or the biological complex, or the expression vector as described herein, wherein the antisense IncRNA consists of a first and a second region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 1 and thousands of nucleotides, and wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and thousands of nucleotides, and a cell penetrating peptide. In another embodiment, the second region has a length of between 1 and 5000. In another embodiment, the second region has a length of between 1 and 2000. In another embodiment, the second region has a length of between 1 and 800. In an embodiment, the antisense IncRNA comprises a first region and a second region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 20 and 3000 nucleotides, and wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and 5000 nucleotides. In an embodiment, the expression vector further comprises a nucleic acid sequence encoding a target mRNA of the IncRNA.

[00027] In another aspect, the present invention relates to a conjugate comprising the antisense IncRNA, or the biological complex, or the expression vector as described herein, wherein the IncRNA comprises a first region, a second region, and a third region. In other embodiments, the present invention relates to a conjugate comprising the antisense IncRNA, or the biological complex, or the expression vector as described herein, wherein the IncRNA comprises a first region, a second region, and a third region, wherein the first, second and third regions comprises between 1 and thousands of nucleotides. In other embodiments, the present invention relates to a conjugate comprising the antisense IncRNA, or the biological complex, or the expression vector as described herein, wherein the IncRNA comprises a first region, a second region, and a third region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 1 and thousands of nucleotides, wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and thousands of nucleotides, and wherein the third region is 5' of the first region, wherein the third region is not complementary to the mRNA and wherein the third region has a length of between 1 and thousands of nucleotides. In another embodiment, the second region has a length of between 1 and 5000. In another embodiment, the second region has a length of between 1 and 2000. In another embodiment, the second region has a length of between 1 and 800. In an embodiment, the third region has a length of between 1 and 5000. In another embodiment, the third region has a length of between 1 and 2000. In another embodiment, the third region has a length of between 1 and 800. In an embodiment, the second region and the third region have a length of between 1 and 5000. In an embodiment, the antisense IncRNA comprises a first region, a second region, and a third region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 20 and 3000 nucleotides, wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and 5000 nucleotides, and wherein the third region is 5' of the first region, wherein the third region is not complementary to the mRNA and wherein the third region has a length of between 1 and 5000 nucleotides.

[00028] In another aspect, the present invention relates to a pharmaceutical composition comprising the antisense IncRNA, or the biological complex, or the expression vector, or the conjugate as described herein. In an embodiment, the expression vector further comprises a nucleic acid sequence encoding a target mRNA of the IncRNA.

[00029] In another aspect, the present invention relates to a pharmaceutical composition comprising the antisense IncRNA, or the biological complex, or the expression vector, or the conjugate as described herein, wherein the antisense IncRNA or the antisense IncRNA of the biological complex or expression vector consists of a first region and a second region. In an embodiment, the present invention relates to a pharmaceutical composition comprising the antisense IncRNA, or the biological complex, or the expression vector as described herein, wherein the antisense IncRNA consists of a first and a second region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 1 and thousands of nucleotides, and wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and thousands of nucleotides. In another embodiment, the second region has a length of between 1 and 5000. In another embodiment, the second region has a length of between 1 and 2000. In another embodiment, the second region has a length of between 1 and 800. In an embodiment, the antisense IncRNA comprises a first region and a second region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 20 and 3000 nucleotides, and wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and 5000 nucleotides. In an embodiment, the expression vector further comprises a nucleic acid sequence encoding a target mRNA of the IncRNA.

[00030] In another aspect, the present invention relates to a pharmaceutical composition comprising the antisense IncRNA, or the biological complex, or the expression vector, or the conjugate as described herein, wherein the IncRNA comprises a first region, a second region, and a third region. In other embodiments, the present invention relates a pharmaceutical composition comprising the antisense IncRNA, or the biological complex, or the expression vector, or the conjugate as described herein, wherein the IncRNA comprises a first region, a second region, and a third region, wherein the first, second and third regions comprises between 1 and thousands of nucleotides. In other embodiments, the present invention relates to a pharmaceutical composition comprising the antisense IncRNA, or the biological complex, or the expression vector, or the conjugate as described herein, wherein the IncRNA comprises a first region, a second region, and a third region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 1 and thousands of nucleotides, wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and thousands of nucleotides, and wherein the third region is 5' of the first region, wherein the third region is not complementary to the mRNA and wherein the third region has a length of between 1 and thousands of nucleotides. In another embodiment, the second region has a length of between 1 and 5000. In another embodiment, the second region has a length of between 1 and 2000. In another embodiment, the second region has a length of between 1 and 800. In an embodiment, the third region has a length of between 1 and 5000. In another embodiment, the third region has a length of between 1 and 2000. In another embodiment, the third region has a length of between 1 and 800. In an embodiment, the second region and the third region have a length of between 1 and 5000.ln an embodiment, the antisense IncRNA comprises a first region, a second region, and a third region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 20 and 3000 nucleotides, wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and 5000 nucleotides, and wherein the third region is 5' of the first region, wherein the third region is not complementary to the mRNA and wherein the third region has a length of between 1 and 5000 nucleotides.

[00031] In another aspect, the present invention relates to the antisense IncRNA, or the biological complex, or the expression vector, or the conjugate as described herein for use as a medicament. In an embodiment, the expression vector further comprises a nucleic acid sequence encoding a target mRNA of the IncRNA.

[00032] In another aspect, the present invention relates to the antisense IncRNA, or the biological complex, or the expression vector, or the conjugate as described herein, for use as a medicament, wherein the antisense IncRNA or the antisense IncRNA of the biological complex or expression vector consists of a first region and a second region. In an embodiment, the present invention relates to the antisense IncRNA, or the biological complex, or the expression vector, or the conjugate as described herein, for use as a medicament, wherein the antisense IncRNA consists of a first and a second region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 1 and thousands of nucleotides, and wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and thousands of nucleotides. In another embodiment, the second region has a length of between 1 and 5000. In another embodiment, the second region has a length of between 1 and 2000. In another embodiment, the second region has a length of between 1 and 800. In an embodiment, the antisense IncRNA comprises a first region and a second region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 20 and 3000 nucleotides, and wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and 5000 nucleotides. In an embodiment, the expression vector further comprises a nucleic acid sequence encoding a target mRNA of the IncRNA.

[00033] In another aspect, the present invention relates to the antisense IncRNA, or the biological complex, or the expression vector, or the conjugate as described herein, for use as a medicament, wherein the IncRNA comprises a first region, a second region, and a third region. In other embodiments, the present invention relates to the antisense IncRNA, or the biological complex, or the expression vector, or the conjugate as described herein, for use as a medicament, wherein the IncRNA comprises a first region, a second region, and a third region, wherein the first, second and third regions comprises between 1 and thousands of nucleotides. In other embodiments, the present invention relates to the antisense IncRNA, or the biological complex, or the expression vector, or the conjugate as described herein, for use as a medicament, wherein the IncRNA comprises a first region, a second region, and a third region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 1 and thousands of nucleotides, wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and thousands of nucleotides, and wherein the third region is 5' of the first region, wherein the third region is not complementary to the mRNA and wherein the third region has a length of between 1 and thousands of nucleotide. In another embodiment, the second region has a length of between 1 and 5000. In another embodiment, the second region has a length of between 1 and 2000. In another embodiment, the second region has a length of between 1 and 800. In an embodiment, the third region has a length of between 1 and 5000. In another embodiment, the third region has a length of between 1 and 2000. In another embodiment, the third region has a length of between 1 and 800. In an embodiment, the second region and the third region have a length of between 1 and 5000. In an embodiment, the antisense IncRNA comprises a first region, a second region, and a third region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 20 and 3000 nucleotides, wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and 5000 nucleotides, and wherein the third region is 5' of the first region, wherein the third region is not complementary to the mRNA and wherein the third region has a length of between 1 and 5000 nucleotides.

[00034] In another aspect, the present invention relates to the antisense IncRNA, or the biological complex, or the expression vector, or the conjugate as described herein for use in the treatment or prevention of a disease, preferably by enhancing or decreasing nuclear export of a therapeutically relevant mRNA. In an embodiment, the expression vector further comprises a nucleic acid sequence encoding a target mRNA of the IncRNA. In an embodiment, the disease is cancer. In an embodiment, the antisense IncRNA, the biological complex, the expression vector, or the conjugate enhances nuclear export of a tumor suppressor mRNA and/or inhibits the nuclear export of oncogene mRNA.

[00035] In another aspect, the present invention relates to the antisense IncRNA, or the biological complex, or the expression vector, or the conjugate as described herein, for use in the treatment or prevention of a disease, preferably by enhancing nuclear export of a therapeutically relevant mRNA, the increased expression of which has a therapeutic benefit, wherein the antisense IncRNA or the antisense IncRNA of the biological complex or expression vector or conjugate consists of a first region and a second region. In an embodiment, the present invention relates to the antisense IncRNA, or the biological complex, or the expression vector, or the conjugate as described herein, for use in the treatment or prevention of a disease, preferably by enhancing nuclear export of a therapeutically relevant mRNA, the increased expression of which has a therapeutic benefit, wherein the antisense IncRNA consists of a first and a second region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 1 and thousands of nucleotides, and wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and thousands of nucleotides. In another embodiment, the second region has a length of between 1 and 5000. In another embodiment, the second region has a length of between 1 and 2000. In another embodiment, the second region has a length of between 1 and 800. In an embodiment, the antisense IncRNA comprises a first region and a second region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 20 and 3000 nucleotides, and wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and 5000 nucleotides. In an embodiment, the expression vector further comprises a nucleic acid sequence encoding a target mRNA of the IncRNA. In an embodiment, the disease is cancer. In an embodiment, the antisense IncRNA, the biological complex, the expression vector, or the conjugate enhances nuclear export of a tumor suppressor mRNA.

[00036] In another aspect, the present invention relates to the antisense IncRNA, or the biological complex, or the expression vector, or the conjugate as described herein, for use in the treatment or prevention of a disease, preferably by inhibiting the nuclear export of a therapeutically relevant mRNA, the expression of which is causally linked to the disease, wherein the IncRNA comprises a first region, a second region, and a third region. In other embodiments, the present invention relates to the antisense IncRNA, or the biological complex, or the expression vector, or the conjugate as described herein, for use in the treatment or prevention of a disease, preferably by inhibiting the nuclear export of a therapeutically relevant mRNA, the expression of which is causally linked to the disease, wherein the IncRNA comprises a first region, a second region, and a third region, wherein the first, second and third regions comprises between 1 and thousands of nucleotides. In other embodiments, the present invention relates to the antisense IncRNA, or the biological complex, or the expression vector, or the conjugate as described herein, for use in the treatment or prevention of a disease, preferably by inhibiting the nuclear export of a therapeutically relevant mRNA, the expression of which is causally linked to the disease, wherein the IncRNA comprises a first region, a second region, and a third region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 1 and thousands of nucleotides, wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and thousands of nucleotides, and wherein the third region is 5' of the first region, wherein the third region is not complementary to the mRNA and wherein the third region has a length of between 1 and thousands of nucleotide. In another embodiment, the second region has a length of between 1 and 5000. In another embodiment, the second region has a length of between 1 and 2000. In another embodiment, the second region has a length of between 1 and 800. In an embodiment, the third region has a length of between 1 and 5000. In another embodiment, the third region has a length of between 1 and 2000. In another embodiment, the third region has a length of between 1 and 800. In an embodiment, the second region and the third region have a length of between 1 and 5000. In an embodiment, the antisense IncRNA comprises a first region, a second region, and a third region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 20 and 3000 nucleotides, wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and 5000 nucleotides, and wherein the third region is 5' of the first region, wherein the third region is not complementary to the mRNA and wherein the third region has a length of between 1 and 5000 nucleotides.

[00037] In another aspect, the present invention relates to a method of treatment comprising administering the antisense IncRNA, or the biological complex, or the expression vector, or the conjugate as described herein to a subject in need thereof. In an embodiment, the expression vector further comprises a nucleic acid sequence encoding a target mRNA of the IncRNA.

[00038] In another aspect, the present invention relates to a method of treatment comprising administering the antisense IncRNA, or the biological complex, or the expression vector, or the conjugate as described herein, to a subject in need thereof, wherein the antisense IncRNA or the antisense IncRNA of the biological complex or expression vector or the conjugate consists of a first region and a second region. In an embodiment, the present invention relates to a method of treatment comprising administering the antisense IncRNA, or the biological complex, or the expression vector, or the conjugate as described herein, to a subject in need thereof, wherein the antisense IncRNA consists of a first and a second region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 1 and thousands of nucleotides, and wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and thousands of nucleotides. In another embodiment, the second region has a length of between 1 and 5000. In another embodiment, the second region has a length of between 1 and 2000. In another embodiment, the second region has a length of between 1 and 800. In an embodiment, the antisense IncRNA comprises a first region and a second region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 20 and 3000 nucleotides, and wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and 5000 nucleotides. In an embodiment, the expression vector further comprises a nucleic acid sequence encoding a target mRNA of the IncRNA.

[00039] In another aspect, the present invention relates to a method of treatment comprising administering the antisense IncRNA, or the biological complex, or the expression vector, or the conjugate as described herein, to a subject in need thereof, wherein the IncRNA comprises a first region, a second region, and a third region. In other embodiments, the present invention relates to a method of treatment comprising administering the antisense IncRNA, or the biological complex, or the expression vector, or the conjugate as described herein, to a subject in need thereof, wherein the IncRNA comprises a first region, a second region, and a third region, wherein the first, second and third regions comprises between 1 and thousands of nucleotides. In other embodiments, the present invention relates to a method of treatment comprising administering the antisense IncRNA, or the biological complex, or the expression vector, or the conjugate as described herein, to a subject in need thereof, wherein the IncRNA comprises a first region, a second region, and a third region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 1 and thousands of nucleotides, wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and thousands of nucleotides, and wherein the third region is 5' of the first region, wherein the third region is not complementary to the mRNA and wherein the third region has a length of between 1 and thousands of nucleotide. In another embodiment, the second region has a length of between 1 and 5000. In another embodiment, the second region has a length of between 1 and 2000. In another embodiment, the second region has a length of between 1 and 800. In an embodiment, the third region has a length of between 1 and 5000. In another embodiment, the third region has a length of between 1 and 2000. In another embodiment, the third region has a length of between 1 and 800. In an embodiment, the second region and the third region have a length of between 1 and 5000. In an embodiment, the antisense IncRNA comprises a first region, a second region, and a third region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 20 and 3000 nucleotides, wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and 5000 nucleotides, and wherein the third region is 5' of the first region, wherein the third region is not complementary to the mRNA and wherein the third region has a length of between 1 and 5000 nucleotides.

[00040] In another aspect, the present invention relates to the use of the antisense IncRNA, or the biological complex, or the expression vector, or the conjugate as described herein, in increasing mRNA vaccine efficacy, wherein the antisense IncRNA or the antisense IncRNA of the biological complex or expression vector or the conjugate consists of a first region and a second region. In an embodiment, the present invention relates to the use of the antisense IncRNA, or the biological complex, or the expression vector, or the conjugate as described herein, in increasing mRNA vaccine efficacy, wherein the antisense IncRNA consists of a first and a second region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 1 and thousands of nucleotides, and wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and thousands of nucleotides. In another embodiment, the second region has a length of between 1 and 5000. In another embodiment, the second region has a length of between 1 and 2000. In another embodiment, the second region has a length of between 1 and 800. In an embodiment, the antisense IncRNA comprises a first region and a second region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 20 and 3000 nucleotides, and wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and 5000 nucleotides.

BRIEF DESCRIPTION OF THE FIGURES

[00041] FIG. 1 shows that dsRNAs are mostly located in the cytoplasm. FIG. 1A: Nucleo- cytoplasmic fractionation experiment eliminates the nuclear content of cells. Western blot analysis shows the presence of the cytosolic protein Zwfl in both fractions and indicates successful fractionation by the absence of the nuclear protein Nopl in the cytoplasmicfraction. FIG. IB: Genome wide analysis of the compartmental presence of different RNA species. Data were filtered for the indicated RNA species. On the y-axis, the depicted Iog2-fold change of the cytoplasmic fraction compared to total lysate indicates the nucleo-cytoplasmic distribution. FIG. 1C: The presence of an antisense RNA increases the probability of its mRNA to be cytoplasmic enriched. The average nucleo- cytoplasmic distribution of mRNAs is shown, based on their relative asRNA expression. FIG. ID: The average cytoplasmic presence of an RNA increases with the probability to form double strands. For this analysis, data was used and RNAs grouped based on the Iog2 fold change gathered with an artificial RNAi system determining dsRNA 12 and applied with their nucleo-cytoplasmic distribution. FIG. IE: dsRNAs are trapped in the nucleus of the export mutant mex67-5 xpol-1. RNA seq analysis of the average nucleo-cytoplasmic distribution in mex67-5 xpol-1 at 37°C for lh of RNAs based on their probability to form dsRNA as determined via RNAi seq. FIG. IF: Overexpression of the antisense RNA of PHO85 (SUT412) results in similar amounts of sense and asRNA. A wild type strain was either transformed with an empty vector or with a plasmid carrying SUT412 under the GALI promoter. Both strains were grown o/N under inducing conditions (2% galactose) to log phase. Subsequently, cells were lysed, the RNA was isolated and used in qPCR. n=5 FIG. 1G: Overexpression of asPHO85 leads to an increased presence of the PHO85 mRNA in the cytoplasm relative to wild type (dotted line). Nucleo-cytoplasmic fractionation experiment, subsequent RNA isolation and consecutive qPCR is shown for the total PHO85 and for the unspliced transcript only. n=5

[00042] FIG. 2 shows that dsRNAs are exported faster than ssRNAs through preferential binding of Mex67. FIG. 2A: dsRNAs are exported to reach the ribosome. IF experiment with J2 antibody (primary) and a Cy3-labelled secondary antibody (left) and FISH with a Cy3-labelled oligo d(T) probe (right) are shown in the indicated strains that were shifted to 37°C for lh. FIG. 2B: dsRNA contacts ribosomes before ssRNAs. Cells were grown to log phase before an export block was induced through shifting mex67-5 to 37°C for lh. The block was subsequently released by lowering the temperature back to 25°C. RIP experiments with Rps2-GFP were carried out after harvesting cells at the indicated time points. The extracted RNA was analyzed via qPCR of three ssRNAs and dsRNAs. n=3 FIG. 2C: More molecules of Mex67 can bind to dsRNA than to ssRNA. Electro mobility shift assay (EMSA) was carried out with FAM-labeled ssRNAs or dsRNAs. Increasing amounts of recombinant purified TAP- tagged Mex67-Mtr2 heterodimer was added to the RNA and complex formation is visualized on a native gel. FIG. 2D: Competition assay reveals a preferential binding of Mex67 to dsRNA. Cy3-labeled ssRNA (red) were pre-incubated with low concentrations of Mex67 for complex formation. Subsequently, increasing amounts of a FAM-labeled dsRNA (green) was added as a competitor (left). Increasing amounts of Cy3-labelled ssRNA was added to pre-bound FAM-labelled dsRNA with Mex67 (right).

[00043] FIG. 3 shows that dsRNA formation is essential for cells that change their expression program. FIG. 3A: Absence of the transcription factor Set2 leads to increased dsRNA production. IF with Cy3-marked J2-antibody (left) and FISH with a Cy3-labelled oligo d(T) probe (right) are shown. FIG. 3B: Quantification of the signal intensities depicted in FIG. 3A. Signal intensity of 30 cells was determined via Fiji. FIG. 3C: The antisense RNA level of the Set2-responsive RNA SEG2 is significantly increased in set2A. n=5 FIG. 3D: Increased presence of SEG2 in the cytosol of set2A cells. Nucleo- cytoplasmic fractionation experiment in set2A was carried out, followed by qPCR of SEG2 and SEG2as. n=4 FIG. 3E: asRNA production increases during stress. Genome wide RNA analysis of cells shifted to 0.6M NaCI 31 were used to compare sense and antisense RNAs (Iog2 fold change) to unstressed conditions. FIG. 3F: dsRNA amounts increase under stress conditions. Cy3-marked J2-IF (left) and Cy3-labelled oligo d(T) FISH (right) are shown in wild type cells, exposed to the indicated stress conditions. FIG. 3G: Directing the dsRNA degrading bacterial RNase III into the nucleus of yeast cells is lethal. 10-fold serial dilutions of wild type strains containing the indicated plasmids were spotted onto glucose (no induction) or galactose plates (with induction) and incubated for 3 days. FIG. 3H: J2-IF and localization of the GFP- and transport signal-tagged RNase III fusion proteins in yeast cells. Plasmid containing wild type cells were grown to log phase before the RNase III expression was induced through the addition of galactose for 6h. FIG. 31: The stress-induced dsRNA is degraded by cytoplasmic RNasel 11. Cy3-marked J2-IF (left) and Cy3-labelled oligo d(T) FISH (right) are shown for the indicated strains either without stress or after a 30 min incubation with 0.7M NaCI. FIG. 3J: Cytoplasmic RNase III is not tolerated in cellular stress situations. 10-fold serial dilutions of wild type cells containing either a constitutively expressed RNase lll-NES from the ADH1 promoter or the RNAi plasmids, which also eliminates cytoplasmic dsRNA, were spotted onto the indicated plates and incubated for 3 days at 30°C.

[00044] FIG. 4 shows that Dbp2 induces dsRNA formation. FIG. 4A: Dbp2 binds to dsRNA. Western blot of J2-ColP is shown. Heml5 served as a negative control. FIG. 4B: Deletion of DBP2 results in the loss of dsRNA. Cy3-marked J2-IF (left) and Cy3-labelled oligo d(T) FISH (right) are shown for indicated strains. Cells were shifted to the non-permissive temperature of dbp2A before harvest. FIG. 4C: Overexpression of SUT412 is not affected in dbp2A. SUT412 expression was induced o/N via galactose addition followed by RNA-isolation and qPCR. RNA levels are shown relative to PHO85 mRNA. FIG. 4D: The increased presence of the PHO85 mRNA in the cytoplasm after SUT412 overexpression is not seen in dbp2A. Cells were shifted to the non-permissive temperature of 25°C for 2h. Nucleo-cytoplasmic fractionation experiment after SUT412 induction, subsequent RNA isolation and consecutive qPCR is shown for the PHO85 transcript. n=5. FIG. 4E: Model for the preferential export of dsRNAs. Top: mRNAs are transcribed by RNA polymerase II and eventually bound by Mex67 leading to export and translation in the cytoplasm. Bottom: Preferential expression during cellular adaptation. Dbp2 and Yral mediate dsRNA formation of mRNAs and their asRNAs. Preferential binding of Mex67 to dsRNAs leads to the favored nuclear export and thus, translation of the regarding mRNA. As translation leads to the separation of the dsRNA, the corresponding asRNA is likewise scanned by ribosomes, however, then subsequently degraded by NMD. In this way preferential gene expression is established.

[00045] FIG. 5 relates to the findings shown in FIG. 1. FIG. 5A: Nucleo-cytoplasmic fractionation experiment in Wild type and the export mutant mex67-5 xpol-1 after a lh temperature shift to 37°C. Western blot analysis of successful fractionation is shown. FIG. 5B: dsRNAs show no increased stability. Data analysis determining the half-life of mRNAs [Chan, 2018] was applied with the Iog2 fold change in RNAi-seq according to [Wery, 2016 #3797], FIG. 5C: The half-life of nuclear transcripts is higher than that of the cytoplasmic mRNAs. Data analyzing the half-life of mRNAs [Chan, 2018 #4329] was applied with the nucleo-cytoplasmic distribution of RNAs identified in FIG. IB. FIG. 5D: Highly expressed genes are more nuclear. The nucleo-cytoplasmic distribution of RNAs obtained in FIG. 1A and FIG. IB was applied with their expression level in RPKM. FIG. 5E: Half-life and expression level of mRNAs correlate. The half-life of mRNA measured by Chan et al. (2018) was applied with their expression level determined in FIG. 2B. FIG. 5F: Western blot of the nucleo-cytoplasmic fractionation experiment used in (FIG. IF and FIG. 1G) is shown.

[00046] FIG. 6. relates to the findings depicted in FIG. 2. FIG. 6A: The J2-antibody precipitates dsRNAs in yeast. qPCRs with example mRNAs are shown after J2-IP. n=3. FIG. 6B: The amount of dsRNAs increase when they are retained in the nucleus. qPCRs are shown in the indicated strains after a lh temperature shift to 37°C. n=3 FIG. 6C: dsRNA contacts ribosomes before ssRNAs. Rps3-GFP was precipitated at different timepoints before and after export release and is shown in western blot.

[00047] FIG. 7 extends the findings shown in FIG. 3. FIG. 7A: Western blot of the nucleo- cytoplasmic fractionation experiment used in FIG. 3E. FIG. 7B: Directing the dsRNA degrading bacterial RNase III into the nucleus of yeast cells is lethal. 10-fold serial dilutions of wild type strains containing the indicated plasmids were spotted onto either glucose (no induction) or galactose containing agar plates (with induction) and incubated for 3 days at the indicated temperatures, (c) Localization of the GFP- and transport signal-tagged RNase III fusion proteins in yeast cells. Plasmid containing wild type cells were grown to log phase before the RNase III expression was induced through the addition of galactose for 6h. FIG. 7C: J2-IF in cells expressing GFP- and transport signal- tagged RNase III fusion proteins. Cells were treated like in FIG. 7C.

[00048] FIG. 8. relates to the findings shown in FIG. 4. 10-fold serial dilutions of wild type and dbp2A strains were spotted onto YPD plates and incubated for 3 days at the indicated temperatures. [00049] FIG. 9 shows a Data Table of Oligos.

[00050] FIG. 10 shows a Data Table of Yeast Strains. Yeast strain source reference: 1- Brune et al., 2005; 2- Baierlein et al., 2013; 3- Gadal et al., 2001; 4- Drinnenberg et al., 2009

[00051] FIG. 11 shows a Data Table of Plasmids. Plasmid source reference: 5- Milkereit et al., 2003; 6- Grosse et al., 2021.

[00052] FIG. 12 shows a graphical layout of a RNP complex of an mRNA and an antisense (as)RNA. FIG. 12A: Boosting antisense RNA. The sense RNA and the antisense RNA overlap and hybridize at their 5' ends (first region) and have both overhanging 3' ends (second region). The figure shows a boost-RNA according to the present invention. FIG. 12B: Inhibiting antisense RNA. The sense RNA and the antisense RNA overlap and hybridize at the first region of the antisense RNA. The antisense RNA further has a second region (3' overhang) and a non-complementary third region at the 5' end of the asRNA The figure shows an inhibiting asRNA according to the present invention. [00053] FIG. 13 Boosting gene expression of yeast genes. FIG. 13A: Top: Western blot show the boosted gene expression of Pho85 in yeast after ectopic asRNA expression. Hem 15 served as a negative control. Bottom: Quantification of mRNA, protein and asRNA after induction of the asRNA. FIG. 13B: Top: Western blot show the boosted gene expression of MTR4 in yeast after ectopic asRNA expression. Hem 15 served as a negative control. Bottom: Quantification of mRNA, protein and asRNA after induction of the asRNA. FIG. 14 Boosting gene expression of yeast genes. FIG. 14A: Example Western blot of transfected human HCT cells expressing an asRNA for BRCA1. Bcral expression is shown. FIG. 14B: Protein expression is boosted for the housekeeping gene GAPDH and BCRA1, MAD2L1, and SMAD4 that all have no annotated asRNAs. Overlapping sequences were designed with 760bp (GAPDH), 94bp (BRCA1), 78bp (MAD1L1) and 411bp (SMAD4) nucleotides, respectively and at least 717bp overhang in addition to poly(A) tail. FIG. 14C: Scheme of the overlapping and overhanging sequences for the used antisense constructs. 5' and 3' UTRs are shown in gray for the mRNA and the antisense only overlaps the 5' UTR of the respective mRNA. Depending on the transcript that is expressed, the overlap for MAD2L1 and SMAD4 can be longer up to 468bp and 411bp, respectively, as annotations exist for different transcript lengths.

DETAILED DESCRIPTION OF THE INVENTION

[00054] Principles provided by the instant Invention

[00055] The inventors of the present invention found that specific antisense RNAs (asRNAs) boost gene expression, and thus termed them boost-RNAs. Interestingly, transcribed mRNAs are not efficiently expressed without significant levels of the respective boost-RNA. Only when the boost- RNA is expressed, the mRNA is efficiently exported into the cytoplasm and translated at ribosomes. The inventors discovered that (sense) mRNA and asRNA form a double strand in the nucleus immediately after transcription and the resulting double stranded RNA is preferentially exported into the cytoplasm and delivered to the ribosomes for translation, resulting in an increased protein level of the mRNA encoded protein. Thus, specific asRNAs can boost gene expression of individual genes. The inventors also identified the enzymes responsible for forming the RNA double strand: the nuclear helicase Dbp2 and its co-factor Yral (human DDX5/p68 and ALY/REV/NXF1, respectively). Importantly, the invention shows that expression of boost-RNA is sufficient to increase the protein level of a given gene.

[00056] Notably, the RNA double strand is not completely overlapping (e.g. a partial double stranded RNA). Only parts of the mRNA overlaps with the boost-RNA, while other parts form overhangs. This structure is crucial for nuclear export. When certain extensions are added to the boost RNA the export and thus expression of the mRNA is blocked. Thus, the boost-RNA system can be utilized at in at least the following ways: a.) to increase nuclear export of target mRNA b) to boost specific gene expression c) when used with certain sequence additions to the boost RNA, to decrease nuclear export of target mRNA, d) when used with certain sequence additions to the boost RNA, to inhibit gene expression for specific genes.

[00057] The boost-RNA system as described herein has advantages over other known gene expression approaches. For example, in contrast to the CRISPR-Cas system, in which the target genome is manipulated, the boost-RNA system does not involve any genomic manipulations. It can be based on transient plasmid-based expression or delivery of antisense IncRNAs, either for expression of the naturally occurring asRNA boosting gene expression or for inhibiting asRNAs (block- RNAs) that reduce the gene expression.

[00058] The boost-RNA system has also an advantage over siRNA-mediated gene repression since it acts already immediately after transcription in the nucleus while siRNAs target transcripts only later in the cytoplasm. Furthermore, siRNAs cannot be used to boost gene expression.

[00059] Furthermore, the boost-RNA system can be applied to address various potential applications in health care and biotechnology (e.g. therapeutic applications). These include the restoration of gene expression in pathologies where loss of specific gene expression is involved in the disease (e.g. tumor suppressor genes), downregulation of gene expression in situations where gene overexpression is relevant (e.g. oncogenes), or optimization of protein production in biotechnology applications (e.g. production of recombinant proteins or mRNA vaccination).

[00060] Antisense long noncoding RNAs

[00061] The present invention generally relates to an antisense IncRNA comprising a first region and a second region, wherein the first region is complementary to a portion of a target mRNA, and wherein the second region is not complementary to the target mRNA.

[00062] The terms "polynucleotide", "nucleotide seguence", "nucleic gcid" and "oligonucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides (DNA) or ribonucleotides (RNA), or analogs thereof. Polynucleotides may have any three dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, exons, introns, messenger RNA (mRNA), non-coding RNA (ncRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), antisense RNA (asRNA), long non-coding RNA (IncRNA), antisense non-coding RNA (asncRNA), antisense long non-coding RNA (asIncRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence. A polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. [00063] As used herein, an "antisense RNA (asRNA)", or "antisense non-coding RNA (asncRNA)", or "antisense long non-coding RNA (asIncRNA)" refers to an antisense transcript that is a single stranded RNA that is complementary to another nucleic acid. A non-limiting example of the nucleic acid that an asRNA, asncRNA, or asIncRNA can be complementary to is mRNA. Furthermore, in another non-limiting example, the complementarity between the asRNA, asncRNA, or asIncRNA and mRNA (target mRNA) is partial complementary, and the asRNA, asncRNA, or asIncRNA and target mRNA form a partial double strand complex.

[00064] The term “long non-coding RNA (IncRNA)" generally refers to a class of RNA molecules comprising more than 200 nucleotides, which do not encode proteins. However, as used herein, an "antisense long non-coding RNA (asIncRNA)" refers to an RNA molecule that is at least 30 nucleotides in length, at least 50 nucleotides, at least 100 nucleotides, at least 150 nucleotides, or at least 200 nucleotides. In an embodiment, the asIncRNA has a length of between 1 and thousands of nucleotides. Whenever in the description it is referred to "IncRNA" or "asnlcRNA" or "antisense RNA or "antisense IncRNA" with no further information to "boost-RNA" or "block-RNA", the term refers to both, the boost-RNA and the block-RNA.

[00065] As used herein, "complementary" or "complementarity" refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick base pairing or other non-traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). As used herein, "perfectly complementary" means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. As used herein, "partial complementary" refers to a degree of complementarity that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%, over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300 or more nucleotides, i.e. over a region of thousands of nucleotides

[00066] As used herein, "partial double strand complex", "partial double stranded complex", or "partial RNA double strand" refers to a double stranded nucleic acid complex formed by two nucleic acids that have partial complementary and are thus not perfectly complementary. In a partial double strand complex, the two nucleic acids are not completely overlapping, and thus parts of the nucleic acids overlap, while other parts of the nucleic acids form overhangs. The overhangs present in a partial double strand complex can be 3' overhangs and/or 5' overhangs, and both or only one of the nucleic acids in the partial double strand complex can have 3' and/or 5' overhangs. A non-limiting example of the types of nucleic acids that can form a partial double strand complex are an antisense IncRNA with mRNA.

[00067] As used herein, the term "boost-RNA" refers to an antisense long-noncoding RNA (asIncRNA), asRNA, or asncRNA that is capable of forming a partial double-strand complex with a target mRNA and increasing nuclear export of the target mRNA via the partial double strand complex that is formed.

[00068] As used herein, the term "block-RNA " refers to an antisense long-noncoding RNA (asIncRNA), asRNA, or asncRNA that is capable of forming a partial double-strand complex with a target mRNA and decreasing or inhibiting nuclear export of the target mRNA via the partial double strand complex that is formed. As described herein, block-RNA comprises a second region that is between 1 and thousands of nucleotides in length and a third region that is 5' of the first region which is not complementary to the target mRNA and which has a length of between 1 and several thousand nucleotides, area block-RNA is capable of forming a partial double-strand complex with a target mRNA and decreasing nuclear export of the target mRNA via the partial double strand complex that is formed.

[00069] Boost-RNA

[00070] In one embodiment, the present invention relates to an antisense IncRNA comprising a first region and a second region, wherein the first region is complementary to a portion at the 5' end of a target mRNA, and wherein the second region is not complementary to the target mRNA, and the second region is between 1 and several thousand nucleotides in length, between 1 and thousands of nucleotides in length, between 1 to 10000 nucleotides in length, between 1 to 9 000 nucleotides in length, between 1 and 8000 nucleotides in length, between 1 and 7000 nucleotides in length, between 1 and 6000 nucleotides in length, between 1 and 5000 nucleotides in length, between 1 and 4000 nucleotides in length, between 1 and 3000 nucleotides in length, between 1 and 2000 nucleotides in length, or between 1 and 1000 nucleotides in length. In an embodiment, the second region is between 1 and 900 nucleotides in length, between 1 and 800 nucleotides in length, between 1 and 700 nucleotides in length, between 1 and 600 nucleotides in length, between 1 and 500 nucleotides in length, between 1 and 400 nucleotides in length, between 1 and 300 nucleotides in length, between 1 and 200 nucleotides in length, between 1 and 100 nucleotides in length. In an embodiment, the second region is 1 to 5 nucleotides in length, or 1 to 3 nucleotides in length. In an embodiment, the second region is between 30 and 9000 nucleotides in length. In an embodiment, the second region is between 100 and 1500 nucleotides in length. In an embodiment, the antisense IncRNA comprises a first region and a second region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 20 and 3000 nucleotides, and wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA 1 and wherein the second region has a length of between 1 and 5000 nucleotides. Preferably, the second region is between 1 and thousands of nucleotides in length. In an embodiment, the first region is complementary to the 5' end of the target mRNA. In an embodiment, the first region is complementary to the 5' UTR of the target mRNA. In another embodiment, the second region is 3' of the first region. In an embodiment, the first region has a length of between 1 and thousands of nucleotides. In an embodiment, the first region has a length of between 100 and 300 nucleotides. In another embodiment, the first region has a length of 1000 to 3000 nucleotides. In another embodiment, the first region has a length of 1 to 16000 nucleotides. In an embodiment, the second region additionally comprises a poly(A) tail at its 3' end. In an embodiment, the second region has a length of at least 717 nucleotides in addition to a poly(A) tail. In another embodiment, the antisense IncRNA is capable of forming a partial double strand complex with the mRNA thereby increasing nuclear export of the mRNA. In an embodiment, the antisense IncRNA has a length of at least 30 nucleotides, at least 50 nucleotides, at least 100 nucleotides, at least 150 nucleotides, or at least 200 nucleotides. In another embodiment, the IncRNA has a length of thousands of nucleotides. In an embodiment, the IncRNA has a length of between 100 and 3000 nucleotides. In an embodiment, the IncRNA has a length of between 100 and 16000 nucleotides. In an embodiment, the IncRNA has a length of between 100 and 14000 nucleotides. In an embodiment, the IncRNA has a length of between 100 and 12000 nucleotides. In an embodiment, the IncRNA has a length of between 100 and 10000 nucleotides. In an embodiment, the IncRNA has a length of between 100 and 8000 nucleotides. In an embodiment, the IncRNA has a length of between 100 and 6000 nucleotides. In an embodiment, the IncRNA has a length of between 100 and 4000 nucleotides. In an embodiment, the IncRNA has a length of between 100 and 2000 nucleotides. In an embodiment, the IncRNA has a length of between 100 and 1000 nucleotides. In another embodiment, the antisense IncRNA is capable of forming, with the target mRNA, a complex that is optimized for binding double-strand formation mediating proteins and/or binding export mediating proteins. In an embodiment, the complex comprises one or more double-strand formation mediating proteins and/or one or more export mediating proteins. In an embodiment, the double-strand formation mediating proteins and export mediating proteins are Dbp2 and/or Yral, and Mex67 and/or Mtr2, respectively. In an embodiment, the double-strand formation mediating protein is Dbp2. In an embodiment, the double-strand formation mediating protein is Yral. In an embodiment, the export mediating protein is Mex67. In an embodiment, the export mediating protein is Mtr2. In an embodiment, the double-strand formation mediating proteins and export mediating proteins are DDX5 and/or ALY/REV/NXF2, and TAP and/or pl5, respectively. In an embodiment, the double-strand formation mediating protein is DDX5. In an embodiment, the double-strand formation mediating protein is ALY/REV/NXF2. In an embodiment, the export mediating protein is TAP. In an embodiment, the export mediating protein is pl5. Without being bound by theory, based on the invention, the formation of a partial double strand complex between the antisense IncRNA and a target mRNA results in increased nuclear export of the target mRNA, thereby increasing expression of the target mRNA; thus the antisense IncRNA is termed a "boost- RNA".

[00071] Block-RNA

[00072] In another aspect, the present invention relates to an antisense IncRNA comprising a first region, a second region and a third region, wherein the first region is complementary to a portion of a target mRNA, and wherein the second region is not complementary to the target mRNA, and wherein the third region is not complementary to the target mRNA. In an embodiment, the second region is 3' of the first region. In an embodiment, the third region is 5' of the first region. In an embodiment, the first region is complementary to the 5' end of the target mRNA. In an embodiment, the first region is complementary to the 5' UTR of the target mRNA. In another embodiment, the second region is 3' of the first region. In an embodiment, the block RNA comprises only the first and the third region. In an embodiment, the block RNA consists of only the first and the third region. In another embodiment, the antisense IncRNA is capable of forming a partial double strand complex with the mRNA thereby decreasing nuclear export of the mRNA. In an embodiment, the antisense IncRNA has a length of at least 30 nucleotides, at least 50 nucleotides, at least 100 nucleotides, at least 150 nucleotides, or at least 200 nucleotides. In another embodiment, the IncRNA has a length of thousands of nucleotides. In an embodiment, the IncRNA has a length of between 1 and 20000 nucleotides. In an embodiment, the IncRNA has a length of between 100 and 20000 nucleotides. In an embodiment, the IncRNA has a length of between 100 and 18000 nucleotides. In an embodiment, the IncRNA has a length of between 100 and 16000 nucleotides. In an embodiment, the IncRNA has a length of between 100 and 14000 nucleotides. In an embodiment, the IncRNA has a length of between 100 and 12000 nucleotides. In an embodiment, the IncRNA has a length of between 100 and 10000 nucleotides. In an embodiment, the IncRNA has a length of between 100 and 8000 nucleotides. In an embodiment, the IncRNA has a length of between 100 and 6000 nucleotides. In an embodiment, the IncRNA has a length of between 100 and 4000 nucleotides. In an embodiment, the IncRNA has a length of between 100 and 2000 nucleotides. In an embodiment, the IncRNA has a length of between 100 and 1000 nucleotides. In an embodiment, the second region of the antisense IncRNA is more than 10 nucleotides in length. In another embodiment, the second region of the antisense IncRNA is more than 15 nucleotides in length. In another embodiment, the second region of the antisense IncRNA is more than 20 nucleotides in length. In another embodiment, the second region of the antisense IncRNA is more than 25 nucleotides in length. In another embodiment, the second region of the antisense IncRNA is more than 30 nucleotides in length. In another embodiment, the second region of the antisense IncRNA is more than 40 nucleotides in length. In an embodiment, the second region is up to 50 nucleotides in length. In a further embodiment, the second region is between 10 and 50, 20 and 50, 30 and 50 or 40 and 50 nucleotides in length. In another embodiment, the second region is between 1 and several thousand nucleotides in length, between 1 and thousands of nucleotides in length, between 1 to 10000 nucleotides in length, between 1 to 9 000 nucleotides in length, between 1 and 8000 nucleotides in length, between 1 and 7000 nucleotides in length, between 1 and 6000 nucleotides in length, between 1 and 5000 nucleotides in length, between 1 and 4000 nucleotides in length, between 1 and 3000 nucleotides in length, between 1 and 2000 nucleotides in length, or between 1 and 1000 nucleotides in length. In an embodiment, the second region is between 1 and 900 nucleotides in length, between 1 and 800 nucleotides in length, between 1 and 700 nucleotides in length, between 1 and 600 nucleotides in length, between 1 and 500 nucleotides in length, between 1 and 400 nucleotides in length, between 1 and 300 nucleotides in length, between 1 and 200 nucleotides in length, between 1 and 100 nucleotides in length. In an embodiment, the second region is 1 to 5 nucleotides in length, or 1 to 3 nucleotides in length. In an embodiment, the second region is between 30 and 9000 nucleotides in length. In an embodiment, the second region is between 100 and 1500 nucleotides in length. Preferably, the second region is between 1 and thousands of nucleotides in length.

[00073] In an embodiment, the third region comprises at least 20 nucleotides. In a further embodiment, the third region is between 3 and 20, 5 and 20, 10 and 20 or 15 and 20 nucleotides in length. In another embodiment, the third region is between 1 and several thousand nucleotides in length, between 1 and thousands of nucleotides in length, between 1 to 10000 nucleotides in length, between 1 to 9 000 nucleotides in length, between 1 and 8000 nucleotides in length, between 1 and 7000 nucleotides in length, between 1 and 6000 nucleotides in length, between 1 and 5000 nucleotides in length, between 1 and 4000 nucleotides in length, between 1 and 3000 nucleotides in length, between 1 and 2000 nucleotides in length, or between 1 and 1000 nucleotides in length. In an embodiment, the third region is between 1 and 900 nucleotides in length, between 1 and 800 nucleotides in length, between 1 and 700 nucleotides in length, between 1 and 600 nucleotides in length, between 1 and 500 nucleotides in length, between 1 and 400 nucleotides in length, between 1 and 300 nucleotides in length, between 1 and 200 nucleotides in length, between 1 and 100 nucleotides in length. In an embodiment, the third region is 1 to 5 nucleotides in length, or 1 to 3 nucleotides in length. In an embodiment, the third region is between 30 and 9000 nucleotides in length. In an embodiment, the third region is between 100 and 1500 nucleotides in length. In an embodiment, the antisense IncRNA comprises a first region, a second region, and a third region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 20 and 3000 nucleotides, wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and 5000 nucleotides, and wherein the third region is 5' of the first region, wherein the third region is not complementary to the mRNAand wherein the third region has a length of between 1 and 5000 nucleotides. Preferably, the third region is between 1 and thousands of nucleotides in length. Without being bound by theory, based on the invention, the formation of a partial double strand complex between the antisense IncRNA and target mRNA results in decreased nuclear export of the target mRNA, thereby decreasing expression of the target mRNA; thus the antisense IncRNA is termed a "Block-R/VA".

[00074] Identification of antisense long non-coding RNAs

[00075] In another aspect, the invention relates to the identification of the antisense IncRNAs. Identification of boost RNAs (e.g. asRNAs, asIncRNA, or asncRNAs) of the invention comprises three main steps: 1.) Isolation of double-stranded RNA (dsRNA) 2.) Strand specific RNA-sequencing 3.) Annotation.

[00076] Isolgtion of dsRNA

[00077] dsRNAs can be isolated by techniques known to the person skilled in the art. In one embodiment, dsRNAs can be isolated by purification over a cellulose matrix. In one embodiment, dsRNAs can be isolated by using an antibody that specifically detects dsRNA, termed J2. In one embodiment, the dsRNA is isolated by J2-RIP (RNA co-immunoprecipitation).

[00078] Briefly and without being bound by theory, dsRNA isolation by J2-RIP is performed as followed. RNA is isolated with Trizol. After the first precipitation a DNasel treatment is conducted followed by a second precipitation overnight. The obtained RNA is eluted in RNase free water. 90 pg of RNA and 1-5 pg of J2-antibody, preferably 3 pg of J2-antibody, preferably the J2-antibody is purchased from SCICONS, are then incubated in 500 pl PBST (lx PBS, 0.5 % Tween-20) for 120 min at 4 °C. After the first incubation the RNA is transferred to prewashed G-sepharose beads and incubated for another 120 min at 4 °C. The beads are centrifuged down for 1 min at 4 krpm and 4 °C. Then supernatant is again cleared with the J2-antibody and the same procedure resulting in the unbound fraction (UB). The beads are washed five times with PBST. Finally, the RNA is purified from the UB and the eluates via Trizol-chloroform (Ambion® RNA by Life technologies™) extraction and forwarded for RNA-sequencing.

[00079] Strond Specific RNA-seguencing

[00080] Strand specific RNA-sequencing can be performed by techniques known to the person skilled in the art. In one embodiment, the strand specific RNA-sequencing kitTruSeqTM from Illumina is used. Samples are prepared with the "TruSeq RNA Sample Prep Kit v2" according to the manufacturer's protocol (Illumina). This kit allows the strand specific sequencing through RNA- specific primers, necessary to determine sense or antisense strands. In one embodiment, single read (50 bp) sequencing is conducted using a HiSeq 4000 (Illumina). Fluorescence images are transformed to BCL files with the Illumina BaseCaller software and samples are demultiplexed to FASTQ files with bcl2fastq (version 2.17). Sequences are aligned to a genome reference sequence using the STAR software ([Dobin, 2013 #3378]; version 2.5) allowing for 2 mismatches. Genome reference sequences can be derived from NONCODE v.6, or GENCODE v.38. Subsequently, abundance measurement of reads overlapping with exons or introns is conducted with featurecounts ([Liao, 2014 #3379], subread version 1.5.0-pl, Ensembl (EF4.68) supplemented with the coordinates of UTRs, CUTs and SUTs [Granovskaia, 2010 #4425;Xu, 2009 #4424;Yassour, 2010 #4426] and Xrnl-sensitive unstable transcripts [Tuck, 2013 #3110;van Dijk, 2011 #3793], Data is processed in the R/Bioconductor environment (www.bioconductor.org, R version 3.6.1) using the DESeq2 package ([Love, 1998 #631]; version 1.24.0).

[00081] Annotation

[00082] RNA-sequencing analysis can be performed by techniques known to the person skilled in the art. In one embodiment, overlapping features respectively sense and antisense pairs are identified with BEDTools intersect [Quinlan, 2010 #4427] requiring overlaps to occur on the opposite strand with a minimum overlap of 0.5. In one embodiment, overlapping features respectively sense and antisense pairs are identified with BEDTools intersect [Quinlan, 2010 #4427] requiring overlaps to occur on the opposite strand with a minimum overlap of 0.3.

[00083] Through the approach as described above, strand-specific analysis of RNA-sequencing using strand specific RNA-sequencing kits, such as TruSeqTM from Illumina, can be used to gain a comprehensive view on all existing boost RNAs in human cells. Through this approach, systematic identification and annotation of all existing boost-RNAs expressed in human cells is possible, also under different growth conditions or in different diseases.

[00084] Biological Complex

[00085] In another aspect, the present invention relates to a biological complex comprising an antisense IncRNA as described herein and a target mRNA, wherein the IncRNA and target mRNA form a partial double strand complex.

[00086] The terms "polynucleotide", "nucleotide sequence", "nucleic acid" and "oligonucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides (DNA) or ribonucleotides (RNA), or analogs thereof. Polynucleotides may have any three dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, exons, introns, messenger RNA (mRNA), non-coding RNA (ncRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), antisense RNA (asRNA), long non-coding RNA (IncRNA), antisense non-coding RNA (asncRNA), antisense long non-coding RNA (asIncRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence. A polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs.

[00087] As used herein, an "antisense RNA (asRNA)", or "antisense non-coding RNA (asncRNA)", or "antisense long non-coding RNA (asIncRNA)" refers to an antisense transcript that is a single stranded RNA that is complementary to another nucleic acid. A non-limiting example of the nucleic acid that an asRNA, asncRNA, or asIncRNA can be complementary to is mRNA. Furthermore, in another non-limiting example, the complementarity between the asRNA, asncRNA, or asIncRNA and mRNA (target mRNA) is partial complementary, and the asRNA, asncRNA, or asIncRNA and target mRNA form a partial double strand complex.

[00088] The term “long non-coding RNA (IncRNA)" generally refers to a class of RNA molecules comprising more than 200 nucleotides, which do not encode proteins. However, as used herein, an "antisense long non-coding RNA (asIncRNA)" may also refer to an RNA molecule that is at least 30 nucleotides in length, at least 50 nucleotides, at least 100 nucleotides, at least 150 nucleotides, or at least 200 nucleotides.

[00089] As used herein, "complementary" or "complementarity" refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick base pairing or other non-traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). As used herein, "perfectly complementary" means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. As used herein, "partial complementary" refers to a degree of complementarity that is at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%, over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300 or up to thousands of nucleotides.

[00090] As used herein, "partial double strand complex", "partial double stranded complex", or "partial RNA double strand" refers to a double stranded nucleic acid complex formed by two nucleic acids that have partial complementary and are thus not perfectly complementary. In a partial double strand complex, the two nucleic acids are not completely overlapping, and thus parts of the nucleic acids overlap, while other parts of the nucleic acids form overhangs. The overhangs present in a partial double strand complex can be 3' overhangs and/or 5' overhangs, and both or only one of the nucleic acids in the partial double strand complex can have 3' and/or 5' overhangs. A non-limiting example of the types of nucleic acids that can form a partial double strand complex are an antisense IncRNA with mRNA.

[00091] As used herein, the term "boost-RNA" refers to an antisense long-noncoding RNA (asIncRNA), asRNA, or asncRNA that is capable of forming a partial double-strand complex with a target mRNA and increasing nuclear export of the target mRNA via the partial double strand complex that is formed.

[00092] As used herein, the term "block-RNA" refers to an antisense long-noncoding RNA (asIncRNA), asRNA, or asncRNA that is capable of forming a partial double-strand complex with a target mRNA and decreasing or inhibiting nuclear export of the target mRNA via the partial double strand complex that is formed. Compared to the boost-RNA, the block-RNA has an additional third region, which is 5' of the first region. The block-RNA thus forms two overhangs in the double-strand complex, one on each side of the complementary first region.

[00093] In one embodiment, the antisense IncRNA is at most 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% complementary to the target mRNA. In an embodiment, the formed partial double strand complex is at most 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% overlapping. In an embodiment, the formed partial double strand complex comprises 3' overhangs and/or 5' overhangs. In an embodiment, the formed partial double strand complex comprises 3' overhangs. In an embodiment, the formed partial double strand complex comprises 5' overhangs. In an embodiment, the formed partial double strand complex comprises 3' overhangs and 5' overhangs. In an embodiment, the 3' overhangs are from the antisense IncRNA. In an embodiment, the 5' overhangs are from the antisense IncRNA. In an embodiment, the 3' overhangs are from the target mRNA. In an embodiment, the 5' overhangs are from the target mRNA. In an embodiment, the 3' overhangs are at least 1 nucleotide long, 2 nucleotides long, 3 nucleotides long, 4 nucleotides long, 5 nucleotides long, 10 nucleotides long, 15 nucleotides long, 20 nucleotides long, 25 nucleotides long, or 30 nucleotides long. In an embodiment, the 3' overhangs are more than 30 nucleotides long. In an embodiment, the 3' overhangs are 50 nucleotides long. In an embodiment, the 3' overhangs are between 1 and thousands of nucleotides long. In an embodiment, the 3' overhangs are 10-5000 nucleotides long. In an embodiment, the 3' overhangs are 10-4000 nucleotides long. In an embodiment, the 3' overhangs are 10-3000 nucleotides long. In an embodiment, the 3' overhangs are 10-2000 nucleotides long. In an embodiment, the 3' overhangs are 10-1000 nucleotides long. In an embodiment, the 3' overhangs are 10-900 nucleotides long. In an embodiment, the 3' overhangs are 10-800 nucleotides long. In an embodiment, the 3' overhangs are 10-700 nucleotides long. In an embodiment, the 3' overhangs are 10-600 nucleotides long. In an embodiment, the 3' overhangs are 10-500 nucleotides long. In an embodiment, the 3' overhangs are 10-400 nucleotides long. In an embodiment, the 3' overhangs are 10-300 nucleotides long. In an embodiment, the 3' overhangs are 10-200 nucleotides long. In an embodiment, the 3' overhangs are 10-100 nucleotides long. In an embodiment, the 3' overhangs are 1-50 nucleotides long. In an embodiment, the 5' overhangs are at least 1 nucleotide long, 2 nucleotides long, 3 nucleotides long, 4 nucleotides long, 5 nucleotides long, 10 nucleotides long, 15 nucleotides long, 20 nucleotides long, 25 nucleotides long, or 30 nucleotides long. In an embodiment, the 5' overhangs are more than 30 nucleotides long. In an embodiment, the 5' overhangs are 50 nucleotides long. In an embodiment, the 5' overhangs are between 1 and thousands of nucleotides long. In an embodiment, the 5' overhangs are 10-5000 nucleotides long. In an embodiment, the 5' overhangs are 10-4000 nucleotides long. In an embodiment, the 5' overhangs are 10-3000 nucleotides long. In an embodiment, the 5' overhangs are 10-2000 nucleotides long. In an embodiment, the 5' overhangs are 10-1000 nucleotides long. In an embodiment, the 5' overhangs are 10-900 nucleotides long. In an embodiment, the 5' overhangs are 10-800 nucleotides long. In an embodiment, the 5' overhangs are 10-700 nucleotides long. In an embodiment, the 5' overhangs are 10-600 nucleotides long. In an embodiment, the 5' overhangs are 10-500 nucleotides long. In an embodiment, the 5' overhangs are 10-400 nucleotides long. In an embodiment, the 5' overhangs are 10-300 nucleotides long. In an embodiment, the 5' overhangs are 10-200 nucleotides long. In an embodiment, the 5' overhangs are 10-100 nucleotides long. In an embodiment, the 5' overhangs are 1-50 nucleotides long. In another embodiment, the biological complex is optimized by binding double-strand formation mediating proteins and/or binding export mediating proteins. In an embodiment, the biological complex further comprises one or more double-strand formation mediating proteins and/or one or more export mediating proteins. In an embodiment, the double-strand formation mediating proteins and export mediating proteins are Dbp2, Yral, Mex67, and/or Mtr2. In an embodiment, the double-strand formation mediating protein is Dbp2. In an embodiment, the double-strand formation mediating protein is Yral. In an embodiment, the export mediating protein is Mex67. In an embodiment, the export mediating protein is Mtr2. In an embodiment, the double-strand formation mediating proteins and export mediating proteins are DDX5, ALY/REV/NXF2, TAP, and/or pl5. In an embodiment, the double-strand formation mediating protein is DDX5. In an embodiment, the double-strand formation mediating protein is ALY/REV/NXF2. In an embodiment, the export mediating protein is TAP. In an embodiment, the export mediating protein is pl5.

[00094] "Boosting" Biological Complex

[00095] In another aspect, the present invention relates to a biological complex comprising an antisense IncRNA (boost-RNA) as described herein and a target mRNA, wherein the IncRNA and mRNA form a partial double strand complex, wherein the second region of the antisense IncRNA is between 1 and several thousands of nucleotides in length. In an embodiment, the second region is 1 to 5 nucleotides in length, or 1 to 3 nucleotides in length. In another embodiment, the second region is between 1 and thousands of nucleotides in length, between 1 to 10 000 nucleotides in length, between 1 to 9 000 nucleotides in length, between 1 and 8000 nucleotides in length, between 1 and 7000 nucleotides in length, between 1 and 6000 nucleotides in length, between 1 and 5000 nucleotides in length, between 1 and 4000 nucleotides in length, between 1 and 3000 nucleotides in length, between 1 and 2000 nucleotides in length, or between 1 and 1000 nucleotides in length. In an embodiment, the second region is between 1 and 900 nucleotides in length, between 1 and 800 nucleotides in length, between 1 and 700 nucleotides in length, between 1 and 600 nucleotides in length, between 1 and 500 nucleotides in length, between 1 and 400 nucleotides in length, between 1 and 300 nucleotides in length, between 1 and 200 nucleotides in length, between 1 and 100 nucleotides in length. In an embodiment, the second region is 1 to 5 nucleotides in length, or 1 to 3 nucleotides in length. In an embodiment, the second region is between 30 and 9000 nucleotides in length. In an embodiment, the second region is between 100 and 1500 nucleotides in length. Preferably, the second region is between 1 and thousands of nucleotides in length. In an embodiment, the antisense IncRNA comprises a first region and a second region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 20 and 3000 nucleotides, and wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and 5000 nucleotides. In an embodiment, the antisense IncRNA is at most 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% complementary to the target mRNA. In an embodiment, the formed partial double strand complex is at most 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% overlapping. In an embodiment, the formed partial double strand complex comprises 3' overhangs. In an embodiment, the formed partial double strand complex comprises a 5' overhang of the target mRNA. In an embodiment, the 3' overhangs are from the antisense IncRNA. In an embodiment, the 3' overhangs are from the target mRNA. In an embodiment, the 5' overhangs are from the target mRNA. In an embodiment, the 3' overhangs are at least 1 nucleotide long, 2 nucleotides long, 3 nucleotides long, 4 nucleotides long, 5 nucleotides long, 10 nucleotides long, 15 nucleotides long, 20 nucleotides long, 25 nucleotides long, or 30 nucleotides long. In an embodiment, the 3' overhangs are more than 30 nucleotides long. In another embodiment, the 3' overhang is between 1 and several thousand nucleotides in length, between 1 and thousands of nucleotides in length, between 1 to 10000 nucleotides in length, between 1 to 9 000 nucleotides in length, between 1 and 8000 nucleotides in length, between 1 and 7000 nucleotides in length, between 1 and 6000 nucleotides in length, between 1 and 5000 nucleotides in length, between 1 and 4000 nucleotides in length, between 1 and 3000 nucleotides in length, between 1 and 2000 nucleotides in length, or between 1 and 1000 nucleotides in length. In an embodiment, the 3' overhang is between 1 and 900 nucleotides in length, between 1 and 800 nucleotides in length, between 1 and 700 nucleotides in length, between 1 and 600 nucleotides in length, between 1 and 500 nucleotides in length, between 1 and 400 nucleotides in length, between 1 and 300 nucleotides in length, between 1 and 200 nucleotides in length, between 1 and 100 nucleotides in length. In an embodiment, the 3' overhang is 1 to 5 nucleotides in length, or 1 to 3 nucleotides in length. In an embodiment, the 3' overhang is between 30 and 9000 nucleotides in length. In an embodiment, the 3' overhang is between 100 and 1500 nucleotides in length. Preferably, the 3' overhang is between 1 and thousands of nucleotides in length. In an embodiment, the 5' overhang of the mRNA is at least 1 nucleotide long, 2 nucleotides long, 3 nucleotides long, 4 nucleotides long, 5 nucleotides long, 10 nucleotides long, 15 nucleotides long, 20 nucleotides long, 25 nucleotides long, or 30 nucleotides long. In an embodiment, the 5' overhang of the mRNA is more than 30 nucleotides long. In another embodiment, the 5' overhang of the mRNA is between 1 and several thousand nucleotides in length, between 1 and thousands of nucleotides in length, between 1 to 10000 nucleotides in length, between 1 to 9 000 nucleotides in length, between 1 and 8000 nucleotides in length, between 1 and 7000 nucleotides in length, between 1 and 6000 nucleotides in length, between 1 and 5000 nucleotides in length, between 1 and 4000 nucleotides in length, between 1 and 3000 nucleotides in length, between 1 and 2000 nucleotides in length, or between 1 and 1000 nucleotides in length. In an embodiment, the 5' overhang is between 1 and 900 nucleotides in length, between 1 and 800 nucleotides in length, between 1 and 700 nucleotides in length, between 1 and 600 nucleotides in length, between 1 and 500 nucleotides in length, between 1 and 400 nucleotides in length, between 1 and 300 nucleotides in length, between 1 and 200 nucleotides in length, between 1 and 100 nucleotides in length. In an embodiment, the 5' overhang of the mRNA is between 30 and 9000 nucleotides in length. In an embodiment, the 5' overhang of the mRNA is between 100 and 1500 nucleotides in length. Preferably, the 5' overhang of the mRNA is between 1 and thousands of nucleotides in length. In another embodiment, the biological complex is optimized for binding double-strand formation mediating proteins and/or binding export mediating proteins. In an embodiment, the biological complex further comprises one or more double-strand formation mediating proteins and/or one or more export mediating proteins. In an embodiment, the double-strand formation mediating proteins and export mediating proteins are Dbp2, Yral, Mex67, and/or Mtr2. In an embodiment, the doublestrand formation mediating protein is Dbp2. In an embodiment, the double-strand formation mediating protein is Yral. In an embodiment, the export mediating protein is Mex67. In an embodiment, the export mediating protein is Mtr2. In an embodiment, the double-strand formation mediating proteins and export mediating proteins are DDX5, ALY/REV/NXF2, TAP, and/or pl5. In an embodiment, the double-strand formation mediating protein is DDX5. In an embodiment, the doublestrand formation mediating protein is DDX17. In an other embodiment, the double-strand formation mediating protein is a helicase. In an embodiment, the double-strand formation mediating protein is ALY/REV/NXF2. In an embodiment, the export mediating protein is TAP. In an embodiment, the export mediating protein is pl5.

[00096] "Blocking" Biological Complex

[00097] In another aspect, the present invention relates to a biological complex comprising an antisense IncRNA (block-RNA) as described herein and the target mRNA, wherein the IncRNA and mRNA form a partial double strand complex, wherein the antisense IncRNA comprises a first region that is complementary to the target mRNA, a second region that is 3' of the first region and which is not complementary to the target mRNA, and a third region that is 5' of the first region and which is not complementary to the target mRNA (i.e. the biological complex comprises a block-RNA). In another embodiment, the second region is between 1 and several thousand nucleotides in length, between 1 and thousands of nucleotides in length, between 1 to 10 000 nucleotides in length, between 1 to 9000 nucleotides in length, between 1 and 8000 nucleotides in length, between 1 and 7000 nucleotides in length, between 1 and 6000 nucleotides in length, between 1 and 5000 nucleotides in length, between 1 and 4000 nucleotides in length, between 1 and 3000 nucleotides in length, between 1 and 2000 nucleotides in length, or between 1 and 1000 nucleotides in length. In an embodiment, the second region is between 1 and 900 nucleotides in length, between 1 and 800 nucleotides in length, between 1 and 700 nucleotides in length, between 1 and 600 nucleotides in length, between 1 and 500 nucleotides in length, between 1 and 400 nucleotides in length, between 1 and 300 nucleotides in length, between 1 and 200 nucleotides in length, between 1 and 100 nucleotides in length. In an embodiment, the second region is 1 to 5 nucleotides in length, or 1 to 3 nucleotides in length. In an embodiment, the second region is between 30 and 9000 nucleotides in length. In an embodiment, the second region is between 100 and 1500 nucleotides in length. Preferably, the second region is between 1 and thousands of nucleotides in length. In another embodiment, the third region comprises at least 3 nucleotides, at least 5 nucleotides, at least 10 nucleotides, at least 15 nucleotides, or at least 20 nucleotides. In an embodiment, the third region comprises at least 20 nucleotides. In a further embodiment, the third region is between 3 and 20, 5 and 20, 10 and 20 or 15 and 20 nucleotides in length. In another embodiment, the third region is between 1 and several thousand nucleotides in length, between 1 and thousands of nucleotides in length, between 1 to 10000 nucleotides in length, between 1 to 9000 nucleotides in length, between 1 and 8000 nucleotides in length, between 1 and 7000 nucleotides in length, between 1 and 6000 nucleotides in length, between 1 and 5000 nucleotides in length, between 1 and 4000 nucleotides in length, between 1 and 3000 nucleotides in length, between 1 and 2000 nucleotides in length, or between 1 and 1000 nucleotides in length. In an embodiment, the third region is between 1 and 900 nucleotides in length, between 1 and 800 nucleotides in length, between 1 and 700 nucleotides in length, between 1 and 600 nucleotides in length, between 1 and 500 nucleotides in length, between 1 and 400 nucleotides in length, between 1 and 300 nucleotides in length, between 1 and 200 nucleotides in length, between 1 and 100 nucleotides in length. In an embodiment, the third region is 1 to 5 nucleotides in length, or 1 to 3 nucleotides in length. In an embodiment, the third region is between 30 and 9000 nucleotides in length. In an embodiment, the third region is between 100 and 1500 nucleotides in length. Preferably, the third region is between 1 and thousands of nucleotides in length. In an embodiment, the antisense IncRNA comprises a first region, a second region, and a third region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 20 and 3000 nucleotides, wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and 5000 nucleotides, and wherein the third region is 5' of the first region, wherein the third region is not complementary to the mRNA and wherein the third region has a length of between

1 and 5000 nucleotides. In an embodiment, the antisense IncRNA is at most 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% complementary to the target mRNA. In an embodiment, the formed partial double strand complex is at most 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% overlapping. In an embodiment, the formed partial double strand complex comprises 3' overhangs and/or 5' overhangs. In an embodiment, the formed partial double strand complex comprises 3' overhangs. In an embodiment, the formed partial double strand complex comprises 5' overhangs. In an embodiment, the formed partial double strand complex comprises 3' overhangs and 5' overhangs. In an embodiment, the 3' overhangs are from the antisense IncRNA. In an embodiment, the 5' overhangs are from the antisense IncRNA. In an embodiment, the 3' overhangs are from the target mRNA. In an embodiment, the 5' overhangs are from the target mRNA. In an embodiment, the 3' overhangs are at least 1 nucleotide long, 2 nucleotides long, 3 nucleotides long, 4 nucleotides long, 5 nucleotides long, 10 nucleotides long, 15 nucleotides long, 20 nucleotides long, 25 nucleotides long, or 30 nucleotides long. In an embodiment, the 3' overhangs are more than 30 nucleotides long. In an embodiment, the 5' overhangs are at least 1 nucleotide long,

2 nucleotides long, 3 nucleotides long, 4 nucleotides long, 5 nucleotides long, 10 nucleotides long, 15 nucleotides long, 20 nucleotides long, 25 nucleotides long, or 30 nucleotides long. In an embodiment, the 5' overhangs are more than 30 nucleotides long. In another embodiment, the 5' overhang is between 1 and several thousand nucleotides in length, between 1 and thousands of nucleotides in length, between 1 to 10000 nucleotides in length, between 1 to 9 000 nucleotides in length, between 1 and 8000 nucleotides in length, between 1 and 7000 nucleotides in length, between 1 and 6000 nucleotides in length, between 1 and 5000 nucleotides in length, between 1 and 4000 nucleotides in length, between 1 and 3000 nucleotides in length, between 1 and 2000 nucleotides in length, or between 1 and 1000 nucleotides in length. In an embodiment, the 5' overhang is between 1 and 900 nucleotides in length, between 1 and 800 nucleotides in length, between 1 and 700 nucleotides in length, between 1 and 600 nucleotides in length, between 1 and 500 nucleotides in length, between 1 and 400 nucleotides in length, between 1 and 300 nucleotides in length, between 1 and 200 nucleotides in length, between 1 and 100 nucleotides in length. In an embodiment, the 5' overhang is 1 to 5 nucleotides in length, or 1 to 3 nucleotides in length. In an embodiment, the 5' overhang is between 30 and 9000 nucleotides in length. In an embodiment, the 5' overhang is between 100 and 1500 nucleotides in length. Preferably, the 5' overhang is between 1 and thousands of nucleotides in length.

[00098] Expression Vectors

[00099] In another aspect, the present invention relates to an expression vector comprising a nucleic acid sequence encoding an antisense IncRNA as described herein.

[000100] As used herein, a "vector" is a tool that allows or facilitates the transfer of an entity from one environment to another. It is a replicon, such as a plasmid, phage, or cosmid, into which another DNA or RNA (coding and/or non-coding RNA) segment may be inserted so as to bring about the replication of the inserted segment. Generally, a vector is capable of replication when associated with the proper control elements. In general, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially doublestranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular); nucleic acid molecules that comprise DNA, RNA (coding and/or non-coding RNA), or both; and other varieties of polynucleotides known in the art. One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA or RNA (coding and/or non coding) segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, wherein virally-derived DNA or RNA (coding and/or non-coding) sequences are present in the vector for packaging into a virus (e.g. retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses (AAVs)). One example of a viral vector is a lentivirus vector (or also referred to as a lentiviral vector). Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non- episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes or the transcription of non-coding RNA to which they are operatively-linked. Vectors are referred to herein as "expression vectors".

[000101] In an embodiment, the expression vector further comprises a nucleic acid sequence encoding a target mRNA of the IncRNA.

[000102] In an embodiment, the present invention relates to an expression vector comprising a nucleic acid sequence encoding an antisense IncRNA as described herein, having a first region and a second region (i.e. an expression vector encoding a boost-RNA). In an embodiment, the second region of the antisense IncRNA is 1 to 10 nucleotides in length, 1 to 5 nucleotides in length, or 1 to 3 nucleotides in length, or wherein the second region is between 1 and several thousand nucleotides in length, between 1 and thousands of nucleotides in length, between 1 to 10 000 nucleotides in length, between 1 to 9 000 nucleotides in length, between 1 and 8000 nucleotides in length, between 1 and 7000 nucleotides in length, between 1 and 6000 nucleotides in length, between 1 and 5000 nucleotides in length, between 1 and 4000 nucleotides in length, between 1 and 3000 nucleotides in length, between 1 and 2000 nucleotides in length, or between 1 and 1000 nucleotides in length. In an embodiment, the second region is between 1 and 900 nucleotides in length, between 1 and 800 nucleotides in length, between 1 and 700 nucleotides in length, between 1 and 600 nucleotides in length, between 1 and 500 nucleotides in length, between 1 and 400 nucleotides in length, between 1 and 300 nucleotides in length, between 1 and 200 nucleotides in length, between 1 and 100 nucleotides in length.. In an embodiment, the second region of the asRNA is between 30 and 9000 nucleotides in length. In an embodiment, the second region is between 100 and 1500 nucleotides in length. Preferably, the second region is between 1 and thousands of nucleotides in length. In an embodiment, the antisense IncRNA comprises a first region and a second region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 20 and 3000 nucleotides, and wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and 5000 nucleotides. In another embodiment, the present invention relates to an expression vector comprising a nucleic acid sequence encoding an antisense IncRNA as described herein having a second region and a third region (i.e. an expression vector encoding a block-RNA). In an embodiment, the second region of the IncRNA is more than 10 nucleotides in length, more than 15 nucleotides in length, more than 20 nucleotides in length, more than 25 nucleotides in length, or more than 30 nucleotides in length, 1 to 10 nucleotides in length, 1 to 5 nucleotides in length, or 1 to 3 nucleotides in length, or wherein the second region is between 1 and several thousand nucleotides in length, between 1 and thousands of nucleotides in length, between 1 to 10 000 nucleotides in length, between 1 to 9 000 nucleotides in length, between 1 and 8000 nucleotides in length, between 1 and 7000 nucleotides in length, between 1 and 6000 nucleotides in length, between 1 and 5000 nucleotides in length, between 1 and 4000 nucleotides in length, between 1 and 3000 nucleotides in length, between 1 and 2000 nucleotides in length, or between 1 and 1000 nucleotides in length. In an embodiment, the second region is between 1 and 900 nucleotides in length, between 1 and 800 nucleotides in length, between 1 and 700 nucleotides in length, between 1 and 600 nucleotides in length, between 1 and 500 nucleotides in length, between 1 and 400 nucleotides in length, between 1 and 300 nucleotides in length, between 1 and 200 nucleotides in length, between 1 and 100 nucleotides in length.. Additionally, the IncRNA encoded by the expression vector comprises a third region, wherein the third region of the IncRNA is least 3 nucleotides, at least 5 nucleotides, at least 10 nucleotides, at least 15 nucleotides, or at least 20 nucleotides, or the third region comprises at least 20 nucleotides. In a further embodiment, the third region is between 3 and 20, 5 and 20, 10 and 20 or 15 and 20 nucleotides in length. In another embodiment, the third region is between 1 and several thousand nucleotides in length, between 1 and thousands of nucleotides in length, between 1 to 10 000 nucleotides in length, between 1 to 9 000 nucleotides in length, between 1 and 8000 nucleotides in length, between 1 and 7000 nucleotides in length, between 1 and 6000 nucleotides in length, between 1 and 5000 nucleotides in length, between 1 and 4000 nucleotides in length, between 1 and 3000 nucleotides in length, between 1 and 2000 nucleotides in length, or between 1 and 1000 nucleotides in length. In an embodiment, the third region is between 1 and 900 nucleotides in length, between 1 and 800 nucleotides in length, between 1 and 700 nucleotides in length, between 1 and 600 nucleotides in length, between 1 and 500 nucleotides in length, between 1 and 400 nucleotides in length, between 1 and 300 nucleotides in length, between 1 and 200 nucleotides in length, between 1 and 100 nucleotides in length. In an embodiment, the third region is 1 to 5 nucleotides in length, or 1 to 3 nucleotides in length. In an embodiment, the third region is between 30 and 9000 nucleotides in length. In an embodiment, the third region is between 100 and 1500 nucleotides in length. Preferably, the third region is between 1 and thousands of nucleotides in length. In an embodiment, the third region comprises at least 3 nucleotides. In an embodiment, the third region comprises at least 5 nucleotides. In an embodiment, the third region comprises at least 10 nucleotides. In an embodiment, the third region comprises at least 15 nucleotides. In an embodiment, the third region comprises at least 20 nucleotides. In an embodiment, the third region comprises between 1 and thousands of nucleotides. In an embodiment, the antisense IncRNA comprises a first region, a second region, and a third region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 20 and 3000 nucleotides, wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and 5000 nucleotides, and wherein the third region is 5' of the first region, wherein the third region is not complementary to the mRNA and wherein the third region has a length of between 1 and 5000 nucleotides.

[000103] In another embodiment, the present invention relates to an expression vector comprising a nucleic acid sequence encoding an antisense IncRNA as described herein, wherein the antisense IncRNA comprises a second region that is 3' of the first region and which is not complementary to the target mRNA and a third region that is 5' of the first region and which is not complementary to the target mRNA. In an embodiment, the third region comprises at least 3 nucleotides, at least 5 nucleotides, at least 10 nucleotides, at least 15 nucleotides, or at least 20 nucleotides. In an embodiment, the third region comprises at least 5 nucleotides. In an embodiment, the third region comprises at least 10 nucleotides. In an embodiment, the third region comprises at least 15 nucleotides. In an embodiment, the third region comprises at least 20 nucleotides. In an embodiment, the antisense IncRNA comprises a first region, a second region, and a third region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 20 and 3000 nucleotides, wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and 5000 nucleotides, and wherein the third region is 5' of the first region, wherein the third region is not complementary to the mRNA and wherein the third region has a length of between 1 and 5000 nucleotides.

[000104] The following embodiments relate to the expression vector aspect of the invention and all the embodiments as described above.

[000105] In one embodiment, the expression vector comprises a nucleic acid sequence encoding one or more antisense IncRNAs as described herein. In one embodiment, the expression vector comprises a nucleic acid sequence encoding 2, 3, or 4 antisense IncRNAs as described herein. In one embodiment, the expression vector comprises a nucleic acid sequence encoding one or more antisense IncRNAs as described herein, wherein each nucleic acid sequence encodes the same IncRNA. In one embodiment, the expression vector comprises a nucleic acid sequence encoding one or more antisense IncRNAs as described herein, wherein each nucleic acid sequence encodes a different IncRNA. In one embodiment, the expression vector further comprises a nucleic acid sequence encoding a target mRNA of the antisense IncRNA. In one embodiment, when the expression vector comprises more than one nucleic acid sequence encoding more than one antisense IncRNA as described herein, the expression vector further comprises at least one nucleic acid sequence encoding a target mRNA of at least one of the antisense IncRNAs. In one embodiment, when the expression vector comprises 2, 3, or 4 nucleic acid sequences encoding antisense IncRNAs as described herein, the expression vector further comprises the same number of nucleic acid sequences wherein each of the nucleic acid sequences encodes a target mRNA for at least one of the 1, 3, or 4 antisense IncRNAs.

[000106] The term "regulatory element" or "regulatory seguence" is intended to include promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g. transcription termination signals, such as polyadenylation signals and poly-U sequences), which as understood by the person skilled in the art, are components of an expression vector. Such regulatory elements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissuespecific regulatory sequences). A tissue-specific promoter may direct expression primarily in a desired tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e.g. liver, pancreas), or particular cell types (e.g. lymphocytes). Regulatory elements may also direct expression in a temporal-dependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific. Encompassed by the term "regulatory element" are promoters. A vector can comprise one or more pol III promoter (e.g. 1, 2,3,4, 5, or more pol III promoters), one or more pol II promoters (e.g. 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g. 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof. Examples of pol III promoters include, but are not limited to, U6 and Hl promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985 )], the SV40 promoter, the dihydrofolate reductase promoter, the p-actin promoter, the phosphoglycerol kinase, (PGK) promoter, and the EFla promoter. In an embodiment, the promoter can be a RNA polymerase I promoter. In an embodiment, the promoter can be a RNA polymerase II promoter. In an embodiment, the promoter can be a RNA polymerase III promoter. In an embodiment, the promoter can be a bacterial promoter. In an embodiment, the promoter can be a phage T7 promoter. With regards to promoters, mention is made of PCT publication WO 2011/028929 and U.S. application 12/511,940 , the contents of which are incorporated by reference herein in their entirety. Also encompassed by the term "regulatory element" are enhancer elements, such as WPRE; CMV enhancers; the R-U5' segment in LTR of HTLV- 1 ( Mol. Cell. Biol., Vol. 8(1), p. 466-472,1988 ); SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit P-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, etc., and such factors will be known and accounted for by the person skilled in the art. [000107] In one embodiment, the one or more nucleic acid sequences encoding the one or more antisense IncRNAs as described herein, are controlled by the same regulatory elements. In one embodiment, the one or more nucleic acid sequences encoding the one or more antisense IncRNAs as described herein, are each controlled by different regulatory elements. In one embodiment, the one or more nucleic acid sequences encoding the one or more antisense IncRNAs as described herein, and the one or more nucleic acid sequences encoding a target mRNA of the one or more antisense IncRNA(s) are controlled by the same regulatory elements. In one embodiment, the one or more nucleic acid sequences encoding the one or more antisense IncRNAs as described herein, and the one or more nucleic acid sequences encoding a target mRNA of the one or more antisense IncRNA(s) are controlled by different regulatory elements. In one embodiment, the one or more nucleic acid sequences encoding the one or more antisense IncRNAs as described herein, and the one or more nucleic acid sequences encoding a target mRNA of the one or more antisense IncRN A(s) are controlled by different regulatory elements, wherein the mRNA and the antisense RNA at least partially overlap on the expression vector, wherein the different regulatory elements drive the expression of a sense and the antisense RNA, wherein one regulatory element is downstream or upstream of another regulatory element, and wherein the one or more nucleic acid sequences encoding the one or more antisense IncRNAs as described herein, and the one or more nucleic acid sequences encoding a target mRNA of the one or more antisense IncRNA(s) are positioned on opposite strands in between the different regulatory elements. In one embodiment, the one or more nucleic acid sequences encoding the one or more antisense IncRNAs as described herein, and the one or more nucleic acid sequences encoding a target mRNA of the one or more antisense IncRNA(s) are controlled by the same regulatory elements, wherein the regulatory element is a divergent promoter, wherein the divergent promotor is flanked upstream or downstream with the one or more nucleic acid sequences encoding the one or more antisense IncRNAs as described herein and upstream or downstream with the one or more nucleic acid sequences encoding a target mRNA of the one or more antisense IncRNA(s). In one embodiment, the one or more nucleic acid sequences encoding the one or more antisense IncRNAs are all controlled by one set of regulatory elements, and the one or more nucleic acid sequences encoding a target mRNA of the one or more antisense IncRNA(s) are all controlled by another different set of regulatory elements.

[000108] In some embodiments, the expression vector can comprise one or more polymerase III (pol III) promoters (e.g. 1, 2,3,4, 5, or more pol III promoters), one or more polymerase II (pol II) promoters (e.g. 1, 2, 3, 4, 5, or more pol II promoters), one or more polymerase I (pol I) promoters (e.g. 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof. In one embodiment, the expression vector comprises one or more CMV promoters and one or more SV40 promoters. In one embodiment, the expression vector comprises a CMV promoter. In one embodiment, the expression vector comprises one or more CMV promoters. In one embodiment, the expression vector comprises a SV40 promoter. In one embodiment, the expression vector comprises a viral promoter. In one embodiment, the expression vector comprises a human EFl promoter. In one embodiment, the expression vector comprises a DOX promoter. In one embodiment, the expression vector comprises a TET promoter. In one embodiment, the expression vector comprises one or more SV40 promoters. In one embodiment, the expression vector comprises a nucleic acid sequence encoding an antisense IncRNA as described herein and further comprises a nucleic acid sequence encoding a target mRNA of the IncRNA, wherein the expression of the antisense IncRNA and the target mRNA is controlled by the same promoter. In one embodiment, the expression vector comprises a nucleic acid sequence encoding an antisense IncRNA as described herein and further comprises a nucleic acid sequence encoding a target mRNA of the IncRNA, wherein the expression of the antisense IncRNA and target mRNA is controlled by different promoters.

[000109] The term "expression cassette” refers to a distinct component of a vector comprising one or more genes (mRNA) and/or one or more non-coding RNAs, and regulatory sequences or elements. An expression cassette is composed of one or more genes (mRNA) and/or non-coding RNA, and the regulatory elements or sequences controlling their expression. An expression cassette comprises three components: a promoter sequence, an open reading frame, and a 3' untranslated region. As such, an expression cassette can serve to control the expression of the one or more genes (mRNA) and/or the one or more non-coding RNAs within the expression cassette. It will be appreciated by those skilled in the art that the design of an expression casette can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, etc., and such factors will be known and accounted for by the person skilled in the art.

[000110] In one embodiment, the expression vector comprises one or more expression cassettes. In one embodiment, a single expression cassette controls the expression of the one or more nucleic acid sequences encoding the one or more antisense IncRNA(s) as described herein. In one embodiment, a different expression cassette controls the expression of each of the one or more nucleic acid sequences encoding the one or more antisense IncRNAs as described herein. In one embodiment, a single expression cassette controls the expression of the one or more nucleic acid sequences encoding the one or more target mRNA of the one or more antisense IncRNA(s) as described herein. In one embodiment, a different expression cassette controls the expression of each of the one or more nucleic acid sequences encoding the one or more target mRNA of the one or more antisense IncRNA(s) as described herein. In one embodiment, a single expression cassette controls the expression of the one or more nucleic acid sequences encoding the one or more antisense IncRNAs as described herein, and the one or more nucleic acid sequences encoding the one or more target mRNA of the one or more antisense IncRNA. In one embodiment, an expression cassette controls the expression of the one or more nucleic acid sequences encoding the one or more antisense IncRNAs as described herein, and a different expression cassette controls the expression of the one or more nucleic acid sequences encoding the one or more target mRNA of the one or more antisense IncRNA.

[000111] In one embodiment, the expression vector is a viral vector. In one embodiment, the viral vector is a retroviral vector. In one embodiment, the viral vector is a lentiviral vector. In one embodiment, the viral vector is an AAV vector. In one embodiment, the expression vector is a plasmid.

[000112] An expression vector is capable of driving expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include, but are not limited to, pCDM8 ( Seed, 1987. Nature 329: 840 ), pMT2PC ( Kaufman, et al., 1987. EMBO J. 6: 187-195 ), or pcDNA (e.g. pcDNA™3.1⁽⁺⁾ from ThermoFisher Scientific). When used in mammalian cells, the expression vector's control functions are typically provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus (CMV), simian virus 40 (SV40), and others disclosed herein and known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. A mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissues-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements arc known in the art. Non-limiting examples of suitable tissuespecific promoters include the albumin promoter (liver-specific; Pinkert, et al., 1987. Genes Dev. 1: 268-277), lymphoid-specific promoters ( Calame and Eaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Baneiji, et al., 1983. Cell 33: 729-740; Queen and Baltimore, 1983. Cell 33: 741- 748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985. Science 230: 912-91), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, e.g., the murine box promoters (Kessel and Gruss, 1990. Science 249: 374-379) and the a-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546). With regards to prokaryotic and eukaryotic vectors, mention is made of U.S. Patent 6,750,059, the contents of which are incorporated by reference herein in their entirety. Tissue-specific regulatory elements are known in the art and in this regard, mention is made of U.S. Patent 7,776,321, the contents of which are incorporated by reference herein in their entirety. [000113] In one embodiment, the expression vector is a mammalian expression vector. In some embodiments, the mammalian expression vector can comprise one or more pol III promoter (e.g. 1,2, 3,4, 5, or more pol III promoters), one or more pol II promoters (e.g. 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g. 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof. In one embodiment, the mammalian expression vector comprises one or more CMV promoters and one or more SV40 promoters. In one embodiment, the mammalian expression vector comprises a CMV promoter. In one embodiment, the mammalian expression vector comprises a SV40 promoter. In one embodiment, the mammalian expression vector is a pcDNA vector. In one embodiment, the pcDNA vector is pcDNA3.1(+). In one embodiment, the expression vector is a lentiviral expression vector. In one embodiment, the expression vector is pcDNA3. In one embodiment, the expression vector is pcDNA4. In one embodiment, the expression vector is pcDNA5/FRT/TO. In one embodiment, the expression vector is a retroviral expression vector. In one embodiment, the expression vector is pCMV. In one embodiment, the expression vector is pEF. In one embodiment, the expression vector is pSG5. In one embodiment, the expression vector is pMSCV. In one embodiment, the expression vector is a lentiviral expression vector. In one embodiment, the expression vector is pBABE. In one embodiment, the expression vector is pLVTH.

[000114] An expression vector according to the invention can be a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerivisae include, but are not limited to, pYepSecl ( Baldari, et al., 1987. EMBO J. 6: 229-234 ), pMFa ( Kuijan and Herskowitz, 1982. Cell 30: 933-943 ), pJRY88 ( Schultz et al., 1987. Gene 54: 113-123 ), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (Invitrogen Corp, San Diego, Calif.). In some embodiments, the expression vector is a yeast expression vector.

[000115] In one embodiment, the expression vector further comprises one or more nucleic acid sequences encoding double-strand formation mediating genes (mRNA). In one embodiment, the expression vector further comprises one or more nucleic acid sequences encoding export mediating genes (mRNA). In one embodiment, the expression vector further comprises one or more nucleic acid sequences encoding double-strand formation mediating genes (mRNA) and export mediating genes (mRNA). In one embodiment, the expression vector further comprises nucleic acid sequences encoding a Dbp2, Yral, Mex67, and/or Mtr2 gene. In one embodiment, the expression vector further comprises a nucleic acid sequence encoding a Dbp2 gene. In one embodiment, the expression vector further comprises a nucleic acid sequence encoding a Yral gene. In one embodiment, the expression vector further comprises a nucleic acid sequence encoding a Mex67 gene. In one embodiment, the expression vector further comprises a nucleic acid sequence encoding a Mtr2 gene. In one embodiment, the expression vector further comprises nucleic acid sequences encoding a DDX5, ALY/REV/NXF2, TAP, and/or pl5. In one embodiment, the expression vector further comprises a nucleic acid sequence encoding a DDX5 gene. In one embodiment, the expression vector further comprises a nucleic acid sequence encoding a ALY/REV/NXF2 gene. In one embodiment, the expression vector further comprises a nucleic acid sequence encoding a TAP gene. In one embodiment, the expression vector further comprises a nucleic acid sequence encoding a pl5.

[000116] In one embodiment, the one or more nucleic acid sequences encoding the export mediating genes are controlled by the same regulatory elements that control the expression of the nucleotide sequences encoding the antisense IncRNAs described herein. In one embodiment, the one or more nucleic acid sequences encoding the export mediating genes are not controlled by the same regulatory elements that control the expression of the nucleotide sequences encoding the antisense IncRNAs described herein. In one embodiment, the one or more nucleic acid sequences encoding the export mediating genes are controlled by the same regulatory elements that control the expression of the nucleotide sequences encoding the target mRNA of the antisense IncRNAs described herein. In one embodiment, the one or more nucleic acid sequences encoding the export mediating genes are not controlled by the same regulatory elements that control the expression of the nucleotide sequences encoding the target mRNA of the antisense IncRNAs described herein. In one embodiment, the one or more nucleic acid sequences encoding the export mediating genes are controlled by the same regulatory elements that control the expression of the nucleotide sequences encoding the antisense IncRNAs described herein and/or the regulatory elements that control the expression of the nucleotide sequences encoding the target mRNA of the antisense IncRNAs described herein. In one embodiment, the one or more nucleic acid sequences encoding the export mediating genes are not controlled by the same regulatory elements that control the expression of the nucleotide sequences encoding the antisense IncRNAs described herein and/or the regulatory elements that control the expression of the nucleotide sequences encoding the target mRNA of the antisense IncRNAs described herein.

[000117] In one embodiment, the one or more nucleic acid sequences encoding the export mediating genes are controlled by the same expression cassette that controls the expression of the nucleotide sequences encoding the antisense IncRNAs described herein. In one embodiment, the one or more nucleic acid sequences encoding the export mediating genes are not controlled by the same expression cassette that controls the expression of the nucleotide sequences encoding the antisense IncRNAs described herein. In one embodiment, the one or more nucleic acid sequences encoding the export mediating genes are controlled by the same expression cassette that control the expression of the nucleotide sequences encoding the target mRNA of the antisense IncRNAs described herein. In one embodiment, the one or more nucleic acid sequences encoding the export mediating genes are not controlled by the same expression cassette that controls the expression of the nucleotide sequences encoding the target mRNA of the antisense IncRNAs described herein. In one embodiment, the one or more nucleic acid sequences encoding the export mediating genes are controlled by the same expression cassette that controls the expression of the nucleotide sequences encoding the antisense IncRNAs described herein and/or the expression cassette that controls the expression of the nucleotide sequences encoding the target mRNA of the antisense IncRNAs described herein. In one embodiment, the one or more nucleic acid sequences encoding the export mediating genes are not controlled by the same expression cassette that controls the expression of the nucleotide sequences encoding the antisense IncRNAs described herein and/or the expression cassette that controls the expression of the nucleotide sequences encoding the target mRNA of the antisense IncRNAs described herein.

[000118] Use of the antisense long noncoding RNA, Biological Complex, or Expression Vector

[000119] Many biotechnological applications require efficient and maximum gene expression, for example for the production of recombinant proteins (in yeast or human cells). Simultaneous expression of the antisense IncRNA as described herein and the target mRNA will enhance gene expression to optimize protein production. In a non-limiting example, as described herein throughout the disclosure, e.g. plasmids are produced on which a gene expression cassette for the expression of the desired mRNA and a gene cassette for the expression of the boost-RNA is available. After transfection of e.g. a human or yeast target cell with the plasmids, mRNA and its boost-RNA are then co-expressed, form a double stranded RNA complex in the cell nucleus and are preferentially exported, which results in the significant increase of mRNA translation. In this way, gene expression can be optimized and the produced protein will be available in significantly increased amounts.

[000120] Increasing Nuclear Export and Expression of Target mRNA

[000121] Another aspect of the invention is directed to the use of the antisense IncRNA (boost- RNA) as described herein, the biological complex comprising the antisense IncRNA (boost-RNA) as described herein, or the expression vector encoding the antisense IncRNA (boost-RNA) as described herein, for increasing export of the endogenous or exogenous target mRNA from the nucleus of a eukaryotic cell via a partial double strand complex with the IncRNA. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell. In one embodiment, the cell is a mouse cell. In one embodiment, the cell is a rat cell. In one embodiment, the cell is a plant cell. In one embodiment, the cell is a non-mammalian cell. In one embodiment the cell is a yeast cell. In one embodiment the cell is a bacterial cell. In one embodiment, the increased export of the target mRNA from the nucleus is increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% when compared to the nuclear export of the target mRNA without the antisense IncRNA as described herein. In one embodiment, the increased export of the target mRNA from the nucleus is increased by 100%, 125%, 150%, 175%, 200%, 300%, or more when compared to the nuclear export of the target mRNA without the antisense IncRNA as described herein. In one embodiment, the target mRNA is a gene of interest. In one embodiment, the gene of interest is a tumor suppressor gene. In one embodiment the tumor suppressor gene is not a haplo- insufficient. In one embodiment, the tumor suppressor gene is a haplo-insufficient tumor suppressor (hTS) gene. In one embodiment the hTS gene is PTEN, SMAD4, LKB1, CDKN1B, or NMPl. In one embodiment the hTS gene is PTEN. In one embodiment the hTS gene is SMAD4. In one embodiment the hTS gene is LKB1. In one embodiment the hTS gene is CDKN1B. In one embodiment the hTS gene is NMPl. In one embodiment the gene of interest is CFTR.

[000122] Another aspect of the invention is directed to the use of the antisense IncRNA (boost- RNA) as described herein, the biological complex comprising the antisense IncRNA (boost-RNA) as described herein, or the expression vector encoding the antisense IncRNA (boost-RNA) as described herein, for increasing expression of the target mRNA in a eukaryotic cell by increasing the nuclear export of the target mRNA via a partial double strand complex with the IncRNA. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell. In one embodiment the cell is a mouse cell. In one embodiment the cell is a rat cell. In one embodiment, the cell is a plant cell. In one embodiment, the cell is a non-mammalian cell. In one embodiment the cell is a yeast cell. In one embodiment the cell is a bacterial cell. In one embodiment, the increased expression of the target mRNA is increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% when compared to the expression of the target mRNA without the antisense IncRNA as described herein. In one embodiment, the increased expression of the target mRNA is increased by 100%, 125%, 150%, 175%, 200%, 300%, or more when compared to the expression of the target mRNA without the antisense IncRNA as described herein. In one embodiment, the target mRNA is a gene of interest. In one embodiment, the use of the antisense IncRNA (boost-RNA) upregulates and/or restores normal expression of the gene of interest. In one embodiment, the gene of interest is a tumor suppressor gene. In one embodiment the tumor suppressor gene is not haplo-insufficient. In one embodiment, the tumor suppressor gene is a haplo- insufficient tumor suppressor (hTS) gene. In one embodiment the hTS gene is PTEN, SMAD4, LKB1, CDKN1B, or NMPl. In one embodiment the hTS gene is PTEN. In one embodiment the hTS gene is SMAD4. In one embodiment the hTS gene is LKB1. In one embodiment the hTS gene is CDKN1B. In one embodiment the hTS gene is NMPl. In one embodiment the gene of interest is CFTR.

[000123] Decreasing Nuclear Export and Expression of Target mRNA

[000124] Another aspect of the invention relates to the use of antisense IncRNA (block-RNA) as described herein, the biological complex comprising an antisense IncRNA (block-RNA) as described herein, or the expression vector encoding an antisense IncRNA (block-RNA) as described herein, for decreasing export of the target mRNA from the nucleus of a eukaryotic cell via a partial double strand complex with the IncRNA. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell. In one embodiment, the cell is a mouse cell. In one embodiment, the cell is a rat cell. In one embodiment, the cell is a plant cell. In one embodiment, the cell is a non-mammalian cell. In one embodiment the cell is a yeast cell. In one embodiment, the cell is a bacterial cell. In one embodiment, the decreased export of the target mRNA from the nucleus is decreased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% when compared to the nuclear export of the target mRNA without the antisense IncRNA as described herein. In one embodiment, the target mRNA is a gene of interest. In one embodiment, the gene of interest is an oncogene. In one embodiment, the oncogene gene is a is MYC, HER2, RAS, or FOS. In one embodiment the oncogene is MYC. In one embodiment the oncogene is HER2. In one embodiment the oncogene is RAS. In one embodiment the oncogene is FOS.

[000125] Another aspect of the invention relates to the use of antisense IncRNA (block-RNA) as described herein, the biological complex comprising an antisense IncRNA (block-RNA) as described herein, or the expression vector encoding an antisense IncRNA (block-RNA) as described herein, for decreasing expression of the target mRNA in a eukaryotic cell by decreasing the nuclear export of the target mRNA via a partial double strand complex with the IncRNA. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell. In one embodiment, the cell is a mouse cell. In one embodiment, the cell is a rat cell. In one embodiment, the cell is a plant cell. In one embodiment, the cell is a non-mammalian cell. In one embodiment the cell is a yeast cell. In one embodiment, the cell is a bacterial cell. In one embodiment, the decreased expression of the target mRNA is decreased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% when compared to the expression of the target mRNA without the antisense IncRNA as described herein. In one embodiment, the use of the antisense IncRNA (block-RNA) downregulates and/or inhibits the expression of the gene of interest. In one embodiment, the gene of interest is an oncogene. In one embodiment, the oncogene gene is a is MYC, HER2, RAS, or FOS. In one embodiment the oncogene is MYC. In one embodiment the oncogene is HER2. In one embodiment the oncogene is RAS. In one embodiment the oncogene is FOS.

[000126] Methods of Altering Nuclear Export and Expression of the Target mRNA

[000127] Methods for increosing nucleor export ond expression of the torget mRNA

[000128] In one aspect, the present invention relates to a method of increasing export of a target mRNA from the nucleus of a eukaryotic cell, the method comprising transfecting a cell with the antisense IncRNA (boost-RNA), or the biological complex, or the expression vector as described herein. In one embodiment, the method is an in vitro method. In one embodiment, the present invention relates to a method of increasing export of a target mRNA from the nucleus of a eukaryotic cel I, the method comprising transfecting a cell with the antisense IncRNA, or the biological complex, or the expression vector as described herein, wherein the antisense IncRNA is a boost-RNA. In one embodiment, the expression vector further comprises a nucleic acid sequence encoding a target mRNA of the IncRNA. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell. In one embodiment, the cell is a mouse cell. In one embodiment, the cell is a rat cell. In one embodiment, the cell is a plant cell. In one embodiment, the cell is a non-mammalian cell. In one embodiment the cell is a yeast cell. In one embodiment, the cell is a bacterial cell. In one embodiment, the increased export of the target mRNA from the nucleus is increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% when compared to the nuclear export of the target mRNA without the antisense IncRNA as described herein. In one embodiment, the increased export of the target mRNA from the nucleus is increased by 100%, 125%, 150%, 175%, 200%, 300%, or more when compared to the nuclear export of the target mRNA without the antisense IncRNA as described herein. In one embodiment, the target mRNA is a gene of interest. In one embodiment, the gene of interest is a tumor suppressor gene. In one embodiment the tumor suppressor gene is not haplo-insufficient. In one embodiment, the tumor suppressor gene is a haplo-insufficient tumor suppressor (hTS) gene. In one embodiment the hTS gene is PTEN, SMAD4, LKB1, CDKN1B, or NMPl. In one embodiment the hTS gene is PTEN. In one embodiment the hTS gene is SMAD4. In one embodiment the hTS gene is LKB1. In one embodiment the hTS gene is CDKN1B. In one embodiment the hTS gene is NMPl. In one embodiment the gene of interest is a CFTR.

[000129] In another aspect, the present invention relates to a method of increasing expression of a target mRNA from the nucleus of a eukaryotic cell, the method comprising transfecting a cell with the antisense IncRNA (boost-RNA), or the biological complex, or the expression vector as described herein. In one embodiment, the method is an in vitro method. In one embodiment, the present invention relates to a method of increasing expression of a target mRNA in a eukaryotic cell, the method comprising transfecting a cell with the antisense IncRNA, or the biological complex, or the expression vector as described herein, wherein the antisense IncRNA or the antisense IncRNA of the biological complex or expression vector is a boost-RNA as described herein. In one embodiment, the expression vector further comprises a nucleic acid sequence encoding a target mRNA of the IncRNA. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell. In one embodiment, the cell is a mouse cell. In one embodiment, the cell is a rat cell. In one embodiment, the cell is a plant cell. In one embodiment, the cell is a non-mammalian cell. In one embodiment the cell is a yeast cell. In one embodiment, the cell is a bacterial cell. In one embodiment, the increased expression of the target mRNA is increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% when compared to the expression of the target mRNA without the antisense IncRNA as described herein. In one embodiment, the increased expression of the target mRNA is increased by 100%, 125%, 150%, 175%, 200%, 300%, or more when compared to the expression of the target mRNA without the antisense IncRNA as described herein. In one embodiment, the increased export of the target mRNA from the nucleus is increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% when compared to the nuclear export of the target mRNA without the antisense IncRNA as described herein . In one embodiment, the increased export of the target mRNA from the nucleus is increased by 100%, 125%, 150%, 175%, 200%, 300%, or more when compared to the nuclear export of the target mRNA without the antisense IncRNA as described herein. In one embodiment, the target mRNA is a gene of interest. In one embodiment, the use of the antisense IncRNA (boost-RNA) upregulates and/or restores normal expression of the gene of interest. In one embodiment, the gene of interest is a tumor suppressor gene. In one embodiment the tumor suppressor gene is not haplo- insufficient. In one embodiment, the tumor suppressor gene is a haplo-insufficient tumor suppressor (hTS) gene. In one embodiment the hTS gene is PTEN, SMAD4, LKB1, CDKN1B, or NMPl. In one embodiment the hTS gene is PTEN. In one embodiment the hTS gene is SMAD4. In one embodiment the hTS gene is LKB1. In one embodiment the hTS gene is CDKN1B. In one embodiment the hTS gene is NMPl. In one embodiment the gene of interest is a CFTR.

[000130] In yet another embodiment, the boost antisense IncRNA, the complex, the expression vector, and the conjugate comprising the boost antisense IncRNA, respectively, are used in a method of enhancing the production of recombinant proteins. Such recombinant proteins may be used in biotechnological applications and for medical purposes. Thus the invention also relates to a method of enhancing the production of recombinant proteins, wherein the method comprises increasing expression and/or export of a target mRNA from the nucleus of a host cell, comprising transfecting the host cell with the antisense IncRNA (boost-RNA), or the biological complex, or the expression vector comprising the boost-RNA, respectively, wherein the target mRNA encodes the desired recombinant protein. In an embodiment, the host cell is a bacterial cell or a yeast cell. In an embodiment, the expression vector comprises a copy of the desired mRNA. In an embodiment, the method is suitable for large scale production of recombinant proteins. In an embodiment, the method comprises isolating and/ or purifying the recombinant protein.

[000131] The following embodiments can apply to the method embodiments according to the invention disclosed herein.

[000132] The skilled person in the art will be aware of various different transfection methods, and it will be appreciated by those skilled in the art that the choice of transfection method can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, what is being transfected (e.g. the antisense IncRNA, the biological complex, or the expression vector as described herein), and such factors will be known and accounted for by the person skilled in the art. The following are non-limiting examples of transfection methods that may be used by the skilled person. In one embodiment, the cell is transfected via biological transfection. In one embodiment, the cell is transfected via physical transfection. In one embodiment, the cell is transfected via chemical transfection. In one embodiment, the cell is transfected by microinjection, electroporation, sonoporation, biolistics, calcium phosphate-mediated transfection, cationic transfection, liposome transfection, dendrimer transfection, heat shock transfection, nucleofection transfection, magnetofection, lipofection, impalefection, optical transfection, proprietary agent-enhanced uptake of nucleic acids, and delivery via liposomes, immunoliposomes, virosomes, or artificial virions. In one embodiment, the cell is transfected by nucleofection.

[000133] Methods for decreasing nuclear export and expression of the target mRNA

[000134] In another aspect, the present invention relates to a method of decreasing export of a target mRNA from the nucleus of a eukaryotic cell, the method comprising transfecting a cell with the antisense IncRNA (block-RNA), or the biological complex, or the expression vector as described herein. In one embodiment, the method is an in vitro method. In one embodiment, the antisense IncRNA or the antisense IncRNA of the biological complex or expression vector is a block-RNA as described herein. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell. In one embodiment, the cell is a mouse cell. In one embodiment, the cell is a rat cell. In one embodiment, the cell is a plant cell. In one embodiment, the cell is a non-mammalian cell. In one embodiment the cell is a yeast cell. In one embodiment, the cell is a bacterial cell. In one embodiment, the decreased export of the target mRNA from the nucleus is decreased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% when compared to the nuclear export of the target mRNA without the antisense IncRNA as described herein. In one embodiment, the target mRNA is a gene of interest. In one embodiment, the gene of interest is an oncogene. In one embodiment, the oncogene gene is a is MYC, HER2, RAS, or FOS. In one embodiment the oncogene is MYC. In one embodiment the oncogene is HER2. In one embodiment the oncogene is RAS. In one embodiment the oncogene is FOS.

[000135] In another aspect, the present invention relates to a method of decreasing expression of a target mRNA in a eukaryotic cell, the method comprising transfecting a cell with the antisense IncRNA (block-RNA), or the biological complex, or the expression vector as described herein. In one embodiment, the method is an in vitro method. In one embodiment, the IncRNA or the antisense IncRNA of the biological complex or expression vector is a block-RNA as described herein. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell. In one embodiment, the cell is a mouse cell. In one embodiment, the cell is a rat cell. In one embodiment, the cell is a plant cell. In one embodiment, the cell is a non-mammalian cell. In one embodiment the cell is a yeast cell. In one embodiment, the cell is a bacterial cell. In one embodiment, the decreased expression of the target mRNA is decreased by 5%, 10%, 15%>, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% when compared to the expression of the target mRNA without the antisense IncRNA as described herein. In one embodiment, the decreased export of the target mRNA from the nucleus is decreased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% when compared to the nuclear export of the target mRNA without the antisense IncRNA as described herein. In one embodiment, the target mRNA is a gene of interest. In one embodiment, the use of the antisense IncRNA (block-RNA) downregulates and/or inhibits the expression of the gene of interest. In one embodiment, the gene of interest is an oncogene. In one embodiment, the oncogene gene is a is MYC, HER2, RAS, or FOS. In one embodiment the oncogene is MYC. In one embodiment the oncogene is HER2. In one embodiment the oncogene is RAS. In one embodiment the oncogene is FOS.

[000136] The following embodiments can apply to the method embodiments according to the invention disclosed herein.

[000137] The skilled person in the art will be aware of various different transfection methods, and it will be appreciated by those skilled in the art that the choice of transfection method can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, what is being transfected (e.g. the antisense IncRNA, the biological complex, or the expression vector as described herein), and such factors will be known and accounted for by the person skilled in the art. The following are non-limiting examples of transfection methods that may be used by the skilled person. In one embodiment, the cell is transfected via biological transfection. In one embodiment, the cell is transfected via physical transfection. In one embodiment, the cell is transfected via chemical transfection. In one embodiment, the cell is transfected by microinjection, electroporation, sonoporation, biolistics, calcium phosphate-mediated transfection, cationic transfection, liposome transfection, dendrimer transfection, heat shock transfection, nucleofection transfection, magnetofection, lipofection, impalefection, optical transfection, proprietary agent-enhanced uptake of nucleic acids, and delivery via liposomes, immunoliposomes, virosomes, or artificial virions. In one embodiment, the cell is transfected by nucleofection.

[000138] Cells or conjugates

[000139] In another aspect, the present invention is directed to a cell transfected with the antisense IncRNA, or the biological complex, or the expression vector as described herein. The transfected cell thus comprises the antisense IncRNA, or the biological complex, or the expression vector as described herein.

[000140] In one embodiment, the present invention relates to a cell transfected with the antisense IncRNA, or the biological complex, or the expression vector as described herein, wherein the antisense IncRNA or the antisense IncRNA of the biological complex or expression vector is a boost-RNA as described herein. In one embodiment, the expression vector further comprises a nucleic acid sequence encoding a target mRNA of the IncRNA.

[000141] In another embodiment, the present invention relates to a cell transfected with the antisense IncRNA, or the biological complex, or the expression vector as described herein, wherein the IncRNA or the antisense IncRNA of the biological complex or expression vector is a block-RNA as described herein.

[000142] The following embodiments can apply to the cell aspects of the instant invention described herein.

[000143] In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell. In one embodiment, the cell is a mouse cell. In one embodiment, the cell is a rat cell. In one embodiment, the cell is a plant cell. In one embodiment, the cell is a non-mammalian cell. In one embodiment the cell is a yeast cell. In one embodiment, the cell is a bacterial cell.

[000144] In another aspect, the present invention relates to a conjugate comprising the antisense IncRNA, or the biological complex, or the expression vector as described herein, and a cell penetrating peptide.

[000145] In one embodiment, the present invention relates to a conjugate comprising the antisense IncRNA, or the biological complex, or the expression vector as described herein, wherein the antisense IncRNA or the antisense IncRNA of the biological complex or expression vector is a boost-RNA as described herein, and a cell penetrating peptide. In one embodiment, the expression vector further comprises a nucleic acid sequence encoding a target mRNA of the IncRNA.

[000146] In another embodiment, the present invention relates to a conjugate comprising the antisense IncRNA, or the biological complex, or the expression vector as described herein, wherein the IncRNA or the antisense IncRNA of the biological complex or expression vector is a block-RNA, and a cell penetrating peptide.

[000147] As used herein, a "cell penetrating peptide” refers to a short peptide that facilitates cellular intake and uptake of molecules. The "cargo" (e.g. the conjugate) is associated with the peptide either through chemical linkage via covalent bonds or through non-covalent interactions. The skilled person in the art will be aware of various different types of cell penetrating peptides, and it will be appreciated by those skilled in the art that the choice of cell penetrating peptide can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, the cargo (e.g. the antisense IncRNA, the biological complex, or the expression vector as described herein), and such factors will be known and accounted for by the person skilled in the art. The following are non-limiting examples of cell penetrating peptides. In one embodiment, the cell penetrating peptide is cationic. In one embodiment, the cell penetrating peptide is amphipathic. In one embodiment, the cell penetrating peptide is hydrophobic. In one embodiment, the cell penetrating peptide is chimeric. In one embodiment, the cell penetrating peptide is synthetic. In one embodiment, the cell penetrating peptide is protein derived. In one embodiment, the cell penetrating peptide is linear. In one embodiment, the cell penetrating peptide is cyclic.

[000148] Therapeutic Applications

[000149] Many genetic disorders are associated with partial under-expression of genes. For example, the lack of expression of various tumor suppressor genes is a key driver of tumorigenesis and progression. In some cancers, the expression of classic tumor suppressor genes is completely absent, e.g. due to homozygous gene deletions. Another type of tumor suppressor genes that play an important role in cancer are so-called haplo-insufficient tumor suppressor genes (hTS), which show a partial loss of the gene expression. These hTS often show the loss of one allele while a wild-type allele is still present in the cancer cells. With the help of boost-RNA expression, the gene expression of the wild type allele can be increased in such a way that a normal expression level of the gene is restored ("rescue of hTS").

[000150] Through the systematic identification and annotation of all boost RNAs expressed in human cells as described herein, selection of a specific boost RNA for specific hTS genes is possible. Examples of specific hTS genes are PTEN, SMAD4, LKB1, CDKN1B and NMP1, which are underexpressed in various tumor types such as prostate, lung, cervical, breast and colon cancer as they preserved one wild type allele. In a non-limiting example, the respective boost-RNA can be cloned into an expression vector under the control of a CMV or SV40 promoter (e.g. by using pcDNA vectors) and the cancer cells are transfected with the plasmid, leading to expression of the boost-RNA. This will result in an increase in gene expression of the hTS sense gene from the wild type allele and can yield a restoration of normal expression of the gene in question without interfering with the genome. [000151] Many genetic disorders are associated with overexpression of genes. Through the systematic identification and annotation of all boost-RNAs expressed in human cells as described herein, selection of specific block-RNAs for these disorders is possible. For example, overexpressed oncogenes, such as MYC, HER2, RAS and FOS are known to be driving forces for tumor development and progression. A specific block-RNA, which is individual to the targeted mRNA will form a double strand with the mRNA of the oncogenes and inhibit its nuclear export. This in turn will lead to reduced oncogene expression. As described above, in a non-limiting example, the delivery of the inhibitory block-RNAs can be expressed with standard expression systems or delivered via standard delivery methods that will be known to the person skilled in the art and/or as described herein.

[000152] The therapeutic applications described herein can be administered in the form of pharmaceutical compositions herein (see "Pharmaceutical Compositions”). The pharmaceutical compositions according to the present disclosure are generally applied in a "pharmaceutically effective amount" and in "a pharmaceutically acceptable preparation" . The term pharmaceutically acceptable refers to the non-toxicity of a material which does not interact with the action of the active component of the pharmaceutical composition.

[000153] The term "pharmaceutically effective amount" or "therapeutically effective amount" refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses. In the case of the treatment of a particular disease, the desired reaction preferably relates to inhibition of the course of the disease. This comprises slowing down the progress of the disease and, in particular, interrupting or reversing the progress of the disease. The desired reaction in a treatment of a disease may also be delay of the onset or a prevention of the onset of the disease or the condition. An effective amount of the compositions described herein will depend on the condition to be treated, the severeness of the disease, the individual parameters of the subject, including age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, the doses administered of the compositions described herein may depend on various such parameters. In the case that a reaction in a subject is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.

[000154] The term "co-administering" or "co-administered” as used herein means a process whereby different compounds or compositions (e.g., the antisense IncRNA, the biological complex, the expression vector, or the conjugate as described herein with a known cancer drug) are administered to the same subject. The different compounds or compositions may be administered simultaneously, at essentially the same time, or sequentially.

[000155] Medical Use of boost-RNAs

[000156] In one aspect, the present invention relates to the antisense IncRNA, or the biological complex, the expression vector, or the conjugate as described herein for use as a medicament, wherein the antisense IncRNA or the antisense IncRNA of the biological complex, expression vector, or conjugate is a boost-RNA as described herein. In one embodiment, the expression vector further comprises a nucleic acid sequence encoding a target mRNA of the IncRNA.

[000157] In another aspect, the present invention relates to the antisense IncRNA, or the biological complex, or the expression vector, or the conjugate as described herein for use in the treatment or prevention of a disease, wherein the antisense IncRNA or the antisense IncRNA of the biological complex, expression vector, or conjugate is a boost-RNA, preferably by enhancing nuclear export of a therapeutically relevant mRNA, the increased expression of which has a therapeutic benefit. In one embodiment, the expression vector further comprises a nucleic acid sequence encoding a target mRNA of the IncRNA. In an embodiment, the disease is a heart disease. In an embodiment, the disease is fibrosis. In an embodiment, the disease is inflammation. In an embodiment, the disease is a neurodegenerative disease. In an embodiment, the disease is an age- related inherited disease. In one embodiment, the disease is cystic fibrosis. In one embodiment, the antisense IncRNA, or the biological complex, or the expression vector, or the conjugate enhances nuclear export of an mRNA linked to cystic fibrosis. In one embodiment, the gene linked to cystic fibrosis is CFTR. In one embodiment, the disease is cancer. In one embodiment, the antisense IncRNA, or the biological complex, or the expression vector, or the conjugate enhances nuclear export of a tumor suppressor mRNA. In one embodiment, the tumor suppressor gene is a haplo-insufficient tumor suppressor (hTS) gene. In one embodiment, the gene is not haplo-insufficient. In one embodiment the hTS gene is PTEN, SMAD4, LKB1, CDKN1B, or NMPl. In one embodiment the hTS gene is PTEN. In one embodiment the hTS gene is SMAD4. In one embodiment the hTS gene is LKB1. In one embodiment the hTS gene is CDKN1B. In one embodiment the hTS gene is NMPl. In one embodiment, the increased expression of the tumor suppressor gene is increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% when compared to the expression of the tumor suppressor gene without the antisense IncRNA as described herein. In one embodiment, the increased expression of the tumor suppressor gene is increased by 100%, 125%, 150%, 175%, 200%, 300%, or more when compared to the expression of the tumor suppressor gene without the antisense IncRNA as described herein. In one embodiment, the increased export of the tumor suppressor gene from the nucleus is increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% when compared to the nuclear export of the tumor suppressor gene without the antisense IncRNA as described herein. In one embodiment, the increased export of the tumor suppressor gene from the nucleus is increased by 100%, 125%, 150%, 175%, 200%, 300%, or more when compared to the nuclear export of the tumor suppressor gene without the antisense IncRNA as described herein. In one embodiment, the expression of the hTS gene is restored to normal expression levels.

[000158] Medical use of block-RNAs

[000159] In another aspect, the present invention relates to the antisense IncRNA, or the biological complex, the expression vector, or the conjugate as described herein for use as a medicament, wherein the IncRNA or the antisense IncRNA of the biological complex, expression vector, or conjugate is a block-RNA as described herein.

[000160] In another aspect, the present invention relates to the antisense IncRNA, or the biological complex, the expression vector, or the conjugate as described herein for use in the treatment or prevention of a disease, wherein the IncRNA or the antisense IncRNA of the biological complex, expression vector, or conjugate is a block-RNA described herein., preferably by inhibiting the nuclear export of a therapeutically relevant mRNA, the expression of which is causally linked to the disease. In one embodiment, the disease is cancer. In an embodiment, the disease is a heart disease. In an embodiment, the disease is fibrosis. In an embodiment, the disease is inflammation. In an embodiment, the disease is a neurodegenerative disease. In an embodiment, the disease is an age-related inherited disease. In an embodiment, the disease is cystic fibrosis. In one embodiment, the antisense IncRNA, or the biological complex, or the expression vector, or the conjugate inhibits expression of an mRNA linked to cystic fibrosis. In one embodiment, the gene linked to cystic fibrosis is CFTR. In one embodiment, the antisense IncRNA, or the biological complex, or the expression vector, or the conjugate inhibits expression of oncogene mRNA. In one embodiment, the oncogene gene is MYC, HER2, RAS, or FOS. In one embodiment the oncogene is MYC. In one embodiment the oncogene is HER2. In one embodiment the oncogene is RAS. In one embodiment the oncogene is FOS. In one embodiment, the decreased expression of the oncogene is decreased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% when compared to the expression of the oncogene without the antisense IncRNA as described herein. In one embodiment, the decreased export of the oncogene from the nucleus is decreased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% when compared to the nuclear export of the oncogene without the antisense IncRNA as described herein.

[000161] The following embodiments can apply to the medical use aspects according to the invention described herein.

[000162] In one embodiment, the cancer is lung cancer. In one embodiment, the cancer is breast cancer. In one embodiment, the cancer is brain cancer. In one embodiment, the cancer is liver cancer. In one embodiment, the cancer is prostate cancer. In one embodiment, the cancer is cervical cancer. In one embodiment, the cancer is colon cancer.

[000163] In one embodiment, the antisense IncRNA, the biological complex, the expression vector, or the conjugate may be administered intravenously, intraarterially, subcutaneously, intradermally or intramuscularly. In certain embodiments, the antisense IncRNA, the biological complex, the expression vector, or the conjugate may be administered locally or systemically. Systemic administration may include enteral administration, which involves absorption through the gastrointestinal tract, or parenteral administration. As used herein, "parenteral administration" refers to the administration in any manner other than through the gastrointestinal tract, such as by intravenous injection. In one embodiment, the antisense IncRNA, the biological complex, the expression vector, or the conjugate may be administered through intramuscular administration. In another embodiment, the antisense IncRNA, the biological complex, the expression vector, or the conjugate may be administered through systemic administration, e.g., intravenous administration. In one embodiment, the antisense IncRNA, the biological complex, the expression vector, or the conjugate is administered through oral administration. In one embodiment, the antisense IncRNA, the biological complex, the expression vector, or the conjugate may be administered through infusion. In one embodiment, the antisense IncRNA, the biological complex, the expression vector, or the conjugate may be administered as a pharmaceutical composition as described herein. In one embodiment, the antisense IncRNA, the biological complex, the expression vector, or the conjugate are administered in a sufficient amount. In one embodiment, the antisense IncRNA, the biological complex, the expression vector, or the conjugate may be co-administered with other pharmaceutical compositions, vaccines, medications, medicaments, or pharmaceutical drugs known in the art. In one embodiment, the antisense IncRNA, the biological complex, the expression vector, or the conjugate may be co-administered with a cancer drug known in the art. In one embodiment, the antisense IncRNA, the biological complex, the expression vector, or the conjugate may be co-administered with other pharmaceutical compositions, vaccines, medications, medicaments, or pharmaceutical drugs known art in the same formulation. In one embodiment, the antisense IncRNA, the biological complex, the expression vector, or the conjugate may be co-administered with other pharmaceutical compositions, vaccines, medications, medicaments, or pharmaceutical drugs known in the art as separate formulations. The skilled person in the art will be aware of various different types of pharmaceutical compositions, vaccines, medications, medicaments, or pharmaceutical drugs, and it will be appreciated by those skilled in the art that the choice of co-administered pharmaceutical compositions, vaccines, medications, medicaments, or pharmaceutical drugs will depend on various factors, e.g. disease to be treated, stage of disease, subject's age, subject's physical health, etc., and such factors will be known and accounted for by the person skilled in the art.

[000164] Methods of treatment

[000165] In another aspect, the present invention relates to a method of treatment comprising administering the antisense IncRNA, the biological complex, the expression vector, or the conjugate as described herein, to a subject in need thereof.

[000166] The term "subject" as used herein refers to an individual or subject for treatment, in particular a diseased individual or subject. The subject can be a mammal, plant, animal, nonmammalian animal, rodent. The subject can be a primate. The subject can preferably be a human. The subject can be a patient. The subject can be a human patient. The subject can be yeast. The subject can be fungi.

[000167] The term "co-administering" or “co-administered" as used herein means a process whereby different compounds or compositions (e.g., the antisense IncRNA, the biological complex, the expression vector, or the conjugate as described herein with a known cancer drug) are administered to the same subject. The different compounds or compositions may be administered simultaneously, at essentially the same time, or sequentially. [000168] In one embodiment, the present invention relates to a method of treatment comprising administering the antisense IncRNA, the biological complex, the expression vector, or the conjugate as described herein to the subject, wherein the antisense IncRNA or the antisense IncRNA of the biological complex, expression vector, or conjugate is a boost-RNA as described herein. In one embodiment, the expression vector further comprises a nucleic acid sequence encoding a target mRNA of the IncRNA.

[000169] In another embodiment, the present invention relates to a method of treatment comprising administering the antisense IncRNA, the biological complex, the expression vector, or the conjugate as described herein to a subject in need thereof, wherein the antisense IncRNA or the antisense IncRNA of the biological complex, expression vector, or conjugate is a block-RNA.

[000170] The following embodiments may apply to the method of treatment aspects according to the invention described herein.

[000171] In one embodiment, the antisense IncRNA, the biological complex, the expression vector, or the conjugate may be administered intravenously, intraarterially, subcutaneously, intradermally or intramuscularly. In certain embodiments, the antisense IncRNA, the biological complex, the expression vector, or the conjugate may be administered locally or systemically. Systemic administration may include enteral administration, which involves absorption through the gastrointestinal tract, or parenteral administration. As used herein, "parenteral administration" refers to the administration in any manner other than through the gastrointestinal tract, such as by intravenous injection. In one embodiment, the antisense IncRNA, the biological complex, the expression vector, or the conjugate may be administered through intramuscular administration. In another embodiment, the antisense IncRNA, the biological complex, the expression vector, or the conjugate may be administered through systemic administration, e.g., intravenous administration. In one embodiment, the antisense IncRNA, the biological complex, the expression vector, or the conjugate is administered through oral administration. In one embodiment, the antisense IncRNA, the biological complex, the expression vector, or the conjugate may be administered through infusion. In one embodiment, the antisense IncRNA, the biological complex, the expression vector, or the conjugate may be administered as a pharmaceutical composition as described herein. In one embodiment, the subject has cancer. In one embodiment, the subject has lung cancer. In one embodiment, the subject has breast cancer. In one embodiment, the subject has brain cancer. In one embodiment, the subject has liver cancer. In one embodiment, the subject has prostate cancer. In one embodiment, the subject has lung cancer. In one embodiment, the subject has cervical cancer. In one embodiment, the subject has colon cancer. In one embodiment, the subject has heart disease. In one embodiment, the subject has fibrosis. In one embodiment, the subject has inflammation. In one embodiment, the subject has a neurodegenerative disease. In one embodiment, the subject has an age-related inherited disease. In one embodiment, the subject has cystic fibrosis. In one embodiment, the antisense IncRNA, the biological complex, the expression vector, or the conjugate may be co-administered with other pharmaceutical compositions, vaccines, medications, medicaments, or pharmaceutical drugs known in the art. In one embodiment, the antisense IncRNA, the biological complex, the expression vector, or the conjugate may be co-administered with a cancer drug known in the art. In one embodiment, the antisense IncRNA, the biological complex, the expression vector, or the conjugate may be co-administered with other pharmaceutical compositions, vaccines, medications, medicaments, or pharmaceutical drugs known art in the same formulation. In one embodiment, the antisense IncRNA, the biological complex, the expression vector, or the conjugate may be co-administered with other pharmaceutical compositions, vaccines, medications, medicaments, or pharmaceutical drugs known in the art as separate formulations. The skilled person in the art will be aware of various different types of pharmaceutical compositions, vaccines, medications, medicaments, or pharmaceutical drugs, and it will be appreciated by those skilled in the art that the choice of co-administered pharmaceutical compositions, vaccines, medications, medicaments, or pharmaceutical drugs will depend on various factors, e.g. disease to be treated, stage of disease, subject's age, subject's physical health, etc., and such factors will be known and accounted for by the person skilled in the art.

[000172] Pharmaceutical Compositions

[000173] The antisense IncRNA, the biological complex, the expression vector, or the conjugate as described herein may be administered in pharmaceutical compositions or medicaments and may be administered in the form of any suitable pharmaceutical composition.

[000174] The term "pharmaceutical composition" relates to a formulation comprising a therapeutically effective agent (e.g. the antisense IncRNA, the biological complex, the expression vector, or the conjugate as described herein), preferably together with pharmaceutically acceptable carriers, diluents and/or excipients. The pharmaceutical composition is useful for treating, preventing, or reducing the severity of a disease or disorder by administration of the pharmaceutical composition to a subject. A pharmaceutical composition is also known in the art as a pharmaceutical formulation.

[000175] The pharmaceutical compositions of the present disclosure may comprise one or more adjuvants or may be administered with one or more adjuvants. The term "adjuvant" relates to a compound which prolongs, enhances or accelerates an immune response. Adjuvants comprise a heterogeneous group of compounds such as oil emulsions (e.g., Freund's adjuvants), mineral compounds (such as alum), bacterial products (such as Bordetella pertussis toxin), or immune- stimulating complexes. Examples of adjuvants include, without limitation, LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines, such as monokines, lymphokines, interleukins, chemokines. The cytokines may be IL1, I L2, I L3, I L4, ILS, I L6, I L7, I L8, I L9, IL10, IL12, IFNa, I FNy, GM- CSF, LT-a. Further known adjuvants are aluminium hydroxide, Freund's adjuvant or oil such as Montanide® ISA51. Other suitable adjuvants for use in the present disclosure include lipopeptides, such as Pam3Cys.

[000176] The pharmaceutical compositions according to the present disclosure are generally applied in a "pharmaceutically effective amount" and in "a pharmaceutically acceptable preparation" . The term pharmaceutically acceptable refers to the non-toxicity of a material which does not interact with the action of the active component of the pharmaceutical composition.

[000177] The term "pharmaceutically effective amount" or "therapeutically effective amount" refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses. In the case of the treatment of a particular disease, the desired reaction preferably relates to inhibition of the course of the disease. This comprises slowing down the progress of the disease and, in particular, interrupting or reversing the progress of the disease. The desired reaction in a treatment of a disease may also be delay of the onset or a prevention of the onset of the disease or the condition. An effective amount of the compositions described herein will depend on the condition to be treated, the severeness of the disease, the individual parameters of the subject, including age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, the doses administered of the compositions described herein may depend on various such parameters. In the case that a reaction in a subject is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.

[000178] The pharmaceutical compositions of the present disclosure may contain salts, buffers, preservatives, and optionally other therapeutic agents. In one embodiment, the pharmaceutical compositions of the present disclosure comprise one or more pharmaceutically acceptable carriers, diluents and/or excipients.

[000179] Suitable preservatives for use in the pharmaceutical compositions of the present disclosure include, without limitation, benzalkonium chloride, chlorobutanol, paraben and thimerosal. The term "excipient" as used herein refers to a substance which may be present in a pharmaceutical composition of the present disclosure but is not an active ingredient. Examples of excipients, include without limitation, carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or colorants.

[000180] The term "diluent" relates a diluting and/or thinning agent. Moreover, the term "diluent" includes any one or more of fluid, liquid or solid suspension and/or mixing media. Examples of suitable diluents include ethanol, glycerol and water. [000181] The term "carrier" refers to a component which may be natural, synthetic, organic, inorganic in which the active component is combined in order to facilitate, enhance or enable administration of the pharmaceutical composition. A carrier as used herein may be one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to subject. Suitable carrier include, without limitation, sterile water, Ringer, Ringer lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide/glycolide copolymers or polyoxyethylene/polyoxy-propylene copolymers. In one embodiment, the pharmaceutical composition of the present disclosure includes isotonic saline.

[000182] Pharmaceutically acceptable carriers, excipients or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985).

[000183] Pharmaceutical carriers, excipients or diluents can be selected with regard to the intended route of administration and standard pharmaceutical practice.

[000184] In one aspect, the present invention relates to a pharmaceutical composition comprising the antisense IncRNA, the biological complex, the expression vector, or the conjugate as described herein.

[000185] In one embodiment, the present invention relates to a pharmaceutical composition comprising the antisense IncRNA, the biological complex, the expression vector, or the conjugate as described herein, wherein the antisense IncRNA or the antisense IncRNA of the biological complex, expression vector, or conjugate is a boost-RNA as described herein. In one embodiment, the expression vector further comprises a nucleic acid sequence encoding a target mRNA of the IncRNA. [000186] In another embodiment, the present invention relates to a pharmaceutical composition comprising the antisense IncRNA, the biological complex, the expression vector, or the conjugate as described herein, wherein the antisense IncRNA or the antisense IncRNA of the biological complex, expression vector, or conjugate is a block-RNA as described herein.

[000187] The following embodiments apply to the pharmaceutical aspect and embodiments of the invention as described above.

[000188] In one embodiment, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and/or excipients. In one embodiment, pharmaceutical compositions described herein may be administered intravenously, intraarterially, subcutaneously, intradermally or intramuscularly. In certain embodiments, the pharmaceutical composition is formulated for local administration or systemic administration. Systemic administration may include enteral administration, which involves absorption through the gastrointestinal tract, or parenteral administration. As used herein, "parenteral administration" refers to the administration in any manner other than through the gastrointestinal tract, such as by intravenous injection. In one embodiment, the pharmaceutical composition is formulated for intramuscular administration. In another embodiment, the pharmaceutical composition is formulated for systemic administration, e.g., for intravenous administration. In one embodiment, the pharmaceutical composition is formulated for oral administration. In one embodiment, the pharmaceutical composition is formulated for infusion.

[000189] mRNA vaccines

[000190] Delivery of mRNA molecules as vaccines is already very successful. However, this method can further be improved because gene expression upon providing only the mRNA to be expressed is not efficient. A co-delivery of the mRNA together with its respective boost-RNA can greatly increase the efficiency of gene expression in targeted cells. Similar to the mRNAs, the required boost-RNAs can be synthesized in vitro and purified. To form the required double strand in vitro, mRNA and boost-RNA can be annealed in the presence of the double stranded RNA forming enzymes Dbp2 and its co-factor Yral (or its human homologues - human DDX5/p68 and ALY/REV/NXF1, respectively), which can be recombinantly expressed and added. The double-stranded RNA can be packaged into the desired delivery systems. The efficiency of expression of the desired gene in this setup is significantly higher than by using the mRNA alone. This procedure thus optimizes the generation of expression competent vaccination RNA particles.

[000191] In another aspect, the present invention relates to the use of the antisense IncRNA, the biological complex, the expression vector, or the conjugate as described herein, in increasing mRNA vaccine efficacy.

[000192] In another aspect, the present invention relates to the use of the antisense IncRNA, the biological complex, the expression vector, or the conjugate as described herein, wherein the antisense IncRNA or the antisense IncRNA of the biological complex, expression vector, or conjugate is a boost-RNA as described herein, in increasing mRNA vaccine efficacy. In one embodiment, the expression vector further comprises a nucleic acid sequence encoding a target mRNA of the IncRNA.

EXAMPLES

[000193] Example 1 - Boost RNAs accelerate mRNA nuclear export in Saccharomyces cerevisiae

[000194] To identify the cellular localization of mRNAs and their antisense (as)RNAs we fractionated cells of the eukaryotic model organism Saccharomyces cerevisiae and analyzed the RNA amounts of the cytoplasmic fraction relative to their total cellular amounts via RNAseq, resulting in a global picture of the nucleo-cytoplasmic RNA distribution (FIG. 1A, FIG. IB). As expected, we detect most rRNAs in the cytoplasmic fraction, as they are part of the translating ribosomes. In contrast we see that snoRNAs were underrepresented in the cytoplasm, as they function in the nucleus. Surprisingly, we found that the relative number of bulk mRNAs in the cytoplasm was on average lower than calculated for the nucleus, while the opposite was true for IncRNAs, in particular asRNAs (FIG. IB). Interestingly, when looking at sense and antisense pairs regarding their compared RPKM values, we noticed whenever the amount of the asRNA was equal or in excess over the sense transcript, these mRNAs were on average mostly detected in the cytoplasm, whereas in case the amount of the antisense was below that of the sense, the regarding mRNA pool was on average nuclear (FIG. 1C). [000195] To analyze the possible formation of dsRNAs, we took advantage of a data set created by Wery and colleagues that identified dsRNAs through an RNAi based screen, in which the dsRNA degrading RNAi system was artificially established in S. cerevisiae, as it had lost this system during evolution (Wery et al., 2016). It allowed identifying formed RN A double strands upon Dicer expression through detection of the accumulating degradation products. Grouping RNAs based on that dataset and applying it to our fractionation experiment showed that the more an RNA is prone to form a double strand the more it is localized to the cytoplasm in wild type cells (FIG. ID).

[000196] The comparatively strong presence of dsRNAs in the cytoplasm under normal conditions created the idea that dsRNAs might be preferentially exported into the cytoplasm. However, it is also conceivable that dsRNAs are more stable, for instance because they might be more efficiently translated. Several global studies exist that have determined the half-life of RNAs. We utilized the newest experimental data set which used a refined pseudouridine labeling technique (Chan et al., 2018) to investigate, whether dsRNAs are more stable than ssRNAs or vice versa. We found that the stability for ss and dsRNAs is comparable (FIG. 5B). Remarkably, mRNAs with a long half-life are generally more nuclear (FIG. 5C), as are highly expressed RNAs (FIG. 5D) showing a notably correlation between both (R=0.56, FIG. 5E). For this reason, we started to think that dsRNAs might indeed leave the nucleus faster than ssRNAs.

[000197] mRNAs are exported via the heterodimer Mex67-Mtr2 (human TAP-pl5) (Braun et al., 2001; Segref et al., 1997). It is recruited to matured mRNAs after capping, splicing and polyadenylation via the guard proteins, which monitor the maturation process (Soheilypour and Mofrad, 2018; Zander and Krebber, 2017; Zenklusen and Stutz, 2001). Also ncRNAs are anticipated to use Mex67 for nuclear export, because their interaction with ncRNAs was shown in high through put analyses (Tuck and Tollervey, 2013; Tudek et al., 2018). Another mRNA export factor that supports Mex67-mediated mRNA export is Crml/Xpol. This Ran-dependent karyopherin additionally supports the export of ncRNAs such as the telomerase RNA TLC1 or snRNAs by contacting the Cap-binding complex (CBC) (Becker et al., 2019; Wu et al., 2014). To confirm that IncRNA transport is dependent on these export factors, we repeated the cytoplasmic fractionation experiment in the double mutant mex67-5 xpol-1 after a lh temperature shift to 37°C at which mRNA export is blocked. Based on the RNAi-seq data set all groups of RNAs, including the dsRNAs were shifted to the nucleus and are thereby revealed their dependence for both export receptors (FIG. IE, FIG. 5A).

[000198] Notably, asRNAs are expressed at 10-fold lower levels compared to the coding transcripts (Villegas and Zaphiropoulos, 2015). If dsRNAs would have an advantage in nuclear export, tuning the asRNA expression artificially up might increase the cytoplasmic localization of the sense transcript. To test that, we chose to analyze the expression of the cyclin-dependent kinase Pho85. Under normal conditions the PHO85 transcript is highly expressed and rather nuclear, while its asRNA (SUT412) is barely detectable. To challenge our idea, we overexpressed the asPHO85 from the strong GALI promoter (P_PGALI:SUT412) to reach equal amounts of sense and antisense RNA (FIG. IF). Subsequent nucleo-cytoplasmic fractionation experiments revealed that the PHO85 transcript significantly shifted its distribution to the cytoplasm through the elevated asRNA expression (FIG. 1G, FIG. 5F). Notably, allowing dsRNA formation to occur did not inhibit splicing of the PHO85 transcript, but rather accelerated the splicing reaction (FIG. 1G). Furthermore, it also seemed to have no effect on the nuclear mRNA quality control, because we detected no increased leakage of unspliced PHO85 mRNA in the cytoplasm (FIG. 1G). Together, we suggest that dsRNA formation might be advantageous for nuclear processing and export.

[000199] To further validate our findings, we made use of an antibody that has been generated to recognize dsRNA, termed J2 (Schonborn et al., 1991; Xie et al., 2019). First, we carried out coimmunoprecipitation (co-IP) experiments and subsequent qPCRs to confirm the binding to several transcripts that were shown to form double strands in yeast (FIG. 6A). As mRNA, IncRNAs and potentially dsRNAs are exported via Mex67-Mtr2 and Crml/Xpol (FIG. IE), we analyzed the possible accumulation of dsRNAs in mex67-5 xpol-1 after a lh temperature shift to 37°C by using the J2 antibody and found that this was indeed the case (FIG. 6B). Moreover, we established a protocol for immunofluorescence (IF) with the J2 antibody, which reflected the cytoplasmic localization of dsRNA in wild type, and a nuclear trapping in mex67-5 xpol-1 cells (FIG. 2A). Together, these results show that dsRNAs are formed in the nucleus and utilize Mex67 and Xpol for their export into the cytoplasm.

[000200] IncRNAs were suggested to be eliminated through the cytoplasmic NMD system in which ribosomes are involved in the identification of these RNAs as non-coding (de Andres-Pablo et al., 2017). If dsRNAs require translation for their strand detachment, one would expect their accumulation in the cytoplasm upon translational inhibition. We chose two different mutants to test this hypothesis. Deletion of the export adapter Nmd3 leads to ribosomal subunit export defects and mutation in RPL10 to subunit joining defects (Hedges et al., 2005; Ho et al., 2000). In both cases, a J2- IF showed a strong increase of dsRNA in the cytoplasm (FIG. 2A), confirming that translation is required for dsRNA detachment and visualizing this necessity. [000201] To support our hypothesis that dsRNAs are preferentially exported and thus would reach ribosomes faster than ssRNAs, we did the following experiment. First, we blocked RNA export by shifting mex67-5 to 37 °C for 1 h allowing ss- and dsRNAs to accumulate in the nucleus. Subsequently, we released the export block by shifting the cultures back to 25°C. Usually Mex67 and also mutant mex67-5 proteins are mostly detectable at the nuclear rim. However, upon the temperature shift, mex67-5 becomes non-functional, detaches from the rim and mislocalizes to the cytoplasm. This loss of function is reversible upon shifting the cultures back to 25°C, at which mex67- 5 returns to the rim and nuclear transport is re-established (Segref et al., 1997). We used this system to re-boot nuclear export and monitor the arrival of the RNAs at the ribosomes. For this purpose, we precipitated Rps2 from mex67-5 cells and purified the co-precipitated RNA at the indicated time points after release (FIG. 6C). Selecting three ss and dsRNAs, we found that dsRNAs reached the ribosome by far first. The presence of dsRNAs was significantly and above-average increased at the ribosomes after 10 min, while ssRNAs only returned slowly to their initial level (FIG. 2B, FIG. 6C), suggesting that dsRNAs are preferentially exported by Mex67 and translated at the ribosome. But how is this preferential export facilitated by the same transport factors? One possibility is that more Mex67 is bound to dsRNA, which increases the probability to pass the hydrophobic interior of the NPC. As suggested earlier the more export proteins cover an RNA, the faster transport is initiated (Soheilypour and Mofrad, 2018; Azimi et al., 2014). Thus, we carried out electrophoretic mobility shift assays (EMSAs). First, we purified recombinant Mex67-Mtr2, which was shown to directly bind to mRNAs (Zander et al., 2016). We then added the heterodimer in the indicated molarity excess to FAM-labeled ssRNA or dsRNA and incubated the sample for 15 min before it was separated through gel electrophoresis. The ssRNA was fully upshifted after the addition of a 2 molar excess and reached its saturation at 5 molar excess of Mex67 (FIG. 2C). Additional molecules did not bind and the complex was not further upshifted. In contrast, dsRNA showed a binding saturation only upon the addition of a 10 molar excess and a full upshift after 3 molar excess of Mex67, suggesting that dsRNA has a higher binding capacity for Mex67 than ssRNA. In fact, competition assays moreover revealed a preferential binding of Mex67 to dsRNA. We used a Cy3-labeled ssRNA (red), pre-incubated it with a fourfold excess of Mex67 to allow all Mex67 and ssRNA molecules to form a complex and added increasing amounts of a FAM-labeled dsRNA (green) as a competitor. As shown in FIG. 2D, Mex67 dissociated from the ssRNA and associated with the dsRNA. When we repeated the experiment the other way around with Mex67 pre-bound dsRNA, it was evident that ssRNA did not act as a good competitor, as only very high concentrations resulted in a very slight decrease of the dsRNA-Mex67 complex (FIG. 2E). Together the results suggest that Mex67 binds preferentially and with more molecules to dsRNA and this explains why these duplexes are favored for export. [000202] Interestingly, the general presence of asRNA transcripts at every gene locus is quite striking and therefore, every gene could benefit from the ds-mediated preferential gene expression when this is desired. In fact, such fundamental regulatory role in preferential nuclear export of generated sense antisense RNA pairs might explain their categorical existence. This novel layer to regulate gene expression can influence the cellular protein content and support changes in the gene expression programs. One such expression program regulator is the histone methyltransferase Set2. Set2 was identified as a suppressor of asRNA transcription through chromatin modification (Venkatesh et al., 2016). The downregulated transcripts were named SRATs (Set2-repressed antisense transcripts) and are positioned antisense to protein-coding genes that are mostly involved in stress response and aging. To investigate whether these asRNAs are paired with their mRNAs, we carried out J2-IF experiments in set2A and found a doubling of the overall amount of dsRNA in the cytoplasm (FIG. 3A, FIG. 3B), suggesting that the induced SRATs form double strands with the sense counterpart and are exported as dsRNA. Cell fractionation experiments and qPCRs with a Set2- responsive transcript, SEG2, confirmed this observation and showed an upregulation of the asSEG2 upon SET2 deletion (FIG. 3C), which subsequently increased the presence of SEG2 mRNAs in the cytoplasm (FIG. 3D, FIG. 7A). Not only this specific example of the SRATs showed a regulation of asRNA expression, but this is also reflected in the general asRNA expression upon osmotic stress (FIG. 3E).

[000203] Remarkably, our analysis, of asRNA expression that was based on the RNAseq data of Lahtvee and colleagues (Lahtvee et al., 2016), reveal increased levels of asRNAs on average, while the mean of the sense RNAs rather stayed the same and showed specific sense transcript upregulation (FIG. 3E).

[000204] To visualize the change in the expression program we determined the dsRNA formation in cells upon stress via J2 FISH experiments in salt- or ethanol shocked cells (FIG. 3F). While bulk mRNAs rather accumulate in the nucleus and dissociate from Mex67 (Zander etal., 2016), dsRNA is not retained but increasingly detected in the cytoplasm of stressed cells. Our novel data opens up the possibility that the cause of this dissociation is the generation of newly made dsRNA that titrate Mex67 away from ssRNAs.

[000205] If this mechanism would be of a general nature, one would expect that destroying dsRNA formation would be hazardous to cells. Therefore, we expressed E. coll RNase III in yeast to degrade dsRNA. We controlled the expression with the inducible GALI promoter and directed the protein to different compartments. It was already shown, that RNase III does not degrade rRNA or generally destabilizes mRNA, therefore a dsRNA specific activity can be assumed (Pines, 1988). When tagged with a nuclear localization signal (NLS), RNase III expression was lethal to cells (FIG. 3G, FIG. 3H). Similarly, expressing a shuttling RNase III that in addition to the NLS contained a nuclear export signal (NES) was equally toxic to cells (FIG. 3G). This fusion protein shuttles between nucleus and cytoplasm and was mostly visible at the nuclear rim (Fig. 3h). Interestingly, a construct that restricted the RNase to the cytoplasm by the presence of an NES was tolerated (FIG. 3G, FIG. 3H). Detection of dsRNA with the J2 antibody furthermore revealed that all three constructs were functional in yeast as all of them decreased the amount of dsRNAs in the cells (FIG. 3H). But they were most efficiently eliminated from all NLS-containing RNase III enzymes that were able to reach the nucleus. In consequence these cells died. Therefore, we used the cytoplasmic NES-containing RNase III for further experiments (FIG. 3G), which had milder effects and was tolerated in cells under stable expression conditions with the ADH1 promoter. We found that the increased presence of dsRNA upon salt stress was prevented when the RNaselll-NES was expressed, while bulk mRNAs were not affected and accumulated in the nucleus as also shown earlier (Zander eto/., 2016) (FIG. 31). As a consequence, even the NES-containing RNase III affected cell viability under these challenging conditions (FIG. 3J). Reassuringly, a similar growth inhibition upon stress was also detected when the RNAi system was artificially expressed in yeast, which also degrades cytoplasmic dsRNAs (FIG. 3J).

[000206] After realizing the importance for the dsRNA formation in the nucleus, the important question, which enzyme mediates the double strand formation, remained. Predestined for such function are RNA-helicases, in particular those of the DEAD-box type because they are known for their strong unwinding activity on duplexes and most importantly, because they principally work also in the other direction to form duplexes (Linder and Jankowsky, 2011; Putnam and Jankowsky, 2013). For few of them, strand annealing activity has been described from which Dbp2 is the only one that is localized to the nucleus (Putnam and Jankowsky, 2013). Earlier studies have characterized Dbp2 (p68 in multicellular organisms) in detail and have shown that this helicase efficiently not only unwinds dsRNA, but that it also catalyzes its formation (Cloutier et al., 2012; Ma et al., 2013). Interestingly, Yral was shown to decrease the efficiency of Dbp2's ATP-dependent duplex unwinding activity by directly binding to the helicase (Cloutier et al., 2012; Ma et al., 2013). As Yral was shown to support mRNA export through interaction with Mex67-Mtr2 (Zenklusen et al., 2001), a model is conceivable in which Dbp2 promotes dsRNA formation when associated with Yral, which in turn promotes Mex67 binding.

[000207] A role of Dbp2 in dsRNA formation for nuclear export seems thus possible and was investigated in the next experiments. First, we confirmed Dbp2 binding to dsRNA in vivo via J2-IP (FIG. 4A). To analyze whether this helicase is indeed responsible for the nuclear dsRNA formation, we used a DBP2 knock out strain that is cold sensitive for J2-IF. Interestingly, dbp2A can only barely grow at 37°C (Ma et al., 2013) and FIG. 4. Clearly, depletion of the enzyme results in a significant reduction of the dsRNA formation and a nuclear accumulation of the poly(A)⁺ RNA (FIG. 4B). To investigate whether the loss of dsRNA formation in dbp2A would reduce the positive effect of the asRNA overexpression on the PHO85 expression, we repeated the experiment from FIG. 1H in dbp2A cells. Clearly, in the absence of Dbp2 the previous effect that overexpression of the asRNA shifted the localization of the sense RNA to the cytoplasm (FIG. 4C, FIG. 4D). Although the asRNA was still highly enriched it was not able to manipulate the localization of the sense RNA, suggesting that Dbp2 is a key factor for dsRNA formation and preferential export.

[000208] In addition, we expressed (with a regular expression vector for yeast cells) a PHO85 asRNA, which is complementary to the 5' end of the mRNA over a range of 881 nucleotides at its 5'end and contains a 567bp long overhang at its 3'end from the strong Galatose indicible promoter GALI. The mRNA, asRNA and the protein level were measured after 0, 10, 15, 20, 30 and 60 minutes. The result is that the asRNA is expressed and boosts the protein expression nearly 4-fold through increased expression of the endogenous mRNA. A similar experiment was done with the MTR4 asRNA. In this case we used a lOObp long region of asRNA that is complementary to the 5'end of the mRNA and a 494bp overhang at its 3'end. The asRNA was again expressed from a GALI inducible yeast vector. The result of the experiment is that the boosting asRNA almost doubled the protein expression as depicted in Figures 13A and 13B.

[000209] Example 2 - Boost RNAs accelerate mRNA nuclear export in human cells

[000210] We expressed (with a regular mammalian expression vector) a GAPDH, BRCA1, MAD2L1 and Smad4 asRNA from the CMV promoter. All constructs were designed to be complementary to the 5' end of the mRNA over a range of 76, 94, 78-468 and 140-411 nucleotides, respectively at their 5'ends. The asRNAs contain overhangs at the 3'end of 717bp, 717bp, 717-1109bp and 717-984bp, respectively. The result is that the asRNAs expression doubles the protein production of Mad2Ll and Smad4 and increases the expression of Gapdh and Brcal ~1,3 and "'1,7-fold, respectively, as depicted in Figures 14A-14C.

[000211] In conclusion, our findings reveal a new layer of regulated gene expression. Pervasive transcription creates asRNA, which is used for Dbp2-mediated dsRNA formation. These dsRNAs are preferentially exported and subsequently, the respective sense transcripts preferentially expressed (FIG. 4E). This mechanism is particularly important for effective cellular adaptation and adds preferential export as a new layer of gene expression regulation. Furthermore, it could explain how pervasive transcription controls gene expression and why so many asRNAs are detected in the cytoplasm.

METHODS

[000212] For the yeast experiments the strain S288C was used and the plasmids were standard yeast expression vectors as published in (Coban et al. 2024, Nature), carrying the indicated asRNA template DNA. For the human cell culture experiments, HCT cells were transfected with a pCDNA3.1 vector that contained the indicated asRNA template DNAs. For cloning of the asRNAs, specifically designed oligonucleotides were ordered from Sigma for PCRs that generated the template DNA strands for asRNA expression.

[000213] Fluorescent in situ hybridization experiments (FISH). The experiments were essentially performed as described earlier (Hackmann et al., 2011). To detect poly(A)⁺ RNA a Cy3- labelled oligo d(T)₅o probe (Sigma) was used. Cells were grown to mid log phase (~lxl0⁷ cells/ml) before they were shifted to 37 °C for 1 h or treated with NaCI or ethanol as indicated. Fixation was carried out by adding formaldehyde to a final concentration of 4 % for 40 min at room temperature, followed by washing once with 0.1 M potassium phosphate buffer pH 6.5 and P-Solution (0.1 M potassium phosphate buffer pH 6.5, 1.2 M sorbitol). Cells were spheroplasted by adding zymoylase (final ImM), subsequently washed twice in P-Solution permeabilized in P-Solution and 0.5 % Triton® X-100, pre-hybridized with Hybmix (50 % deionized formamide, 5x SSC, lx Denhardts, 500 pg/ml tRNA, 500 pg/ml salmon sperm DNA, 50 pg/ml heparin, 2.5 mM EDTA pH 8.0, 0.1 % Tween® 20, 10 % dextran sulfate) for 1 h on a polylysine coated slide at 37 °C and hybridized with the Cy3-labelled oligo d(T)₅o probe in Hybmix (1:200) over night at 37 °C. After hybridization, cells were washed with 2x SSC and lx SSC at room temperature, each for 1 h and 0.5x SSC at 37 °C and room temperature, each for 30 min. DNA was stained with DAPI (Sigma) for 2 min and washed once with 0,5 % Tween in lx PBS and twice with lx PBS each for 5 min. Microscopy studies were carried out with a Leica AF6000 microscope and pictures were obtained by using the LEICA DFC360FX camera and the LAS AF 2.7.3.9 software (Leica) and quantified by using the Fiji-software.

[000214] smFISH The experiment was mainly conducted as described above. Cells were grown in 2 % raffinose to logarithmic growth phase. The expression of the PHO85 mRNA and its asRNA were induced by the addition of 2% galactose. Cells were harvested after the indicated times and fixed for 20 min in 3.7% formaldehyde. The probes used are shown in SI Table 5. They were incubated for 3 h at 37 °C. Thereafter, the washing steps with SSC were carried out for 15 min each as described in the FISH protocol. The quantification of the signal was carried out with the Fiji software. For the determination of the nuclear signal, the DAPI signal was used as a reference. The boundary of the total cell was determined in Nomarski optic. The cytoplasmic signal was calculated by subtracting the nuclear from the total signal. The background signal was measured three times per image and subtracted from the measured signal of the cell as follows: Integrated Density - (selected area) x (mean fluorescence of background readings), which resulted in the final signal strength that was used for all images. For every timepoint cells of 3 biological independent repetition were quantified.

[000215] Immunofluorescence (IF). Cells were grown, harvested and treated as described in the FISH experiment. After permeabilization cells were blocked in ABB (0.1 M Tris pH 9.0, 0.2 M NaCI, 5 % FCS, 0,3 % Tween, 500 pg/ml tRNA) and incubated for 1 h at 37 °C, followed by ABB with the addition of 1/200 pl of the J2-antibody (1 pg/pl) from Scicons (Schonborn et al., 1991) and 0,2 % Triton for 2 h at 37 °C. Subsequently, cells were washed with 0.5 % Triton in lxPBS for 15min, twice with lx PBS for 15 min and finally with ABB for 30 min. The secondary Cy3 conjugated anti-mouse antibody in ABB (1:200) was then incubated for lh at room temperature. Thereafter the cells were washed with 0.5 % Tween in lx PBS for 10 min and twice in lx PBS for 10 min. Nuclei were stained with DAPI (Sigma) and mounting was carried out as described in the FISH experiment.

[000216] GFP-microscopy. Cells were grown in glucose (2 %) containing medium until early log phase (0,5 x 10^A7 cells/ml), washed once with 1 ml sterile H₂O, transferred into galactose (2 %) containing medium and grown for 6 h. Next, cells were fixed with 4 % formaldehyde for 1 min at room temperature and washed twice with 1 ml P-Solution (0.1 M potassium phosphate buffer pH 6.5, 1.2 M sorbitol) before adding 20 pl on a polylysine-coated slide for 15 min at room temperature. Permeabilization, DNA staining, microscopy and quantification was carried out as described in the FISH experiment.

[000217] J2-RNA co-immunoprecipitation experiments (RIP). All yeast strains were grown to mid log phase (2xl0⁷ cells/ml). For the RNA-co-IP (RIP) experiment depicted in FIG. 6B, cells were shifted to 37 °C for 2 h. Afterwards the cells were UV-crosslinked at 254 nm for 7 min, harvested and lysed in RIP buffer (25 mM Tris HCI pH 7.5, 150 mM NaCI, 2 mM MgCI₂0.5 % (v/v) Triton X-100, 0.2 mM PMSF, 0.5 mM DTT, 10 U RiboLock™ RNase Inhibitor (Thermo Fisher Scientific) and protease inhibitor (Roche)) by using the FastPrep®-24 machine (MP Biomedicals) with the interval 3-times for 30 sec at 5.5 m/s. After centrifugation, 30 pl of the supernatant was taken for input control and the rest was either incubated with or without 3 pl of the J2-antibody (1 pg/pl) (Schonborn et al., 1991) from Scicons for 30 min at 4 °C. After the first incubation the lysates were transferred to prewashed G-sepharose beads and incubated for another 90 min at 4 °C. The beads were then washed five times with RIP buffer (0,25 % Triton). For FIG. 4A the supernatant was removed and SDS loading dye (125 mM Tris pH 6.8, 2 % SDS, 10 % glycerol, 5 % 2-mercaptoethanol, bromphenolblue) was added. Subsequently, samples were incubated at 95 °C for 5 min and loaded onto an SDS-gel followed by western blotting. For FIG. 6A and FIG. 6B the RNA was purified from the lysates and the eluates via Trizol-chloroform (Ambion® RNA by Life technologies™) extraction followed by strand specific cDNA- synthesis and qPCR.

[000218] Protein isolation and purification. Transformed Rosetta 2 E. coll cells were grown in 200 ml LB medium with ampicillin (100 pg/ml) and chloramphenicol (25 pg/ml) overnight, diluted to ODgoo = 0.1 in 1200 ml terrific both media (28,8 g yeast extract, 24 g Trypton, 9 ml 50% glycerin, 17mM KH2PO4, 72 mM K2HPO4) and lOOpg/ml ampicillin. The diluted cells were incubated at 32°C and 130 rpm for 3 hours, followed by 37 °C and 130 rpm for 1 hour. For protein induction, 1.2 ml of 1 M IPTG was added and the culture was further incubated at 16 °C and 130 rpm overnight. After induction, cells were washed in 200 ml IMAC loading buffer (50 mM Na^PC , 500 mM NaCI, 10 mM Imidazol, pH 7.8) and finally resuspended in 75 ml IMAC loading buffer with Roche complete Protease inhibitor (1 tablet/50 ml). Cells were lysed by using a microfluidizer with the setting 3-times at 700 bar. Thereafter, the lysate was centrifuged at 15,000 x g for 90 min. Cleared lysate was loaded onto a 5 ml HisFF column and subsequently washed with IMAC exchange buffer, then 1 M LiCI, again with IMAC exchange buffer and finally with IMAC loading buffer. The proteins were eluted with IMAC elution buffer (50 mM NaH₂PO₄, 500 mM NaCI, 400 mM Imidazol, pH 7.8) and dialysed against heparin base buffer (40 mM HEPES KOH, 100 mM KCI, pH 7.5) overnight. After dialysis the eluate was loaded onto a heparin column and again eluted with heparin elution buffer (40 mM HEPES-KOH, 100 mM KCI, 2 M NaCI, pH 7.5). Finally, the eluate was dialyzed in dialysis buffer (30 mM HEPES-KOH, 160 mM KCI, pH 7.6) for 2 days. Protein concentration was determined by measuring the OD at 280nm.

[000219] Transfection of human cells. HCT116 cells were transfected to express specific antisense RNAs. For this, cells were seeded in a 6-well plate for several hours to around 70% confluency. For transfection 2 pg plasmid DNA was mixed with 6 pl ScreenFect A (ScreenFect) and 60 pl dilution buffer, incubated for 20 min and added to the cells after exchanging the media to media without antibiotics. After overnight incubation the media was exchanged again to media containing penicillin/streptomycin and transfected cell cultivated for 24 hours at 37°C 5% CO2. Cells were collected by adding PBS/EDTA and half of the pellet was used for RNA isolation and half as a protein lysate.

[000220] J2 hybridization in human cells. J2 immounfluorescence was carried out to localize dsRNA in cells. (HCT116 cells were cultivated to 90% confluency on 10 mm microscope slides and the cell cycle arrested by a double thymidine block. Thymidine was washed out and cell cycle released for 1 hour prior to 100 nM or 400 nm APH treatment for 3h. )(RPE and cancer cells were cultivated to 90% confluency on 10 mm microscope slides). Cells were fixed in 2% PFA containing PBS for 10 min at RT and subsequently for 10 min in 100% MeOH at -20°C. Cells were washed with lx PBS and permeabilized with 0.5% triton containing PBS for 5 min. Unspecific binding was prevented by incubating cells for 5 min in 5% FCS and 500pg/ml yeast tRNA containing PBS. Microscope slides were placed face down in a drop containing J2 antibody (1:400) in 2% FCS 0.2% tween in PBS and incubated overnight at 4°C. Slides were washed with 0.5% tween containing PBS and three times in lx PBS for each 5 min. Subsequently slides were put face down into a drop of Cy3 labeled secondary antibody (1:400) in 2% FCS containing PBS for 2h at RT. Microscope slides were washed once in lx PBS and DNA stained with DAPI (1:8000). Microscope slides were washed as described before and dried for at least lh prior to sealing slides face down in a drop of mountening medium.

[000221] Lysis and preparation of human cells for western blot and quantification. Protein lysates of HCT116 were prepared to quantify protein levels of respective target proteins via Western blot. Therefore, HCT116 cells were transfected with the respective antisense plasmids and grown in a 6-well plate. 48h after transfection cells were collected in PBS/EDTA and pelleted via centrifugation at 4000 rpm for 5 min at 4°C. Half of the cells were mixed with 50 pl Boehringer buffer (50 mM Tris- HCI pH 7.4, 150 mM NaCI, 5 mM EGTA pH 8.0, 5 mM EDTA pH 8.0, 1% NP40, 0.1% SDS) containing 0.1V phosphatase inhibitor (PhosSTOP, Roche) and 0.04V protease inhibitor (complete protease inhibitor cocktail, Roche) and cells lysed for 25 min on ice. Lysate was spun down for 25 min at 13.000 rpm at 4°C and the supernatant was transferred into a new tube. Protein concentration was quantified spectrophotometric using the DC Protein Assay (BioRad). 40 pg used for WB ...

[000222] Electrophoretic mobility shift assay (EMSA). Either ordered FAB or CY3 labeled RNAs (Sigma Aldrich) were used. Every RNA had the equal amount of C, G, T and A (FIG. 11). dsRNAs were formed by incubating 20 pM of the labeled and 20,5 pM of the complementary non labeled RNA in dialysis buffer (30 mM HEPES-KOH, 160 mM, KCI pH 7.6) in a total volume of 100 pl at 65 °C for 5 min and immediate subsequent cool down on ice. dsRNAs or ssRNAs and Mex67-Mtr2 were incubated in the given molarity with 2 pl Ribolock™ RNase Inhibitor (Thermo Fisher Scientific) in dialysis buffer resulting at a final volume of 20 pl at 30 °C for 15min. For the competition assay the competitor RNA was added after the first incubation and further incubated at 30 °C for 15 min. Finally, a 6x loading dye (10 mM Tris pH 7.6, 60 % glycerol, 60 mM EDTA, 0.03% bromophenol blue) was added and the samples were loaded onto a 0.5 % agarose gel with lx TAE (40 mM Tris, 1 mM EDTA, 0,1 % Acidic acid) pH 9.5 running in lx TAE pH 9.5. Complexes were separated by running the gel at 300 V and 4 °C for 40 min. In gel detection was carried out with the Fusion FX7 Edge 18.06c (Vilber) using the filter F-595 YR and Epi-Light module C530 or filter F-710 and Epi-Light module C640 together with the Evolution-Capt. Edge Software.

[000223] Cytoplasmic fractionation. For detection of RNAs in the cytoplasm, cells were grown to mid log-phase (2xl0⁷ cells/ml), washed once with 1ml YPD/ 1 M Sorbitol/ 2 mM DTT and resuspended in YPD/ 1 M Sorbitol/ 1 mM DTT with the addition of zymolyase (lOOmg/ml) to spheroplast cells. Prior to cytoplasmic fractionation, 200 pl of cell suspension were taken for total lysate control. For FIG. 1A and associated, after spheroblasting, cells were diluted in 50 ml YPD/ 1 M Sorbitol for 30 min at 25 °C before they were shifted to 37 °C for 1 h. After shifting, 10 ml were taken for total cell lysation. Next, cells were cooled down on ice and centrifuged for 5 min at 2 krpm. For cytoplasmic fractionation, the cell pellets were resuspended in 500 pl Ficoll buffer (18 % Ficoll 400, 10 mM HEPES pH 6.0) and cells were lysed by addition of 1 ml buffer A (50 mM NaCI, 1 mM MgCL, 10 mM HEPES pH 6.0) and lpl Ribolock™ RNase Inhibitor (Thermo Fisher Scientific). The suspension was vortexed and centrifuged for 10 min at 2 krpm. The resulting supernatant refelects the cytoplasmic fraction. To verify correct fractionation, samples were analyzed in western blots for the presence of the cytoplasmic Zwfl and the nucleolar Nopl proteins. RNA was isolated using the Nucleo-Spin RNA Kit (Macherey and Nagel). [000224] Export release assay. The mex67-5xpol-l RPS3-GFP strain was grown to mid log phase (2xl0⁷ cells/ml) and shifted to 37 °C for 2 h. Cells were harvested either directly after shifting (0 min) or after shifting them back to 25 °C for 5 min, 10 min, 15 min, 30 min and 60 min. The cell pellets were frozen in liquid nitrogen and subsequent RIP experiments were carried out as described in J2- RNA co-immunoprecipitation experiments with the exception that GFP Trap beads were used and no antibody was added. After the final washing step, the beads were split in half for RNA isolation with Trizol and subsequent qPCRs and for SDS-page and western blot analysis. For qPCR measurements the single stranded RNAs RPS17A, RPS6A and TDH1 and the dsRNA FRE5, HPF1 and PRY3 were analyzed. dsRNA targets were chosen based on three criteria: The asRNA had a higher RPKM then the sense RNA, they were identified as dsRNA in RNAi seq experiment (Wery et al., 2016) and enriched after J2 pulldown (FIG. 6A).

[000225] Strand specific cDNA synthesis. To exclusively measure either mRNA or asRNA in qPCR, gene specific primers were used in cDNA synthesis (Nippon genetics). Additionally, actinomycin D was added together with the reverse transcriptase as it prevents unspecific transcription from DNA and thereby secures strand specific transcription as reported previously (Xie et al., 2019; Perocchi et al., 2007).

[000226] Drop dilution analysis. Cells were grown to log phase (2xl0⁷ cells/ml) and diluted to 1x10^s cells/ml. 10-fold serial dilutions to lxlO³ cells/ml were prepared and 8 pl of each dilution was spotted onto selective plates. The plates were incubated for 3 days at the indicated temperatures and conditions. Pictures were taken after 2 or 3 days with the Intelli Scan 1600 (Quanto technology) and the SilverFast Ai program.

[000227] RNA-sequencing. The sequencing of RNA samples was conducted at the Microarray and Deep-Sequencing Facility Gottingen (Transcriptome and Genome Analysis Laboratory, TAL). Samples were prepared with the "TruSeq RNA Sample Prep Kit v2" according to the manufacturer's protocol (Illumina). Single read (50 bp) sequencing was conducted using a HiSeq 4000 (Illumina). Fluorescence images were transformed to BCL files with the Illumina BaseCaller software and samples were demultiplexed to FASTQ files with bcl2fastq (version 2.17). Sequences were aligned to the genome reference sequence of Saccharomyces cerevisiae (sacCer3, obtained from UCSC, https://hgdownload.cse.ucsc.edu/goldenPath/sacCer3/bigZips/) using the STAR software (Dobin et al., 2013; version 2.5) allowing for 2 mismatches. Subsequently, abundance measurement of reads overlapping with exons or introns was conducted with featurecounts (Liao et al., 2014), subread version 1.5.0-pl, Ensembl (EF4.68) supplemented with the coordinates of UTRs, CUTs and SUTs (Granovskaia et al., 2010; Xu et al., 2009; Yassour et al., 2010) and Xrnl-sensitive unstable transcripts (van Dijk et al., 2011; Tuck and Tollervey, 2013). Data was processed in the R/Bioconductor environment (www.bioconductor.org, R version 3.6.1) using the DESeq2 package (Love et al., 1998); version 1.24.0). Overlapping features respectively sense and antisense pairs were identified with BEDTools intersect (Quinlan and Hall, 2010) requiring overlaps to occur on the opposite strand with a minimum overlap of 0.5. The sequencing data and abundance measurement files have been submitted to the NCBI Gene Expression Omnibus (GEO) database.

[000228] J2-RIP for RNA-seq

[000229] All yeast strains were grown to mid log phase (2xl0⁷ cells/ml). total RNA was isolated with the TRIzol™-reagent. After the first ethanol precipitation, a DNasel treatment was conducted followed by a second precipitation overnight. The obtained RNA was eluted in RNase free water. 90 pg of RNA and 3 pg of the J2-antibody were then incubated in 500 pl PBST for 120 min at 4 °C (lx PBS, 0.5 % Tween-20). After the first incubation, the RNA-antibody mix was transferred to prewashed G- sepharose beads and incubated for another 120 min at 4 °C. The beads were centrifuged for 1 min at

4 krpm and 4 °C. The supernatant was transferred a second time together with 3 pg of the J2 antibody to freshly washed beads and incubated for 120 min. Subsequently, these beads were centrifuged and the supernatant was used as the unbound fraction. The beads from the first incubation were washed

5 times with 1 ml PBST. Between each step, the beads were centrifuged for 1 min at 4 krpm and 4 °C. Finally, the RNA was purified from the unbound fraction and from the eluates via TRIzol-chloroform (Ambion® RNA by Life technologies™) extraction and forwarded to RNA-sequencing. We repeated the experiment three times, which showed a high reproducibility

[000230] J2 dot-blot Cells were grown to log-phase and shifted, if necessary, as indicated. RNA isolation was carried out with TRIzol™-reagent. 1 pg of the isolated RNA was applied onto a Nylon membrane, which was blocked in PBST (lx PBS, 1% Tween-20), 0.05 mg/ml ssDNA, and 5% (w/v) nonfat dried milk before. Subsequently, the J2-antibody (anti-dsRNA in PBST, 1:5000) was added and incubated for 2 h at room temperature. Finally, two washing steps with PBST, each for 15 min at room temperature, were carried out before the HRP-coupled goat anti-mouse secondary antibody was added in PBST for 1 h. Finally, the membrane was washed again three times with PBST for 10 min at room temperature, before the ECL detection was carried out with the Fusion FX7 Edge 18.06c (Vilber). Quantification was finalized with the analysis software Bio-ID from Vilber Lourmat.

[000231] Accession number. Cytoplasmic fractionation-Seq data have been deposited at the NCBI gene expression omnibus (GEO; www.ncbi.nlm.nih.gov/geo/) with the GEO accession number GSE188455.

[000232] Quantification. All experiments shown in this work were carried out at least three times independently. Error bars represent the standard deviation. P values were calculated using a two-tailed, two-sample unequal variance t-test. P values are indicated as follows: ***p < 0.001, **p < 0.01, *p < 0.05. For quantification of cells with the displayed phenotypes (FIG. 3B) 30 cells were counted for each experiment. REFERENCES

Azimi, M., Bulat, E., Weis, K. & Mofrad, M. R. An agent-based model for mRNA export through the nuclear pore complex. Molecular biology of the cell 25, 3643-3653, doi:10.1091/mbc.E14-06-1065 (2014).

Baierlein, C. et al. Monosome Formation during Translation Initiation Requires the Serine/Arginine- Rich Protein Npl3. Mol. Cell. Biol. 33, 4811 (2013).

Becker, D. et al. Nuclear Pre-snRNA Export Is an Essential Quality Assurance Mechanism for Functional Spliceosomes. Cell reports 27 , 3199-3214 e3193, doi:10.1016/j.celrep.2019.05.031 (2019).

Braun, I. C., Herold, A., Rode, M., Conti, E. & Izaurralde, E. Overexpression of TAP/pl5 heterodimers bypasses nuclear retention and stimulates nuclear mRNA export. The Journal of biological chemistry 276, 20536-20543 (2001).

BRUNE, C., MUNCHEL, S. E., FISCHER, N., PO DTE LEJ NIKOV, A. V. & WEIS, K. Yeast poly(A)-binding protein Pabl shuttles between the nucleus and the cytoplasm and functions in mRNA export. RNA 11, 517-531 (2005).

Chan, L. Y., Mugler, C. F., Heinrich, S., Vallotton, P. & Weis, K. Non-invasive measurement of mRNA decay reveals translation initiation as the major determinant of mRNA stability. Elife 7, doi:10.7554/eLife.32536 (2018).

Cloutier, S. C., Ma, W. K., Nguyen, L. T. & Tran, E. J. The DEAD-box RNA helicase Dbp2 connects RNA quality control with repression of aberrant transcription. Journal of Biological Chemistry 287, 26155- 26166, doi:10.1074/jbc.M112.383075 (2012). de Andres-Pablo, A., Morillon, A. & Wery, M. LncRNAs, lost in translation or licence to regulate? Current genetics 63, 29-33, doi:10.1007/s00294-016-0615-l (2017).

Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15-21, doi:10.1093/bioinformatics/bts635 (2013).

Drinnenberg, I. A. et al. RNAi in Budding Yeast. Science (80-. ). 326, 544-550 (2009).

Gadal, O. et al. Nuclear Export of 60S Ribosomal Subunits Depends on Xpolp and Requires a Nuclear Export Sequence-Containing Factor, Nmd3p, That Associates with the Large Subunit Protein RpIlOp. Mol. Cell. Biol. 21, 3405 (2001).

Granovskaia, M. V. et al. High-resolution transcription atlas of the mitotic cell cycle in budding yeast. Genome Biol 11, R24, doi:10.1186/gb-2010-ll-3-r24 (2010).

Grosse, S., Lu, Y.-Y., Coban, L, Neumann, B. & Krebber, H. Nuclear SR-protein mediated mRNA quality control is continued in cytoplasmic nonsense-mediated decay. https://doi.org/10.1080/15476286.2020.1851506 18, 1390-1407 (2021).

Hackmann, A., Gross, T., Baierlein, C. & Krebber, H. The mRNA export factor Npl3 mediates the nuclear export of large ribosomal subunits. EM BO reports 12, 1024-1031, doi:10.1038/embor.2011.155 (2011).

Hedges, J., West, M. & Johnson, A. W. Release of the export adapter, Nmd3p, from the 60S ribosomal subunit requires RpIlOp and the cytoplasmic GTPase Lsglp. The EMBO journal 24, 567-579 (2005). Ho, J. H., Kallstrom, G. & Johnson, A. W. Nmd3p is a Crmlp-dependent adapter protein for nuclear export of the large ribosomal subunit. The Journal of cell biology 151, 1057-1066 (2000).

Lahtvee, P. J., Kumar, R., Hallstrom, B. M. & Nielsen, J. Adaptation to different types of stress converge on mitochondrial metabolism. Molecular biology of the cell 27, 2505-2514, doi:10.1091/mbc.E16-03-0187 (2016).

Liao, Y., Smyth, G. K. & Shi, W. featurecounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923-930, doi:10.1093/bioinformatics/btt656 (2014).

Linder, P. & Jankowsky, E. From unwinding to clamping - the DEAD box RNA helicase family. Nature reviews 12, 505-516, doi:10.1038/nrm3154 (2011).

Love, D. C., Sweitzer, T. D. & Hanover, J. A. Reconstitution of HIV-1 rev nuclear export: independent requirements for nuclear import and export. Proceedings of the National Academy of Sciences of the United States of America 95, 10608-10613 (1998).

Ma, W. K., Cloutier, S. C. & Tran, E. J. The DEAD-box protein Dbp2 functions with the RNA-binding protein Yral to promote mRNP assembly. Journal of molecular biology 425, 3824-3838, doi:10.1016/j.jmb.2013.05.016 (2013).

Milkereit, P. et al. A Noc Complex Specifically Involved in the Formation and Nuclear Export of Ribosomal 40 S Subunits *. J. Biol. Chem. 278, 4072-4081 (2003).

Perocchi, F., Xu, Z., Clauder-Munster, S. & Steinmetz, L. M. Antisense artifacts in transcriptome microarray experiments are resolved by actinomycin D. Nucleic acids research 35, el28, doi:10.1093/nar/gkm683 (2007).

Putnam, A. A. & Jankowsky, E. DEAD-box helicases as integrators of RNA, nucleotide and protein binding. Biochimica et biophysica acta 1829, 884-893, doi:10.1016/j.bbagrm.2013.02.002 (2013).

Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841-842, doi:10.1093/bioinformatics/btq033 (2010).

Schein, A., Zucchelli, S., Kauppinen, S., Gustincich, S. & Carninci, P. Identification of antisense long noncoding RNAs that function as SINEUPs in human cells. Sci Rep 6, 33605, doi:10.1038/srep33605 (2016).

Schonborn, J. et al. Monoclonal antibodies to double-stranded RNA as probes of RNA structure in crude nucleic acid extracts. Nucleic acids research 19, 2993-3000, doi:10.1093/nar/19.11.2993 (1991).

Segref, A. et al. Mex67p, a novel factor for nuclear mRNA export, binds to both poly(A)⁺ RNA and nuclear pores. The EMBO journal 16, 3256-3271 (1997).

Simone, R. et al. MIR-NATs repress MAPT translation and aid proteostasis in neurodegeneration. Nature 594, 117-123, doi:10.1038/s41586-021-03556-6 (2021).

Soheilypour, M. & Mofrad, M. R. K. Quality control of mRNAs at the entry of the nuclear pore: Cooperation in a complex molecular system. Nucleus 9, 202-211, doi:10.1080/19491034.2018.1439304 (2018).

Tuck, A. C. & Tollervey, D. A Transcriptome-wide Atlas of RNP Composition Reveals Diverse Classes of mRNAs and IncRNAs. Cell 154, 996-1009, doi:10.1016/j. cell.2013.07.047 (2013). Tudek, A. et al. A Nuclear Export Block Triggers the Decay of Newly Synthesized Polyadenylated RNA. Cell reports 24, 2457-2467 e2457, doi:10.1016/j.celrep.2018.07.103 (2018). van Dijk, E. L. et al. XUTs are a class of Xrnl-sensitive antisense regulatory non-coding RNA in yeast. Nature 475, 114-117, doi:10.1038/naturel0118 (2011).

Venkatesh, S., Li, H., Gogol, M. M. & Workman, J. L. Selective suppression of antisense transcription by Set2-mediated H3K36 methylation. Nature communications 7, 13610, doi:10.1038/ncommsl3610 (2016).

Villegas, V. E. & Zaphiropoulos, P. G. Neighboring gene regulation by antisense long non-coding RNAs. International journal of molecular sciences 16, 3251-3266, doi:10.3390/ijmsl6023251 (2015).

Wery, M. et al. Nonsense-Mediated Decay Restricts LncRNA Levels in Yeast Unless Blocked by Double-Stranded RNA Structure. Molecular cell 61, 379-392, doi:10.1016/j.molcel.2015.12.020 (2016).

Wu, H., Becker, D. & Krebber, H. Telomerase RNA TLC1 Shuttling to the Cytoplasm Requires mRNA Export Factors and Is Important for Telomere Maintenance. Cell reports 8, 1630-1638, doi:10.1016/j.celrep.2014.08.021 (2014).

Xie, B. et al. The anti-cancer drug 5-fluorouracil affects cell cycle regulators and potential regulatory long non-coding RNAs in yeast. RNA Biol 16, 727-741, doi:10.1080/15476286.2019.1581596 (2019).

Xu, Z. et al. Bidirectional promoters generate pervasive transcription in yeast. Nature 457, 1033- 1037, doi:10.1038/nature07728 (2009).

Yassour, M. et al. Strand-specific RNA sequencing reveals extensive regulated long antisense transcripts that are conserved across yeast species. Genome Biol 11, R87, doi:10.1186/gb-2010-ll-8- r87 (2010).

Zander, G. & Krebber, H. Quick or quality? How mRNA escapes nuclear quality control during stress. RNA Biol 14, 1642-1648, doi:10.1080/15476286.2017.1345835 (2017).

Zander, G. et al. mRNA quality control is bypassed for immediate export of stress-responsive transcripts. Nature 540, 593-596, doi:10.1038/nature20572 (2016).

Zenklusen, D. & Stutz, F. Nuclear export of mRNA. FEBS Lett 498, 150-156. (2001).

Zenklusen, D., Vinciguerra, P., Strahm, Y. & Stutz, F. The yeast hnRNP-Like proteins Yralp and Yra2p participate in mRNA export through interaction with Mex67p. Molecular and cellular biology 21, 4219-4232. (2001).

Claims

1. An antisense long non-encoding RNA (IncRNA) comprising a first region and a second region, wherein the first region is complementary to a portion of a target mRNA, and wherein the second region is not complementary to the target mRNA.

2. The antisense IncRNA according to claim 1, wherein the first region is complementary to the 5' end of the target mRNA.

3. The antisense IncRNA according to claim 1 or 2, wherein the second region is 3' of the first region.

4. The antisense IncRNA according to any of claims 1 to 3, wherein the antisense IncRNA is capable of forming a partial double strand complex with the mRNA thereby promoting or inhibiting the nuclear export of the mRNA.

5. The antisense IncRNA according to any one of claims 1 to 4, wherein the overlap of the first region with the 5' end of the target mRNA has a length of between 1 and thousands of nucleotides.

6. The antisense IncRNA according to any of claims 1 to 5, wherein the antisense IncRNA has a length of at least 30 nucleotides, at least 50 nucleotides, at least 100 nucleotides, at least 150 nucleotides, or at least 200 nucleotides.

7. The antisense IncRNA according to any one of claims 1 to 6, wherein the antisense IncRNA is capable of forming, with the target mRNA, a complex that is optimized for binding double-strand formation mediating proteins and/or binding export mediating proteins, preferably to Dbp2, Yral, Mex67, Mtr2, or to its human homologues, comprising DDX5, ALY/REV/NXF2, TAP, pl5.

8. The antisense IncRNA according to any one of claims 1 to 7, wherein the second region has a length of between 1 and thousands of nucleotides.

9. The antisense IncRNA according to any one of claims 1 to 8, wherein the antisense IncRNA further comprises a third region that is 5' of the first region and which is not complementary to the target mRNA.

10. The antisense IncRNA according to claim 9, wherein the third region comprises between 1 and thousands of nucleotides.

11. A biological complex comprising an antisense IncRNA according to any one of claims 1 to 10 and the target mRNA, wherein the antisense IncRNA and mRNA form a partial double strand complex.

12. A biological complex comprising an antisense IncRNA according to claim 8 and the target mRNA, wherein the antisense IncRNA and mRNA form a partial double strand complex.

13. A biological complex comprising an antisense IncRNA according to any one of claims 9 or 10 and the target mRNA, wherein the antisense IncRNA and mRNA form a partial double strand complex.

14. An expression vector comprising a nucleic acid sequence encoding an antisense IncRNA according to any one of claims 1 to 10.

15. An expression vector comprising a nucleic acid sequence encoding an antisense IncRNA according to claim 8.

16. An expression vector comprising a nucleic acid sequence encoding an antisense IncRNA according to any one of claims 9 or 10.

17. An expression vector according to claim 14, further comprising a nucleic acid sequence encoding a target mRNA of the antisense IncRNA.

18. An expression vector according to claim 15, further comprising a nucleic acid sequence encoding a target mRNA of the antisense IncRNA.

19. Use of the antisense IncRNA according to claim 8, the biological complex according to claim 12, or the expression vector according to claim 15 or claim 18 for increasing export of the target mRNA from the nucleus of a eukaryotic cell via a partial double strand complex with the antisense IncRNA.

20. Use of the antisense IncRNA according to claim 8, the biological complex according to claim 12, or the expression vector according to claim 15 or claim 18 for increasing expression of the target mRNA in a eukaryotic cell by increasing nuclear export of the target mRNA via a partial double strand complex with the antisense IncRNA.

21. Use of the antisense IncRNA according to any one of claims 9 or 10, the biological complex according to claim 13, the expression vector according to claim 16 for decreasing export of the target mRNA from the nucleus of a eukaryotic cell via a partial double strand complex with the antisense IncRNA.

22. Use of the antisense IncRNA according to any one of claims 9 or 10, the biological complex according to claim 13, or the expression vector according to claim 16 for decreasing expression of the target mRNA in a eukaryotic cell by decreasing the nuclear export of the target mRNA via a partial double strand complex with the antisense IncRNA.

23. A method of increasing export of a target mRNA from the nucleus of a eukaryotic cell, the method comprising transfecting a cell with the antisense IncRNA according to claim 8, the biological complex according to claim 12, or the expression vector according to claim 15 or claim 18.

24. A method of increasing expression of a target mRNA in a eukaryotic cell, the method comprising transfecting a cell with the antisense IncRNA according to claim 8, the biological complex according to claim 12, or the expression vector according to claim

15 or claim 18.

25. A method of decreasing export of a target mRNA from the nucleus of a eukaryotic cell, the method comprising transfecting a cell with the antisense IncRNA according to any one of claims 9 or 10, the biological complex according to claim 13, or the expression vector according to claim 16.

26. A method of decreasing expression of a target mRNA in a eukaryotic cell, the method comprising transfecting a cell with the antisense IncRNA according to any one of claims 9 or 10, the biological complex according to claim 13, or the expression vector according to claim 16.

27. The method of any of claims 23-26, wherein the method is an in vitro method.

28. A cell transfected with the antisense IncRNA according to any one of claims 1 to 10, the biological complex according to claim 11, or the expression vector according to claim 14 or claim 17.

29. A cell transfected with the antisense IncRNA according to claim 8, the biological complex according to claim 12, or the expression vector according to claim 15 or claim 18.

30. A cell transfected with the antisense IncRNA according to any one of claims 9 or 10, the biological complex according to claim 13, or the expression vector according to claim 16.

31. A conjugate comprising the antisense IncRNA according to any one of claims 1 to 10, the biological complex according to claim 11, or the expression vector according to claim 14 or claim 17, and a cell penetrating peptide.

32. A conjugate comprising the antisense IncRNA according to claim 8, the biological complex according to claim 12, or the expression vector according to claim 15 or claim 18, and a cell penetrating peptide.

33. A conjugate comprising the antisense IncRNA according to any one of claims 9 or 10, the biological complex according to claim 13, or the expression vector according to claim 16, and a cell penetrating peptide.

34. A pharmaceutical composition comprising the antisense IncRNA according to any one of claims 1 to 10, the biological complex according to claim 11, the expression vector according to claim 4 or claim 17, or the conjugate according to claim 31.

35. A pharmaceutical composition comprising the antisense IncRNA according to claim 8, the biological complex according to claim 12, the expression vector according to claim 15 or claim 18, or the conjugate according to claim 32.

36. A pharmaceutical composition comprising the antisense IncRNA according to any one of claims 9 or 10, the biological complex according to claim 13, the expression vector according to claim 16, or the conjugate according to claim 33.

37. The antisense IncRNA according to any one of claims 1 to 10, the biological complex according to claim 11, the expression vector according to claim 14 or claim 17, or the conjugate according to claim 31 for use as a medicament.

38. The antisense IncRNA according to claim 8, the biological complex o according to claim 12, the expression vector according to claim 15 or claim 18, or the conjugate according to claim 32 for use as a medicament.

39. The antisense IncRNA according to any one of claims 9 or 10, the biological complex of claim 13, the expression vector according to claim 16, or the conjugate according to claim 33 for use as a medicament.

40. The antisense IncRNA according to any one of claims 1 to 10, the biological complex according to claim 11, the expression vector according to claim 14 or claim 17, or the conjugate according to claim 31 for use in the treatment or prevention of a disease, preferably by enhancing or decreasing nuclear export of a therapeutically relevant mRNA.

41. The antisense IncRNA for use according to claim 40, for use in the treatment or prevention of cancer, preferably by enhancing nuclear export of a tumor suppressor mRNA and/or inhibiting the nuclear export of oncogene mRNA.

42. The antisense IncRNA according to claim 8, the biological complex according to claim 12, the expression vector according to claim 15 or claim 18, or the conjugate according to claim 32 for use in the treatment or prevention of a disease, preferably by enhancing nuclear export of a therapeutic mRNA, the increased expression of which has a therapeutic benefit.

43. The antisense IncRNA for the use according to claim 42, for use in the treatment or prevention of cancer, preferably by enhancing nuclear export of a tumor suppressor mRNA.

44. The antisense IncRNA according to any one of claims 9 or 10, the biological complex according to claim 13, the expression vector according to claim 16, or the conjugate according to claim 33 for use in the treatment or prevention of a disease, preferably by inhibiting the nuclear export of a therapeutically relevant mRNA, the expression of which is causally linked to the disease.

45. The antisense IncRNA for the use according to claim 44, for use in the treatment or prevention of cancer, preferably by inhibiting nuclear expression of oncogene mRNA.

46. A method of treatment comprising administering the antisense IncRNA according to any one of claims 1 to 10, the biological complex according to claim 11, the expression vector according to claim 14 or claim 17, or the conjugate according to claim 31 to a subject in need thereof.

47. A method of treatment comprising administering the antisense IncRNA according to claim 8, the biological complex according to claim 12, the expression vector according to claim 15 or claim 18, or the conjugate according to claim 32 to a subject in need thereof.

48. A method of treatment comprising administering the antisense IncRNA according to any one of claims 9 or 10, the biological complex according to claim 13, the expression vector according to claim 16, or the conjugate according to claim 33 to a subject in need thereof.

49. Use of the antisense IncRNA according to claim 8, the biological complex according to claim 12, or the expression vector according to claim 15 or claim 18, or the conjugate according to claim 32 for increasing mRNA vaccine efficacy.

50. A method for enhancing the production of recombinant proteins, wherein the method comprises increasing the expression and/or export of a target mRNA from the nucleus of a host cell, comprising transfecting the host cell with the antisense IncRNA according to claim 8, or the biological complex according to claim 12, or the expression vector comprising the boost-RNA according to claim 15 or 18, wherein the target mRNA encodes the desired recombinant protein.

51. The antisense long non-encoding RNA (IncRNA) according to claims 1-10, wherein the first region has a length of between 20 and 3000 nucleotides.

52. The antisense long non-encoding RNA (IncRNA) according to claims 1-10, wherein the second region has a length of between 1 and 5000 nucleotides.

53. The antisense long non-encoding RNA (IncRNA) according to claims 9-10, wherein the third region has a length of between 1 and 5000 nucleotides.