Artificial nucleic acid molecules

Abstract

The invention relates to an artificial nucleic acid molecule comprising at least one open reading frame and at least one 3′-untranslated region element (3′-UTR element) comprising a nucleic acid sequence which is derived from the 3′-UTR of a FIG4 gene or from a variant of the 3′-UTR of a FIG4 gene. The invention further relates to the use of such an artificial nucleic acid molecule in gene therapy and/or genetic vaccination. Furthermore, the invention relates to the use of a 3′-UTR element comprising a nucleic acid sequence which is derived from the 3′-UTR of a FIG4 gene or from a variant of the 3′-UTR of a FIG4 gene for the stabilization and/or prolongation of protein expression from a nucleic acid sequence comprising such 3′-UTR element.

1 . An artificial nucleic acid molecule comprising
 a. at least one open reading frame (ORF); and   b. at least one 3′-untranslated region element (3′-UTR element) comprising a nucleic acid sequence which is derived from the 3′-UTR of a FIG4 gene or from a variant of the 3′-UTR of a FIG4 gene.   
     
     
         2 . The artificial nucleic acid molecule according to  claim 1 , wherein the open reading frame (ORF) does not encode a reporter gene or is not derived from a reporter gene, wherein the reporter gene is preferably not selected from the group consisting of light emitting proteins, preferably not luciferase, fluorescent proteins, preferably not red, blue or green fluorescent proteins; enzymatic reporters; the lacZ gene from  E. coli  (beta-galactosidase); alkaline phosphatase; secreted embryonic alkaline phosphatase (SEAP); chloramphenicol acetyl transferase (CAT); hormones and cytokines. 
     
     
         3 . The artificial nucleic acid molecule according to  claim 1  or  2 , wherein the open reading frame (ORF) and the 3′-UTR element are heterologous to each other. 
     
     
         4 . The artificial nucleic acid molecule according to any one of  claims 1  to  3 , wherein the open reading frame (ORF) does not encode a FIG4 gene or is not derived from a FIG4 gene, preferably not an eukaryotic FIG4 gene, more preferably not a mammalian FIG4 gene, most preferably not a human FIG4 gene. 
     
     
         5 . The artificial nucleic acid molecule according to any one of  claims 1  to  4 , wherein the at least one 3′-UTR element stabilizes/prolongs protein production from said artificial nucleic acid molecule. 
     
     
         6 . The artificial nucleic acid molecule according to any one of  claims 1  to  5 , wherein the at least one 3′-UTR element comprises a nucleic acid sequence which is derived from the 3′-UTR of a vertebrate FIG4 gene or from a variant thereof, preferably from the 3′-UTR of a mammalian FIG4 gene or from a variant thereof, more preferably from the 3′-UTR of a primate FIG4 gene, in particular of the human FIG4 gene, or from a variant thereof, even more preferably from the 3′-UTR of the human FIG4 gene according to GenBank Accession number NM_014845.5 or from a variant thereof. 
     
     
         7 . The artificial nucleic acid molecule according to any one of  claims 1 - 6 , wherein the at least one 3′-UTR element comprises or consists of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the nucleic acid sequence according to SEQ ID No. 1 or SEQ ID No. 2, or wherein the at least one 3′-UTR element comprises or consists of a fragment of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the nucleic acid sequence according to SEQ ID No. 1 or SEQ ID No. 2. 
     
     
         8 . The artificial nucleic acid molecule according to  claim 7 , wherein the fragment exhibits a length of at least about 50 nucleotides, preferably of at least about 75 nucleotides, more preferably of at least about 100 nucleotides, even more preferably of at least about 125 nucleotides, most preferably of at least about 150 nucleotides. 
     
     
         9 . The artificial nucleic acid molecule according to any one of  claims 1 - 8 , wherein the at least one 3′-UTR element exhibits a length of at least about 50 nucleotides, preferably of at least about 75 nucleotides, more preferably of at least about 100 nucleotides, even more preferably of at least about 125 nucleotides, most preferably of at least about 150 nucleotides. 
     
     
         10 . The artificial nucleic acid molecule according to any one of  claims 1 - 9  further comprising
 c. a poly(A) sequence and/or a polyadenylation signal. 
 
     
     
         11 . The artificial nucleic acid molecule according to  claim 10 , wherein the poly(A) sequence or the polyadenylation signal is located 3′ of the 3′-UTR element. 
     
     
         12 . The artificial nucleic acid molecule according to  claim 10  or  11 , wherein the polyadenylation signal comprises the consensus sequence NN(U/T)ANA, with N=A or U, preferably AA(U/T)AAA or A(U/T)(U/T)AAA. 
     
     
         13 . The artificial nucleic acid molecule according to any one of  claims 10 - 12 , wherein the polyadenylation signal, preferably the consensus sequence NNUANA, is located less than about 50 nucleotides downstream of the 3′-end of the 3′-UTR element. 
     
     
         14 . The artificial nucleic acid molecule according to any one of  claims 10 - 13 , wherein the poly(A) sequence has a length of about 20 to about 300 adenine nucleotides, preferably of about 40 to about 200 adenine nucleotides, more preferably of about 50 to about 100 adenine nucleotides, even more preferably of about 60 to about 70 adenine nucleotides. 
     
     
         15 . The artificial nucleic acid molecule according to any one of  claims 1 - 14 , further comprising a 5′-cap structure, a poly(C) sequence, a histone stem-loop, and/or an IRES-motif. 
     
     
         16 . The artificial nucleic acid molecule according to any one of  claims 1 - 15 , wherein the histone stem-loop comprises a sequence according to SEQ ID NO: 5. 
     
     
         17 . The artificial nucleic acid molecule according to any one of  claims 1 - 16 , wherein the nucleic acid comprises an additional 5′-element, preferably a 5′-UTR, a promoter, or a 5′-UTR and a promoter containing-sequence. 
     
     
         18 . The artificial nucleic acid molecule according to  claim 17 , wherein the 5′-UTR is a 5′-TOP UTR. 
     
     
         19 . The artificial nucleic acid molecule according to any one of  claims 1 - 18 , wherein the artificial nucleic acid molecule, preferably the open reading frame, is at least partially G/C modified, preferably wherein the G/C content of the open reading frame is increased compared to the wild type open reading frame. 
     
     
         20 . The artificial nucleic acid molecule according to any one of  claims 1 - 19 , wherein the open reading frame comprises a codon-optimized region, preferably, wherein the open reading frame is codon-optimized. 
     
     
         21 . The artificial nucleic acid molecule according to any one of  claims 1 - 20 , which is an RNA, preferably an mRNA molecule. 
     
     
         22 . A vector comprising
 a. an open reading frame and/or a cloning site; and   b. at least one 3′-untranslated region element (3′-UTR element) comprising a nucleic acid sequence which is derived from the 3′-UTR of a FIG4 gene or from a variant of the 3′-UTR of a FIG4 gene.   
     
     
         23 . The vector according to  claim 22 , wherein the open reading frame (ORF) does not encode a reporter gene or is not derived from a reporter gene, wherein the reporter gene is preferably not selected from the group consisting of light emitting proteins, preferably not luciferase, fluorescent proteins, preferably not red, blue or green fluorescent proteins; enzymatic reporters; the lacZ gene from  E. coli  (beta-galactosidase); alkaline phosphatase; secreted embryonic alkaline phosphatase (SEAP); chloramphenicol acetyl transferase (CAT); hormones and cytokines. 
     
     
         24 . The vector according to  claim 22  or  23 , wherein the open reading frame (ORF) and the 3′-UTR element are heterologous to each other. 
     
     
         25 . The artificial nucleic acid molecule according to any one of  claims 22  to  24 , wherein the open reading frame (ORF) does not encode a FIG4 gene or is not derived from a FIG4 gene, preferably not an eukaryotic FIG4 gene, more preferably not a mammalian FIG4 gene, most preferably not a human FIG4 gene. 
     
     
         26 . The vector according to any one of  claims 22  to  25 , wherein the at least one 3′-UTR element comprises or consists of a nucleic acid sequence which is derived from the 3′-UTR of a vertebrate FIG4 gene or from a variant thereof, preferably from the 3′-UTR of a mammalian FIG4 gene or from a variant thereof, more preferably from the 3′-UTR of a primate FIG4 gene, in particular of the human FIG4 gene, or from a variant thereof, even more preferably from the 3′-UTR of the human FIG4 gene according to GenBank Accession number NM_014845.5 or from a variant thereof. 
     
     
         27 . The vector according to any one of  claims 22  to  26 , wherein the at least one 3′-UTR element comprises or consists of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the nucleic acid sequence according to SEQ ID No. 1 or SEQ ID No. 2, or wherein the at least one 3′-UTR element comprises or consists of a fragment of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the nucleic acid sequence according to SEQ ID No. 1 or SEQ ID No. 2. 
     
     
         28 . The vector according to  claim 27 , wherein the fragment exhibits a length of at least about 50 nucleotides, preferably of at least about 75 nucleotides, more preferably of at least about 100 nucleotides, even more preferably of at least about 125 nucleotides, most preferably of at least about 150 nucleotides. 
     
     
         29 . The vector according to any one of  claims 22 - 28 , wherein the at least one 3′-UTR element exhibits a length of at least about 50 nucleotides, preferably of at least about 75 nucleotides, more preferably of at least about 100 nucleotides, even more preferably of at least about 125 nucleotides, most preferably of at least about 150 nucleotides. 
     
     
         30 . The vector according to any one of  claims 22 - 29  further comprising
 c. a poly(A) sequence and/or a polyadenylation signal. 
 
     
     
         31 . The vector according to  claim 30 , wherein the poly(A) sequence or the polyadenylation signal is located 3′ of the 3′-UTR element. 
     
     
         32 . The vector according to  claim 30  or  31 , wherein the polyadenylation signal comprises the consensus sequence NN(U/T)ANA, with N=A or U, preferably AA(U/T)AAA or A(U/T)(U/T)AAA. 
     
     
         33 . The vector according to any one of  claims 30 - 32 , wherein the polyadenylation signal, preferably the consensus sequence NNUANA, is located less than about 50 nucleotides downstream of the 3′-end of the 3′-UTR element. 
     
     
         34 . The vector according to any one of  claims 30 - 33 , wherein the poly(A) sequence has a length of about 20 to about 300 adenine nucleotides, preferably of about 40 to about 200 adenine nucleotides, more preferably of about 50 to about 100 adenine nucleotides, more preferably of about 60 to about 70 adenine nucleotides. 
     
     
         35 . The vector according to any one of  claims 22 - 34 , further comprising a poly(C) sequence, a histone stem-loop, and/or an IRES-motif. 
     
     
         36 . The vector according to any one of  claims 22 - 35 , further comprising a 5′-element, preferably a 5′-UTR, a promoter, or a 5′-UTR5′-UTR and a promoter containing-sequence. 
     
     
         37 . The vector according to  claim 36 , wherein the 5′-UTR is a 5′-TOP UTR. 
     
     
         38 . The vector according to any one of  claims 22 - 37 , which is at least partially G/C modified, preferably wherein the open reading frame is at least partially G/C modified, preferably wherein the G/C content of the open reading frame is increased compared to the wild type open reading frame. 
     
     
         39 . The vector according to any one of  claims 22 - 38 , wherein the open reading frame comprises a codon-optimized region, preferably wherein the open reading frame is codon-optimized. 
     
     
         40 . The vector according to any one of  claims 22 - 39 , which is a DNA vector. 
     
     
         41 . The vector according to any one of  claims 22 - 40 , which is a plasmid vector or a viral vector, preferably a plasmid vector. 
     
     
         42 . The vector according to any one of  claims 22 - 41 , which comprises an artificial nucleic acid molecule according to any one of  claims 1 - 21 . 
     
     
         43 . The vector according to any one of  claims 22 - 42 , which is a circular molecule. 
     
     
         44 . The vector according to  claim 43 , wherein the poly(A) sequence, the poly(C) sequence, the histone stem loop or the 3′-UTR element of the coding strand is followed in 5′→3′ direction by a restriction site for linearization of the circular vector molecule. 
     
     
         45 . A cell comprising the artificial nucleic acid molecule according to any one of  claims 1 - 21  or the vector according to any one of  claims 22 - 44 . 
     
     
         46 . The cell according to  claim 45 , which is a mammalian cell. 
     
     
         47 . The cell according to  claim 45  or  46 , which is a cell of a mammalian subject, preferably an isolated cell of a mammalian subject, preferably of a human subject. 
     
     
         48 . A pharmaceutical composition comprising the artificial nucleic acid molecule according to any one of  claims 1 - 21 , the vector according to any one of  claims 22 - 44 , or the cell according to any one of  claims 45 - 47 . 
     
     
         49 . The pharmaceutical composition according to  claim 48 , further comprising one or more pharmaceutically acceptable vehicles, diluents and/or excipients and/or one or more adjuvants. 
     
     
         50 . The artificial nucleic acid molecule according to any one of  claims 1 - 21 , the vector according to any one of  claims 22 - 44 , the cell according to any one of  claims 45 - 47 , or the pharmaceutical composition according to  claim 48  or  49  for use as a medicament. 
     
     
         51 . The artificial nucleic acid molecule according to any one of  claims 1 - 21 , the vector according to any one of  claims 22 - 44 , the cell according to any one of  claims 45 - 47 , or the pharmaceutical composition according to  claim 48  or  49  for use as a vaccine or for use in gene therapy. 
     
     
         52 . A method for treating or preventing a disorder comprising administering the artificial nucleic acid molecule according to any one of  claims 1 - 21 , the vector according to any one of  claims 22 - 44 , the cell according to any one of  claims 45 - 47 , or the pharmaceutical composition according to  claim 48  or  49  to a subject in need thereof. 
     
     
         53 . A method of treating or preventing a disorder comprising transfection of a cell with an artificial nucleic acid molecule according to any one of  claims 1 - 21  or with the vector according to any one of  claims 22 - 44 . 
     
     
         54 . The method according to  claim 53 , wherein transfection of a cell is performed in vitro/ex vivo and the transfected cell is administered to a subject in need thereof, preferably to a human patient. 
     
     
         55 . The method according to  claim 54 , wherein the cell, which is to be transfected in vitro, is an isolated cell of the subject, preferably of the human patient. 
     
     
         56 . The method according to any one of  claims 52 - 55 , which is a vaccination method or a gene therapy method. 
     
     
         57 . A method for stabilizing and/or prolonging protein production from an artificial nucleic acid molecule, preferably from an mRNA molecule or a vector, the method comprising the step of associating the nucleic acid molecule, preferably the mRNA molecule or the vector, with an 3′-UTR element, wherein the 3′-UTR element comprises or consists of a nucleic acid sequence which is derived from the 3′-UTR of a FIG4 gene or from a variant of the 3′-UTR of a FIG4 gene. 
     
     
         58 . Use of an 3′-UTR element for stabilizing and/or prolonging protein production from a nucleic acid molecule, preferably from an mRNA molecule or a vector, wherein the 3′-UTR element comprises or consists of a nucleic acid sequence which is derived from the 3′-UTR of a FIG4 gene or from a variant of the 3′-UTR of a FIG4 gene. 
     
     
         59 . The method according to  claim 57  or the use according to  claim 58 , wherein the 3′-UTR element comprises or consists of a nucleic acid sequence which is derived from the 3′-UTR of a vertebrate FIG4 gene or from a variant thereof, preferably from the 3′-UTR of a mammalian FIG4 gene or from a variant thereof, more preferably from the 3′-UTR of a primate FIG4 gene, in particular of the human FIG4 gene, or from a variant thereof, even more preferably from the 3′-UTR of the human FIG4 gene according to GenBank Accession number NM_014845.5 or from a variant thereof. 
     
     
         60 . The method or the use according to any one of  claims 57 - 59 , wherein the 3′-UTR element comprises or consists of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the nucleic acid sequence according to SEQ ID No. 1 or SEQ ID No. 2, or wherein the 3′-UTR element comprises or consists of a fragment of a nucleic acid sequence that has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the nucleic acid sequence according to SEQ ID No. 1 or SEQ ID No. 2. 
     
     
         61 . The method or the use according to  claim 60 , wherein the fragment exhibits a length of at least about 50 nucleotides, preferably of at least about 75 nucleotides, more preferably of at least about 100 nucleotides, even more preferably of at least about 125 nucleotides, most preferably of at least about 150 nucleotides. 
     
     
         62 . The method or the use according to any one of  claims 57 - 61 , wherein the 3′-UTR element exhibits a length of at least about 50 nucleotides, preferably of at least about 75 nucleotides, more preferably of at least about 100 nucleotides, even more preferably of at least about 125 nucleotides, most preferably of at least about 150 nucleotides. 
     
     
         63 . A kit or kit of parts comprising an artificial nucleic acid molecule according to any one of  claims 1 - 21 , a vector according to any one of  claims 22 - 44 , a cell according to any one of  claims 45 - 47 , and/or a pharmaceutical composition according to  claim 48  or  49 . 
     
     
         64 . The kit according to  claim 63  further comprising instructions for use, cells for transfection, an adjuvant, means for administration of the pharmaceutical composition, a pharmaceutically acceptable carrier and/or a pharmaceutically acceptable solution for dissolution or dilution of the artificial nucleic acid molecule, the vector, the cells or the pharmaceutical composition.