Nucleic acid comprising or coding for a histone stem-loop and a poly(a) sequence or a polyadenylation signal for increasing the expression of an encoded therapeutic protein

Abstract

The present invention relates to a nucleic acid sequence, comprising or coding for a coding region, encoding at least one peptide or protein comprising a therapeutic protein or a fragment, variant or derivative thereof, at least one histone stem-loop and a poly(A) sequence or a polyadenylation signal. Furthermore the present invention provides the use of the nucleic acid for increasing the expression of said encoded peptide or protein, particularly for the use in gene therapy. It also discloses its use for the preparation of a pharmaceutical composition, e.g. for use in gene therapy, particularly in the treatment of diseases which are in need of a treatment with a therapeutic peptide or protein, preferably as defined herein. The present invention further describes a method for increasing the expression of a peptide or protein comprising a therapeutic protein or a fragment, variant or derivative thereof, using the nucleic acid comprising or coding for a histone stem-loop and a poly(A) sequence or a polyadenylation signal.

1 . A pharmaceutical composition comprising nucleic acid encoding:
 a) a polypeptide coding region encoding IL-12;   b) at least one histone stem-loop; and   c) a poly(A) sequence or polyadenylation signal,   
       wherein said mRNA does not include a histone downstream element (HDE). 
     
     
         2 . The composition of  claim 1 , wherein the polyadenylation sequence comprises the consensus sequence NN(U/T)ANA, AA(U/T)AAA or A(U/T)(U/T)AAA. 
     
     
         3 . The composition of  claim 1 , wherein the nucleic acid is a mRNA. 
     
     
         4 . The composition of  claim 1 , comprising a poly(A) sequence. 
     
     
         5 . The composition of  claim 1 , wherein the G/C content of the polypeptide coding region is increased compared with the G/C content of the wild-type nucleic acid of IL-12. 
     
     
         6 . The composition of  claim 1 , wherein the mRNA comprises a 5′ cap structure. 
     
     
         7 . The composition of  claim 4 , wherein the poly(A) sequence comprises about 25 to about 400 adenosine nucleotides. 
     
     
         8 . The composition of  claim 1 , wherein the nucleic acid molecule additionally comprises a poly(C) sequence of about 10 to about 200 cytosine nucleotides. 
     
     
         9 . The method of  claim 1 , wherein the mRNA further comprises a stabilizing sequence from the alpha globin 3′ UTR. 
     
     
         10 . The composition of  claim 3 , wherein the mRNA is modified by introduction of a non-native nucleotide compared with a native mRNA sequence or by covalent coupling of the mRNA with a further chemical moiety. 
     
     
         11 . The composition of  claim 10 , wherein the mRNA comprises a chemical modification relative to a naturally occurring mRNA. 
     
     
         12 . The composition of  claim 10 , wherein the non-native nucleotide is selected from the group consisting of 2-amino-6-chloropurineriboside-5′-triphosphate, 2-aminoadenosine-5′-triphosphate, 2-thiocytidine-5′-triphosphate, 2-thiouridine-5′-triphosphate, 4-thiouridine-5′-triphosphate, 5-aminoallylcytidine-5′-triphosphate, 5-aminoallyluridine-5′-triphosphate, 5-bromocytidine-5′-triphosphate, 5-bromouridine-5′-triphosphate, 5-iodocytidine-5′-triphosphate, 5-iodouridine-5′-triphosphate, 5-methylcytidine-5′-triphosphate, 5-methyluridine-5′-triphosphate, 6-azacytidine-5′-triphosphate, 6-azauridine-5′-triphosphate, 6-chloropurineriboside-5′-triphosphate, 7-deazaadenosine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate, 8-azaadenosine-5′-triphosphate, 8-azidoadenosine-5′-triphosphate, benzimidazole-riboside-5′-triphosphate, N1-methyladenosine-5′-triphosphate, N1-methylguanosine-5′-triphosphate, N6-methyladenosine-5′-triphosphate, 06-methylguanosine-5′-triphosphate, pseudouridine-5′-triphosphate, or puromycin-5′-triphosphate, and xanthosine-5′-triphosphate. 
     
     
         13 . A method of treating a subject comprising administering an effective amount of a pharmaceutical composition in accordance with  claim 1  to the subject. 
     
     
         14 . The method of  claim 13 , wherein the subject has a cancer. 
     
     
         15 . The method of  claim 14  wherein the cancer is a sarcoma, melanoma, lung cancer, ovarian cancer, leukemia, lymphoma, brain and central nervous system tumors, testicular cancer, prostate cancer, pancreatic cancer, or breast cancer. 
     
     
         16 . The method of  claim 13 , wherein the pharmaceutical composition is administered by injection. 
     
     
         17 . The method of  claim 14 , wherein the pharmaceutical composition is administered by intralesional injection.