PHARMACEUTICAL COMPOSITION CONTAINING A STABILISED mRNA OPTIMISED FOR TRANSLATION IN ITS CODING REGIONS

Abstract

The present invention relates to a pharmaceutical composition comprising a modified mRNA that is stabilized by sequence modifications and optimised for translation. The pharmaceutical composition according to the invention is particularly well suited for use as an inoculating agent, as well as a therapeutic agent for tissue regeneration. In addition, a process is described for determining sequence modifications that promote stabilization and translational efficiency of modified mRNA of the invention.

1 - 28 . (canceled) 
     
     
         29 . A method of making a stabilized mRNA comprising:
 a. obtaining a wild type mRNA nucleic acid sequence encoding an antigen with a wild type amino acid sequence;   b. increasing the guanine/cytosine (G/C) content of the wild type nucleic mRNA nucleic acid sequence to obtain a modified mRNA nucleic acid sequence; and   c. obtaining the stabilized mRNA wherein the stabilized mRNA comprises the modified mRNA nucleic acid sequence and wherein:
 i. the stabilized mRNA is less susceptible to exonuclease digestion than the wild type mRNA; and 
 ii. the stabilized mRNA encodes the antigen with a wild type amino acid sequence. 
   
     
     
         30 . The method of claim  1  wherein the G/C content of the modified mRNA nucleic acid sequence is increased at least 7% relative to the wild type mRNA nucleic acid sequence. 
     
     
         31 . The method of claim  1  wherein the G/C content of the modified mRNA nucleic acid sequence is increased at least 15% relative to the wild type mRNA nucleic acid sequence. 
     
     
         32 . The method of claim  1  wherein the G/C content of the modified mRNA nucleic acid sequence is maximized relative to the wild type mRNA nucleic acid sequence. 
     
     
         33 . The method of claim  1  wherein the wild type mRNA nucleic acid sequence comprises at least one codon which is recognized by a rare cellular tRNA; and wherein the at least one codon is replaced in the modified mRNA nucleic acid sequence with a replacement codon which is recognized by an abundant cellular tRNA. 
     
     
         34 . The method of claim  1  wherein the stabilized mRNA comprises a 5′ cap and a poly-A tail. 
     
     
         35 . The method of claim  1  wherein the stabilized mRNA has no destabilizing sequences. 
     
     
         36 . The method of claim  1  wherein the stabilized mRNA comprises an analog of a naturally occurring nucleotide. 
     
     
         37 . The method of claim  1  wherein the antigen is a tumor peptide or polypeptide. 
     
     
         38 . The method of claim  1  wherein the stabilized mRNA further encodes at least one cytokine. 
     
     
         39 . The method of claim  9  wherein the tumor is a malignant melanoma, colon carcinoma, lymphoma, sarcoma, small-cell lung carcinoma, or blastoma. 
     
     
         40 . The method of claim  9  wherein the tumor is a non-small cell lung cancer. 
     
     
         41 . The method of claim  9  wherein the peptide or polypeptide is selected from the group consisting of 707-AP, AFP, ART-4, BAGE, β-catenin/m, Bcr-abl, CAMEL, CAP-1, CASP-8, CDC27/m, CDK4/m, CEA, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gp100, HAGE, HER-2/neu, HLA-A*0201-R170I, HPV-E7, HSP70-2M, HAST-2, hTERT, iCE, KIAA0205, LAGE, LDLR/FUT, MAGE, MART-1/melan-A, MCIR, myosin/m, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, NY-ESO-1, p190 minor bcr-abl, Pml/RARα, PRAME, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1, SART-3, TEL/AML1, TPI/m, TRP-1, TRP-2, TRP-2/INT2 and WT1. 
     
     
         42 . The method of claim  9  wherein the peptide or polypeptide is selected from the group consisting of CEA, MUC1, NY-ESO-1, PSA and MAGE. 
     
     
         43 . The method of claim  1  wherein the stabilized mRNA is formulated in a pharmaceutical composition.