Novel Codon-Optimized CFTR MRNA

Abstract

The present invention provides, among other things, improved methods and pharmaceutical compositions for treating cystic fibrosis based on codon optimized mRNA encoding a Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein.

1 . A pharmaceutical composition for treating cystic fibrosis, comprising a codon optimized mRNA encoding a Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein and wherein the codon optimized CFTR mRNA comprises a polynucleotide sequence of SEQ ID NO: 1. 
     
     
         2 - 4 . (canceled) 
     
     
         5 . The pharmaceutical composition of  claim 1 , wherein the codon optimized CFTR mRNA encoding the CFTR protein is encapsulated within a nanoparticle. 
     
     
         6 . The pharmaceutical composition of  claim 5 , wherein the nanoparticle is a liposome. 
     
     
         7 . The pharmaceutical composition of  claim 6 , wherein the liposome comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids. 
     
     
         8 . The pharmaceutical composition of  claim 6 , wherein the liposome comprises no more than three distinct lipid components. 
     
     
         9 - 10 . (canceled) 
     
     
         11 . A method of large scale production of codon optimized mRNA encoding a Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein, comprising in vitro synthesizing codon optimized CFTR mRNA using a SP6 RNA polymerase,
 wherein at least 80% of the synthesized codon optimized CFTR mRNA molecules are full-length and wherein at least 100 mg of codon optimized mRNA is synthesized at a single batch, and   wherein the codon optimized CFTR mRNA comprises a polynucleotide sequence of SEQ ID NO: 1.   
     
     
         12 . (canceled) 
     
     
         13 . The method of  claim 11 , wherein the in vitro synthesis of codon optimized CFTR mRNA results in a secondary polynucleotide species that constitutes less than 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1% of the total mRNA synthesized. 
     
     
         14 . The method of  claim 11 , wherein at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the synthesized codon optimized CFTR mRNA molecules are full-length. 
     
     
         15 . (canceled) 
     
     
         16 . The method of  claim 11 , wherein at least 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 5 g, 10 g, 25 g, 50 g, 75 g, 100 g, 150 g, 200 g, 250 g, 500 g, 750 g, 1 kg, 5 kg, 10 kg, 50 kg, 100 kg, 1000 kg, or more of codon optimized CFTR mRNA is synthesized at a single batch. 
     
     
         17 .- 20 . (canceled) 
     
     
         21 . The method of  claim 11 , wherein the method further comprises a step of capping and/or tailing of the synthesized codon optimized CFTR mRNA. 
     
     
         22 . (canceled) 
     
     
         23 . A method of treating cystic fibrosis, comprising administering to a subject in need of treatment a composition comprising a codon optimized mRNA encoding an Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein, wherein the codon optimized CFTR mRNA comprises a polynucleotide sequence at least 85% identical to SEQ ID NO: 1. 
     
     
         24 . The method of  claim 23 , wherein the codon optimized CFTR mRNA comprises SEQ ID NO: 1. 
     
     
         25 .- 26 . (canceled) 
     
     
         27 . The method of  claim 23 , wherein the codon optimized CFTR mRNA is encapsulated within a nanoparticle. 
     
     
         28 . The method of  claim 27 , wherein the nanoparticle is a liposome. 
     
     
         29 . The method of  claim 28 , wherein the liposome comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids. 
     
     
         30 - 32 . (canceled) 
     
     
         33 . The method of  claim 23 , wherein the codon optimized CFTR mRNA is administered to the subject via pulmonary delivery. 
     
     
         34 . The method of  claim 33 , wherein the pulmonary delivery is nebulization. 
     
     
         35 . A pharmaceutical composition for treating cystic fibrosis, comprising an mRNA encoding a Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein and wherein the mRNA encoding the CFTR protein comprises a polynucleotide sequence comprising any one of SEQ ID NO: 21-40. 
     
     
         36 - 40 . (canceled) 
     
     
         41 . The pharmaceutical composition of  claim 35 , wherein the mRNA is encapsulated in a nanoparticle, and wherein the nanoparticle is a liposome. 
     
     
         42 . The pharmaceutical composition of  claim 41 , wherein the liposome comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids. 
     
     
         43 - 44 . (canceled)