Polynucleotides encoding interleukin-12 (il12) and uses thereof

Abstract

The present disclosure relates to polynucleotides comprising an open reading frame of linked nucleosides encoding human interleukin-12 (IL12), functional fragments thereof, and fusion proteins comprising IL12. In some embodiments, the open reading frame is sequence-optimized. In particular embodiments, the disclosure provides sequence-optimized polynucleotides comprising nucleotides encoding the polypeptide sequence of human IL12, or sequences having high sequence identity with those sequence optimized polynucleotides.

1 .- 157 . (canceled) 
     
     
         158 . A lipid nanoparticle (LNP) comprising a messenger RNA (mRNA) comprising an open reading frame (ORF) encoding a fusion protein comprising a human IL-12B polypeptide fused directly or by a linker to a human IL-12A polypeptide, and wherein the LNP comprises a molar ratio of about 20-60% ionizable amino lipid; 5-25% phospholipid; 25-55% sterol; and 0.5-15% PEG-modified lipid. 
     
     
         159 . The LNP of  claim 158 , wherein the IL-12B polypeptide is fused directly to the IL-12A polypeptide without a linker. 
     
     
         160 . The LNP of  claim 158 , wherein the IL-12B polypeptide is fused directly to the IL-12A polypeptide with a peptide linker. 
     
     
         161 . The LNP of  claim 158 , wherein the fusion protein comprises a heterologous signal peptide. 
     
     
         162 . The LNP of  claim 158 , wherein the fusion protein comprises a human IL-12B signal peptide or a human IL-12A signal peptide. 
     
     
         163 . The LNP of  claim 158 , wherein the ORF comprises from 5′ to 3′ a nucleotide sequence encoding the IL-12B polypeptide, a nucleotide sequence encoding a peptide linker, and a nucleotide sequence encoding the IL-12A polypeptide. 
     
     
         164 . The LNP of  claim 158 , wherein the ORF comprises from 5′ to 3′ a nucleotide sequence encoding the IL-12B polypeptide, and a nucleotide sequence encoding the IL-12A polypeptide. 
     
     
         165 . The LNP of  claim 158 , wherein the ORF comprises from 5′ to 3′ a nucleotide sequence encoding the IL-12A polypeptide, a nucleotide sequence encoding a peptide linker, and a nucleotide sequence encoding the IL-12B polypeptide. 
     
     
         166 . The LNP of  claim 158 , wherein the ORF comprises from 5′ to 3′ a nucleotide sequence encoding the IL-12A polypeptide, and a nucleotide sequence encoding the IL-12B polypeptide. 
     
     
         167 . The LNP of  claim 163 , wherein the ORF comprises a nucleotide sequence encoding a signal peptide located at the 5′ terminus of the ORF. 
     
     
         168 . The LNP of  claim 164 , wherein the ORF comprises a nucleotide sequence encoding a signal peptide located at the 5′ terminus of the ORF. 
     
     
         169 . The LNP of  claim 165 , wherein the ORF comprises a nucleotide sequence encoding a signal peptide located at the 5′ terminus of the ORF. 
     
     
         170 . The LNP of  claim 166 , wherein the ORF comprises a nucleotide sequence encoding a signal peptide located at the 5′ terminus of the ORF. 
     
     
         171 . The LNP of  claim 158 , wherein the mRNA comprises (i) a 3′ UTR; (ii) a 5′ UTR and (iii) a polyA tail. 
     
     
         172 . The LNP of  claim 171 , wherein the 3′ UTR comprises a microRNA binding site, and wherein the microRNA binding site is a miR-122-3p or a miR-122-5p binding site. 
     
     
         173 . The LNP of  claim 158 , wherein the mRNA comprises a chemically modified nucleotide. 
     
     
         174 . The LNP of  claim 173 , wherein the mRNA is fully modified with chemically modified uridines. 
     
     
         175 . The LNP of  claim 158 , wherein the mRNA comprises a 3′ UTR comprising a microRNA binding site, wherein the microRNA binding site is a miR-122-3p or a miR-122-5p binding site, and wherein the mRNA is fully modified with chemically modified uridines. 
     
     
         176 . A lipid nanoparticle (LNP) comprising a messenger RNA (mRNA) comprising an open reading frame (ORF) encoding a human IL-12 polypeptide, and wherein the LNP comprises a molar ratio of about 20-60% ionizable amino lipid; 5-25% phospholipid; 25-55% sterol; and 0.5-15% PEG-modified lipid.