POLYNUCLEOTIDES ENCODING INTERLEUKIN-12 (IL12) AND USES THEREOF
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.