LIVER SPECIFIC DELIVERY OF MESSENGER RNA
Disclosed herein are compositions and methods of modulating the expression of gene or the production of a protein by transfecting target cells with nucleic acids. The compositions disclosed herein demonstrate a high transfection efficacy and are capable of ameliorating diseases associated with protein or enzyme deficiencies.
1 .- 126 . (canceled)
127 . A method of delivery of messenger RNA (mRNA) for in vivo production of protein, comprising
administering systemically to a subject in need of delivery a composition comprising an mRNA encoding a protein, encapsulated within a liposome such that the administering of the composition results in the prolonged stable expression of the protein encoded by the mRNA in the liver;
wherein the protein encoded by the mRNA is an enzyme; and
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 and has a size less than about 100 nm.
128 . The method of claim 127 , wherein the one or more non-cationic lipids comprise DOPE.
129 . The method of claim 127 , wherein the one or more cationic lipids comprise DLinDMA, CLinDMA, or DLin-K-XTC2-DMA.
130 . The method of claim 127 , wherein the one or more PEG-modified lipids comprise DMG-PEG-2000 or C8-PEG-2000.
131 . The method of claim 127 , wherein the mRNA is of at least 30 kDa.
132 . The method of claim 127 , wherein the enzyme is a liver-specific enzyme.
133 . The method of claim 132 , wherein the enzyme is selected from ornithine transcarbamylase (OTC), carbamyl phosphate synthetase (CPS), argininosuccinate synthetase 1 (ASS1) argininosuccinate lyase (ASL), and arginase (ARG).
134 . The method of claim 127 , wherein the mRNA is modified to enhance stability.
135 . The method of claim 127 , wherein the mRNA is modified to include a modified nucleotide, an alteration to the 5′ or 3′ untranslated region, a cap structure or a poly A tail.
136 . The method of claim 135 , wherein the modified nucleotide is pseudouridine.
137 . The method of claim 127 , wherein the mRNA is unmodified.
138 . The method of claim 127 , wherein the protein is retained within the cytosol of the liver cells after expression.
139 . The method of claim 127 , wherein the protein is secreted extracellularly after expression.
140 . The method of claim 127 , wherein the protein is systemically distributed.
141 . The method of claim 127 , wherein the composition is administered intravenously.
142 . The method of claim 127 , wherein the expression of the protein encoded by the mRNA in the liver is detectable at least 4 hours post administration.
143 . The method of claim 141 , wherein the expression of the protein encoded by the mRNA in the liver is detectable at least 4 hours post administration.
144 . The method of claim 127 , wherein the one or more cationic lipids carry a net positive charge at physiological pH.
145 . The method of claim 144 , wherein the one or more cationic lipids constitute about 20-70% mol % of the total lipids present in the liposome.
146 . The method of claim 145 , wherein the one or more PEG-modified lipids comprise a polyethylene glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C 6 -C 20 length.
147 . The method of claim 146 , wherein the one or more PEG-modified lipids constitute up to about 20% by molar of the total lipids present in the liposome.
148 . The method of claim 127 , wherein the subject is suffering from or susceptible to a metabolic disorder resulting from deficiency of the enzyme encoded by the mRNA.
149 . The method of claim 148 , wherein the composition is administered bi-weekly.
150 . The method of claim 148 , wherein the composition is administered monthly.