NON-VIRAL DELIVERY OF DNA FOR PROLONGED POLYPEPTIDE EXPRESSION IN VIVO
The compositions and methods described herein relate to lipid nanoparticle encapsulation of circular or linear DNA sequences, including DNA vectors, for delivery into a subject such that prolonged expression in vivo occurs. Lipid nanoparticles containing DNA can be administered to a subject to express therapeutic polypeptides.
1 . A pharmaceutical composition comprising a DNA sequence formulated in a lipid nanoparticle comprising an ionizable amino lipid, a phospholipid, a structural lipid, and a PEG-lipid,
wherein the ionizable amino lipid is Compound II or its N-oxide, or a salt or isomer thereof, or Compound A or its N-oxide, or a salt or isomer thereof,
wherein the DNA sequence is a plasmid DNA (pDNA) or a close-ended DNA (ceDNA), and wherein the DNA sequence comprises a tissue specific regulatory region, a nucleotide sequence encoding a polypeptide, a terminator of transcription, and a poly(A) signal.
2 . The pharmaceutical composition of claim 1 , wherein the DNA sequence further comprises a scaffold/matrix attachment region positioned downstream from the terminator of transcription.
3 . The pharmaceutical composition of claim 2 , wherein the scaffold/matrix attachment region is derived from the human interferon beta gene.
4 . The pharmaceutical composition of any one of claims 1 to 3 , wherein the phospholipid is DSPC.
5 . The pharmaceutical composition of any one of claims 1 to 3 , wherein the structural lipid is cholesterol.
6 . The pharmaceutical composition of any one of claims 1 to 3 , wherein the PEG-lipid is Compound I.
7 . The pharmaceutical composition of any one of claims 1 to 3 , wherein the ionizable amino lipid is Compound II or its N-oxide, or a salt or isomer thereof, the phospholipid is DSPC, the structural lipid is cholesterol, and the PEG-lipid is Compound I.
8 . The pharmaceutical composition of any one of claims 1 to 3 , wherein the ionizable amino lipid is Compound A or its N-oxide, or a salt or isomer thereof, the phospholipid is DSPC, the structural lipid is cholesterol, and the PEG-lipid is Compound I.
9 . The pharmaceutical composition of any one of claims 1 to 8 , wherein the lipid nanoparticle comprises:
(i) 40-50 mol % of the ionizable amino lipid, 30-45 mol % of the structural lipid, 5-15 mol % of the phospholipid, and 1-5 mol % of the PEG-lipid; or
(ii) 45-50 mol % of the ionizable amino lipid, 35-45 mol % of the structural lipid, 8-12 mol % of the phospholipid, and 1.5 to 3.5 mol % of the PEG-lipid.
10 . The pharmaceutical composition of any one of claims 1 to 9 , wherein the tissue specific regulatory region comprises a tissue specific enhancer.
11 . The pharmaceutical composition of claim 10 , wherein the tissue specific enhancer is a liver specific enhancer.
12 . The pharmaceutical composition of any one of claims 1 to 9 , wherein the tissue specific regulatory region comprises a tissue specific promoter.
13 . The pharmaceutical composition of claim 12 , wherein the tissue specific promoter is a liver specific promoter.
14 . The pharmaceutical composition of any one of claims 1 to 9 , wherein the tissue specific regulatory region comprises a tissue specific enhancer and a tissue specific promoter.
15 . The pharmaceutical composition of claim 14 , wherein the tissue specific enhancer and the tissue specific promoter are a liver specific enhancer and a liver specific promoter.
16 . The pharmaceutical composition of claim 13 or 15 , wherein the liver specific promoter is the human TTR promoter.
17 . The pharmaceutical composition of claim 13 or 15 , wherein the liver specific promoter is the human AAT promoter.
18 . The pharmaceutical composition of claim 11 or 15 , wherein the liver specific enhancer is the human ApoE HCR1 enhancer.
19 . The pharmaceutical composition of claim 11 or 15 , wherein the liver specific enhancer is the human TTR enhancer.
20 . The pharmaceutical composition of any one of claims 1 to 19 , wherein the polypeptide is a secreted polypeptide
21 . The pharmaceutical composition of any one of claims 1 to 19 , wherein the polypeptide is an intracellular polypeptide
22 . The pharmaceutical composition of any one of claims 1 to 19 , wherein the polypeptide is a transmembrane polypeptide.
23 . The pharmaceutical composition of any one of claims 1 to 22 , wherein the DNA sequence is unmodified.
24 . The pharmaceutical composition of any one of claims 1 to 23 , wherein the DNA sequence is a plasmid DNA (pDNA).
25 . The pharmaceutical composition of claim 24 , wherein the plasmid is a bacterial plasmid.
26 . The pharmaceutical composition of any one of claims 1 to 23 , wherein the DNA sequence is a close-ended DNA (ceDNA).
27 . The pharmaceutical composition of any one of claims 1 to 26 , wherein the polypeptide is expressed for at least four weeks in vivo upon administration of the pharmaceutical composition to a subject.
28 . The pharmaceutical composition of any one of claims 1 to 26 , wherein the polypeptide is expressed for at least 6 months in vivo upon administration of the pharmaceutical composition to a subject.
29 . The pharmaceutical composition of claim 27 or 28 , wherein the polypeptide is expressed in the liver in vivo upon administration of the pharmaceutical composition to the subject.
30 . A method of expressing a polypeptide in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition of any one of claims 1 to 26 .
31 . The method of claim 30 , wherein the polypeptide is expressed for at least four weeks in vivo upon administration of the pharmaceutical composition to the subject.
32 . The method of claim 30 , wherein the polypeptide is expressed for at least 6 months in vivo upon administration of the pharmaceutical composition to the subject.
33 . The method of claim 31 or 32 , wherein the polypeptide is expressed in the liver in vivo upon administration of the pharmaceutical composition to the subject.