Therapeutic circular DNA forms
The disclosure provides, for example, double stranded DNA (dsDNA) molecules comprising one or more chemically modified nucleobases. In some embodiments, the dsDNA molecule is circular and comprises a first strand and a second strand, wherein the first strand comprises one or more chemically modified nucleobases, and the second strand is free of chemically modified nucleobases. In some embodiments, the dsDNA molecule comprises a promoter sequence and an effector sequence that encodes an effector.
1 . A pharmaceutical composition comprising a lipid nanoparticle (LNP) that comprises a double stranded DNA (dsDNA) molecule, wherein:
(a) the dsDNA molecule is circular;
(b) the dsDNA molecule comprises a first strand and a second strand, wherein:
at least 50% of thymine or uracil positions in the first strand of the dsDNA molecule comprise 5-hydroxymethyluracil, and
the second strand is free of chemically modified nucleobases; and
(c) the dsDNA molecule comprises an effector sequence that encodes an effector, wherein the effector is selected from the group consisting of: a transcription factor, a chromatin remodeling factor, an antigen, a peptide, a hormone, an enzyme, an antibody, a receptor ligand, a receptor, a clotting factor, and a membrane protein.
2 . The pharmaceutical composition of claim 1 , wherein the first strand is a sense strand and the second strand is an antisense strand.
3 . The pharmaceutical composition of claim 1 , wherein the first strand is an antisense strand and the second strand is a sense strand.
4 . The pharmaceutical composition of claim 1 , wherein the dsDNA molecule further comprises a promoter sequence operably linked to the effector sequence.
5 . The pharmaceutical composition of claim 1 , wherein the effector is a chimeric antigen receptor (CAR) or a T cell receptor.
6 . The pharmaceutical composition of claim 1 , wherein at least 75% of thymine or uracil positions in the first strand of the dsDNA molecule comprise 5-hydroxymethyluracil.
7 . The pharmaceutical composition of claim 1 , wherein at least 90% of thymine or uracil positions in the first strand of the dsDNA molecule comprise 5-hydroxymethyluracil.
8 . The pharmaceutical composition of claim 1 , wherein the dsDNA molecule has a length of between 500-1000, 1000-2000, 2000-3000, 3000-4000, or 4000-5000 nucleotides.
9 . The pharmaceutical composition of claim 1 , wherein the dsDNA molecule further comprises one or more additional chemically modified nucleotides that comprise a backbone modification.
10 . The pharmaceutical composition of claim 1 , wherein the dsDNA molecule further comprises one or more additional chemically modified nucleotides that comprise a chemically modified sugar.
11 . The pharmaceutical composition of claim 1 , wherein the first strand of the dsDNA molecule further comprises a second type of chemically modified nucleobase.
12 . The pharmaceutical composition of claim 1 , wherein at least 99% of sugars of the dsDNA molecule are deoxyribose sugars.
13 . The pharmaceutical composition of claim 1 , wherein the longest stretch of unmodified nucleotides in the first strand is no more than 100 nucleotides.
14 . The pharmaceutical composition of claim 1 , wherein the dsDNA molecule is resistant to endonuclease digestion.
15 . The pharmaceutical composition of claim 1 , wherein when the dsDNA molecule is introduced to a cell, the cell exhibits a level of the effector that is at least 80% of the level of the effector in a control cell of the same type that was contacted with an unmodified circular dsDNA molecule having the same sequence as the dsDNA molecule but comprising no chemically modified nucleobases.
16 . The pharmaceutical composition of claim 1 , wherein when the dsDNA molecule is contacted to a human cell, the cell exhibits a level of cyclic AMP-GMP (cGAMP) that is less than 10% of the level of cGAMP in a control cell of the same type that was contacted with an unmodified circular dsDNA molecule having the same sequence as the dsDNA molecule but comprising no chemically modified nucleobases.
17 . The pharmaceutical composition of claim 1 , wherein at least 70% by mass of total DNA in the composition is the dsDNA molecule.
18 . The pharmaceutical composition of claim 1 , which further comprises a protein or a second nucleic acid molecule encoding the protein.
19 . The pharmaceutical composition of claim 18 , wherein the protein is an enzyme, a DNA-binding protein, an RNA-binding protein, a nuclear protein, a cytoplasmic protein, a kinase, a phosphatase, a structural protein, an antigen, or an antibody.
20 . A method of delivering an effector to a target cell or a subject in need thereof, the method comprising:
contacting the target cell with or administering to the subject the pharmaceutical composition of claim 1 ,
thereby delivering the effector to the target cell or the subject.
21 . A method of treating a cell, tissue, or subject in need thereof, the method comprising:
administering to the cell, tissue, or subject the pharmaceutical composition of claim 1 ;
thereby treating the cell, tissue, or subject.
22 . The pharmaceutical composition of claim 1 , wherein the pharmaceutical composition is free of one or more of: endotoxin, mononucleotides, and protein.