DNA compositions and related methods
The disclosure provides, for example, single stranded, covalently closed DNA that does not form a double stranded structure longer than 100 base pairs. The ssDNA may encode an effector sequence, for instance a therapeutic protein. The ssDNA may comprise a nuclear targeting sequence (NTS). In some embodiments, the ssDNA shows decreased activation of the innate immune system compared to an otherwise similar dsDNA.
1. A pharmaceutical formulation comprising:
a lipid nanoparticle (LNP) comprising a single stranded DNA (ssDNA), wherein the ssDNA:
(a) encodes only one protein, which is a therapeutic protein,
(b) is covalently closed,
(c) does not form a double stranded structure longer than 40 base pairs,
(d) is at least 200 nucleotides in length, and
(e) does not comprise a protelomerase target sequence;
wherein:
(i) at least 95% by mass of total DNA in the composition is the ssDNA that is covalently closed;
(ii) the pharmaceutical formulation is free of polypeptides; and
(iii) the therapeutic protein is expressed when the ssDNA is introduced into a target cell.
2. The pharmaceutical formulation of claim 1 , wherein the ssDNA comprises a promoter sequence operably linked to a sequence encoding the therapeutic protein.
3. The pharmaceutical formulation of claim 1 , wherein the therapeutic protein 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.
4. The pharmaceutical formulation of claim 1 , wherein the ssDNA has a GC content of 30-40%, 40-50%, 50-60%, or 60-70%.
5. The pharmaceutical formulation of claim 1 , wherein the ssDNA further comprises a nuclear targeting sequence (NTS).
6. The pharmaceutical formulation of claim 1 , wherein the ssDNA further comprises a maintenance sequence.
7. The pharmaceutical formulation of claim 1 , wherein the ssDNA further comprises a second strand motif (SSM).
8. The pharmaceutical formulation of claim 1 , wherein the ssDNA comprises between 200 and 3,000 nucleotides.
9. The pharmaceutical formulation of claim 1 , wherein the ssDNA comprises between 500 and 2,000 nucleotides.
10. The pharmaceutical formulation of claim 1 , wherein the ssDNA is a sense ssDNA strand.
11. The pharmaceutical formulation of claim 1 , wherein the ssDNA comprises at least one nucleotide modification.
12. The pharmaceutical formulation of claim 11 , wherein the nucleotide modification is 5-formylcytosine.
13. The pharmaceutical formulation of claim 1 , wherein the pharmaceutical formulation is formulated for parenteral administration.
14. The pharmaceutical formulation of claim 1 , wherein the pharmaceutical formulation is formulated for topical administration.
15. The pharmaceutical formulation of claim 1 , wherein the pharmaceutical formulation is free of one or more of: endotoxin, mononucleotides, modified mononucleotides, and double stranded DNA.
16. The pharmaceutical formulation of claim 1 , wherein the ssDNA lacks one or both of a bacteriophage packaging site and a bacteriophage origin of replication, or wherein the ssDNA does not encode a bacteriophage capsid gene.
17. A method of delivering a therapeutic protein to a subject, comprising administering to the subject the pharmaceutical formulation of claim 1 .
18. The method of claim 17 , wherein the method does not result in substantial integration of the ssDNA into the genome of the subject.
19. A pharmaceutical formulation comprising:
an LNP comprising a single stranded DNA (ssDNA), wherein the ssDNA:
(a) encodes only one protein, which is a therapeutic protein,
(b) is covalently closed,
(c) does not form a double stranded structure longer than 100 base pairs,
(d) is at least 200 nucleotides in length, and
(e) does not comprise a protelomerase target sequence;
wherein:
(i) at least 95% by mass of total DNA in the composition is the ssDNA that is covalently closed;
(ii) the pharmaceutical formulation is free of viral protein; and
(iii) the therapeutic protein is expressed when the ssDNA is introduced into a target cell.
20. The pharmaceutical formulation of claim 3 , wherein the antibody is a monoclonal antibody, Fab fragment, or single-chain Fv.
21. A pharmaceutical formulation comprising:
an LNP comprising a single stranded DNA (ssDNA), wherein the ssDNA:
(a) encodes a therapeutic protein,
(b) is covalently closed,
(c) does not form a double stranded structure longer than 100 base pairs,
(d) is at least 200 nucleotides in length, and
(e) does not comprise a protelomerase target sequence;
wherein:
(i) at least 95% by mass of total DNA in the composition is the ssDNA that is covalently closed;
(ii) the pharmaceutical formulation is free of polypeptides;
(iii) the therapeutic protein is expressed when the ssDNA is introduced into a target cell; and
(iv) the ssDNA does not encode viral protein.
22. A pharmaceutical formulation comprising:
an LNP comprising a single stranded DNA (ssDNA), wherein the ssDNA:
(a) encodes a therapeutic protein,
(b) is covalently closed,
(c) does not comprise a region of intramolecular complementarity longer than 100 base pairs,
(d) is at least 200 nucleotides in length, and
(e) does not comprise a protelomerase target sequence;
wherein:
(i) at least 95% by mass of total DNA in the composition is the ssDNA that is covalently closed;
(ii) the pharmaceutical formulation is free of polypeptides;
(iii) the therapeutic protein is expressed when the ssDNA is introduced into a target cell;
(iv) the therapeutic protein 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; and
(v) the ssDNA does not encode viral protein.