Closed-ended linear duplex DNA for non-viral gene transfer
Aspects of the disclosure relate to a nucleic acid comprising a heterologous nucleic acid insert flanked by interrupted self-complementary sequences, wherein one self-complementary sequence is interrupted by a cross-arm sequence forming two opposing, lengthwise-symmetric stem-loops, and wherein the other of the self-complementary sequences is interrupted by a truncated cross-arm sequence. Methods of delivering the nucleic acid to a cell are also provided.
1. A method of preventing or treating a condition, disease, or disorder associated with the blood in a mammalian subject having the condition, disease, or disorder associated with the blood, comprising administering a closed-ended linear duplex DNA (ceDNA) to the subject, wherein the ceDNA comprises a nucleic acid insert comprising a transgene encoding a Factor VIII (FVIII), Factor IX (FIX), or von Willebrand factor (VWF) protein or RNA;
wherein the insert is flanked by at least two adeno-associated virus (AAV) inverted terminal repeat (ITR) sequences;
wherein the at least two ITR sequences are asymmetric and covalently linked with respect to one another, each sequence having an operative terminal resolution site and a rolling circle replication protein binding element (RBE);
wherein a first ITR sequence is interrupted by a cross-arm sequence forming two opposing, lengthwise-symmetric stem-loops formed by interrupted palindromic sequences B-B′ and C-C′, each of the opposing lengthwise-symmetric stem-loops having a stem portion in the range of 5 to 15 base pairs in length and a loop portion having 2 to 5 unpaired deoxyribonucleotides;
wherein a second ITR sequence is interrupted by a truncated cross-arm sequence having one or more deletions of between 11 and 20 nucleotides in a palindromic sequence loop region B-B′ and/or a palindromic sequence loop region C-C′; and
wherein the ceDNA is administered to the subject in an amount sufficient to treat or prevent the condition, disease, or disorder associated with the blood in the subject.
2. The method of claim 1 , wherein the transgene encodes Factor VIII (FVIII).
3. The method of claim 1 , wherein the transgene encodes Factor IX (FIX).
4. The method of claim 1 , wherein
(i) the ITRs are in the range of 40 to 1000 nucleotides in length;
(ii) the cross-arm sequence has a Gibbs free energy (ΔG) of unfolding under physiological conditions in the range of −12 kcal/mol to −30 kcal/mol;
(iii) the RBE comprises the sequence 5′-GCTCGCTCGCTC-3′ (SEQ ID NO: 1);
(iv) the operative terminal resolution site comprises a sequence 5′-TT-3′ and/or the 3′ end of the operative terminal resolution site is 15 to 25 nucleotides from the 5′ end of the rolling circle replication protein binding element;
(v) the truncated cross-arm sequence forms two opposing, lengthwise-asymmetric stem-loops; and/or
(vi) the nucleic acid insert is a promoterless construct as a substrate for gene editing selected from a substrate for TALENS, a substrate for zinc finger nucleases (ZFNs), a substrate for meganucleases, a substrate for Cas9, and a substrate for another gene editing protein.
5. The method of claim 4 , wherein one of the opposing, lengthwise-asymmetric stem-loops has a stem portion in the range of 8 to 10 base pairs in length and a loop portion having 2 to 5 unpaired deoxyribonucleotides or the one lengthwise-asymmetric stem-loop has a stem portion less than 8 base pairs in length and a loop portion having 2 to 5 deoxyribonucleotides.
6. The method of claim 4 , wherein the ITRs are in the range of 100 to 160 nucleotides in length.
7. The method of claim 1 , wherein the ceDNA is prepared with a pharmaceutically acceptable excipient or carrier.
8. The method of claim 1 , wherein the ceDNA is administered by a lipid nanoparticle.
9. The method of claim 1 , wherein the ceDNA is administered to the subject in a therapeutically effective amount to transfect a desired tissue and to provide sufficient levels of gene transfer and expression.
10. The method of claim 1 , wherein the ceDNA is administered by intramuscular injection, intravenous administration, administration into the bloodstream by injection into a vein, an artery, or any other vascular conduit, or isolated limb perfusion.
11. The method of claim 1 , wherein the ceDNA is administered by a portal vein injection.
12. A method of preventing or treating a condition, disease, or disorder associated with the blood in a mammalian subject having the condition, disease, or disorder associated with the blood, comprising administering a host cell comprising a closed-ended linear duplex DNA (ceDNA) to the subject, wherein the ceDNA comprises a nucleic acid insert comprising a transgene encoding a Factor VIII (FVIII), Factor IX (FIX), or von Willebrand Factor (VWF) protein or RNA;
wherein the insert is flanked by at least two adeno-associated virus (AAV) inverted terminal repeat (ITR) sequences;
wherein the at least two ITR sequences are asymmetric and covalently linked with respect to one another, each sequence having an operative terminal resolution site and a rolling circle replication protein binding element (RBE);
wherein a first ITR sequence is interrupted by a cross-arm sequence forming two opposing, lengthwise-symmetric stem-loops formed by interrupted palindromic sequences B-B′ and C-C′, each of the opposing lengthwise-symmetric stem-loops having a stem portion in the range of 5 to 15 base pairs in length and a loop portion having 2 to 5 unpaired deoxyribonucleotides;
wherein a second ITR sequence is interrupted by a truncated cross-arm sequence having one or more deletions of between 11 and 20 nucleotides in a palindromic sequence loop region B-B′ and/or a palindromic sequence loop region C-C′; and
wherein the host cell is administered to the subject in an amount sufficient to treat or prevent the condition, disease, or disorder associated with the in the subject.
13. The method of claim 1 , wherein the transgene encodes von Willebrand Factor (VWF).
14. The method of claim 1 , wherein the condition, disease, or disorder associated with the blood is anemia, deep vein thrombosis, hemophilia, Henoch-Schönlein Purpura, pulmonary embolism, thalassemia, or von Willebrand disease.
15. The method of claim 14 , wherein the hemophilia is hemophilia A, hemophilia B, or hemophilia C.
16. The method of claim 1 , wherein the transgene encodes Factor VIII (FVIII) and the condition, disease, or disorder associated with the blood is Hemophilia A.
17. The method of claim 1 , wherein the transgene encodes von Willebrand Factor (VWF) and the condition, disease, or disorder associated with the blood is von Willebrand disease.
18. The method of claim 12 , wherein
(i) the ITRs are in the range of 40 to 1000 nucleotides in length;
(ii) the cross-arm sequence has a Gibbs free energy (ΔG) of unfolding under physiological conditions in the range of −12 kcal/mol to −30 kcal/mol;
(iii) the RBE comprises the sequence 5′-GCTCGCTCGCTC-3′ (SEQ ID NO: 1);
(iv) the operative terminal resolution site comprises a sequence 5′-TT-3′ and/or the 3′ end of the operative terminal resolution site is 15 to 25 nucleotides from the 5′ end of the rolling circle replication protein binding element;
(v) the truncated cross-arm sequence forms two opposing, lengthwise-asymmetric stem-loops; and/or
(vi) the nucleic acid insert is a promoterless construct as a substrate for gene editing selected from a substrate for TALENS, a substrate for zinc finger nucleases (ZFNs), a substrate for meganucleases, a substrate for Cas9, and a substrate for another gene editing protein.
19. The method of claim 12 , wherein the condition, disease, or disorder associated with the blood is anemia, deep vein thrombosis, hemophilia, Henoch-Schönlein Purpura, pulmonary embolism, thalassemia, or von Willebrand disease.
20. The method of claim 12 , wherein the transgene encodes Factor VIII.