US 12,252,703 B2
Closed-ended linear duplex DNA for non-viral gene transfer
Robert M. Kotin, Worcester, MA (US); and Sylvain Cecchini, Worcester, MA (US)
Assigned to University of Massachusetts, Westborough, MA (US); and Voyager Therapeutics, Cambridge, MA (US)
Filed by University of Massachusetts, Westborough, MA (US); and Voyager Therapeutics, Inc., Cambridge, MA (US)
Filed on Jun. 1, 2021, as Appl. No. 17/335,499.
Application 17/335,499 is a continuation of application No. 16/081,337, granted, now 11,066,679, previously published as PCT/US2017/020828, filed on Mar. 3, 2017.
Claims priority of provisional application 62/406,913, filed on Oct. 11, 2016.
Claims priority of provisional application 62/394,720, filed on Sep. 14, 2016.
Claims priority of provisional application 62/303,047, filed on Mar. 3, 2016.
Prior Publication US 2021/0355507 A1, Nov. 18, 2021
Prior Publication US 2022/0195456 A9, Jun. 23, 2022
Int. Cl. C12N 15/86 (2006.01); A61K 48/00 (2006.01); A61P 1/16 (2006.01); A61P 3/00 (2006.01); A61P 3/08 (2006.01); A61P 7/04 (2006.01); A61P 11/12 (2006.01); A61P 27/02 (2006.01); C07K 14/005 (2006.01); C12N 15/09 (2006.01); C12N 15/63 (2006.01); C12N 15/64 (2006.01); C12N 15/66 (2006.01)
CPC C12N 15/86 (2013.01) [A61K 48/005 (2013.01); A61K 48/0075 (2013.01); A61P 1/16 (2018.01); A61P 3/00 (2018.01); A61P 3/08 (2018.01); A61P 7/04 (2018.01); A61P 11/12 (2018.01); A61P 27/02 (2018.01); C07K 14/005 (2013.01); C12N 15/09 (2013.01); C12N 15/63 (2013.01); C12N 15/64 (2013.01); C12N 15/66 (2013.01); A61K 48/00 (2013.01); C12N 2710/14043 (2013.01); C12N 2750/14122 (2013.01); C12N 2750/14143 (2013.01); C12N 2750/14151 (2013.01)] 20 Claims
 
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.