Closed-ended linear duplex DNA for non-viral gene transfer
Inventors
Kotin, Robert M. • Cecchini, Sylvain
Assignees
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Publication Number
US-12252703-B2
Publication Date
2025-03-18
Expiration Date
Abstract
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.
Core Innovation
The invention relates to methods of preventing or treating a condition, disease, or disorder associated with the blood in a mammalian subject by administering closed-ended linear duplex DNA (ceDNA). The ceDNA comprises a nucleic acid insert encoding a transgene comprising Factor VIII (FVIII), Factor IX (FIX), or von Willebrand factor (VWF) protein or RNA, and the insert is flanked by at least two adeno-associated virus (AAV) inverted terminal repeat (ITR) sequences.
The ITR sequences are asymmetric and covalently linked with respect to one another, and each ITR sequence has an operative terminal resolution site and a rolling circle replication protein binding element (RBE). A first ITR sequence is interrupted by a cross-arm sequence that forms two opposing, lengthwise-symmetric stem-loops formed by interrupted palindromic sequences B-B′ and C-C′, and a second ITR sequence is interrupted by a truncated cross-arm sequence having one or more deletions in palindromic sequence loop regions B-B′ and/or C-C′.
The described approach includes delivering the ceDNA as the nucleic acid construct itself or in a host cell comprising the ceDNA, with administration in an amount sufficient to treat or prevent the blood-associated condition. The disclosed insert architecture supports therapeutic use directed to gene transfer and expression for blood-related diseases.
Claims Coverage
Two independent claims are present. Both cover preventing or treating blood-associated conditions by using ceDNA carrying specific transgene inserts (FVIII, FIX, or VWF) flanked by asymmetric, covalently linked AAV ITRs with defined cross-arm and stem-loop interruption architectures; one claim administers ceDNA directly and the other administers a host cell comprising the ceDNA.
CeDNA for treating blood-associated conditions with asymmetric covalently linked AAV ITRs
A method of preventing or treating a condition, disease, or disorder associated with the blood by administering closed-ended linear duplex DNA (ceDNA) comprising a nucleic acid insert encoding Factor VIII (FVIII), Factor IX (FIX), or von Willebrand factor (VWF) protein or RNA, wherein the insert is flanked by at least two asymmetric, covalently linked AAV inverted terminal repeat (ITR) sequences each having an operative terminal resolution site and a rolling circle replication protein binding element (RBE), with a first ITR interrupted by a cross-arm forming two opposing lengthwise-symmetric stem-loops formed by interrupted palindromic sequences B-B′ and C-C′, and a second ITR interrupted by a truncated cross-arm sequence with deletions in palindromic loop regions B-B′ and/or C-C′; administering ceDNA in an amount sufficient to treat or prevent the blood-associated condition.
Host cell comprising ceDNA with asymmetric covalently linked AAV ITRs for treating blood-associated conditions
A method of preventing or treating a condition, disease, or disorder associated with the blood by administering a host cell comprising closed-ended linear duplex DNA (ceDNA) with a nucleic acid insert encoding Factor VIII (FVIII), Factor IX (FIX), or von Willebrand factor (VWF) protein or RNA, wherein the insert is flanked by at least two asymmetric, covalently linked AAV inverted terminal repeat (ITR) sequences each having an operative terminal resolution site and a rolling circle replication protein binding element (RBE), with a first ITR interrupted by a cross-arm forming two opposing lengthwise-symmetric stem-loops formed by interrupted palindromic sequences B-B′ and C-C′, and a second ITR interrupted by a truncated cross-arm sequence with deletions in palindromic loop regions B-B′ and/or C-C′; administering the host cell in an amount sufficient to treat or prevent the blood-associated condition.
Across both independent claims, the core coverage is the same ceDNA-architecture concept: a closed-ended linear duplex DNA nucleic acid insert encoding FVIII/FIX/VWF, flanked by at least two asymmetric, covalently linked AAV ITRs with specified operative terminal resolution sites and RBEs, including defined cross-arm interruptions. The principal distinction is whether the ceDNA is administered directly or via a host cell comprising the ceDNA.
Stated Advantages
Improved genetic stability.
Reduced immunogenicity and immune interference enabling repeat dosing.
Documented Applications
Preventing or treating a blood-associated condition in a mammalian subject by administering ceDNA encoding Factor VIII (FVIII), Factor IX (FIX), or von Willebrand factor (VWF) protein or RNA, including anemia, deep vein thrombosis, hemophilia, Henoch-Schönlein Purpura, pulmonary embolism, thalassemia, and von Willebrand disease.
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