NUCLEIC ACID MOLECULES AND USES THEREOF FOR NON-VIRAL GENE THERAPY
The present disclosure provides nucleic acid molecules comprising a first inverted terminal repeat (ITR), a second ITR, and a genetic cassette encoding a target sequence. In some embodiments, the target sequence encodes a miRNA and/or a therapeutic protein. In certain embodiments, the therapeutic protein comprises a clotting factor, a growth factor, a hormone, a cytokine, an antibody, a fragment thereof, and a combination thereof. In some embodiments, the first ITR and/or the second ITR is an ITR of a non-adeno-associated virus (AAV). The present disclosure also provides methods of treating a metabolic disorder of the liver in a subject comprising administering to the subject the nucleic acid molecule or a polypeptide encoded thereby.
1 - 76 . (canceled)
77 . A method of expressing a clotting factor in a subject in need thereof, or treating a clotting factor deficiency in a subject in need thereof, comprising administering to the subject a nucleic acid molecule comprising a first inverted terminal repeat (ITR) and a second ITR flanking a genetic cassette comprising a heterologous polynucleotide sequence, wherein the first ITR and/or second ITR comprises a nucleotide sequence at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a nucleotide sequence set forth in SEQ ID NO: 180, 181, 183, 184, 185, 186, 187 or 188, or a functional derivative thereof,
wherein the heterologous polynucleotide sequence encodes a clotting factor.
78 . A method of treating a disease or disorder in a subject in need thereof, or expressing a heterologous polynucleotide sequence in a subject in need thereof, comprising administering to the subject a nucleic acid molecule comprising a first inverted terminal repeat (ITR) and a second ITR flanking a genetic cassette comprising a heterologous polynucleotide sequence, wherein the first ITR and/or second ITR comprises a nucleotide sequence at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a nucleotide sequence set forth in SEQ ID NO: 180, 181, 183, 184, 185, 186, 187 or 188, or a functional derivative thereof.
79 - 80 . (canceled)
81 . The method of claim 77 , wherein the nucleic acid molecule is administered intravenously, transdermally, intradermally, subcutaneously, orally, pulmonarily, or any combination thereof.
82 - 89 . (canceled)
90 . The method of claim 78 , wherein the disease or disorder comprises a bleeding disorder.
91 . (canceled)
92 . A method of treating a metabolic disorder of the liver in a subject in need thereof, comprising administering to the subject a nucleic acid molecule comprising a first inverted terminal repeat (ITR) and a second ITR flanking a genetic cassette comprising a heterologous polynucleotide sequence encoding a liver-associated metabolic enzyme that is deficient in the subject, wherein the first ITR and/or second ITR are an ITR of a non-adeno-associated virus (non-AAV).
93 . The method of claim 92 , wherein the first ITR and/or second ITR comprises a nucleotide sequence at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a nucleotide sequence set forth in SEQ ID NO: 180, 181, 183, 184, 185, 186, 187 or 188, or a functional derivative thereof.
94 - 123 . (canceled)
124 . The method of claim 78 , wherein:
the first ITR comprises the nucleotide sequence set forth in SEQ ID NO: 180 and the second ITR comprises the nucleotide sequence set forth in SEQ ID NO: 181;
the first ITR comprises the nucleotide sequence set forth in SEQ ID NO: 183 and the second ITR comprises the nucleotide sequence set forth in SEQ ID NO: 184;
the first ITR comprises the nucleotide sequence set forth in SEQ ID NO: 185 and the second ITR comprises the nucleotide sequence set forth in SEQ ID NO: 186;
the first ITR comprises the nucleotide sequence set forth in SEQ ID NO: 187 and the second ITR comprises the nucleotide sequence set forth in SEQ ID NO: 188; and/or
the first ITR and the second ITR are reverse complements of each other.
125 . The method of claim 78 , further comprising a promoter.
126 . The method of claim 78 , wherein the heterologous polynucleotide sequence further comprises an intronic sequence and/or the heterologous polynucleotide sequence encodes a clotting factor, a growth factor, a hormone, a cytokine, an antibody, a fragment thereof, or any combination thereof.
127 . The method of claim 78 , wherein the genetic cassette further comprises a post-transcriptional regulatory element, a 3′UTR poly(A) tail sequence, and/or an enhancer sequence.
128 . The method of claim 78 , wherein the genetic cassette comprises a single stranded nucleic acid; and/or the genetic cassette comprises a double stranded nucleic acid.
129 . The method of claim 78 , wherein the nucleic acid molecule comprises from 5′ to 3′: the first ITR, the genetic cassette, and the second ITR; wherein the genetic cassette comprises a tissue-specific promoter sequence, an intronic sequence, the heterologous polynucleotide sequence, a post-transcriptional regulatory element, and a 3′UTR poly(A) tail sequence.
130 . The method of claim 78 , wherein the nucleic acid molecule is formulated with a delivery agent.
131 . the method of claim 78 , wherein the nucleic acid molecule is formulated for intravenous, transdermal, intradermal, subcutaneous, pulmonary, or oral delivery, or any combination thereof.
132 . The method of claim 77 , wherein the clotting factor is selected from the group consisting of factor I (FI), factor II (FII), factor III (FIII), factor IV (FVI), factor V (FV), factor VI (FVI), factor VII (FVII), factor VIII (FVIII), factor IX (FIX), factor X (FX), factor XI (FXI), factor XII (FXII), factor XIII (FVIII), Von Willebrand factor (VWF), prekallikrein, high-molecular weight kininogen, fibronectin, antithrombin III, heparin cofactor II, protein C, protein S, protein Z, Protein Z-related protease inhibitor (ZPI), plasminogen, alpha 2-antiplasmin, tissue plasminogen activator (tPA), urokinase, plasminogen activator inhibitor-1 (PAI-1), plasminogen activator inhibitor-2 (PAI2), and any combination thereof.
133 . The method of claim 132 , wherein the clotting factor is FVIII.
134 . The method of claim 90 , wherein the bleeding disorder is hemophilia A.
135 . The method of claim 92 , wherein the metabolic disorder of the liver is selected from the group consisting of phenylketonuria (PKU), a urea cycle disease, a lysosomal storage disorder, and a glycogen storage disease.
136 . The method of claim 92 , wherein the heterologous polynucleotide sequence encodes a gene selected from dystrophin X-linked, MTM1 (myotubularin), tyrosine hydroxylase, AADC, cyclohydrolase, SMN1, FXN (frataxin), GUCY2D, RS1, CFH, HTRA, ARMS, CFB/CC2, CNGA/CNGB, Prf65, ARSA, PSAP, IDUA (MPS I), IDS (MPS II), PAH, GAA (acid alpha-glucosidase), and any combination thereof.
137 . The method of claim 78 , wherein the nucleic acid molecule comprises from 5′ to 3′: the first ITR, the genetic cassette, and the second ITR; wherein the genetic cassette comprises a tissue-specific promoter sequence, an intronic sequence, the heterologous polynucleotide sequence, a post-transcriptional regulatory element, and a 3′UTR poly(A) tail sequence.