GENETIC CONSTRUCTS FOR GENE EDITING
The present disclosure provides methods and compositions concerning the disclosed genetic constructs, including those that target mutated portions of a dystrophin gene for excision, thereby restoring functional dystrophin protein expression. The disclosure further provides methods and compositions for treating Duchenne muscular dystrophy.
1 .- 67 . (canceled)
68 . A genetic construct, comprising:
i) a first inverted terminal repeat (ITR) nucleotide sequence;
ii) an RNA polymerase II (Pol II)-driven promoter operably linked to a transgene;
iii) a first RNA polymerase III (Pol III)-driven promoter operably linked to a first guide RNA (gRNA) nucleotide sequence as set forth in SEQ ID NO: 14;
iv) a second Poll III-driven promoter operably linked to a second gRNA nucleotide sequence as set forth in SEQ ID NO: 31,
wherein the first and second Pol III-driven promoters and the first and second gRNAs are in a reverse orientation to the Pol II-driven promoter and the transgene; and
v) a second ITR nucleotide sequence,
wherein said genetic construct is encoded in a single polynucleotide.
69 . The genetic construct of claim 68 , wherein the first ITR nucleotide sequence and the second ITR nucleotide sequence are set forth in SEQ ID NO: 99.
70 . The genetic construct of claim 68 , wherein the Pol II promoter is a modified CK8 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 100.
71 . The genetic construct of claim 68 , wherein the Pol II promoter is a minimal CK8 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 101.
72 . The genetic construct of claim 68 , wherein the transgene is a Cas9 nucleotide sequence, as set forth in SEQ ID NO: 107.
73 . The genetic construct of claim 68 , wherein the transgene is a Cas9 nucleotide sequence, as set forth in SEQ ID NO: 108.
74 . The genetic construct of claim 68 , wherein the first Pol III-driven promoter and the second Pol III-driven promoter are human U6 (hU6) promoters, comprising the nucleotide sequence set forth in SEQ ID NO: 103.
75 . The genetic construct of claim 68 , wherein the single polynucleotide is at least 99% identical to SEQ ID NO: 140.
76 . The genetic construct of claim 75 , wherein the single polynucleotide is identical to SEQ ID NO: 140.
77 . A vector capable of expressing the genetic construct of claim 68 , wherein the vector is an adeno-associated virus (AAV) vector.
78 . The AAV vector of claim 77 , wherein the vector is a rh.74 AAV vector or a recombinant variant thereof.
79 . The AAV vector of claim 77 , wherein the vector is a MyoAAV-4E vector or a recombinant variant thereof.
80 . A eukaryotic cell comprising the genetic construct of claim 68 .
81 . A kit comprising the genetic construct of claim 68 .
82 . A method of treating a subject having a mutant gene, the method comprising administering to the subject the genetic construct of claim 68 .
83 . A method of treating a disease in a patient in need thereof, the method comprising administering to the patient the genetic construct of claim 68 .
84 . The method of claim 83 , wherein the disease is Duchenne muscular dystrophy.
85 . The method of claim 83 , wherein the disease is Becker muscular dystrophy.
86 . The method of claim 83 , wherein the genetic construct is administered to the patient intramuscularly, intravenously, or a combination thereof.