TREATMENT OF GENETIC NEUROLOGICAL CONDITIONS WITH GENOMIC EDITING
The present disclosure provides methods and compositions concerning the CRISPR/Cas9 systems and associated guide RNAs, which target and excise portions of particular exons of a huntingtin gene, thereby abrogating huntingtin protein expression. The disclosure further provides methods and compositions for treating Huntington's Disease.
1 . A CRISPR based gene editing system comprising one or more polynucleotides, wherein the one or more polynucleotides encode a composition that comprises:
(a) a Cas protein or a fusion protein comprising the Cas protein or its component, and
(b) a gRNA, wherein the gRNA comprises a sequence selected from SEQ ID NOs: 1-120.
2 . The gene editing system of claim 1 , wherein the gRNA targets an exon of a HTT gene selected from any one of exons 1-63, exon 65, and exon 66.
3 . The gene editing system of claim 1 , wherein the Cas protein is a type II Cas enzyme or a type V Cas enzyme.
4 . The gene editing system of claim 3 , wherein the Cas protein is a Cas9 protein.
5 . The gene editing system of claim 4 , wherein the Cas9 protein is a SaCas9 protein; and wherein the gRNA comprises a sequence selected from any one of SEQ ID NOs: 1-40.
6 . The gene editing system of claim 5 , wherein the gRNA comprises SEQ ID NO: 1 or SEQ ID NO: 2.
7 . The gene editing system of claim 6 , wherein the SaCa9 protein recognizes a protospacer-adjacent motif (PAM) comprising SEQ ID NO: 121 or 122.
8 .- 11 . (canceled)
12 . The gene editing system of claim 4 , wherein the Cas9 protein is a KKH-SaCas9 protein; and wherein the gRNA comprises a sequence selected from any one of SEQ ID NOs: 41-80.
13 . (canceled)
14 . The gene editing system of claim 12 , wherein the KKH-SaCa9 protein recognizes a PAM comprising a sequence of SEQ ID NO: 161 or 162.
15 .- 17 . (canceled)
18 . The gene editing system of claim 4 , wherein the Cas9 protein is a SpCas9 protein; and wherein the gRNA comprises a sequence selected from any one of SEQ ID NOs: 81-120.
19 . (canceled)
20 . The gene editing system of claim 18 , wherein the SpCas9 protein recognizes a PAM comprising a sequence of SEQ ID NO: 201-212.
21 .- 54 . (canceled)
55 . The gene editing system of claim 1 , wherein the system introduces a double stranded break at a target nucleic acid sequence.
56 . The gene editing system of claim 4 , wherein the expression of the Cas9 protein is driven by a constitutive promoter or a neuron-specific promoter, wherein the consitutive promoter is a CBh promoter, a EFS promoter, an SCP1 promoter, an SCP3 promoter, or a JeT promoter, and the neuron-specific promoter is a E/hSyn promoter or a E/hMeCP2 promoter.
57 .- 58 . (canceled)
59 . The gene editing system of claim 1 , wherein the Cas protein and the gRNA are encoded by a single vector.
60 . The gene editing system of claim 1 , wherein the Cas protein is encoded by a first vector and the gRNA is encoded by a second vector.
61 . A viral vector expressing the gene editing system of claim 1 .
62 . (canceled)
63 . The viral vector of claim 61 , wherein the viral vector is an adeno-associated virus (AAV) vector, selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-10, AAV-11, AAV-12, AAV-13, AAVrh.74, or a recombinant variant thereof.
64 .- 66 . (canceled)
67 . A cell comprising
the gene editing system of claim 1 .
68 .- 75 . (canceled)
76 . A method of treating Huntington's Disease in a patient in need thereof, the method comprising administering to the patient the gene editing system of claim 1 , wherein the gene editing system is administered to the patient intravenously, intracranially, or a combination thereof.
77 .- 78 . (canceled)
79 . The method of claim 76 , wherein the detectable amount of huntingtin protein is reduced by at least about 50%, at least about 55% at least about 60%, at least about 70%, or at least about 75% as compared to an unmodified control.
80 .- 83 . (canceled)