Materials and methods for treatment of Spinocerebellar Ataxia Type 1 (SCA1) and other Spinocerebellar Ataxia Type 1 Protein (ATXN1) gene related conditions or disorders
The present disclosure provides materials and methods for treating a patient with one or more conditions or disorders associated with ATXN1 whether ex vivo or in vivo. For example, the present disclosure provides materials and methods for treating a patient with Spinocerebellar ataxia type 1 (SCA1). Also provided are materials and methods for editing a ATXN1 gene in a cell by genome editing. The present disclosure also provides materials and methods for altering the contiguous genomic sequence of a ATXN1 gene in a cell. In addition, the present disclosure provides one or more gRNAs for editing a ATXN1 gene. Also provided are therapeutics comprising at least one or more gRNAs for editing a ATXN1 gene. In addition, the present disclosure provides therapeutics for treating patients with a ATXN1 related condition or disorder.
1. A method for editing a Spinocerebellar Ataxia Type 1 Protein (ATXN1) gene in a cell by genome editing comprising: introducing into the cell one or more deoxyribonucleic acid (DNA) endonucleases and one or more gRNAs or one or more sgRNAs to effect one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within or near the ATXN1 gene that results in a permanent deletion in the expanded trinucleotide repeat or replacement of one or more nucleotide bases, or one or more exons and/or introns within or near the ATXN1 gene, thereby restoring the ATXN1 gene function, wherein the one or more gRNA or one or more sgRNA comprises a RNA sequence corresponding to a sequence selected from the group consisting of SEQ ID NOs: 42685, 42504, 101247, 42473, 101253, 101251, 101250, 101085, 101252, 101259, 101139, 101314, 42644, 42421, 101103, 42634, 101054, 101319, 101044, 101152, 101320, 42637, 101060, 101150, 101062, 42605, 101249, 101316, 101061, 101248, 101296, 42686 and 101093.
2. An ex vivo method for treating a patient having an ATXN1 related condition or disorder comprising:
(a) editing a patient specific induced pluripotent stem cell (iPSC) or a mesenchymal stem cell according to the method of claim 1 ;
(b) differentiating the edited cell of (a) into a neuron or glial cell of the Central Nervous System (CNS); and
(c) implanting the neuron or glial cell of the Central Nervous System (CNS) into the patient.
3. An in vivo method for treating a patient with an ATXN1 related condition or disorder comprising: editing the ATXN1 gene in a cell of the patient according to the method of claim 1 , optionally wherein the cell is a cell of the Central Nervous System (CNS) such as a neuron or a glial cell.
4. The method of claim 1 , wherein the one or more DNA endonuclease is provided as:
one or more protein or polypeptide; or
one or more polynucleotide encoding the one or more DNA endonuclease, wherein the one or more polynucleotide is codon optimized, and when the one or more polynucleotide is one or more ribonucleic acid (RNA), the one or more RNA is optionally chemically modified, optionally in the coding region.
5. The method of claim 4 , wherein the one or more protein or polypeptide comprises an amino acid sequence having at least 90% sequence homology to any one of SEQ ID NOs: 1, 8, 28, 30, 33, 35, and 37-620 and/or wherein the one or more polynucleotide encoding the one or more DNA endonuclease comprises a nucleic acid sequence having at least 90% sequence homology to any one of SEQ ID NOs: 2-7, 9-27, 29, 31-32, 34, 36, and 37-620.
6. The method of claim 5 , wherein the one or more DNA endonuclease is flanked at the N-terminus, the C-terminus, or both the N-terminus and C-terminus by one or more nuclear localization signals (NLSs).
7. The method of claim 1 , wherein the one or more gRNA or one or more sgRNA:
is chemically modified;
comprises three 2′-O-methyl-phosphorothioate residues at or near each of its 5′ and 3′ ends;
is pre-complexed with the one or more DNA endonuclease to form one or more ribonucleoproteins (RNPs); or
a combination thereof.
8. The method of claim 1 , wherein:
the one or more DNA endonuclease is formulated in a liposome or lipid nanoparticle;
the one or more DNA endonuclease is formulated in a liposome or a lipid nanoparticle which also comprises the one or more gRNA or one or more sgRNA;
the one or more DNA endonuclease is encoded in an AAV vector particle;
the one or more gRNA or one or more sgRNA is encoded in an AAV vector particle; or
wherein the one or more DNA endonuclease is encoded in an AAV vector particle which also encodes the one or more gRNA or one or more sgRNA.
9. The method of claim 1 , wherein the method further comprises introducing into the cell a donor template comprising at least a portion of the wild-type ATXN1 gene, wherein the donor template:
comprises homologous arms to the genomic locus of the ATXN1 gene;
is a single or double stranded polynucleotide;
is encoded in an AAV vector particle, or
a combination thereof.
10. The method of claim 9 , wherein the at least a portion of the wild-type ATXN1 gene comprises one or more sequences selected from the group consisting of an ATXN1 exon, an ATXN1 intron, and a sequence comprising an exon:intron junction of ATXN1.
11. The method of claim 4 , wherein the one or more polynucleotide encoding one or more DNA endonuclease is formulated into a lipid nanoparticle, and the one or more gRNA or one or more sgRNA is delivered to the cell ex vivo by electroporation and the donor template is delivered to the cell by an adeno-associated virus (AAV) vector, or the one or more polynucleotide encoding one or more DNA endonuclease is formulated into a liposome or lipid nanoparticle which also comprises the one or more gRNA or one or more sgRNA and the donor template.
12. A single-molecule guide RNA comprising at least a spacer sequence that is an RNA sequence corresponding to any one of SEQ ID NOs: 42685, 42504, 101247, 42473, 101253, 101251, 101250, 101085, 101252, 101259, 101139, 101314, 42644, 42421, 101103, 42634, 101054, 101319, 101044, 101152, 101320, 42637, 101060, 101150, 101062, 42605, 101249, 101316, 101061, 101248, 101296, 42686 and 10109.
13. The single-molecule guide RNA of claim 12 , wherein the single-molecule guide RNA:
further comprises a spacer extension region;
further comprises a tracrRNA extension region;
is chemically modified; or
a combination thereof.
14. The single-molecule guide RNA of claim 13 pre-complexed with a DNA endonuclease, wherein the DNA endonuclease is a Cas9 or CpfI endonuclease.
15. The single-molecule guide RNA of claim 14 , wherein the Cas9 or CpfI endonuclease is selected from the group consisting of S. pyogenes Cas9, S. aureus Cas9, N. meningitidis Cas9, S. thermophilus CRISPRI Cas9, S. thermophilus CRISPR 3 Cas9, T. denticola Cas9, L. bacterium ND2006 CpfI and Acidaminococcus sp. BV3L6 CpfI, and variants having at least 90% homology to the endonucleases.
16. The single-molecule guide RNA of claim 15 , wherein the Cas9 or CpfI endonuclease comprises one or more nuclear localization signals (NLSs), wherein the at least one NLS is at or within 50 amino acids of the amino-terminus of the Cas9 or CpfI endonuclease and/or at least one NLS is at or within 50 amino acids of the carboxy-terminus of the Cas9 or CpfI endonuclease.
17. A DNA encoding the single-molecule guide RNA of claim 12 .
18. A therapeutic for treating a patient with an ATXN1 related condition or disorder formed by the method comprising:
introducing one or more DNA endonucleases;
introducing one or more gRNA or one or more sgRNA for editing a ATXN1 gene;
wherein the one or more gRNAs or sgRNAs comprise a spacer sequence comprising a RNA sequence corresponding to a sequence selected from the group consisting of SEQ ID NOs: 42685, 42504, 101247, 42473, 101253, 101251, 101250, 101085, 101252, 101259, 101139, 101314, 42644, 42421, 101103, 42634, 101054, 101319, 101044, 101152, 101320, 42637, 101060, 101150, 101062, 42605, 101249, 101316, 101061, 101248, 101296, 42686 and 10109.