Gene-editing compositions and methods to modulate faah for treatment of neurological disorders
AssigneeCrispr Therapeutics AG
InventorsSeshidhar Reddy Police, Tony Ho, Yanfei Yang, Hemangi Chaudhari, Anandan Paldurai
Life of this patent
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Abstract
The disclosure provides systems (e.g., CRISPR/Cas systems) for introducing an edit in a genomic DNA molecule comprising the fatty acid amide hydrolase gene (FAAH) and/or the FAAH pseudogene (FAAH-OUT). Also provided are methods for use of the systems, nucleic acids, delivery systems, and/or compositions described for genome editing to modulate the expression and/or activity of FAAH, for example, in a method of treating chronic pain.
Claims45
A system for introducing a deletion in a genomic DNA molecule comprising a fatty-acid amide hydrolase gene (FAAH) upstream a FAAH pseudogene (FAAH-OUT) in a cell, the system comprising:
- (i) a site-directed endonuclease in the form of protein, an mRNA encoding the site-directed endonuclease, or a recombinant expression vector comprising a nucleotide sequence encoding the site-directed endonuclease;
- (ii) a first gRNA molecule comprising a spacer sequence corresponding to a first target sequence adjacent a first PAM which is downstream of a 3′ terminus of FAAH and upstream a transcriptional start site of FAAH-OUT in the genomic DNA molecule, wherein when the first gRNA is introduced into a cell with a site-directed endonuclease that recognizes the PAM, the first gRNA combines with the site-directed endonuclease to induce cleavage proximal the first target sequence with a cleavage efficiency of at least 15%, 20%, 25%, or 30%;
- and
- (iii) a second gRNA molecule comprising a spacer sequence corresponding to a second target sequence adjacent a second PAM which is downstream of the FAAH-OUT transcriptional start site and upstream an exon 3 of FAAH-OUT in the genomic DNA molecule, wherein when the second gRNA is introduced into a cell with the site-directed endonuclease, the second gRNA combines with the site-directed endonuclease to induce cleavage proximal the second target sequence with a cleavage efficiency of at least 15%, 20%, 25%, or 30%,
- wherein when the system is introduced to the cell with the site-directed endonuclease, the first gRNA and second gRNA combine with the site-directed endonuclease to induce cleavage proximal the first and second target sequences, to introduce an approximately 2-10 kb deletion in the genomic DNA molecule resulting in a full or a partial removal of a FAAH-OUT promoter (FOP) and a FAAH-OUT conserved (FOC) element, thereby resulting in elimination of FAAH mRNA expression in the cell.
2-100. (canceled)
A nucleic acid molecule comprising:
- (i) a nucleotide sequence encoding a first gRNA comprising a spacer sequence corresponding to a first target sequence adjacent a first PAM which is downstream of a 3′ terminus of FAAH and upstream a transcriptional start site of FAAH-OUT in the genomic DNA molecule, wherein when the first gRNA is introduced into a cell with the site-directed endonuclease, the first gRNA combines with the site-directed endonuclease to induce cleavage proximal the first target sequence with a cleavage efficiency of at least 30%;
- and
- (ii) a and a nucleotide sequence encoding a second gRNA comprising a spacer sequence corresponding to a second target sequence adjacent a second PAM which is downstream of the FAAH-OUT transcriptional start site and upstream an exon 3 of FAAH-OUT in the genomic DNA molecule, wherein when the second gRNA is introduced into a cell with the site-directed endonuclease, the second gRNA combines with the site-directed endonuclease to induce cleavage proximal the second target sequence with a cleavage efficiency of at least 30%,
- wherein when the first and second gRNAs are introduced into a cell with (i) a SluCas9 endonuclease or functional variant thereof or (ii) a SpCas9 endonuclease or functional variant thereof, result in an approximate 2-8 kb deletion in a in a genomic DNA molecule comprising FAAH upstream FAAH-OUT, wherein the deletion results in full removal of a FAAH-OUT promoter (FOP) and a FAAH-OUT conserved (FOC) element in the genomic DNA molecule.
102-119. (canceled)
A system for introducing a mutation in a genomic DNA molecule comprising FAAH in a cell, the system comprising:
- (i) a site-directed endonuclease in the form of protein, an mRNA encoding the site-directed endonuclease, or a recombinant expression vector comprising a nucleotide sequence encoding the site-directed endonuclease;
- and
- (ii) a gRNA molecule comprising a spacer sequence corresponding to a target sequence within or proximal exon 1, exon 2, exon 3, or exon 4 of the FAAH coding sequence,
- wherein when the gRNA is introduced into a cell with the site-directed endonuclease, the gRNA combines with the endonuclease to induce a cleavage proximal the target sequence in the genomic DNA with a cleavage efficiency of at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, wherein the cleavage is a double-stranded DNA break (DSB), whereby repair of the DSB results in a mutation, and wherein the mutation provides reduced cellular expression of FAAH mRNA by at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% in the cell.
125-130. (canceled)
135-168. (canceled)
A nucleic acid molecule comprising a nucleotide sequence encoding one or more gRNA molecules targeting a target site in a genomic DNA molecule comprising a fatty-acid amide hydrolase gene (FAAH) in a cell, the gRNA(s) selected from:
- (i) a gRNA comprising a spacer sequence corresponding to a target sequence consisting of a nucleotide sequence selected from any one of SEQ ID NOs:
- 4, 5, 7, 14, and 20;
- (ii) a gRNA comprising a spacer sequence corresponding to a target sequence consisting of a nucleotide sequence selected from any one of SEQ ID NOs:
- 3, 6, 8-13, 16-19, 21-34;
- (iii) a gRNA molecule comprising a spacer sequence comprising a nucleotide sequence selected from any one of SEQ ID NOs:
- 38, 39, 41, 48, and 54;
- and
- (iv) a gRNA molecule comprising a spacer sequence comprising a nucleotide sequence selected from any one of SEQ ID NOs:
- 37, 40, 42-47, 50-53, 55-68.
(canceled)
A nucleic acid molecule comprising a nucleotide sequence encoding one or more gRNA molecules targeting a target site in a genomic DNA molecule comprising a fatty-acid amide hydrolase gene (FAAH) in a cell, the gRNA(s) selected from:
- (i) a gRNA comprising a spacer sequence corresponding to a target sequence consisting of a nucleotide sequence selected from any one of SEQ ID NOs:
- 149, 150, 151, 152, 153, 155, 156, 158, 159, 160, 161, 162, 163 and 164;
- or
- (ii) a gRNA molecule comprising a spacer sequence comprising a nucleotide sequence selected from any one of SEQ ID NOs:
- 165, 166, 167, 168, 169, 171, 172, 174, 175, 176, 177, 178, 179, and 180.
174-175. (canceled)
185-191. (canceled)
193-194. (canceled)
(canceled)
A system for use with a site-directed endonuclease to introduce a mutation in a genomic DNA molecule comprising FAAH in a cell, the system comprising a recombinant expression vector comprising (i) a nucleotide sequence encoding the site directed endonuclease, and (ii) a nucleotide sequence encoding the gRNA, wherein the gRNA comprises:
- (i) a gRNA molecule comprising a spacer sequence comprising a nucleotide sequence set forth in SEQ ID NO:
- 165, 171, 175, 176 or 177;
- or;
- or
- (ii) a gRNA comprising a spacer sequence corresponding to a target sequence consisting of a nucleotide sequence set forth in SEQ ID NO:
- 149, 155, 159, 160 or 161.
A system for use with a site-directed endonuclease to introduce a mutation in a genomic DNA molecule comprising FAAH in a cell, the system comprising a recombinant expression vector comprising (i) a nucleotide sequence encoding the site directed endonuclease, and (ii) a nucleotide sequence encoding the gRNA, wherein the gRNA comprises:
- (i) a gRNA comprising a spacer sequence corresponding to a target sequence consisting of a nucleotide sequence set forth in SEQ ID NO:
- 29, 30, 31, 32 or 34;
- or
- (ii) a gRNA molecule comprising a spacer sequence comprising a nucleotide sequence set forth in SEQ ID NO:
- 63, 64, 65, 66 or 68.
Dependencies are inferred from the claim text.