US 2024/0175012 A1Published application

Guide rnas and compositions for editing huntingtin gene, and methods related thereto

AssigneesThe University of British Columbia, Incisive Genetics, Inc

InventorsBlair LEAVITT, Austin HILL, Pamela WAGNER

Life of this patent

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Abstract

gRNAs, combinations of gRNAs, polynucleotides and vectors encoding, RNPs, and compositions for effecting CRISPR-mediated gene editing of the huntingtin gene are provided. Also provided are methods for effecting CRISPR-mediated gene editing of the huntingtin gene and/or for preventing, ameliorating, slowing the progression of, and/or treating HD or HD symptoms.

Claims27

Dependent claims

An isolated guide RNA (gRNA) for Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-mediated gene editing, wherein the gRNA comprises a targeting sequence comprising at least or consisting of 17 nucleotides, optionally 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, wherein:

(I) the targeting sequence comprises or consists of:
(i) (i-1) a sequence of at least 17 consecutive nucleotides, optionally 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consequent nucleotides, contained in one or more of SEQ ID NO:
200, 201, 202, 205, 206, and/or 207, and/or (i-2) a sequence of:
(a) SEQ ID NO:
4, 210, 211, 212, or 213,
(b) SEQ ID NO:
5, 220, 221, 222, or 223, or
(c) SEQ ID NO:
6, 230, 231, 232, or 233,
(ii) a sequence of at least 17 nucleotides comprising one or more mutations, optionally one, two, three, four, or five mutations, said mutations relative to the at least 17 consecutive nucleotides, optionally 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consequent nucleotides, of (i), optionally wherein the one or more mutations are at any nucleotide position(s) or are at position(s) other than the 4th to the 7th nucleotide positions from the 3′-end of the at least 17 consecutive nucleotides, or
(iii) a sequence of at least 17 nucleotides which comprises at least 85, 90, 95, 96, 97, 98 or 99% sequence identity to the sequence of at least 17 consecutive nucleotides, optionally 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides, of (i),
optionally wherein the at least 17 consecutive nucleotides are;
(a) immediately upstream of a protospacer adjacent motif (PAM) or protospacer flanking site (PFS) of a CRISPR-associated (Cas) endonuclease in one or more of SEQ ID NO:
200, 201, 202, 205, 206, and/or 207, optionally wherein the Cas endonuclease is Cas9, further optionally wherein the Cas endonuclease is Streptococcus pyogenes Cas9 (SpCas9) and/or the PAM sequence is 5′-NGG-3′ wherein N represents any nucleotide;
or
(b) immediately downstream of the PAM or PFS of a Cas endonuclease in one or more of SEQ ID NO:
200, 201, 202, 205, 206, and/or 207, optionally wherein the Cas endonuclease is Cpf1 and/or the PAM sequence is 5′-TTTN-3′ wherein N represents any nucleotide;
and/or
(II) when the gRNA is complexed with a Cas endonuclease, the targeting sequence guides the Cas endonuclease to allow for cleavage of exon 5 of the huntingtin (HTT) gene or within 30 nucleotides upstream or downstream of the exon 5, optionally wherein:
(i) the cleavage is within one or more of SEQ ID NOS:
200, 201, 202, 205, 206, and/or 207, optionally at any one or more of the following positions:
(i-1) between the 9th and 10th nucleotides from the 5′ end of SEQ ID NO:
200,
(i-2) between the 55th and 56th nucleotides from the 5′ end of SEQ ID NO:
200,
(i-3) between the 72th and 73th nucleotides from the 5′ end of SEQ ID NO:
200,
(i-4) between the 9th and 10th nucleotides from the 3′ end of SEQ ID NO:
205,
(i-5) between the 55th and 56th nucleotides from the 3′ end of SEQ ID NO:
205, and/or
(i-6) between the 72th and 73th nucleotides from the 3′ end of SEQ ID NO:
200;
and/or
(ii) the Cas endonuclease is Cas 9, Cas3, Cas8a2, Cas8b, Cas8c, Cas10, Cas11, Cas12, Cas12a or Cpf1, Cas13, Cas13a, C2c1, C2c3, or C2c2, further optionally Cas9 or Cpf1, yet further optionally SpCas9,
optionally wherein the gRNA comprises one or more of the following features:
(A) (A-1) the gRNA is a single guide RNA (sgRNA) comprising in a single strand:
(i) a CRISPR RNA (crRNA) sequence comprising the targeting sequence and a crRNA backbone sequence, and
(ii) a trans-activating CRISPR RNA (tracrRNA) sequence,
optionally wherein the crRNA sequence and the tracrRNA sequence are linked via a linker optionally comprising SEQ ID NO:
109, further optionally wherein the gRNA comprises the targeting sequence followed by, optionally immediately followed by, a sgRNA backbone sequence of any of SEQ ID NOS:
111-114, optionally wherein the sgRNA backbone sequence is followed by one or more uracils, further optionally 1-10 uracils, or
(A-2) the gRNA is a dual guide RNA (dgRNA) formed by hybridization between:
(i) a crRNA comprising the targeting sequence and a crRNA backbone sequence, and
(ii) a tracrRNA,
optionally wherein:
(a) the targeting sequence is followed by, optionally immediately followed by, a sgRNA backbone sequence of SEQ ID NOS:
115 and the tracrRNA comprises SEQ ID NO:
116, or
(b) the targeting sequence is followed by, optionally immediately followed by, a sgRNA backbone sequence of SEQ ID NOS:
117 and the tracrRNA comprises SEQ ID NO:
118;
(B) the gRNA is synthetic or recombinant;
and/or
(C) the gRNA is a synthetic sgRNA and comprises at least one chemical modification, which is optionally:
(C-1) 2-O-methylation further optionally at first three and last three bases, and/or
(C-2) one or more 3′ phosphorothioate bonds, further optionally between first three and last two bases.

54 more elements

An isolated gRNA for CRISPR-mediated gene editing, wherein the gRNA comprises a targeting sequence comprising at least or consisting of 17 nucleotides, optionally 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, wherein:

(I) the targeting sequence comprises or consists of:
(i) (i-1) a sequence of at least 17 consecutive nucleotides, optionally 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consequent nucleotides, contained in one or more of SEQ ID NO:
300, 301, 302, 305, 306, and/or 307, and/or (i-2) a sequence of:
(a) SEQ ID NO:
7, 310, 311, 312, or 313,
(b) SEQ ID NO:
8, 320, 321, 322, or 323, or
(c) SEQ ID NO:
9, 330, 331, 332, or 333,
(ii) a sequence of at least 17 nucleotides comprising one or more mutations, optionally one, two, three, four, or five mutations, said mutations relative to the at least 17 consecutive nucleotides, optionally 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consequent nucleotides, of (i), optionally wherein the one or more mutations are at any nucleotide position(s) or are at position(s) other than the 4th to the 7th nucleotide positions from the 3′-end of the at least 17 consecutive nucleotides, or
(iii) a sequence of at least 17 nucleotides which comprises at least 85, 90, 95, 96, 97, 98 or 99% sequence identity to the sequence of at least 17 consecutive nucleotides, optionally 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides, of (i),
optionally wherein the at least 17 consecutive nucleotides are;
(a) immediately upstream of a PAM or PFS of a Cas endonuclease in one or more of SEQ ID NO:
300, 301, 302, 305, 306, and/or 307, optionally wherein the Cas endonuclease is Cas9, further optionally wherein the Cas endonuclease is SpCas9 and/or the PAM sequence is 5′-NGG-3′ wherein N represents any nucleotide;
or
(b) immediately downstream of the PAM or PFS of a Cas endonuclease in one or more of SEQ ID NO:
300, 301, 302, 305, 306, and/or 307, optionally wherein the Cas endonuclease is Cpf1 and/or the PAM sequence is 5′-TTTN-3′ wherein N represents any nucleotide;
and/or
(II) when the gRNA is complexed with a Cas endonuclease, the targeting sequence guides the Cas endonuclease to allow for cleavage of exon 8 of the HTT gene or within 30 nucleotides upstream or downstream of the exon 8, optionally wherein:
(i) the cleavage is within one or more of SEQ ID NOS:
300, 301, 302, 305, 306, and/or 307, optionally at any one or more of the following positions:
(i-1) between the 15th and 16th nucleotides from the 5′ end of SEQ ID NO:
300,
(i-2) between the 69th and 70th nucleotides from the 5′ end of SEQ ID NO:
300,
(i-3) between the 112th and 113th nucleotides from the 5′ end of SEQ ID NO:
300,
(i-4) between the 15th and 16th nucleotides from the 3′ end of SEQ ID NO:
305,
(i-5) between the 69th and 70th nucleotides from the 3′ end of SEQ ID NO:
305, and/or
(i-6) between the 112th and 113th nucleotides from the 3′ end of SEQ ID NO:
305;
and/or
(ii) the Cas endonuclease is Cas 9, Cas3, Cas8a2, Cas8b, Cas8c, Cas10, Cas11, Cas12, Cas12a or Cpf1, Cas13, Cas13a, C2c1, C2c3, or C2c2, further optionally Cas9 or Cpf1, yet further optionally SpCas9,
optionally wherein the gRNA comprises one or more of the following features:
(A) (A-1) the gRNA is a sgRNA comprising in a single strand:
(i) a crRNA sequence comprising the targeting sequence and a crRNA backbone sequence, and
(ii) a tracrRNA sequence,
optionally wherein the crRNA sequence and the tracrRNA sequence are linked via a linker optionally comprising SEQ ID NO:
109, further optionally wherein the gRNA comprises the targeting sequence followed by, optionally immediately followed by, a sgRNA backbone sequence of any of SEQ ID NOS:
111-114, optionally wherein the sgRNA backbone sequence is followed by one or more uracils, further optionally 1-10 uracils, or
(A-2) the gRNA is a dgRNA formed by hybridization between:
(i) a crRNA comprising the targeting sequence and a crRNA backbone sequence, and
(ii) a tracrRNA,
optionally wherein:
(a) the targeting sequence is followed by, optionally immediately followed by, a sgRNA backbone sequence of SEQ ID NOS:
115 and the tracrRNA comprises SEQ ID NO:
116, or
(b) the targeting sequence is followed by, optionally immediately followed by, a sgRNA backbone sequence of SEQ ID NOS:
117 and the tracrRNA comprises SEQ ID NO:
118;
(B) the gRNA is synthetic or recombinant;
and/or
(C) the gRNA is a synthetic sgRNA and comprises at least one chemical modification, which is optionally:
(C-1) 2-O-methylation further optionally at first three and last three bases, and/or
(C-2) one or more 3′ phosphorothioate bonds, further optionally between first three and last two bases.

54 more elements

(I) the targeting sequence comprises or consists of:
(i) (i-1) a sequence of at least 17 consecutive nucleotides, optionally 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consequent nucleotides, contained in one or more of SEQ ID NO:
400, 401, 402, 405, 406, and/or 407, or (i-2) a sequence of:
(a) SEQ ID NO:
1, 410, 411, 412, or 413,
(b) SEQ ID NO:
2, 420, 421, 422, or 423,
(c) SEQ ID NO:
3, 430, 431, 432, or 433, or
(d) SEQ ID NO:
440, 441, 442, or 443,
(e) SEQ ID NO:
450, 451, 452, or 453,
(ii) a sequence of at least 17 nucleotides comprising one or more mutations, optionally one, two, three, four, or five mutations, said mutations relative to the at least 17 consecutive nucleotides, optionally 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consequent nucleotides, of (i), optionally wherein the one or more mutations are at any nucleotide position(s) or are at position(s) other than the 4th to the 7th nucleotide positions from the 3′-end of the at least 17 consecutive nucleotides, or
(iii) a sequence of at least 17 nucleotides which comprises at least 85, 90, 95, 96, 97, 98 or 99% sequence identity to the sequence of at least 17 consecutive nucleotides, optionally 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides, of (i),
optionally wherein the at least 17 consecutive nucleotides are;
(a) immediately upstream of a PAM or PFS of a Cas endonuclease in one or more of SEQ ID NO:
400, 401, 402, 405, 406, and/or 407, optionally wherein the Cas endonuclease is Cas9, further optionally wherein the Cas endonuclease is SpCas9 and/or the PAM sequence is 5′-NGG-3′ wherein N represents any nucleotide;
or
(b) immediately downstream of the PAM or PFS of a Cas endonuclease in one or more of SEQ ID NO:
400, 401, 402, 405, 406, and/or 407, optionally wherein the Cas endonuclease is Cpf1 and/or the PAM sequence is 5′-TTTN-3′ wherein N represents any nucleotide;
and/or
(II) when the gRNA is complexed with a Cas endonuclease, the targeting sequence guides the Cas endonuclease to allow for cleavage of exon 31 of the HTT gene or within 30 nucleotides upstream or downstream of the exon 31, optionally wherein:
(i) the cleavage is within one or more of SEQ ID NOS:
400, 401, 402, 405, 406, and/or 407, optionally at any one or more of the following positions:
(i-1) between the 36th and 37th nucleotides from the 5′ end of SEQ ID NO:
400, (i-2) between the 106th and 107th nucleotides from the 5′ end of SEQ ID NO:
400, (i-3) between the 185th and 186th nucleotides from the 5′ end of SEQ ID NO:
400, (i-4) between the 112th and 113th nucleotides from the 5′ end of SEQ ID NO:
400, (i-5) between the 101th and 102th nucleotides from the 5′ end of SEQ ID NO:
400, (i-6) between the 36th and 37th nucleotides from the 3′ end of SEQ ID NO:
405, (i-7) between the 106th and 107th nucleotides from the 3′ end of SEQ ID NO:
405, (i-8) between the 185th and 186th nucleotides from the 3′ end of SEQ ID NO:
405,
(i-9) between the 112th and 113th nucleotides from the 3′ end of SEQ ID NO:
405, and/or
(i-10) between the 101th and 102th nucleotides from the 3′ end of SEQ ID NO:
405;
and/or
(ii) the Cas endonuclease is Cas 9, Cas3, Cas8a2, Cas8b, Cas8c, Cas10, Cas11, Cas12, Cas12a or Cpf1, Cas13, Cas13a, C2c1, C2c3, or C2c2, further optionally Cas9 or Cpf1, yet further optionally SpCas9,
optionally wherein the gRNA comprises one or more of the following features:
(A) (A-1) the gRNA is a sgRNA comprising in a single strand:
(i) a crRNA sequence comprising the targeting sequence and a crRNA backbone sequence, and
(ii) a tracrRNA sequence,
optionally wherein the crRNA sequence and the tracrRNA sequence are linked via a linker optionally comprising SEQ ID NO:
109, further optionally wherein the gRNA comprises the targeting sequence followed by, optionally immediately followed by, a sgRNA backbone sequence of any of SEQ ID NOS:
111-114, optionally wherein the sgRNA backbone sequence is followed by one or more uracils, further optionally 1-10 uracils, or
(A-2) the gRNA is a dgRNA formed by hybridization between:
(i) a crRNA comprising the targeting sequence and a crRNA backbone sequence, and
(ii) a tracrRNA,
optionally wherein:
(a) the targeting sequence is followed by, optionally immediately followed by, a sgRNA backbone sequence of SEQ ID NOS:
115 and the tracrRNA comprises SEQ ID NO:
116, or
(b) the targeting sequence is followed by, optionally immediately followed by, a sgRNA backbone sequence of SEQ ID NOS:
117 and the tracrRNA comprises SEQ ID NO:
118;
(B) the gRNA is synthetic or recombinant;
and/or
(C) the gRNA is a synthetic sgRNA and comprises at least one chemical modification, which is optionally:
(C-1) 2′-O-methylation further optionally at first three and last three bases, and/or
(C-2) one or more 3′ phosphorothioate bonds, further optionally between first three and last two bases.

59 more elements

Dependencies are inferred from the claim text.

Facts

Application: 18/282,711Filed 18 Mar 2022
Priority: 19 Mar 2021
Published: 30 May 2024
Term ends: Not estimatedThe source records don’t support an estimate.

Classification19 codes

C12N 15/11
Recombinant DNA-technology: dNA or RNA fragments; Modified forms thereof ; Non-coding nucleic acids having a biological activity
A61K 31/7088
Carbohydrates; Sugars; Derivatives thereof: compounds having three or more nucleosides or nucleotides
A61K 31/7105
Compounds having three or more nucleosides or nucleotides: natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine,…
A61K 48/005
Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy:…

Family

Application63/163,692Us provisional application · filed 19 Mar 2021
Application63/164,404Us provisional application · filed 22 Mar 2021
US 2024/0175012 A1This patent

From the US filing history. Foreign family members aren’t listed.

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Abstract

(I) the targeting sequence comprises or consists of:
(i) (i-1) a sequence of at least 17 consecutive nucleotides, optionally 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consequent nucleotides, contained in one or more of SEQ ID NO:
200, 201, 202, 205, 206, and/or 207, and/or (i-2) a sequence of:
(a) SEQ ID NO:
4, 210, 211, 212, or 213,
(b) SEQ ID NO:
5, 220, 221, 222, or 223, or
(c) SEQ ID NO:
6, 230, 231, 232, or 233,
(ii) a sequence of at least 17 nucleotides comprising one or more mutations, optionally one, two, three, four, or five mutations, said mutations relative to the at least 17 consecutive nucleotides, optionally 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consequent nucleotides, of (i), optionally wherein the one or more mutations are at any nucleotide position(s) or are at position(s) other than the 4th to the 7th nucleotide positions from the 3′-end of the at least 17 consecutive nucleotides, or
(iii) a sequence of at least 17 nucleotides which comprises at least 85, 90, 95, 96, 97, 98 or 99% sequence identity to the sequence of at least 17 consecutive nucleotides, optionally 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides, of (i),
optionally wherein the at least 17 consecutive nucleotides are;
(a) immediately upstream of a protospacer adjacent motif (PAM) or protospacer flanking site (PFS) of a CRISPR-associated (Cas) endonuclease in one or more of SEQ ID NO:
200, 201, 202, 205, 206, and/or 207, optionally wherein the Cas endonuclease is Cas9, further optionally wherein the Cas endonuclease is Streptococcus pyogenes Cas9 (SpCas9) and/or the PAM sequence is 5′-NGG-3′ wherein N represents any nucleotide;
or
(b) immediately downstream of the PAM or PFS of a Cas endonuclease in one or more of SEQ ID NO:
200, 201, 202, 205, 206, and/or 207, optionally wherein the Cas endonuclease is Cpf1 and/or the PAM sequence is 5′-TTTN-3′ wherein N represents any nucleotide;
and/or
(II) when the gRNA is complexed with a Cas endonuclease, the targeting sequence guides the Cas endonuclease to allow for cleavage of exon 5 of the huntingtin (HTT) gene or within 30 nucleotides upstream or downstream of the exon 5, optionally wherein:
(i) the cleavage is within one or more of SEQ ID NOS:
200, 201, 202, 205, 206, and/or 207, optionally at any one or more of the following positions:
(i-1) between the 9th and 10th nucleotides from the 5′ end of SEQ ID NO:
200,
(i-2) between the 55th and 56th nucleotides from the 5′ end of SEQ ID NO:
200,
(i-3) between the 72th and 73th nucleotides from the 5′ end of SEQ ID NO:
200,
(i-4) between the 9th and 10th nucleotides from the 3′ end of SEQ ID NO:
205,
(i-5) between the 55th and 56th nucleotides from the 3′ end of SEQ ID NO:
205, and/or
(i-6) between the 72th and 73th nucleotides from the 3′ end of SEQ ID NO:
200;
and/or
(ii) the Cas endonuclease is Cas 9, Cas3, Cas8a2, Cas8b, Cas8c, Cas10, Cas11, Cas12, Cas12a or Cpf1, Cas13, Cas13a, C2c1, C2c3, or C2c2, further optionally Cas9 or Cpf1, yet further optionally SpCas9,
optionally wherein the gRNA comprises one or more of the following features:
(A) (A-1) the gRNA is a single guide RNA (sgRNA) comprising in a single strand:
(i) a CRISPR RNA (crRNA) sequence comprising the targeting sequence and a crRNA backbone sequence, and
(ii) a trans-activating CRISPR RNA (tracrRNA) sequence,
optionally wherein the crRNA sequence and the tracrRNA sequence are linked via a linker optionally comprising SEQ ID NO:
109, further optionally wherein the gRNA comprises the targeting sequence followed by, optionally immediately followed by, a sgRNA backbone sequence of any of SEQ ID NOS:
111-114, optionally wherein the sgRNA backbone sequence is followed by one or more uracils, further optionally 1-10 uracils, or
(A-2) the gRNA is a dual guide RNA (dgRNA) formed by hybridization between:
(i) a crRNA comprising the targeting sequence and a crRNA backbone sequence, and
(ii) a tracrRNA,
optionally wherein:
(a) the targeting sequence is followed by, optionally immediately followed by, a sgRNA backbone sequence of SEQ ID NOS:
115 and the tracrRNA comprises SEQ ID NO:
116, or
(b) the targeting sequence is followed by, optionally immediately followed by, a sgRNA backbone sequence of SEQ ID NOS:
117 and the tracrRNA comprises SEQ ID NO:
118;
(B) the gRNA is synthetic or recombinant;
and/or
(C) the gRNA is a synthetic sgRNA and comprises at least one chemical modification, which is optionally:
(C-1) 2-O-methylation further optionally at first three and last three bases, and/or
(C-2) one or more 3′ phosphorothioate bonds, further optionally between first three and last two bases.

54 more elements

Facts

Application

18/282,711Filed 18 Mar 2022

Priority

19 Mar 2021

Published

30 May 2024

Term ends

Not estimatedThe source records don’t support an estimate.

Classification19 codes

C12N 15/11
Recombinant DNA-technology: dNA or RNA fragments; Modified forms thereof ; Non-coding nucleic acids having a biological activity
A61K 31/7088
Carbohydrates; Sugars; Derivatives thereof: compounds having three or more nucleosides or nucleotides
A61K 31/7105
Compounds having three or more nucleosides or nucleotides: natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine,…
A61K 48/005
Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy:…