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US 2021/0060180 A1Published application
Recombinogenic nucleic acid strands in situ
AssigneeSNIPR Technologies Limited
InventorJasper CLUBE
Life of this patent
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Abstract
The invention relates to retrieving or modifying target nucleic acids, such as host cell chromosomal DNA, by homologous recombination with vectors that have been cut to provide recombinogenic nucleic acid strands in situ.
Claims33
1-21. (canceled)
22:
- A method for gene therapy in vitro or in vivo, comprising:
- (a) providing a nucleic acid vector comprising a first nucleic acid strand, wherein the first strand comprises:
- i. a first homology arm (HA1) that is homologous to a first nucleotide sequence (NS1) of a donor strand for homologous recombination between the first strand and the donor strand;
- ii. a second homology arm (HA2) that is homologous to a second nucleotide sequence (NS2) of the donor strand for homologous recombination between the first strand and the donor strand;
- and
- iii. an intervening nucleotide sequence joining the 3′ end of HA1 to the 5′ end of HA2:
- wherein HA1 is 5′ to HA2 in the first strand;
- and NS1 is 3′ to NS2 in the donor strand,
- wherein the donor strand comprises a retrieval sequence (RS) between NS1 and NS2:
- (b) combining the first strand with the donor strand:
- (c) nuclease cutting of a first cut site (CS1) of the first strand at the 5′ end of HA1, or flanking 5′ of the 5′ end of HA1;
- and/or nuclease cutting of a second cut site (CS2) of the first strand at the 3′ end of HA2, or flanking 3′ of the 3′ end of HA2 to provide a cut first strand;
- and
- wherein steps (b) and (c) are carried out in any order or simultaneously:
- (d) carrying out homologous recombination of the cut first strand with the donor strand, whereby gap repair of the cut first strand produces a product vector in which RS is retrieved between HA1 and HA2, and wherein the product of step (d) is a circular vector;
- and
- (e) inserting all or part of said RS of the product vector into a further nucleic acid strand;
- wherein a mutation of the further strand is removed by said insertion of RS into the further strand.
15 more elements
23:
- A method for correcting a genetic mutation in vitro in a target human cell, the method comprising:
- (i) obtaining a first cell which is a clone of the target cell carrying out the method of claim 22 inside the first cell, wherein RS in the product vector of step (d) comprises an uncorrected RS sequence comprising said genetic mutation;
- (ii) using genetic engineering to correct the genetic mutation in the product vector, thereby producing a product vector comprising a corrected RS sequence;
- and
- (iii) inserting the corrected RS sequence into a second cell which is a clone of the target cell, wherein the second cell comprises the further strand comprising the genetic mutation, wherein the corrected RS sequence supplements or replaces the uncorrected RS sequence in the second cell, thereby producing a corrected target cell.
2 more elements
24-27. (canceled)
28:
- A method producing a Chimeric Antigen Receptor T-cell (CAR-T cell) by carrying out the method of claim 22, wherein the further strand is comprised by a T cell.
29-80. (canceled)
81:
- A method for treating a genetic defect caused by a genetic mutation of a target cell in a human or animal subject, comprising administering to the subject the corrected target cell obtained using the method of claim 23.
A method for treating or preventing a disease or condition in a human or animal subject, comprising administering to the subject the RS obtained by the method of claim 22.
83-94. (canceled)
95:
- A method for producing a plurality of vectors comprising a collection of retrieved sequences (RSs) comprising at least two RSs that are different from each other, the method comprising for each vector in the plurality of vectors:
- (a) providing a nucleic acid vector comprising a first nucleic acid strand, wherein the first strand comprises:
- i. a first homology arm (HA1) that is homologous to a first nucleotide sequence (NS1) of a donor strand for homologous recombination between the first strand and the donor strand;
- ii. a second homology arm (HA2) that is homologous to a second nucleotide sequence (NS2) of the donor strand for homologous recombination between the first strand and the donor strand;
- and
- iii. an intervening nucleotide sequence joining the 3′ end of HA1 to the 5′ end of HA2;
- wherein HA1 is 5′ to HA2 in the first strand;
- and NS1 is 3′ to NS2 in the donor strand,
- wherein the donor strand comprises a corresponding retrieval sequence (RS) between NS1 and NS2;
- (b) combining the first strand with the donor strand;
- (c) nuclease cutting of a first cut site (CS1) of the first strand at the 5′ end of HA1, or flanking 5′ of the 5′ end of HA1;
- and/or nuclease cutting of a second cut site (CS2) of the first strand at the 3′ end of HA2, or flanking 3′ of the 3′ end of HA2 to provide a cut first strand;
- and
- wherein steps (b) and (c) are carried out in any order or simultaneously;
- (d) carrying out homologous recombination of the cut first strand with the donor strand,
- whereby gap repair of the cut first strand produces a product vector in which the RS is retrieved between HA1 and HA2, and wherein the product of step (d) is a circular vector;
- thereby obtaining the vector comprising the corresponding RS.
14 more elements
96:
- The method of claim 95, wherein the vectors are BAC or YAC vectors comprising a collection of genomic fragments retrieved from a cell sample.
97:
- The method of claim 95, wherein the method uses a respective cutting site motif to direct each said cutting of step (c), wherein each cutting site is removed by the homologous recombination.
98:
- The method of claim 95, wherein the donor strand is a strand of a chromosome or an episome of a cell.
99:
- The method of claim 95, wherein each RS is at least 10 kb in length.
100:
- The method of claim 95, wherein the nuclease cutting is Cas, meganuclease, zinc finger protein-mediated or TALEN-mediated cutting.
101:
- The method of claim 95, wherein the nuclease cutting is by a Cas nuclease, and wherein in step (a) the vector comprises in 5′ to 3′ direction:
- A. A first protospacer sequence (PS1) comprising CS1;
- B. A first PAM (P1) adjacent to the 3′ end of PS1;
- and
- C. HA1;
- wherein a seed sequence (S) of PS1 joins CS1 to P1;
- and/or
- wherein in step (a) the vector comprises in 5′ to 3′ direction:
- D. HA2;
- E. A second protospacer sequence (PS2) comprising CS2;
- F. A second PAM (P2) adjacent to the 3′ end of PS2;
- wherein a seed sequence (S2) of PS2 joins CS2 to P2.
9 more elements
102:
- The method of claim 101, wherein in step (c) the same guide RNA (gRNA1) is used to guide the nuclease cutting of CS1 and CS2 by the Cas nuclease.
103:
- The method of claim 95, wherein before or during step (d) the donor strand is cut at the 5′end of NS2 or flanking 5′ of the 5′ end of NS2;
- and/or at the 3′ end of NS1 or flanking 3′ of the 3′ end of NS1.
104:
- The method claim 95, wherein in step (b) the vector is a closed circular vector.
105:
- The method of claim 104, wherein in step (b) the vector is supercoiled.
106:
- The method of claim 95, wherein sequence between HA1 and HA2 is deleted from the first strand in step (c) and/or sequence between NS1 and NS2 is deleted from the donor strand in or after step (d).
107:
- The method of claim 106, wherein in step (d) the RS is deleted from the donor strand.
108:
- The method of claim 95, wherein said nuclease cutting is cutting by a Cas nuclease, wherein a protospacer/PAM combination at the 5′ end of HA1 or flanking 5′ of the 5′ end of HA1 is destroyed by step (d), whereby PS1 is rendered non-functional in the first strand;
- and/or wherein a protospacer/PAM combination at the 3′ end of HA2 or flanking 3′ of the 3′ end of HA2 is destroyed by step (d), whereby PS2 is rendered non-functional in the first strand.
109:
- The method of claim 95, wherein in step (d) a donor sequence is inserted into the first strand and/or a sequence is deleted from the first strand, to form a new protospacer/PAM combination or a new marker sequence.
110:
- The method of claim 109, comprising cutting the protospacer of the new combination using a Cas after step (d).
111:
- The method of claim 95, wherein in step (d) a donor sequence is deleted from the donor strand to form a new protospacer/PAM combination or a new marker sequence.
112:
- The method of claim 111, comprising cutting the protospacer of the new combination in the donor strand using a Cas after step (d).
113:
- The method of claim 95, wherein in step (d) the RS is inserted into the first strand and the inserted RS sequence is functional for expression from the modified first strand.
114:
- The method of claim 95, wherein steps (b) to (d) are performed in a host cell, wherein the RS is deleted from the donor strand in or after step (d) and (i) the deletion is lethal to the host cell, or (ii) the method down-regulates cell viability, growth or proliferation, or (iii) the method up-regulates cell viability, growth or proliferation.
115:
- The method of claim 95, wherein the method is carried out in a bacterial or archaeal cell.
116:
- The method of claim 95, wherein steps (b) to (d) are performed in a host cell, wherein the nuclease cutting is Cas-mediated cutting, and in step (c) the Cas-mediated cutting is in a protospacer sequence in the first strand, wherein the protospacer comprises a seed sequence, wherein the seed sequence comprises a nucleotide sequence that is not found in the genome of the host cell.
117:
- The method of claim 95, wherein steps (b) to (d) are performed in a host cell.
118:
- The method of claim 95, wherein the plurality of vectors comprises a first vector and a second vector, and wherein:
- (i) the HA1 and HA2 corresponding to the first vector are different from the HA1 and HA2 corresponding to the second vector;
- (ii) the NS1 and NS2 corresponding to the first vector are different from the NS1 and NS2 corresponding to the second vector;
- and/or
- (iii) the RS corresponding to the first vector is different from the RS corresponding to the second vector.
2 more elements
Dependencies are inferred from the claim text.