Granted Patent B2

US 12,529,041 · App. 17/273,209 · Granted Jan 20, 2026

Inventor: David Bryson (Cambridge, MA)

Assignee: Beam Therapeutics Inc.

C12N9/22C12N9/78C12N15/11C12N15/907C12Y305/04004C07K2319/09C12N2310/20C12N2800/80

Abstract

Claims (63)

1 . A composition comprising:

A)

(a) a first polynucleotide encoding a fusion protein comprising a deaminase and an N-terminal fragment of Cas9,

wherein the N-terminal fragment of Cas9 starts at the N-terminus of Cas9 and is a contiguous sequence that terminates at position 302, 309, 312, or 354 of Cas9 as numbered in SEQ ID NO: 16, and

wherein the N-terminal fragment of Cas9 is fused at the C-terminus to a split intein-N, and

(b) a second polynucleotide encoding a C-terminal fragment of Cas9,

wherein the C-terminal fragment of Cas9 starts:

i) at position S303 when the N-terminal fragment terminates at position 302 of Cas9,

ii) at position T310 when the N-terminal fragment terminates at position 309 of Cas9,

iii) at position T313 when the N-terminal fragment terminates at position 312 of Cas9, or

iv) at position S355 when the N-terminal fragment terminates at position 354 of Cas9,

 wherein the positions of Cas9 are as numbered in SEQ ID NO: 16, and

 wherein the C-terminal fragment of Cas9 is a contiguous sequence that terminates at the C-terminus of Cas9, and

 wherein the C-terminal fragment of Cas9 is fused at the N-terminus to a split intein-C; or

B)

(a) a first polynucleotide encoding an N-terminal fragment of Cas9,

wherein the N-terminal fragment of Cas9 starts at the N-terminus of Cas9 and is a contiguous sequence that terminates at position 302, 309, 312, or 354 of Cas9 as numbered in SEQ ID NO: 16,

wherein the N-terminal fragment of Cas9 is fused at the C-terminus to a split intein-N, and

(b) a second polynucleotide encoding fusion protein comprising a C-terminal fragment of Cas9 and a deaminase,

wherein the C-terminal fragment of Cas9 starts:

i) at position S303 when the N-terminal fragment terminates at position 302 of Cas9,

ii) at position T310 when the N-terminal fragment terminates at position 309 of Cas9,

iii) at position T313 when the N-terminal fragment terminates at position 312 of Cas9, or

iv) at position S355 when the N-terminal fragment terminates at position 354 of Cas9,

wherein the positions of Cas9 are as numbered in SEQ ID NO: 16, and

wherein the C-terminal fragment of Cas9 is a contiguous sequence that terminates at the C-terminus of Cas9, and

wherein the C-terminal fragment of Cas9 is fused at the N-terminus to a split intein-C; or

C)

(a) a first polynucleotide encoding a fusion protein comprising a deaminase and an N-terminal fragment of Cas9,

wherein the N-terminal fragment of Cas9 starts at the N-terminus of Cas9 and is a contiguous sequence that terminates at position 302, 309, 312, 354, 465, or 471 of Cas9 as numbered in SEQ ID NO: 16,

wherein the N-terminal fragment of Cas9 is fused at the C-terminus to a split intein-N, and

(b) a second polynucleotide encoding a C-terminal fragment of Cas9,

wherein the C-terminal fragment of Cas9 starts:

i) at position S303 when the N-terminal fragment terminates at position 302 of Cas9,

ii) at position T310 when the N-terminal fragment terminates at position 309 of Cas9,

iii) at position T313 when the N-terminal fragment terminates at position 312 of Cas9,

iv) at position S355 when the N-terminal fragment terminates at position 354 of Cas9,

v) at position T466 when the N-terminal fragment terminates at position 465 of Cas9, or

vi) at position T472 when the N-terminal fragment terminates at position 471 of Cas9,

 wherein the positions of Cas9 are as numbered in SEQ ID NO: 16, and

 wherein the C-terminal fragment of Cas9 is a contiguous sequence that terminates at the C-terminus of Cas9,

 wherein the N-terminus residue of the C-terminal fragment of Cas9 is a Cys substituted for an Ala, Ser, or Thr, and

 wherein the C-terminal fragment of Cas9 is fused at the N-terminus to a split intein-C.

2 . The composition of claim 1 , further comprising a single guide RNA (sgRNA) or a polynucleotide encoding the sgRNA.

3 . The composition of claim 1 , wherein the first and the second polynucleotides are joined or are expressed separately.

4 . The composition of claim 1 , wherein the deaminase is an adenosine deaminase.

5 . The composition of claim 1 , where the deaminase is a wild-type TadA or TadA7.10.

6 . The composition of claim 1 , wherein the deaminase is a TadA dimer.

7 . The composition of claim 6 , wherein the TadA dimer comprises a wild-type TadA and a TadA 7.10.

8 . The composition of claim 1 , wherein the fusion protein comprises a nucleus localization signal (NLS).

9 . The composition of claim 8 , wherein the NLS is a bipartite NLS.

10 . The composition of claim 1 , wherein the Cas9 has nickase activity or is catalytically inactive.

11 . A composition comprising the fusion protein and the N- or C-terminal fragment of Cas9 of claim 1 .

12 . A vector comprising the first and the second polynucleotide of claim 1 .

13 . A cell comprising the composition of claim 1 .

14 . A method for delivering a base editor system to a cell, the method comprising contacting the cell with the first and second polynucleotides of claim 1 ; and a single guide RNA (sgRNA) or a polynucleotide encoding the sgRNA.

15 . The method of claim 14 , wherein the target polynucleotide is present in the genome of an organism.

16 . The method of claim 15 , wherein the organism is an animal, plant, or bacteria.

17 . A polynucleotide encoding a fusion protein, wherein the fusion protein comprises a deaminase and an N-terminal fragment of Cas9,

wherein the N-terminal fragment of Cas9 starts at the N-terminus of Cas9 and is a contiguous sequence that terminates at position 302, 309, 312, or 354 of Cas9 as numbered in SEQ ID NO: 16, and

wherein the N-terminal fragment of Cas9 is fused at the C-terminus to a split intein-N.

18 . A polynucleotide encoding a fragment of an A-to-G Base Editor fusion protein, the fusion protein comprising one or more deaminases and an N-terminal fragment of Cas9 of claim 1 , wherein the N-terminal fragment is fused at the C-terminus to a split intein-N.

19 . A polynucleotide encoding a fragment of an A-to-G Base Editor fusion protein, the fusion protein comprising one or more deaminases and a C-terminal fragment of Cas9 of claim 1 , wherein the C-terminal fragment is fused at the N-terminus to a split intein-C.

Assignments (4)

Continuity (3)

References Cited (400)