MODIFIED REGULATORY T CELLS AND METHODS OF USING THE SAME
The disclosure features modified regulatory T (T REG ) cells having increased lineage stability, decreased activation of T cells (e.g., conventional T (T CONV ) cells), and/or increased resistance to immune rejection, and methods of producing and using such cells, for example, in the treatment of graft versus host disease (GVHD).
1 . A method for producing a functionally enhanced and/or lineage stabilized regulatory T (T REG ) cell, the method comprising contacting a T REG cell with a base editor, or one or more polynucleotides encoding the base editor, wherein the base editor comprises a polynucleotide programmable DNA binding polypeptide (napDNAbp), and a deaminase, and one or more guide RNAs (gRNAs), or one or more polynucleotides encoding the gRNAs, wherein the one or more gRNAs target the base editor to effect an alteration in a nucleic acid molecule, wherein the nucleic acid molecule encodes a polypeptide and/or comprises a regulatory element associated with expression thereof, and wherein the polypeptide is selected from the group consisting of bone morphogenetic protein/retinoic acid-inducible neural-specific protein 1 (BRINP1), C terminus of HSC70-interacting protein (CHIP), Cluster of Differentiation 70 (CD70), c-JUN kinase 1 (JNK1), protein kinase C theta (PRKCQ), ring finger protein 20 (RNF20), and sirtuin 1 (SIRT1), thereby producing the functionally enhanced and/or lineage stabilized T REG cell.
2 . The method of claim 1 , further comprising contacting the T REG cell with a gRNA that targets the base editor to effect an alteration in a nucleic acid molecule, wherein the nucleic acid molecule encodes a polypeptide and/or comprises a regulatory element associated with expression thereof, and wherein the polypeptide is selected from the group consisting of beta-2 microglobulin (B2M), Cluster of Differentiation 58, and programmed cell death 1 (PD-1).
3 . A method for producing a functionally enhanced and/or lineage stabilized regulatory T (T REG ) cell, the method comprising contacting a T REG cell with a base editor, or one or more polynucleotide encoding the base editor, wherein the base editor comprises a polynucleotide programmable DNA binding polypeptide (napDNAbp), and a deaminase, and two or more guide RNAs (gRNAs), or one or more polynucleotides encoding the gRNAs, wherein each gRNA targets the base editor to effect an alteration in a nucleic acid molecule, wherein each nucleic acid molecule encodes a polypeptide and/or comprises a regulatory element associated with expression of the polypeptide, and wherein a first polypeptide is selected from the group consisting of bone morphogenetic protein/retinoic acid-inducible neural-specific protein 1 (BRINP1), C terminus of HSC70-interacting protein (CHIP), c-JUN kinase 1 (JNK1), protein kinase C theta (PRKCQ), ring finger protein 20 (RNF20), and sirtuin 1 (SIRT1), and wherein a second polypeptide is selected from the group consisting of Cluster of Differentiation 58 (CD58), Cluster of Differentiation 70 (CD70), and programmed cell death 1 (PD-1).
4 . The method of claim 3 further comprising effecting an alteration in a third nucleic acid molecule, wherein the nucleic acid molecule encodes a beta-2 microglobulin (B2M) polypeptide and/or comprises a regulatory element associated with expression thereof.
5 . The method of claim 4 , wherein the method comprises contacting the T REG cell with gRNAs comprising each of the following nucleotide sequences: TSBTx2813, TSBTx2817, TSBTx2834, TSBTx025, and TSBTx845 to thereby reduce or eliminate expression of each of CD70, SIRT1, CD58, PD-1, and B2M in the T REG cell.
6 . The method of claim 1 , wherein the method comprises editing a combination of nucleic acid molecules encoding a combination of two or more polypeptides and/or a regulatory element associated with expression thereof, wherein the combination of polypeptides is selected from the group consisting of: SIRT1, PD-1, CD70, and CD58; SIRT1, PD-1, and CD70; SIRT1, PD-1, and CD58; SIRT1, CD70, and CD58; SIRT1 and PD-1; SIRT1 and CD70; SIRT1 and CD58; SIRT1, PD-1, CD70, CD58, and B2M; SIRT1, PD-1, CD70, and B2M; SIRT1, PD-1, CD58 and B2M; SIRT1, CD70, CD58, and B2M; SIRT1, PD-1, and B2M; SIRT1, CD70, and B2M; SIRT1, CD58, and B2M; RNF20, PD-1, CD70, and CD58; RNF20, PD-1, and CD70; RNF20, PD-1, and CD58; RNF20, CD70, and CD58; RNF20 and PD-1; RNF20 and CD70; RNF20 and CD58; RNF20, PD-1, CD70, CD58, and B2M; RNF20, PD-1, CD70, and B2M; RNF20, PD-1, CD58 and B2M; RNF20, CD70, CD58, and B2M; RNF20, PD-1, and B2M; RNF20, CD70, and B2M; RNF20, CD58, and B2M; SIRT1, RNF20, PD-1, CD70, and CD58; SIRT1, RNF20, PD-1, and CD70; SIRT1, RNF20, PD-1, and CD58; SIRT1, RNF20, CD70, and CD58; SIRT1, RNF20, and PD-1; SIRT1, RNF20, and CD70; SIRT1, RNF20, and CD58; SIRT1, RNF20, PD-1, CD70, CD58, and B2M; SIRT1, RNF20, PD-1, CD70, and B2M; SIRT1, RNF20, PD-1, CD58, and B2M; SIRT1, RNF20, CD70, CD58, and B2M; SIRT1, RNF20, PD-1, and B2M; SIRT1, RNF20, CD70, and B2M; and SIRT1, RNF20, CD58, and B2M.
7 . The method of claim 1 , wherein each guide RNA comprises a sequence selected from the group consisting of TSBTx2810, TSBTx2813, TSBTx2815, TSBTx2813, TSBTx2814, TSBTx2816, TSBTx2834, TSBTx845, TSBTx025, TSBTx2817, TSBTx2817, TSBTx2818, TSBTx2819, TSBTx2820, TSBTx2821, TSBTx2822, TSBTx2823, TSBTx2824, TSBTx2825, TSBTx2826, TSBTx2827, TSBTx2828, TSBTx2830, or TSBTx2831, TSBTx1680, TSBTx1681, TSBTx1682, TSBTx1683, TSBTx1684, TSBTx1685, TSBTx1686, TSBTx1687, TSBTx1688, TSBTx1689, TSBTx1690, TSBTx1691, TSBTx1692, TSBTx1693, TSBTx1694, TSBTx1695, TSBTx1696, TSBTx1697, TSBTx1698, or TSBTx2853, TSBTx2813, TSBTx2817, TSBTx2834, TSBTx025, and TSBTx845.
8 . The method of claim 1 , wherein:
the alteration is associated with a reduction of costimulation of a T CONV cell by the functionally enhanced and/or lineage stabilized T REG cell relative to a reference cell;
the deaminase is a cytidine deaminase or an adenosine deaminase;
the napDNAbp is Cas9 or Cas12;
the base editor further comprises one or more uracil glycosylase inhibitors (UGIs); and/or
the base editor further comprises one or more nuclear localization signals (NLS).
9 . The method of claim 8 , wherein the adenosine deaminase is ABE8.20.
10 . The method of claim 1 , further comprising expressing a chimeric antigen receptor (CAR) in the T REG cell.
11 . A method for producing a functionally enhanced and/or lineage stabilized regulatory T (T REG ) cell, the method comprising contacting a T REG cell with a polynucleotide programmable DNA binding polypeptide (napDNAbp), or one or more polynucleotides encoding the napDNAbp, and one or more guide RNAs (gRNAs), or one or more polynucleotides encoding the gRNAs, that target the napDNAbp to cleave a target nucleic acid molecule and effect an alteration in the target nucleic acid molecule, wherein the target nucleic acid molecule encodes a polypeptide and/or comprises a regulatory element associated with expression thereof, and wherein the polypeptide is selected from the group consisting of bone morphogenetic protein/retinoic acid-inducible neural-specific protein 1 (BRINP1), C terminus of HSC70-interacting protein (CHIP), Cluster of Differentiation 70 (CD70), c-JUN kinase 1 (JNK1), protein kinase C theta (PRKCQ), ring finger protein 20 (RNF20), and sirtuin 1 (SIRT1), thereby producing the functionally enhanced and/or lineage stabilized T REG cell.
12 . A functionally enhanced and/or lineage stabilized regulatory T (T REG ) cell produced according to the method of claim 1 .
13 . A functionally enhanced and/or lineage stabilized regulatory T (T REG ) cell comprising a nucleobase alteration that reduces or eliminates expression of a polypeptide selected from the group consisting of bone morphogenetic protein/retinoic acid-inducible neural-specific protein 1 (BRINP1), C terminus of HSC70-interacting protein (CHIP), Cluster of Differentiation 70 (CD70), c-JUN kinase 1 (JNK1), protein kinase C theta (PRKCQ), ring finger protein 20 (RNF20), and sirtuin 1 (SIRT1).
14 . The functionally enhanced and/or lineage stabilized regulatory T (T REG ) cell of claim 1 , where the T REG cell comprises a nucleobase alteration that reduces or eliminates expression of two or more polypeptides, wherein a first polypeptide is selected from the group consisting of bone morphogenetic protein/retinoic acid-inducible neural-specific protein 1 (BRINP1), C terminus of HSC70-interacting protein (CHIP), c-JUN kinase 1 (JNK1), protein kinase C theta (PRKCQ), ring finger protein 20 (RNF20), and sirtuin 1 (SIRT1), and wherein a second polypeptide is selected from the group consisting of Cluster of Differentiation 58 (CD58), Cluster of Differentiation 70 (CD70), and programmed cell death 1 (PD-1).
15 . The functionally enhanced and/or lineage stabilized regulatory T (T REG ) cell of any claim 14 , wherein the T REG cell comprises an alteration that reduces or eliminates expression of a combination of polypeptides selected from the group consisting of: SIRT1, PD-1, CD70, and CD58; SIRT1, PD-1, and CD70; SIRT1, PD-1, and CD58; SIRT1, CD70, and CD58; SIRT1 and PD-1; SIRT1 and CD70; SIRT1 and CD58; SIRT1, PD-1, CD70, CD58, and B2M; SIRT1, PD-1, CD70, and B2M; SIRT1, PD-1, CD58 and B2M; SIRT1, CD70, CD58, and B2M; SIRT1, PD-1, and B2M; SIRT1, CD70, and B2M; SIRT1, CD58, and B2M; RNF20, PD-1, CD70, and CD58; RNF20, PD-1, and CD70; RNF20, PD-1, and CD58; RNF20, CD70, and CD58; RNF20 and PD-1; RNF20 and CD70; RNF20 and CD58; RNF20, PD-1, CD70, CD58, and B2M; RNF20, PD-1, CD70, and B2M; RNF20, PD-1, CD58 and B2M; RNF20, CD70, CD58, and B2M; RNF20, PD-1, and B2M; RNF20, CD70, and B2M; RNF20, CD58, and B2M; SIRT1, RNF20, PD-1, CD70, and CD58; SIRT1, RNF20, PD-1, and CD70; SIRT1, RNF20, PD-1, and CD58; SIRT1, RNF20, CD70, and CD58; SIRT1, RNF20, and PD-1; SIRT1, RNF20, and CD70; SIRT1, RNF20, and CD58; SIRT1, RNF20, PD-1, CD70, CD58, and B2M; SIRT1, RNF20, PD-1, CD70, and B2M; SIRT1, RNF20, PD-1, CD58, and B2M; SIRT1, RNF20, CD70, CD58, and B2M; SIRT1, RNF20, PD-1, and B2M; SIRT1, RNF20, CD70, and B2M; and SIRT1, RNF20, CD58, and B2M.
16 . A pharmaceutical composition comprising a functionally enhanced and/or lineage stabilized regulatory T (T REG ) cell of claim 14 .
17 . The pharmaceutical composition of claim 17 , wherein the T REG cell overexpresses an inhibitory receptor selected from the group consisting of Human Leukocyte Antigen-E (HLA-E), Human Leukocyte Antigen-G (HLA-G), Programmed Death Ligand 1 (PD-L1), and Cluster of Differentiation 47 (CD47).
18 . A pharmaceutical composition comprising one or more guide RNAs (gRNA) and a polynucleotide encoding a base editor comprising a polynucleotide programmable DNA binding polypeptide (napDNAbp) domain and a deaminase domain, wherein each gRNA comprises a nucleic acid sequence that is complementary to a nucleic acid molecule, wherein each nucleic acid molecule encodes a polypeptide and/or comprises a regulatory element associated with expression of the polypeptide, wherein a first polypeptide is selected from the group consisting of bone morphogenetic protein/retinoic acid-inducible neural-specific protein 1 (BRINP1), C terminus of HSC70-interacting protein (CHIP), c-JUN kinase 1 (JNK1), protein kinase C theta (PRKCQ), ring finger protein 20 (RNF20), and sirtuin 1 (SIRT1), and wherein a second polypeptide is selected from the group consisting of Cluster of Differentiation 58 (CD58), Cluster of Differentiation 70 (CD70), and programmed cell death 1 (PD-1).
19 . A kit comprising the functionally enhanced and/or stabilized T REG cell of claim 13 .
20 . A method of treating an autoimmune or alloimmune disease in a subject, the method comprising administering to the subject an effective amount of a functionally enhanced and/or stabilized T REG cell of claim 13 .
21 . A guide RNA (gRNA) or a polynucleotide encoding the guide RNA, wherein the guide RNA comprises a nucleotide sequence with at least 70% sequence identity to a sequence selected from one or more of those listed in Tables 1A-1C or 2A-2C, or truncations thereof, wherein the gRNA targets a base editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) to effect an alteration in a nucleic acid molecule encoding a polypeptide and/or comprising a regulatory element associated with expression thereof, and wherein the polypeptide is selected from the group consisting of bone morphogenetic protein/retinoic acid-inducible neural-specific protein 1 (BRINP1), C terminus of HSC70-interacting protein (CHIP), Cluster of Differentiation 70 (CD70), c-JUN kinase 1 (JNK1), protein kinase C theta (PRKCQ), ring finger protein 20 (RNF20), and sirtuin 1 (SIRT1), thereby producing a functionally enhanced and/or lineage stabilized T REG cell.
22 . A method for producing a functionally enhanced and/or lineage stabilized regulatory T (T REG ) cell, the method comprising contacting a T REG cell with a base editor, or a polynucleotide encoding the base editor, wherein the base editor comprises a polynucleotide programmable DNA binding polypeptide (napDNAbp), and an adenosine deaminase, and one or more of the following guide RNAs, or one or more polynucleotides encoding the one or more guide RNAs:
a) a guide RNA comprising a nucleotide sequence selected from the group consisting of TSBTx2810, TSBTx2813, and TSBTx2815;
b) a guide RNA comprising a nucleotide sequence selected from the group consisting of TSBTx2834 and TSBTx845;
c) a guide RNA comprising the nucleotide sequence TSBTx025;
d) a guide RNA comprising the nucleotide sequence TSBTx845; and
e) a guide RNA comprising a nucleotide sequence selected from the group consisting of TSBTx2817, TSBTx2818, TSBTx2819, TSBTx2820, TSBTx2821, TSBTx2822, TSBTx2823, TSBTx2824, TSBTx2825, TSBTx2826, TSBTx2827, TSBTx2828, TSBTx2830, and TSBTx2831;
thereby reducing or eliminating expression in the T REG of one or more of the following polypeptides: cluster of differentiation 70 (CD70), cluster of differentiation 58 (CD58), programmed cell death 1 (PD-1), beta-2 microglobulin (B2M), and sirtuin 1 (SIRT1).
23 . A cell prepared according to the method of claim 23 .
24 . A pharmaceutical composition comprising the cell of claim 23 .