US2022275356A1PendingUtilityA1

Base editors with improved precision and specificity

Assignee: MASSACHUSETTS GEN HOSPITALPriority: May 25, 2017Filed: May 9, 2022Published: Sep 1, 2022

Est. expiryMay 25, 2037(~10.8 yrs left)· nominal 20-yr term from priority

Inventors:J. Keith Joung Jason Michael Gehrke

C12N 15/63C12N 9/78C12N 15/102C07K 2319/80C07K 2319/70C12N 2310/20C12N 9/22C12N 15/90C12N 5/0647C12Y 305/04001C12N 15/10C07K 14/435C07K 2319/81C07K 14/4703

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References

Claims

Abstract

Methods and compositions for improving the genome-wide specificities of targeted base editing technologies.

Claims

exact text as granted — not AI-modified

What is claimed is: 
     
         1 . A fusion protein comprising a Cas9-like nickase (nCas9) linked to an engineered variant deaminase hAID, rAPOBEC1*, mAPOBEC3, hAPOBEC3A, hAPOBEC3B, hAPOBEC3C, hAPOBEC3F, hAPOBEC3G, or hAPOBEC3H, with an optional intervening linker, comprising one or more mutations shown in Table 7, 8 and/or 9 in the substrate sequence motif of the deaminase that causes the deaminase to act at a non-canonical motif, and optionally wherein the fusion protein further comprises a uracil glycosylase inhibitor (UGI). 
     
     
         2 . The fusion protein of  claim 1 , wherein the fusion protein comprises an engineered variant of a deaminase domain listed in Table 1 or Table 2 that recognizes a non-cognate sequence motif. 
     
     
         3 . The fusion protein of  claim 1 , wherein the engineered variant of hAID, rAPOBEC1*, mAPOBEC3, hAPOBEC3A, hAPOBEC3B, hAPOBEC3C, hAPOBEC3F, hAPOBEC3G, or hAPOBEC3H comprises one or more mutations shown in Tables 7 or 8. 
     
     
         4 . The fusion protein of  claim 1 , wherein the engineered variant of hAID, rAPOBEC1*, mAPOBEC3, hAPOBEC3A, hAPOBEC3B, hAPOBEC3C, hAPOBEC3F, hAPOBEC3G, or hAPOBEC3H comprises (i) one or more mutations shown in Table 7 and/or 8 and (ii) one or more mutations shown in Table 9. 
     
     
         5 . The fusion protein of  claim 1 , wherein the engineered variant of hAID, rAPOBEC1*, mAPOBEC3, hAPOBEC3A, hAPOBEC3B, hAPOBEC3C, hAPOBEC3F, hAPOBEC3G, or hAPOBEC3H comprises hAPOBEC3A with a mutation at one or more of N57; K60, and/or Y130); or hAID, rAPOBEC1*, mAPOBEC3, hAPOBEC3B, hAPOBEC3C, hAPOBEC3F, hAPOBEC3G, or hAPOBEC3H with a mutation corresponding to N57, K60, or Y130. 
     
     
         6 . The fusion protein of  claim 1 , wherein the engineered variant comprises hAPOBEC3A with a mutation at N57 and Y130. 
     
     
         7 . The fusion protein of  claims 5 - 6 , comprising hAPOBEC3A with one or more of a N57G or N57Q mutation; a K60A or K60D mutation; and/or a Y130F mutation. 
     
     
         8 . The fusion protein of  claim 5 , wherein the engineered variant comprises hAID, rAPOBEC1*, mAPOBEC3, hAPOBEC3B, hAPOBEC3C, hAPOBEC3F, hAPOBEC3Cc or hAPOBEC3H with a mutation corresponding to N57, K60, and/or a mutation corresponding to Y130. 
     
     
         9 . The fusion protein of  claim 5  or  7 , comprising hAID, rAPOBEC1*, mAPOBEC3, hAPOBEC3B, hAPOBEC3C, hAPOBEC3F, hAPOBEC3Cc or hAPOBEC3H with one or more of a mutation corresponding to N57G; K60D; or Y130F. 
     
     
         10 . The fusion protein of  claims 1 - 9 , wherein the engineered variant of hAID, rAPOBEC1*, mAPOBEC3, hAPOBEC3A, hAPOBEC3B, hAPOBEC3C, hAPOBEC3F, hAPOBEC3Cc or hAPOBEC3H comprises hAPOBEC3A with a mutation at A71 and/or 196, or hAID, rAPOBEC1*, mAPOBEC3, hAPOBEC3B, hAPOBEC3C, hAPOBEC3F, hAPOBEC3Cc or hAPOBEC3H with a mutation corresponding to A71 and/or 196 (e.g., as shown in table 10). 
     
     
         11 . A method of treating a subject with beta thalassemia mutation HBB-28 (A&gt;G), the method comprising delivering a therapeutically effective amount of a fusion protein of any of the preceding claims, wherein the deaminase comprises APO3A comprising a mutation at N57G or N57A or N57Q or K60A or K60D or Y130F, and preferably wherein the fusion protein comprises a ssDNA nicking or catalytically-inactive Cas9. 
     
     
         12 . The method of  claim 11 , wherein the fusion protein is delivered as an RNP, mRNA, or plasmid. 
     
     
         13 . The method of  claim 11 , comprising delivering the fusion protein ex vivo to a population of cells comprising CD34+ hematopoietic stem and/or progenitor cells collected from the subject under conditions sufficient for deamination of the mutated, and re-infusing the cells back into the subject. 
     
     
         14 . A method of deaminating a selected cytidine in a nucleic acid, the method comprising contacting the nucleic acid with a fusion protein or base editing system of any of  claims 1 - 10 . 
     
     
         15 . A composition comprising a purified a fusion protein or base editing system of any of  claims 1 - 10 . 
     
     
         16 . The composition of  claim 15 , comprising one or more ribonucleoprotein (RNP) complexes. 
     
     
         17 . A nucleic acid encoding a fusion protein or base editing system of any of  claims 1 - 10 . 
     
     
         18 . A vector comprising the nucleic acid of  claim 17 . 
     
     
         19 . An isolated host cell comprising the nucleic acid of  claim 18 . 
     
     
         20 . The host cell of  claim 19 , which is a stem cell. 
     
     
         21 . The host cell of  claim 19 , which is a hematopoietic stem cell.

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