Compositions and Methods for Kallikrein (KLKB1) Gene Editing

Abstract

Compositions and methods for editing, e.g., introducing double-stranded breaks, within the KLKB1 gene are provided. Compositions and methods for treating subjects having hereditary angioedema (HAE), are provided.

1 . A guide RNA comprising:
 a. a guide sequence at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 15, 8, and 41;   b. a guide sequence comprising at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 15, 8, and 41; or   c. a guide sequence selected from SEQ ID NOs: 15, 8, and 41.   
     
     
         2 . The guide RNA of  claim 1 , further comprising the nucleotide sequence of SEQ ID NO: 202. 
     
     
         3 . The guide RNA of  claim 1 , wherein the guide RNA further comprises a nucleotide sequence selected from SEQ ID NO: 170, 171, 172, and 173 wherein the sequence of SEQ ID NO: 170, 171, 172, or 173 is 3′ of the guide sequence. 
     
     
         4 . The guide RNA of  claim 1 , wherein the guide RNA further comprises a 3′ tail. 
     
     
         5 . The guide RNA of  claim 1 , wherein the guide RNA comprises at least one modification. 
     
     
         6 . The guide RNA of  claim 5 , wherein the modification comprises (a) a 5′ end modification, (b) a 3′ end modification, or (c) a modification in a hairpin region. 
     
     
         7 . (canceled) 
     
     
         8 . (canceled) 
     
     
         9 . The guide RNA of  claim 1 , wherein the modification comprises (a) a 2′-O-methyl (2′-O-Me) modified nucleotide, (b) a phosphorothioate (PS) bond between nucleotides, or (c) a 2′-fluor (2′F) modified nucleotide. 
     
     
         10 . (canceled) 
     
     
         11 . (canceled) 
     
     
         12 . The guide RNA of  claim 1 , further comprising the nucleotide sequence of SEQ ID NO: 171 or 173. 
     
     
         13 . The guide RNA of  claim 12 , wherein the nucleotide sequence of SEO ID NO: 171 is modified according to the pattern of nucleotide sequence of SEQ ID NO: 405: or wherein the nucleotide sequence of SEO ID NO: 173 is modified according to the pattern of SEO ID NOs: 248-255 or 450. 
     
     
         14 . (canceled) 
     
     
         15 . (canceled) 
     
     
         16 . The guide RNA of  claim 12 , wherein the guide sequence comprises a sequence of SEQ ID NO: 15, 8, or 41. 
     
     
         17 . (canceled) 
     
     
         18 . (canceled) 
     
     
         19 . The guide RNA of  claim 1 , wherein the guide RNA is modified according to the pattern of SEQ ID NO: 300, wherein the N's are collectively the guide sequence of  claim 1 . 
     
     
         20 . (canceled) 
     
     
         21 . The guide RNA of  claim 19 , wherein
 (a) the guide sequence is SEQ ID NO: 15 and the guide RNA is modified according to mG*mG*mA* UUGCGUAUGGGACACAA GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCC GUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUm CmGmGmUmGmCmU*mU*mU*mU; or   (b) the guide sequence is SEO ID NO: 8 and the guide RNA is modified according to mU*mA*mC*CCGGGAGUUGACUUUGG GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCC GUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUm CmGmGmUmGmCmU*mU*mU*mU: or   (c) the guide sequence is SEO ID NO: 41 and the guide RNA is modified according to mU*mA*mU*UAUCAAAUCACAUUACC GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCC GUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUm CmGmGmUmGmCmU*mU*mU*mU,   wherein “mA,” “mC,” “mU,” or “mG” denote a nucleotide that has been modified with 2′-O-Me, a * denotes a phosphorothioate bond, and N is a natural nucleotide.   
     
     
         22 . (canceled) 
     
     
         23 . (canceled) 
     
     
         24 . A composition comprising a guide RNA of  claim 1 . 
     
     
         25 . A composition of  claim 24 , further comprising an RNA-guided DNA binding agent or nucleic acid encoding an RNA-guided DNA binding agent. 
     
     
         26 . The composition of  claim 25 , wherein the nucleic acid encoding an RNA-guided DNA binding agent comprises an mRNA comprising an open reading frame (ORF) encoding an RNA guided DNA binding agent. 
     
     
         27 . The composition of  claim 26 , wherein the RNA-guided DNA binding agent is Cas9. 
     
     
         28 . The composition of  claim 27 , wherein the Cas9 is  S. pyogenes  Cas9. 
     
     
         29 . (canceled) 
     
     
         30 . (canceled) 
     
     
         31 . The composition of  claim 24 , wherein the guide RNA is associated with a lipid nanoparticle (LNP). 
     
     
         32 . The composition of  claim 31 , wherein the LNP comprises an ionizable lipid. 
     
     
         33 . The composition of  claim 32 , wherein the ionizable lipid is (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate. 
     
     
         34 . The composition of  claim 31 , wherein the LNP comprises (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate, DSPC, cholesterol, and PEG2k-DMG. 
     
     
         35 . A pharmaceutical composition comprising a guide RNA of  claim 1  and further comprising a pharmaceutical excipient or carrier. 
     
     
         36 . (canceled) 
     
     
         37 . (canceled) 
     
     
         38 . (canceled) 
     
     
         39 . (canceled) 
     
     
         40 . (canceled) 
     
     
         41 . (canceled) 
     
     
         42 . (canceled) 
     
     
         43 . A method of
 (a) inducing a double stranded break or a single stranded break within a KLKB1 gene in a cell;   (b) reducing expression of KLKB1 in a cell;   (c) treating a subject having hereditary angioedema (HAE);   (d) treating or preventing angioedema associated with HAE, bradykinin production and accumulation, bradykinin-induced swelling, angioedema obstruction of the airway, or asphyxiation: or   (e) reducing total plasma kallikrein in a subject,   the method comprising contacting a cell with a guide RNA of  claim 1 .   
     
     
         44 . The method of  claim 43 , wherein the cell is in a subject. 
     
     
         45 . (canceled) 
     
     
         46 . The method of  claim 43 , wherein treating the subject comprises reducing the frequency and/or severity of HAE attacks. 
     
     
         47 . (canceled) 
     
     
         48 . (canceled) 
     
     
         49 . (canceled) 
     
     
         50 . (canceled) 
     
     
         51 . The composition of  claim 27 , wherein the ORF encoding the Cas9 comprises an ORF from an mRNA of SEQ ID NOs: 501-516.