Gene editing systems comprising an rna guide targeting lactate dehydrogenase a (ldha) and uses thereof

Abstract

Provided herein are gene editing systems and/or compositions comprising RNA guides targeting LDHA for use in genetic editing of the LDHA gene. Also provide herein are methods of using the gene editing system for introducing edits to the LDHA gene and/or for treatment of primary hyperoxaluria (PH), and processes for characterizing the gene editing system.

1 . A gene editing system for genetic editing of a lactate dehydrogenase A (LDHA) gene, comprising
 (i) a Cas12i2 polypeptide or a first nucleic acid encoding the Cas12i2 polypeptide, wherein the Cas12i2 polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO: 1166 and comprises one or more mutations relative to SEQ ID NO: 1166;   (ii) an RNA guide or a second nucleic acid encoding the RNA guide, wherein the RNA guide comprises a spacer sequence specific to a target sequence within an LDHA gene, the target sequence being adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5′-TTN-3′, which is located 5′ to the target sequence.   
     
     
         2 . The gene editing system of  claim 1 , wherein the one or more mutations in the Cas12i2 polypeptide are at positions D581, G624, F626, P868, I926, V1030, E1035, and/or S1046 of SEQ ID NO: 1166. 
     
     
         3 . The gene editing system of  claim 2 , wherein the one or more mutations are amino acid substitutions, which optionally is D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, S1046G, or a combination thereof. 
     
     
         4 . The gene editing system of  claim 3 , wherein the Cas12i2 polypeptide comprises:
 (i) mutations at positions D581, D911, I926, and V1030, which optionally are amino acid substitutions of D581R, D911R, I926R, and V1030G;   (ii) mutations at positions D581, I926, and V1030, which optionally are amino acid substitutions of D581R, I926R, and V1030G;   (iii) mutations at positions D581, I926, V1030, and S1046, which optionally are amino acid substitutions of D581R, I926R, V1030G, and S1046G;   (iv) mutations at positions D581, G624, F626, I926, V1030, E1035, and S1046, which optionally are amino acid substitutions of D581R, G624R, F626R, I926R, V1030G, E1035R, and S1046G; or   (v) mutations at positions D581, G624, F626, P868, I926, V1030, E1035, and S1046, which optionally are amino acid substitutions of D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, and S1046G.   
     
     
         5 . The gene editing system of  claim 1 , wherein the Cas12i2 polypeptide comprises the amino acid sequence of SEQ ID NO: 1167, 1168, 1169, 1170, or 1171. 
     
     
         6 . The gene editing system of  claim 1 , which comprises the first nucleic acid encoding the Cas12i2 polypeptide. 
     
     
         7 . The gene editing system of  claim 6 , wherein the first nucleic acid is a messenger RNA (mRNA), and/or is included in a viral vector. 
     
     
         8 . (canceled) 
     
     
         9 . The gene editing system of  claim 1 , wherein the target sequence is within exon 3 or exon 5 of the LDHA gene, and/or comprises: 
       
         
           
                 
                 
               
                     
                   (i) 
                 
                     
                   (SEQ ID NO: 1237) 
                 
                     
                   5′-TAGGACTTGGCAGATGAACT-3′; 
                 
                     
                     
                 
                     
                   (ii) 
                 
                     
                   (SEQ ID NO: 1239) 
                 
                     
                   5′-GATGACATCAACAAGAGCAA-3; 
                 
                     
                     
                 
                     
                   (iii) 
                 
                     
                   (SEQ ID NO: 1245) 
                 
                     
                   5′-TTCATAGTGGATATCTTGAC-3′; 
                 
                     
                     
                 
                     
                   (iv) 
                 
                     
                   (SEQ ID NO: 1248) 
                 
                     
                   5′-TCATAGTGGATATCTTGACC-3′; 
                 
                     
                   or 
                 
                     
                     
                 
                     
                   (v) 
                 
                     
                   (SEQ ID NO: 1249) 
                 
                     
                   5′-CATAGTGGATATCTTGACCT-3′. 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         10 . (canceled) 
     
     
         11 . The gene editing system of  claim 9 , wherein the spacer sequence comprises: 
       
         
           
                 
                 
               
                     
                   (i) 
                 
                     
                   (SEQ ID NO: 1269) 
                 
                     
                   5′-UAGGACUUGGCAGAUGAACU-3′; 
                 
                     
                     
                 
                     
                   (ii) 
                 
                     
                   (SEQ ID NO: 1270) 
                 
                     
                   5′-GAUGACAUCAACAAGAGCAA-3′; 
                 
                     
                     
                 
                     
                   (iii) 
                 
                     
                   (SEQ ID NO: 1271) 
                 
                     
                   5′-UUCAUAGUGGAUAUCUUGAC-3′; 
                 
                     
                     
                 
                     
                   (iv) 
                 
                     
                   (SEQ ID NO: 1272) 
                 
                     
                   5′-UCAUAGUGGAUAUCUUGACC-3′; 
                 
                     
                   or 
                 
                     
                     
                 
                     
                   (v)  
                 
                     
                   (SEQ ID NO: 1273) 
                 
                     
                   5′-CAUAGUGGAUAUCUUGACCU-3. 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         12 . (canceled) 
     
     
         13 . The gene editing system of  claim 1 , wherein the RNA guide comprises the spacer sequence and a direct repeat sequence, which is at least 90% identical to any one of SEQ ID NOs: 1-10 or a fragment thereof that is at least 23-nucleotide in length. 
     
     
         14 - 16 . (canceled) 
     
     
         17 . The gene editing system of  claim 13 , wherein the direct repeat sequence is 5′-AGAAAUCCGUCUUUCAUUGACGG-3′ (SEQ ID NO: 10). 
     
     
         18 . The gene editing system of  claim 1 , wherein the RNA guide comprises the nucleotide sequence of: 
       
         
           
                 
               
                   (i) 
                 
                   (SEQ ID NO: 1214) 
                 
                   5′-AGAAAUCCGUCUUUCAUUGACGGUAGGACUUGGCAGAUGAACU-3′; 
                 
                     
                 
                   (ii) 
                 
                   (SEQ ID NO: 1235) 
                 
                   5′-AGAAAUCCGUCUUUCAUUGACGGGAUGACAUCAACAAGAGCAA-3′; 
                 
                     
                 
                   (iii) 
                 
                   (SEQ ID NO: 1221) 
                 
                   5′-AGAAAUCCGUCUUUCAUUGACGGUUCAUAGUGGAUAUCUUGAC-3′; 
                 
                     
                 
                   (iv) 
                 
                   (SEQ ID NO: 1224) 
                 
                   5′-AGAAAUCCGUCUUUCAUUGACGGUCAUAGUGGAUAUCUUGACC-3′; 
                 
                   or 
                 
                     
                 
                   (v) 
                 
                   (SEQ ID NO: 1225) 
                 
                   5′-AGAAAUCCGUCUUUCAUUGACGGCAUAGUGGAUAUCUUGACCU-3′. 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         19 . The gene editing system of  claim 1 , wherein the system comprises the second nucleic acid encoding the RNA guide, or wherein the nucleic acid encoding the RNA guide is located in a viral vector. 
     
     
         20 . (canceled) 
     
     
         21 . The gene editing system of  claim 7 , wherein the viral vector comprises both the first nucleic acid encoding the Cas12i2 polypeptide and the second nucleic acid encoding the RNA guide. 
     
     
         22 . The gene editing system of  claim 21 , wherein the system comprises the first nucleic acid encoding the Cas12i2 polypeptide, which is located on a first vector, and wherein the system comprises the second nucleic acid encoding the RNA guide, which is located on a second vector; or
 wherein the system comprises one or more lipid nanoparticles (LNPs), which encompass (i), (ii), or both.   
     
     
         23 - 24 . (canceled) 
     
     
         25 . The gene editing system of  claim 22 , wherein the system comprises the LNP, which encompass (i), and wherein the system comprises a viral vector comprising the second nucleic acid encoding the RNA guide; or
 wherein the system comprises the LNP, which encompass (ii), and wherein the system comprises a viral vector comprising the first nucleic acid encoding Cas12i2 polypeptide.   
     
     
         26 . The gene editing system of  claim 25 , wherein the viral vector is an AAV vector. 
     
     
         27 . A gene editing system for genetic editing of a lactate dehydrogenase A (LDHA) gene, comprising
 (i) a Cas12i polypeptide or a first nucleic acid encoding the Cas12i polypeptide, optionally wherein the Cas12i polypeptide is a Cas12i2 polypeptide; and   (ii) an RNA guide or a second nucleic acid encoding the RNA guide, wherein the RNA guide comprises a spacer sequence specific to a target sequence within exon 3 or exon 5 of an LDHA gene, the target sequence being adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5′-TTN-3′, which is located 5′ to the target sequence.   
     
     
         28 - 46 . (canceled) 
     
     
         47 . A pharmaceutical composition comprising the gene editing system set forth in  claim 1 . 
     
     
         48 . A kit comprising the elements (i) and (ii) of the gene editing system set forth in  claim 1 . 
     
     
         49 . A method for editing a lactate dehydrogenase A (LDHA) gene in a cell, the method comprising contacting a host cell with the gene editing system for editing the LDHA gene set forth in  claim 1  to genetically edit the LDHA gene in the host cell. 
     
     
         50 - 52 . (canceled) 
     
     
         53 . A method for treating primary hyperoxaluria (PH) in a subject, comprising administering to a subject in need thereof a gene editing system for editing a lactate dehydrogenase A (LDHA) gene set forth in  claim 1 . 
     
     
         54 - 55 . (canceled) 
     
     
         56 . An RNA guide, comprising (i) a spacer sequence that is specific to a target sequence in a lactate dehydrogenase A (LDHA) gene, wherein the target sequence is adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5′-TTN-3′, which is located 5′ to the target sequence; and (ii) a direct repeat sequence. 
     
     
         57 - 65 . (canceled)