PCSK9 endonuclease variants, compositions, and methods of use
The present disclosure provides improved genome editing compositions and methods for editing a PCSK9 gene. The disclosure further provides genome edited cells for the prevention, treatment, or amelioration of at least one symptom of hypercholesterolemia or a condition associated therewith.
1. A polypeptide comprising a I-Onul homing endonuclease (HE) variant that cleaves a target site in a proprotein convertase subtilisin/kexin type 9 (PCSK9) gene,
wherein the target site is SEQ ID NO: 11, and
wherein the I-Onul HE variant comprises an amino acid sequence that has at least 90% sequence identical identity to the amino acid sequence set forth in any one of SEQ ID NOs: 6-7.
2. The polypeptide of claim 1 , wherein the I-Onul HE variant lacks:
(a) the 1, 2, 3, 4, 5, 6, 7, or 8 N-terminal amino acids compared to a corresponding wild type I-Onul LAGLIDADG homing endonuclease (LHE) comprising the amino acid sequence of SEQ ID NO: 2;
(b) the 4 N-terminal amino acids compared to a corresponding wild type I-Onul LHE comprising the amino acid sequence of SEQ ID NO: 2;
(c) the 8 N-terminal amino acids compared to a corresponding wild type I-Onul LHE comprising the amino acid sequence of SEQ ID NO: 2;
(d) the 1, 2, 3, 4, or 5 C-terminal amino acids compared to a corresponding wild type I-Onul LHE comprising the amino acid sequence of SEQ ID NO: 2;
(e) the C-terminal amino acid compared to a corresponding wild type I-Onul LHE comprising the amino acid sequence of SEQ ID NO: 2; and/or
(f) the 2 C-terminal amino acids compared to a corresponding wild type I-Onul LHE comprising the amino acid sequence of SEQ ID NO: 2.
3. The polypeptide of claim 1 , wherein the I-Onul HE variant cleaves a PCSK9 target site and comprises the following amino acid substitutions:
a) S24C, L26M, R28N, R30W, N32K, K34R, S35T, S36R, V37A, G38K, S40Y, E42A, G44R, Q46E, T48A, Q61R, V68K, A70R, S72I, N75R, A76V, S78K, K80V, T82G, V116L, L138M, T143N, S159P, F168L, E178D, C180Y, F182G, N184E, I186M, S188R, K189T, S190T, K191G, L192T, G193H, Q195T, Q197R, V199R, T203A, K207R, Y223R, K225S, K227R, F232Y, and D236E of any one of SEQ ID NOs: 1-5;
b) S24C, L26M, R28N, R30W, N32K, K34R, S35T, S36R, V37A, G38K, S40Y, E42A, G44R, Q46E, T48A, Q61R, V68K, A70R, S72I, N75R, A76V, S78K, K80V, T82G, V116L, L138M, T143N, S159P, F168L, E178D, C180Y, F182G, N184E, I186M, S188R, S190T, K191G, L192T, G193H, Q195T, Q197R, V199R, T203A, K207R, Y223R, K225S, K227R, F232Y, and D236E of any one of SEQ ID NOs: 1-5; or
c) S24C, L26M, R28N, R30W, N32K, K34R, S35T, S36R, V37A, G38K, S40Y, E42A, G44R, Q46E, T48A, Q61R, V68K, A70R, S72I, N75R, A76V, S78K, K80V, T82G, V116L, L138M, T143N, S159P, F168L, E178D, C180Y, F182G, N184E, I186M, S188R, K189T, S190T, K191G, L192T, G193H, Q195T, Q197R, V199R, K207R, Y223R, K225S, K227R, F232Y, and D236E of any one of SEQ ID NOs: 1-5.
4. The polypeptide of claim 1 , wherein the I-Onul HE variant comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOS: 6-7.
5. The polypeptide of claim 1 , wherein the I-Onul HE variant comprises the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7.
6. The polypeptide of claim 1 , further comprising a transcription activator-like effectors (TALE) DNA binding domain.
7. The polypeptide of claim 6 , wherein the TALE DNA binding domain comprises about 9.5 TALE repeat units to about 15.5 TALE repeat units.
8. The polypeptide of claim 6 , wherein the TALE DNA binding domain binds the PCSK9 polynucleotide sequence set forth in SEQ ID NO: 13.
9. The polypeptide of claim 1 , further comprising a peptide linker and an end-processing enzyme.
10. The polypeptide of claim 9 , wherein the end-processing enzyme comprises Trex2.
11. The polypeptide of claim 1 , wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 9.
12. A polynucleotide, mRNA, cDNA, or vector encoding the polypeptide of claim 1 .
13. A composition comprising a lipid nanoparticle comprising:
(a) an mRNA encoding a polypeptide of claim 1 ; and
(b) an mRNA encoding Trex2.
14. A lipid nanoparticle comprising:
(a) an mRNA encoding a polypeptide of claim 1 ; and
(b) an mRNA encoding Trex2.
15. A composition comprising a lipid nanoparticle comprising:
(a) an mRNA encoding a polypeptide of claim 1 ; and
(b) an mRNA encoding Trex2.