GENOMIC EDITING OF COMPLEMENT
Complement activation occurs via three main pathways: the antibody-dependent classical pathway, the alternative pathway, and the mannose-binding lectin (MBL) pathway. Inappropriate or excessive complement activation is an underlying cause or contributing factor to a number of serious diseases and conditions, and considerable effort has been devoted over the past several decades to exploring various complement inhibitors as therapeutic agents. Methods, systems, and compositions for genomic editing of a gene encoding a complement protein, e.g., C3, are disclosed.
1 . A method of treating a complement-mediated eye disorder in a subject, the method comprising contacting a hepatic cell of the subject with:
(i) a base editor comprising a fusion protein comprising an endonuclease (e.g., a Cas endonuclease) and a deaminase; and
(ii) a gRNA (e.g., a single guide RNA (sgRNA)) comprising a targeting domain comprising a nucleotide sequence that is complementary to a portion of a human C3 gene,
wherein after the contacting step, the cell and/or the subject exhibits reduced expression and/or activity of C3 protein (e.g., reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%), relative to a control, thereby treating the eye disorder.
2 . The method of claim 1 , wherein the portion of the human C3 gene comprises a nucleotide sequence within an exon of SEQ ID NO:1.
3 . The method of claim 1 , wherein the portion of the human C3 gene comprises a nucleotide sequence within an intron of SEQ ID NO:1.
4 . The method of any one of claims 1-3 , wherein the gRNA targets the base editor to one or more base positions recited in Table 2, 3 or 4.
5 . The method of any one of claims 1-4 , wherein after the contacting step, the human C3 gene comprises a base edit, relative to a wildtype human C3 gene, from a C to a T; from a G to an A; from a T to a C; or from an A to a G at one or more base positions recited in Table 2, 3 or 4.
6 . The method of any one of claims 1-5 , wherein after the contacting step, the human C3 gene comprises a genomic edit, relative to a wildtype human C3 gene, of a nonstop codon to a stop codon at one or more base positions recited in Table 2, 3, or 4.
7 . The method of any one of claims 1-6 , wherein the reduced activity of the C3 protein comprises reduced thioester domain activity.
8 . The method of any one of claims 1-7 , wherein after the contacting step, the cell or the subject expresses a mutant C3 protein, and a level or rate of cleavage of the mutant C3 protein by a C3 convertase is reduced (e.g., reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%), relative to level or rate of cleavage of a wildtype C3 protein by the C3 convertase.
9 . The method of any one of claims 1-8 , wherein the Cas endonuclease is a nuclease inactive Cas endonuclease.
10 . The method of any one of claims 1-8 , wherein the Cas endonuclease is a nickase.
11 . The method of claim 10 , wherein the nickase is a Cas9 nickase.
12 . The method of any one of claims 1-11 , wherein the deaminase is a deaminase from the apolipoprotein B mRNA-editing complex (APOBEC) family deaminase.
13 . The method of claim 12 , wherein the APOBEC family deaminase is selected from the group consisting of APOBEC1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3F deaminase, APOBEC3G deaminase, and APOBEC3H deaminase.
14 . The method of any one of claims 1-13 , comprising contacting the hepatic cell with a nucleotide sequence encoding the base editor.
15 . The method of claim 14 , comprising contacting the hepatic cell with a viral vector comprising the nucleotide sequence encoding the base editor.
16 . The method of any one of claims 1-15 , comprising contacting the hepatic cell with a viral vector comprising the gRNA.
17 . The method of claim 15 or 16 , comprising contacting the hepatic cell with a viral vector comprising the nucleotide sequence encoding the base editor and comprising the gRNA.
18 . The method of any one of claims 1-13 , comprising contacting the hepatic cell with a ribonucleoprotein (RNP) complex comprising the base editor and the gRNA.
19 . The method of any one of claims 1-18 , wherein the eye disorder is geographic atrophy or intermediate AMD.
20 . A method of inhibiting or reducing, relative to a control, level of complement C3 in the eye of a subject, the method comprising contacting a hepatic cell of the subject with, or administering to the subject (e.g., systemically or locally to the liver of the subject):
(i) a base editor comprising a fusion protein comprising an endonuclease (e.g., a Cas endonuclease) and a deaminase; and
(ii) a gRNA (e.g., a single guide RNA (sgRNA)) comprising a targeting domain comprising a nucleotide sequence that is complementary to a portion of the human C3 gene,
wherein after the contacting or administering step, the hepatic cell comprises a human C3 gene comprising at least one genomic edit, thereby inhibiting or reducing level of C3 in the eye.
21 . The method of claim 20 , wherein after the contacting or administering step, the cell and/or the subject exhibits reduced expression and/or activity of C3 protein (e.g., reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%), relative to a control.
22 . The method of claim 20 or 21 , wherein the portion of the human C3 gene comprises a nucleotide sequence within an exon of SEQ ID NO:1.
23 . The method of claim 20 or 21 , wherein the portion of the human C3 gene comprises a nucleotide sequence within an intron of SEQ ID NO:1.
24 . The method of any one of claims 20-23 , wherein the gRNA targets the base editor to one or more base positions recited in Table 2, 3 or 4.
25 . The method of any one of claims 20-24 , wherein after the contacting or administering step, the human C3 gene comprises a base edit, relative to a wildtype human C3 gene, from a C to a T; from a G to an A; from a T to a C; or from an A to a G at one or more base positions recited in Table 2, 3 or 4.
26 . The method of any one of claims 20-25 , wherein after the contacting or administering step, the human C3 gene comprises a genomic edit, relative to a wildtype human C3 gene, of a nonstop codon to a stop codon at one or more base positions recited in Table 2, 3, or 4.
27 . The method of any one of claims 20-26 , wherein the reduced activity of the C3 protein comprises reduced thioester domain activity.
28 . The method of any one of claims 20-27 , wherein after the contacting or administering step, the cell or the subject expresses a mutant C3 protein, and a level or rate of cleavage of the mutant C3 protein by a C3 convertase is reduced (e.g., reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%), relative to level or rate of cleavage of a wildtype C3 protein by the C3 convertase.
29 . The method of any one of claims 20-28 , wherein the Cas endonuclease is a nuclease inactive Cas endonuclease.
30 . The method of any one of claims 20-28 , wherein the Cas endonuclease is a nickase.
31 . The method of claim 30 , wherein the nickase is a Cas9 nickase.
32 . The method of any one of claims 20-31 , wherein the deaminase is a deaminase from the apolipoprotein B mRNA-editing complex (APOBEC) family deaminase.
33 . The method of claim 32 , wherein the APOBEC family deaminase is selected from the group consisting of APOBEC1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3F deaminase, APOBEC3G deaminase, and APOBEC3H deaminase.
34 . The method of any one of claims 20-33 , comprising contacting the hepatic cell with or administering a nucleotide sequence encoding the base editor.
35 . The method of claim 34 , comprising contacting the hepatic cell with or administering a viral vector comprising the nucleotide sequence encoding the base editor.
36 . The method of any one of claims 20-35 , comprising contacting the hepatic cell with or administering a viral vector comprising the gRNA.
37 . The method of claim 35 or 36 , comprising contacting the hepatic cell with or administering a viral vector comprising the nucleotide sequence encoding the base editor and comprising the gRNA.
38 . The method of any one of claims 20-33 , comprising contacting the hepatic cell with or administering a ribonucleoprotein (RNP) complex comprising the base editor and the gRNA.
39 . The method of any one of claims 20-38 , wherein the subject has or suffers from a complement-mediated eye disorder.
40 . The method of claim 39 , wherein the complement-mediated eye disorder is geographic atropy or intermediate AMD.
41 . The method of any one of claims 1-40 , wherein the base editor and the gRNA are not locally administered to, or targeted to, the eye of the subject.