COMPOSITIONS AND METHODS FOR ALTERING A NUCLEOBASE IN A LIPOPROTEIN(A) POLYNUCLEOTIDE
The disclosure features compositions and methods for treating cardiovascular disease by introducing one or more alterations into a lipoprotein(a) (LPA) polynucleotide in a cell. In particular embodiments, the disclosure provides a base editor system (e.g., a fusion protein or complex comprising a programable DNA binding protein, a nucleobase editor, and gRNA) for modifying an LPA polynucleotide, where the modification is associated with reduced expression, and/or reduced activity of the LPA polypeptide encoded by the polynucleotide.
1 . A method of editing a nucleobase of a lipoprotein A (LPA) polynucleotide in a cell, the method comprising contacting the LPA polynucleotide with a base editor system comprising a guide RNA, or a polynucleotide encoding said guide RNA, and a base editor comprising a fusion protein or a protein complex comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain, or one or more polynucleotides encoding the base editor, wherein said guide RNA targets said base editor to effect an alteration of the nucleobase of the LPA polynucleotide.
2 . A method of treating atherosclerosis and/or cardiovascular disease in a subject in need thereof, the method comprising contacting a cell of the subject with a base editor system comprising a base editor comprising a fusion protein or protein complex comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain, or one or more polynucleotide encoding the base editor, and a guide RNA, or a polynucleotide encoding said guide RNA, wherein the guide RNA targets the base editor to effect an alteration of a nucleobase of an LPA polynucleotide.
3 . The method of claim 1 , wherein the guide RNA is selected from the guides listed in Tables 1B-1, 1A-1, and 1C-1.
4 . The method of claim 1 , wherein the deaminase domain is an adenosine deaminase comprising an amino acid sequence with at least about 90% identity to the following amino acid sequence or a fragment thereof lacking the N-terminal methionine and comprises one or more amino acid alterations selected from the group consisting of I76Y, V82S, Y123H, Y147R, and Q154R compared to the following amino acid sequence:
TadA*7.10
(SEQ ID NO: 1)
MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG
LHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIG
RVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFR
MPRQVFNAQKKAQSSTD.
5 . The method of claim 4 , wherein the adenosine deaminase comprises the amino acid alterations I76Y, V82S, Y123H, Y147R, and Q154R.
6 . The method of claim 1 , wherein the deaminase domain is a cytidine deaminase comprising an amino acid sequence with at least about 90% identity to the following amino acid sequence or a fragment thereof lacking the N-terminal methionine:
ppAPOBEC1
(SEQ ID NO: 23)
MTSEKGPSTGDPTLRRRIESWEFDVFYDPRELRKETCLLYEIKWGMSRKI
WRSSGKNTTNHVEVNFIKKFTSERRFHSSISCSITWFLSWSPCWECSQAI
REFLSQHPGVTLVIYVARLFWHMDQRNRQGLRDLVNSGVTIQIMRASEYY
HCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQ
NHLAFFRLHLQNCHYQTIPPHILLATGLIHPSVTWR.
7 . The method of claim 1 , wherein alteration of the nucleobase is associated with a reduction in transcription of a polynucleotide sequence encoding the LPA protein; and/or wherein alteration of the nucleobase in the LPA polynucleotide disrupts a splice site.
8 . The method of claim 1 , wherein the alteration introduces a single nucleotide polymorphism (SNP) into the LPA polynucleotide, wherein the SNP is associated with reduced serum concentrations of LPA in a subject and/or reduced incidence of atherosclerosis and/or cardiovascular disease in a subject, and wherein the SNP is selected from the group consisting of, chr6:160531784:T>C, chr6:160532531:C>T, chr6:160548552:G>A, chr6:160532610:A>G, chr6:160591049:A>G, chr6:160635134:G>A, and chr6:160547886:A>G.
9 . The method of claim 2 , wherein the method is associated with at least a 10% reduction of incidence of coronary heart disease in the subject.
10 . The method of claim 2 , wherein the base editor system is administered to the subject using a lipid nanoparticle comprising the guide RNA and an mRNA molecule encoding the base editor.
11 . A modified cell comprising an alteration in a nucleobase of a LPA polynucleotide, wherein the alteration is prepared by the method of claim 1 .
12 . A base editor system comprising a fusion protein or one or more polynucleotides encoding said fusion protein, wherein said fusion protein comprises a nucleic acid programmable DNA binding protein domain (napDNAbp) and a deaminase domain, and a guide RNA, or a guide polynucleotide encoding said guide RNA, wherein said guide RNA targets said base editor to effect an alteration of a nucleobase of an LPA polynucleotide.
13 . The base editor system of claim 12 , wherein the guide RNA is selected from the guides 5 listed in Tables 1B-1, 1A-1, and 1C-1.
14 . The base editor system of claim 12 , wherein the deaminase is an adenosine deaminase comprising an amino acid sequence with at least about 90% identity to the following amino acid sequence or a fragment thereof lacking the N-terminal methionine and comprises one or more amino acid alterations selected from the group consisting of I76Y, V82S, Y123H, Y147R, and Q154R compared to the following amino acid sequence:
TadA*7.10
(SEQ ID NO: 1)
MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG
LHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIG
RVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFR
MPRQVFNAQKKAQSSTD.
15 . The base editor system of claim 12 , wherein the SNP is selected from the group consisting of, chr6:160531784:T>C, chr6:160532531:C>T, chr6:160548552:G>A, chr6:160532610:A>G, chr6:160591049:A>G, chr6:160635134:G>A, and chr6:160547886:A>G.
16 . A polynucleotide or set of polynucleotides encoding the base editor system of claim 12 or a component thereof.
17 . A lipid nanoparticle comprising the base editor system of claim 12 .
18 . A pharmaceutical composition comprising the modified cell of claim 11 , and a pharmaceutically acceptable excipient.
19 . A kit comprising the modified cell of claim 11 , and directions for the use of same.
20 . A guide RNA comprising a sequence listed in Table 1B-1, 1B-2, 1A-1, 1A-2, 1C-1, or 1C-2.