Guide nucleic acids targeting vegfa and uses thereof
AssigneeHuidagene Therapeutics (Singapore) Pte. Ltd.
InventorXing Wang
Life of this patent
Estimating the term…
Abstract
Provided are systems and methods for modifying VEGFA expression.
Claims33
A system comprising:
- (1) a guide nucleic acid comprising
- c) a scaffold sequence capable of forming a complex with a Cas13 endonuclease, and
- d) a guide sequence capable of hybridizing to a target sequence on a transcript of a VEGFA gene, thereby guiding the complex to the transcript, or a polynucleotide encoding the guide nucleic acid;
- and
- (2) the Cas13 endonuclease or a polynucleotide encoding the Cas13 endonuclease;
- wherein the Cas13 endonuclease comprises a sequence having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO:
- 16 or 32, comprises substitutions D160A, D642A, Y666A, and Y677A relative to the wild type of the Cas13 endonuclease of SEQ ID NO:
- 16, and having substantially the same (e.g., at least about 80%, 90%, 95%, 99%, or more) guide sequence-specific RNA endonuclease activity as the Cas13 endonuclease of SEQ ID NO:
- 16;
- wherein the guide sequence comprises (1) a sequence of SEQ ID NO:
- 1 or 2 or a 5′ or 3′ end truncation thereof with 1, 2, 3, 4, 5, or 6 nucleotides truncated at the 5′ or 3′ end;
- or (2) a sequence having a sequence identity of at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% to SEQ ID NO:
- 1 or 2 or a 5′ or 3′ end truncation thereof with 1, 2, 3, 4, 5, or 6 nucleotides truncated at the 5′ or 3′ end;
- or (3) a sequence having at most 1, 2, 3, 4, 5, or 6 nucleotide differences, whether consecutive or not, compared to SEQ ID NO:
- 1 or 2.
The system of any preceding claim, wherein the scaffold sequence has substantially the same secondary structure as the secondary structure of the sequence of SEQ ID NO:
- 5;
- or wherein the scaffold sequence comprises (1) a sequence of SEQ ID NO:
- 5 or a 5′ or 3′ end truncation thereof with 1, 2, 3, 4, 5, or 6, nucleotides truncated at the 5′ or 3′ end;
- or (2) a sequence having a sequence identity of at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% to SEQ ID NO:
- 5 or a 5′ or 3′ end truncation thereof with 1, 2, 3, 4, 5, or 6 nucleotides truncated at the 5′ or 3′ end;
- or (3) a sequence having at most 1, 2, 3, 4, 5, or 6 nucleotide differences, whether consecutive or not, compared to SEQ ID NO:
- 5.
The system of any preceding claim, wherein the guide nucleic acid comprises, from 5′ to 3′, one said scaffold sequence, one said guide sequence, and one said scaffold sequence, wherein scaffold sequences are the same or different.
The system of any preceding claim, wherein the guide nucleic acid comprises, from 5′ to 3′, one said scaffold sequence, one said guide sequence, one said scaffold sequence, one said guide sequence, and one said scaffold sequence, wherein scaffold sequences are the same or different, and wherein guide sequences are the same or different.
The system of any preceding claim, wherein the guide nucleic acid comprises a sequence having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of any one of SEQ ID NOs:
- 17 and 30-31;
- or a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences, whether consecutive or not, compared to the sequence of any one of SEQ ID NOs:
- 17 and 30-31.
The system of any preceding claim, wherein the transcript of the VEGFA gene is a VEGFA pre-mRNA or a VEGFA mRNA.
The system of any preceding claim, wherein the VEGFA gene is human VEGFA gene.
A recombinant adeno-associated virus (rAAV) vector genome comprising:
- (1) a first polynucleotide sequence comprising a sequence comprising or encoding the guide nucleic acid as defined in any preceding claim;
- and
- (2) a second polynucleotide sequence comprising a sequence encoding the Cas13 endonuclease as defined in any preceding claim,
- wherein the rAAV vector genome is adapted to be encapsulated into a rAAV particle.
The rAAV vector genome of any preceding claim, wherein the first polynucleotide sequence comprises a U6 promoter (e.g., SEQ ID NO:
- 4 or 22) operably linked to the sequence comprising or encoding the guide nucleic acid.
The rAAV vector genome of any preceding claim, wherein the second polynucleotide sequence comprises a Cbh promoter (e.g., SEQ ID NO:
- 6) operably linked to the sequence encoding the Cas13 endonuclease.
The rAAV vector genome of any preceding claim, wherein the second polynucleotide comprises a first sequence encoding a first NLS at the 5′ end of the sequence encoding the Cas13 endonuclease and/or a second sequence encoding a second NLS at the 3′ end of the sequence encoding the Cas13 endonuclease.
The rAAV vector genome of any preceding claim, wherein the first NLS and/or the second NLS is a SV40 NLS (e.g., SEQ ID NO:
- 26), a bpSV40 NLS (e.g., SEQ ID NO:
- 15), or a Nucleoplasmin NLS (npNLS).
The rAAV vector genome of any preceding claim, wherein the second polynucleotide sequence comprises a WPRE sequence downstream of the sequence encoding the Cas13 endonuclease;
- optionally wherein the WPRE sequence is selected from the group consisting of Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE), WPRE3 (e.g., SEQ ID NO:
- 11), and a functional variant (e.g., a functional truncation) thereof.
The rAAV vector genome of any preceding claim, wherein the second polynucleotide sequence comprises a sequence encoding a bGH polyA signal (e.g., SEQ ID NO:
- 12) downstream of the sequence encoding the Cas13 endonuclease.
The rAAV vector genome of any preceding claim, wherein the second polynucleotide sequence comprises, from 5′ to 3′, the promoter of SEQ ID NO:
- 6, Kozak sequence of gccacc, an in-frame start codon ATG, the first sequence encoding the first NLS of SEQ ID NO:
- 15, the sequence encoding the Cas13 endonuclease of SEQ ID NO:
- 16 or 32, the second sequence encoding the second NLS of SEQ ID NO:
- 15, an in-frame stop codon, the WPRE sequence of SEQ ID NO:
- 11, and the sequence encoding the polyA signal of SEQ ID NO:
- 12.
The rAAV vector genome of any preceding claim, wherein the rAAV vector genome comprises, from 5′ to 3′, the first polynucleotide sequence and the second polynucleotide sequence;
- or comprises, from 5′ to 3′, the second polynucleotide sequence and the first polynucleotide sequence.
The rAAV vector genome of any preceding claim, wherein the rAAV vector genome comprises, from the 5′ to 3′,
- (1) the 5′ITR of SEQ ID NO:
- 3;
- (2) the promoter of SEQ ID NO:
- 4;
- (3) a sequence encoding the scaffold sequence of SEQ ID NO:
- 5;
- (4) a sequence encoding the guide sequence of SEQ ID NO:
- 1;
- (5) a sequence encoding the scaffold sequence of SEQ ID NO:
- 5;
- (6) a sequence encoding the guide sequence of SEQ ID NO:
- 2;
- (7) a sequence encoding the scaffold sequence of SEQ ID NO:
- 5;
- (8) the promoter of SEQ ID NO:
- 6;
- (9) Kozak sequence of gccacc;
- (10) in-frame start codon ATG;
- (11) the first sequence of SEQ ID NO:
- 8 encoding the first NLS of SEQ ID NO:
- 15;
- (12) the sequence of SEQ ID NO:
- 33 encoding the Cas13 endonuclease of SEQ ID NO:
- 32;
- (13) the second sequence SEQ ID NO:
- 10 encoding the second NLS of SEQ ID NO:
- 15;
- (14) an in-frame stop codon;
- (15) the WPRE sequence of SEQ ID NO:
- 11;
- (16) the sequence encoding a polyA signal of SEQ ID NO:
- 12;
- and
- (17) the 3′ ITR of SEQ ID NO:
- 13;
- wherein the rAAV vector genome comprises an optional linker between any two adjacent components of the components (1) to (17).
The rAAV vector genome of any preceding claim, wherein the rAAV vector genome comprises, consists essentially of, or consists of a polynucleotide having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the sequence of SEQ ID NO:
- 14.
The rAAV vector genome of any preceding claim, wherein the rAAV vector genome comprises, from the 5′ to 3′,
- (1) the 5′ ITR of SEQ ID NO:
- 3;
- (2) the promoter of SEQ ID NO:
- 4;
- (3) a sequence encoding the scaffold sequence of SEQ ID NO:
- 5;
- (4) a sequence encoding the guide sequence of SEQ ID NO:
- 1;
- (5) a sequence encoding the scaffold sequence of SEQ ID NO:
- 5;
- (6) a sequence encoding the guide sequence of SEQ ID NO:
- 2;
- (7) a sequence encoding the scaffold sequence of SEQ ID NO:
- 5;
- (8) the promoter of SEQ ID NO:
- 6;
- (9) Kozak sequence of gccacc;
- (10) in-frame start codon ATG;
- (11) the first sequence of SEQ ID NO:
- 8 encoding the first NLS of SEQ ID NO:
- 15;
- (12) the sequence of SEQ ID NO:
- 33 encoding the Cas13 endonuclease of SEQ ID NO:
- 32;
- (13) the second sequence SEQ ID NO:
- 10 encoding the second NLS of SEQ ID NO:
- 15;
- (14) an in-frame stop codon;
- (15) the WPRE sequence of SEQ ID NO:
- 11;
- (16) the sequence encoding a polyA signal of SEQ ID NO:
- 12;
- and
- (17) the 3′ ITR of SEQ ID NO:
- 13;
- wherein the rAAV vector genome comprises a sequence having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the sequence of SEQ ID NO:
- 14;
- wherein the rAAV vector genome does not contain any nucleotide difference from the sequence of SEQ ID NO:
- 14 in any one of the components (1) to (17);
- wherein the rAAV vector genome contains one or more nucleotide difference or does not contain any nucleotide difference from the sequence of SEQ ID NO:
- 14 between any two adjacent components of the components (1) to (17).
The rAAV vector genome of any preceding claim, wherein the rAAV vector genome comprises, consists essentially of, or consists of the sequence of SEQ ID NO:
- 14.
A recombinant AAV (rAAV) particle comprising the rAAV vector genome of any preceding claim.
The rAAV particle of any preceding claim, wherein the rAAV particle comprising a capsid with a serotype of AAV8.
The rAAV particle of any preceding claim, wherein the AAV8 is wild type AAV8 or a functional variant thereof.
A method for production of a rAAV particle, comprising culturing in a host cell a transgene plasmid comprising the rAAV vector genome of any preceding claim, thereby encapsulating the rAAV vector genome into a capsid with a serotype of AAV8.
A cell comprising a transgene plasmid comprising the rAAV vector genome of any preceding claim.
Use of the cell comprising a transgene plasmid comprising the rAAV vector genome of any preceding claim for the production of rAAV particles.
A pharmaceutical composition comprising (1) the system of any preceding claim or the rAAV particle of any preceding claim and (2) a pharmaceutically acceptable excipient.
A method of downregulating expression of a VEGFA gene, comprising contacting the transcript of the VEGFA gene with the system of any preceding claim, the rAAV particle of any preceding claim, or the pharmaceutical composition of any preceding claim.
A cell or a progeny thereof comprising the system of any preceding claim, or the rAAV particle of any preceding claim.
A cell or a progeny thereof comprising a VEGFA transcript modified by the system of any preceding claim, the rAAV particle of any preceding claim, or the method of any preceding claim.
A method for preventing, diagnosing, and/or treating a VEGFA associated disease in a subject in need thereof, comprising administering to the subject the system of any preceding claim, the rAAV particle of any preceding claim, or the pharmaceutical composition of any preceding claim, wherein the Cas13 endonuclease modifies the transcript of VEGFA gene, leading to downregulated expression of VEGFA gene, thereby treating the VEGFA associated disease.
The method of any preceding claim, wherein the VEGFA associated disease is neovascular age-related macular degeneration (nAMD), Diabetic Macular Edema (DME), or a cancer.
The method of any preceding claim, wherein the subject is not responsive to anti-VEGF therapy, such as, anti-VEGF antibody.
Dependencies are inferred from the claim text.