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US 2020/0370137 A1Published application
Engineering aav
AssigneeSangamo Therapeutics, Inc.
InventorsKyle McGovern, David S. Ojala
Life of this patent
Estimating the term…
Abstract
The present disclosure provides methods and compositions to develop AAV capsids with a desired characteristic compared to a natural AAV serotype. These capsids are useful, for example, for the delivery of genome engineering molecules and gene therapy molecules for the treatment of a subject in need thereof.
Claims89
A method of identifying an AAV capsid variant with a desired characteristic compared to a natural AAV serotype, comprising:
- (1) (i) contacting a cell, cell line, or tissue with a library of AAV variants, wherein each member of the library comprises:
- a) a nucleic acid encoding an AAV variant capsid protein comprising a peptide inserted into a hypervariable and/or surface-exposed loop of the variant capsid protein;
- and
- one or more of:
- b) one or more silent mutations in the nucleic acid sequence flanking the peptide insertion site in the AAV variant capsid protein of a);
- c) a nucleic acid encoding a localization signal;
- d) a nucleic acid comprising a barcode;
- and
- e) a nucleic acid encoding a reporter protein;
- (ii) allowing the AAV variants in said library to transduce the cell, cell line, or tissue;
- (iii) recovering from the cell, cell line, or tissue the AAV variant;
- and
- (iv) identifying the AAV capsid variant with the desired characteristic;
- or (2) (i) contacting a cell, cell line, or tissue with a library of AAV variants, wherein each member of the library comprises:
- a) an AAV variant capsid protein comprising a peptide inserted into a hypervariable and/or surface-exposed loop of the variant capsid protein;
- and
- one or more of:
- b) one or more silent mutations in the nucleic acid encoding the AAV variant capsid protein of a) in the nucleic acid sequence flanking the peptide insertion site;
- c) a nucleic acid encoding a localization signal;
- d) a nucleic acid comprising a barcode;
- and
- e) a nucleic acid encoding a reporter protein;
- (ii) allowing the AAV variants in said library to transduce the cell, cell line, or tissue;
- (iii) recovering from the cell, cell line, or tissue the AAV variant;
- and
- (iv) identifying the AAV capsid variant with the desired characteristic.
23 more elements
A method of identifying an AAV capsid variant with a desired characteristic compared to a natural AAV serotype, comprising:
- (i) inserting a plurality of nucleic acids encoding peptides, into a population of nucleic acids encoding a hypervariable and/or surface-exposed loop of an AAV capsid protein to create a library of nucleic acids encoding AAV variant capsid proteins;
- (ii) linking each nucleic acid encoding an AAV variant capsid protein in said library to one or more of:
- (a) a nucleic acid comprising a barcode,
- (b) a nucleic acid encoding a reporter protein, and
- (c) a nucleic acid encoding a localization signal;
- (iii) manufacturing a library of AAV variants in producer cells by providing adenovirus helper and AAV rep functions in trans;
- (iv) purifying the library of AAV variants;
- (v) contacting a cell, cell line, or tissue with the library of AAV variants;
- (vi) recovering the AAV variants from the target cell, cell line, or tissue;
- and
- (vii) identifying the AAV capsid variant with the desired characteristic.
8 more elements
A library of AAV variants, wherein each member of said library comprises:
- (1) a) a nucleic acid sequence encoding an AAV variant capsid protein comprising a peptide inserted into a hypervariable and/or surface-exposed loop of the variant capsid protein;
- and
- one or more of:
- b) one or more silent mutations in the nucleic acid sequence flanking the peptide insertion site in the AAV variant capsid protein of a);
- c) a nucleic acid encoding a localization signal;
- d) a nucleic acid comprising a barcode;
- and
- e) a nucleic acid encoding a reporter protein;
- or
- (2) a) an AAV variant capsid protein comprising a peptide inserted into a hypervariable and/or surface-exposed loop of the variant capsid protein;
- and
- one or more of:
- b) one or more silent mutations in the nucleic acid encoding the AAV variant capsid protein of a) in the nucleic acid sequence flanking the peptide insertion site;
- c) a nucleic acid encoding a localization signal;
- d) a nucleic acid comprising a barcode;
- and
- e) a nucleic acid encoding a reporter protein.
14 more elements
An AAV variant comprising:
- (1) a) a nucleic acid sequence encoding an AAV variant capsid protein comprising a peptide inserted into a hypervariable and/or surface-exposed loop of the variant capsid protein;
- and
- one or more of:
- b) one or more silent mutations in the nucleic acid sequence flanking the peptide insertion site in the AAV variant capsid protein of a);
- c) a nucleic acid encoding a localization signal;
- d) a nucleic acid comprising a barcode;
- and
- e) a nucleic acid encoding a reporter protein;
- or
- (2) a) an AAV variant capsid protein comprising a peptide inserted into a hypervariable and/or surface-exposed loop of the variant capsid protein;
- and
- one or more of:
- b) one or more silent mutations in the nucleic acid encoding the AAV variant capsid protein of a) in the nucleic acid sequence flanking the peptide insertion site;
- c) a nucleic acid encoding a localization signal;
- d) a nucleic acid comprising a barcode;
- and
- e) a nucleic acid encoding a reporter protein.
14 more elements
A nucleic acid molecule comprising:
- a) a nucleic acid sequence encoding an AAV variant capsid protein comprising a peptide inserted into a hypervariable and/or surface-exposed loop of the variant capsid protein;
- and
- one or more of:
- b) a nucleic acid sequence encoding a variant capsid protein comprising one or more silent mutations in the nucleotide sequence flanking the peptide insertion site in the hypervariable and/or surface-exposed loop of the variant capsid protein;
- c) a nucleic acid sequence encoding a localization signal;
- d) a nucleic acid sequence comprising a barcode;
- and
- e) a nucleic acid sequence encoding a reporter protein.
5 more elements
An AAV vector comprising a nucleic acid molecule encoding a peptide that comprises at least 4 contiguous amino acids of an amino acid sequence set forth in any one of SEQ ID NO:
- 1-32 or SEQ ID NO:68-110.
A peptide comprising an amino acid sequence set forth in any one of SEQ ID NO:
- 1-32 or SEQ ID NO:
- 68-110.
A capsid protein comprising a peptide comprising at least 4 contiguous amino acids of an amino acid sequence set forth in any one of SEQ ID NO:
- 1-32 or SEQ ID NO:68-110.
Dependencies are inferred from the claim text.