Insect cells and methods for engineering the same
Assignee64-X, Inc.
InventorAlexis Rovner
Life of this patent
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Abstract
The present disclosure provides insect cell lines for expression of viral vectors, and methods of making and using the same. Provided methods employ use of identifiers that are capable of being packaged into a viral vector to select and/or identify insect cell lines with engineered sequences associated with beneficial characteristics for viral vectors production. Exemplary viral vectors include AAV vectors.
Claims30
A method, comprising:
- a. producing a plurality of adeno-associated viral (AAV) vectors from a library of insect cells, wherein the library of insect cells comprise a plurality of insect cells, wherein each insect cell individually comprises:
- (i) a nucleic acid sequence comprising a barcode positioned between two functional AAV ITR sequences,
- (ii) one or more library variants that result in one or more perturbations, wherein at least one library variant comprises a gRNA,
- (iii) an RNA-guided nuclease, and
- (iv) one or more nucleic acid sequences essential for production of AAV vectors,
- wherein the plurality of insect cells produce a plurality of AAV vectors, wherein each AAV vector comprises an identifier that corresponds to the identifier of the insect cell from which it was produced;
- and
- b. detecting the barcodes of the plurality of AAV vectors.
A method of selecting insect cells for producing adeno-associated viral (AAV) vector, the method comprising:
- a. transfecting a library of insect cells with one or more sequences essential for the production of an AAV vector, wherein each insect cell of the plurality individually comprise:
- (i) a nucleic acid sequence comprising a barcode positioned between two functional AAV ITR sequences,
- (ii) one or more library variants that result in one or more perturbations, wherein at least one library variant comprises a gRNA, and
- (iii) an RNA-guided nuclease,
- b. producing a plurality of AAV vectors from the library of insect cells, wherein the AAV vectors each comprise a barcode that corresponds to the barcode of the individual insect cell from which it was produced;
- and
- c. detecting the barcodes of the plurality of AAV vectors, wherein the abundance of the barcode correlates with production and/or secretion of AAV vectors by the insect cell.
An insect cell or insect cell population comprising a plurality of insect cells, wherein each insect cell comprises:
- (i) an engineered nucleic acid sequence comprising a barcode positioned between two functional AAV ITR sequences, wherein the nucleic acid sequence is present episomally within the insect cell,
- (ii) one or more library variants that result in one or more perturbations, wherein at least one library variant comprises a gRNA,
- (iii) an RNA-guided nuclease, and
- (iv) one or more nucleic acid sequences essential for production of AAV vectors,
- wherein the insect cell population produces a plurality of AAV vectors, wherein each AAV vector comprises a barcode that corresponds to the barcode of the insect cell from which it was produced.
An insect cell or insect cell population comprising a plurality of insect cells, wherein each insect cell comprises:
- (i) an engineered nucleic acid sequence comprising a barcode positioned between two functional AAV ITR sequences, wherein the nucleic acid sequence is integrated into the insect cell genome positioned between a pair of cis-acting integration sequences,
- (ii) one or more library variants that result in one or more perturbations, wherein at least one library variant comprises a gRNA,
- (iii) an RNA-guided nuclease, and
- (iv) one or more nucleic acid sequences essential for production of AAV vectors,
- wherein the insect cell population produces a plurality of AAV vectors, wherein each AAV vector comprises a barcode that corresponds to the barcode of the insect cell from which it was produced.
Dependencies are inferred from the claim text.