Mammalian cells and methods for engineering the same
Assignee64-X, Inc.
InventorsAlexis Rovner, Gaurab Chakrabarti, David Thompson, Gregory Sieczkiewicz, McKenna Hicks, Joseph Bellucci
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Abstract
The present disclosure provides mammalian 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 mammalian cell lines with engineered sequences associated with beneficial characteristics for viral vectors production. Exemplary viral vectors include AAV vectors.
Claims24
A method, comprising:
- a. producing a plurality of adeno-associated viral (AAV) vectors from a library of mammalian cells, wherein the library of mammalian cells comprise a plurality of mammalian cells, wherein each mammalian cell individually comprises:
- (i) a nucleic acid sequence comprising a barcode positioned between two functional AAV ITR sequences, wherein the nucleic acid sequence is integrated into the mammalian 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 plurality of mammalian cells produce a plurality of AAV vectors, wherein each AAV vector comprises a barcode that corresponds to the barcode of the mammalian cell from which it was produced;
- and
- b. detecting the barcodes of the plurality of AAV vectors.
A method of selecting mammalian cells for producing adeno-associated viral (AAV) vector, the method comprising:
- a. transfecting a library of mammalian cells with one or more sequences essential for the production of an AAV vector, wherein the library of mammalian cells comprise a plurality of mammalian cells, wherein each mammalian cell of the plurality individually comprise:
- (i) a nucleic acid sequence comprising a barcode positioned between two functional AAV ITR sequences, wherein the nucleic acid sequence is integrated into the mammalian 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, and
- (iii) an RNA-guided nuclease,
- b. producing a plurality of AAV vectors from the library of mammalian cells wherein the AAV vectors each comprise a barcode that corresponds to the barcode of the individual mammalian 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 mammalian cell.
(canceled)
A mammalian cell population comprising a plurality of mammalian cells, wherein each mammalian cell of the plurality comprises:
- (i) a nucleic acid sequence comprising a barcode positioned between two functional AAV ITR sequences, wherein the nucleic acid sequence is integrated into the mammalian 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 mammalian cell population produces a plurality of AAV vectors, wherein each AAV vector comprises a barcode that corresponds to the barcode of the mammalian cell from which it was produced.
20-26. (canceled)
An isolated nucleic acid comprising a 5′ and a 3′ viral repeat sequence derived from lentivirus, wherein the sequence between the 5′ and 3′ viral repeat sequences comprises:
- (i) a barcode positioned between two functional adeno-associated viral (AAV) ITR sequences,
- (ii) a selection marker, and
- (iii) one or more library variants, wherein the at least one library variant comprises at least one engineered sequence that comprises at least one gene, at least one ORF, at least one gRNA sequence, at least one non-coding nucleic acid, or a combination and/or a plurality thereof.
(canceled)
A population of nucleic acids comprising a library of variants, wherein each nucleic acid of the population comprises cis-acting integration sequences for introducing into a target mammalian cell and positioned between these cis-acting integration sequence is:
- (i) at least one library variant, and (ii) a barcode positioned between two functional adeno-associated viral (AAV) ITR sequences.
(canceled)
Dependencies are inferred from the claim text.