Methods for assessing integrated nucleic acids
AssigneeJuno Therapeutics, Inc.
InventorAdrian Wrangham BRIGGS
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Abstract
Provided are methods for assessing nucleic acid sequences integrated into a genome of a genetically engineered cell, such as a genetically engineered cell used in cell therapy. Cells are generally genetically engineered to express a recombinant protein, such as a recombinant receptor, via introduction of a polynucleotide and integration of certain sequences in the polynucleotide, such as recombinant sequences, into the genome of the cell. In some aspects, the provided methods can be used to distinguish integrated nucleic acids and non-integrated, residual nucleic acids.
Claims90
A method for assessing genomic integration of a transgene sequence, the method comprising:
- (a) separating a high molecular weight fraction of deoxyribonucleic acid (DNA) of greater than or greater than about 10 kilobases (kb) from DNA isolated from one or more cells, said one or more cells comprising, or are suspected of comprising, at least one engineered cell comprising a transgene sequence encoding a recombinant protein;
- (b) from the high molecular weight fraction, determining the presence, absence or amount of the transgene sequence integrated into the genome of the one or more cell.
A method for assessing a transgene sequence in a biological sample from a subject, the method comprising:
- (a) separating a high molecular weight fraction of deoxyribonucleic acid (DNA) of greater than or greater than about 10 kilobases (kb) from DNA isolated from one or more cells present in a biological sample from a subject, wherein the biological sample comprises, or is suspected of comprising, at least one engineered cell comprising a transgene sequence encoding a recombinant protein;
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- (b) from the high molecular weight fraction, determining the presence, absence or amount of transgene sequence in all or a portion of the biological sample.
A method for assessing genomic integration of a transgene sequence, the method comprising:
- (a) separating, by pulse field gel electrophoresis, a high molecular weight fraction of deoxyribonucleic acid (DNA) of greater than or greater than about 10 kilobases (kb) from DNA isolated from a population of cells, said population of cells comprising a plurality of engineered cells that each comprise, or are suspected of comprising, a transgene sequence encoding a recombinant protein;
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- (b) from the high molecular weight fraction, determining the average or mean copy number per diploid genome or per cell of the transgene sequence integrated into the genome of the plurality of engineered cells of the population of cells.
Dependencies are inferred from the claim text.