DNA-binding domain of CRISPR system, non-fucosylated and partially fucosylated proteins, and methods thereof
Inventors
Prasad, Bhargav • Unnikrishnan, Divya • Hazarika, Jahnabi • Rodrigues, Kavitha Iyer • Ghosh, Maloy • M, Pavithra • D, Pravin Kumar • Bhattacharjee, Sanghamitra • M, Sathyabalan • Srinivasan, Sankaranarayanan • Chatterjee, Sohang • Maity, Sunit • K, Veeresha • Halan, Vivek • B. M., Yogendra Manjunath • Hora, Anuradha • N, Bairavabalakumar • Nair, Karthika • Thanigaivel, Aswini • Maliwalave, Amol • Shenoy, Bharath R • Pendse, Rajeshwari • Pathak, Prabhat Kumar • Kurup, Anisha • Rao, Sahana Bhima
Assignees
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Publication Number
US-10752674-B2
Publication Date
2020-08-25
Expiration Date
Abstract
The present disclosure relates to a method of obtaining a cell where fucosylation pathways are modified, leading to production of partially fucosylated and non-fucosylated protein products, specifically antibodies from the cell. The present disclosure employs the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology. The method of the present disclosure targets the Fut8 gene and GMD gene in a cell. Such products are used in developing therapeutics and biomarkers, and in diagnosis and prognosis of diseases.
Core Innovation
The disclosure describes engineering CHOK1 cells using CRISPR to modify fucosylation pathways. The targeted genes include FUT8 and GMD, and the CRISPR system employs crRNA selected from SEQ ID No. 42, SEQ ID No. 44, and SEQ ID No. 46, with a CRISPR nuclease using Cas9 or Cas9n for gene editing at the FUT8 and/or GMD genomic loci.
The engineered cells produce fucose knockout cells that support production of proteins with fucosylation levels ranging from 0% to 100%, including non-fucosylated, partially fucosylated, and fully fucosylated states. The disclosure further ties control of fucosylation to disruption of the GMD gene and, when applicable, addition of L-Fucose. Proteins emphasized in the disclosure include antibodies and monoclonal antibodies.
The disclosure includes functional screening and confirmation of editing. Fucosylation is evaluated using LCA-FITC flow cytometry, with specificity addressed using Strep-FITC. Genomic sequencing is used to demonstrate INDELs, deletions, frameshifts, and premature stop codons at targeted FUT8 and/or GMD loci, consistent with fucose knockout.
Claims Coverage
The independent claims span three inventive features: specified crRNA sequences, a CRISPR-nuclease complex using those crRNAs with a nuclease, and a vector encoding the crRNA together with Cas9 or Cas9n. Method coverage also includes cleaving the GMD gene at Exon 3 and/or Exon 4 in a selected cell line, with fucose knockout cell outcomes tied to FUT8/GMD disruption.
crRNA selected from SEQ ID No. 42, SEQ ID No. 44, and SEQ ID No. 46
A CRISPR RNA (crRNA) of a CRISPR system, where the crRNA is selected from SEQ ID No. 42, SEQ ID No. 44, and SEQ ID No. 46.
Cleaving a GMD gene sequence at Exon 3 and/or Exon 4 using the specified crRNA
A method using a CRISPR-nuclease complex that cleaves a GMD gene sequence in a selected cell line at Exon 3, Exon 4, or both, where the GMD gene encodes GDP-D-mannose 4,6-dehydratase.
CRISPR-nuclease complex comprising crRNA selected from SEQ ID No. 42, SEQ ID No. 44, SEQ ID No. 46, and combinations thereof, and a nuclease
A CRISPR-nuclease complex comprising a crRNA selected from the group consisting of SEQ ID No. 42, SEQ ID No. 44, SEQ ID No. 46, and combinations thereof, and a nuclease.
Vector encoding crRNA selected from SEQ ID No. 42, SEQ ID No. 44, SEQ ID No. 46, and combinations thereof, and encoding Cas9 or Cas9n nuclease
A vector comprising nucleotide sequence encoding crRNA selected from the group consisting of SEQ ID No. 42, SEQ ID No. 44, SEQ ID No. 46, and combinations thereof, wherein the nucleotide sequence is selected from the group consisting of SEQ ID No. 41, SEQ ID No. 43, SEQ ID No. 45, and combinations thereof, and wherein the vector further comprises a nucleotide sequence encoding a Cas9 or Cas9n nuclease.
The inventive coverage centers on specified crRNAs (SEQ ID No. 42, SEQ ID No. 44, and SEQ ID No. 46), their incorporation into a CRISPR-nuclease complex with a nuclease including Cas9 or Cas9n, and encoding in a vector that includes nucleotide sequences for the crRNA and a Cas9 or Cas9n nuclease. Method coverage is directed to cleaving the GMD gene at Exon 3 and/or Exon 4 in a selected cell line, enabling fucose knockout cells.
Stated Advantages
Produce proteins with fucosylation levels from 0% to 100%.
Enable generation of fucose knockout cells.
Allow functional screening and specificity assessment of fucosylation using LCA-FITC and Strep-FITC.
Confirm targeted genomic editing using genomic sequencing showing INDELs, deletions, frameshifts, and premature stop codons at targeted FUT8/GMD loci.
Documented Applications
Producing fucosylated proteins, including antibodies and monoclonal antibodies, with fucosylation levels from 0% to 100% using engineered fucose knockout cells.
Using engineered cells with modified FUT8/GMD activity to control fucosylation, including via disruption of the GMD gene and, when applicable, addition of L-Fucose.
Screening and confirming fucosylation outcomes using LCA-FITC flow cytometry with Strep-FITC specificity controls.
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