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-11548937-B2
Publication Date
2023-01-10
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 CRISPR RNA (crRNA) selected from the group consisting of SEQ ID No. 38 and SEQ ID No. 40 for use in a CRISPR system. The CRISPR elements are implemented with a CRISPR-nuclease complex that includes a nuclease selected from a Cas9 endonuclease or a Cas9n endonuclease.
Targeting is directed to fucose biosynthesis by selecting FUT8 gene sequence regions, including Exon 7, as CRISPR targets, with the goal of producing fucose knockout cells. The disclosure further provides CRISPR-nuclease complexes and CRISPR systems in which the crRNA is selected from SEQ ID No. 38 and SEQ ID No. 40 and the nuclease is Cas9 or Cas9n.
By cleaving FUT8 gene sequence, including Exon 7 in selected cell types, the system disrupts the FUT8 gene that codes for alpha-1,6 fucosyltransferase. The disclosure also describes targeting fucose biosynthesis through disruption of GMD in addition to FUT8, in order to control fucosylation outcomes and produce non-fucosylated and partially fucosylated protein and antibody products.
It describes control of fucosylation outcomes within a 0% to 100% range by using fucose knockout cell systems and, in the case of partial fucosylation control, combining GMD disruption with L-Fucose supplementation. Functional results include improved knockout clone generation versus prior art and confirmation of knockouts via LCA-FITC flow cytometry and genomic sequencing of target loci for deletions, insertions, frameshifts, and premature stop codons.
Claims Coverage
The independent claims cover three core areas: a CRISPR system crRNA defined by SEQ ID variants, a CRISPR-nuclease complex with the nuclease bound to that crRNA, and a CRISPR vector encoding the crRNA and the nuclease. Across the independent claims, the inventive subject matter is centered on crRNA selection, nuclease selection, and vectorized CRISPR implementation for targeted fucose biosynthesis disruption.
crRNA selected from SEQ ID No. 38 and SEQ ID No. 40
A clustered regularly interspaced short palindromic repeats (CRISPR) RNA (crRNA) selected from the group consisting of SEQ ID No. 38 and SEQ ID No. 40.
CRISPR-nuclease complex with SEQ ID No. 38 or SEQ ID No. 40 crRNA
A CRISPR-nuclease complex comprising a nuclease and crRNA selected from the group consisting of SEQ ID No. 38 and SEQ ID No. 40.
Vector encoding SEQ ID No. 38 or SEQ ID No. 40 crRNA plus Cas9 or Cas9n
A vector comprising a nucleotide sequence encoding crRNA selected from the group consisting of SEQ ID No. 38 and SEQ ID No. 40, wherein the vector further comprises a nucleotide sequence encoding a Cas9 or Cas9n endonuclease.
Overall, the claim coverage ties together SEQ ID No. 38 and SEQ ID No. 40 crRNA definitions with CRISPR-nuclease complexes and vectors encoding Cas9 or Cas9n, and places these elements into embodiments directed to fucose knockout and controlled fucosylation outcomes in expressed proteins.
Stated Advantages
Improved knockout clone generation versus prior art.
Higher ADCC is stated in the document.
Documented Applications
Producing non-fucosylated and partially fucosylated protein and antibody products by disrupting fucose biosynthesis via FUT8 and/or GMD targeting.
Cleaving the FUT8 gene sequence at Exon 7 in selected cell types.
Using fucose knockout cell systems to express fucosylated proteins with fucosylation activity between 0% and 100%.
Confirmation of CRISPR/Cas-induced knockouts at FUT8 (Exon 7) and GMD (Exon 3/4) in CHOK1 cells using LCA-FITC flow cytometry and sequencing-based INDEL identification.
Production of non-fucosylated monoclonal antibody and partially fucosylated antibody connected to FUT8/GMD knockout fucose biosynthesis control.
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