Amann, Thomas

Andersen, Mikael Rørdam

Hansen, Anders Holmgaard

Kildegaard, Helene Faustrup

Min Lee, Gyun

Pristovsek, Nusa

Singh, Ankita

Combining the chinese hamster ovary (CHO) - K1 draft genome1,2, identified CHO glycosyltransferases3 and the power of multiplexing gene knock-outs with CRISPR/Cas94 via co-transfection of Cas9 and one single guiding RNA (sgRNA) per target, we generated 20 Rituximab expressing CHO-S cell lines differing in amount and combination of insertions or deletions (indels) in the targeted genes. Clones harboring 9, 6 and 4 indels were further investigated for growth, Rituximab productivity and secretome N-glycosylation.This resulted in clones with prolonged viabilites, no changes in N-glycan galactose contents but an increase of matured and sialylated N-glycan structures in the secretome. Additionally we point out, that multiplexing an increasing amount of genes most likely results in clones only revealing a few of all possible combinations of the targets and is highly driven by the sgRNA efficiency which can differ from each other by factor 4, even after FACS sorting

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Downloaded from orbit.dtu.dk on: Jan 01, 2018CHO glyco-engineering using CRISPR/Cas9 multiplexing for protein production withhomogeneous N-glycan profilesAmann, Thomas; Hansen, Anders Holmgaard; Pristovsek, Nusa; Singh, Ankita; Min Lee, Gyun;Andersen, Mikael Rørdam; Kildegaard, Helene FaustrupPublication date:2017Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):Amann, T., Hansen, A. H., Pristovsek, N., Singh, A., Min Lee, G., Andersen, M. R., & Kildegaard, H. F. (2017).CHO glyco-engineering using CRISPR/Cas9 multiplexing for protein production with homogeneous N-glycanprofiles. Poster session presented at 25th ESACT Meeting 2017, Lausanne, Switzerland.CHO-glyco-engineering using CRISPR/Cas9 multiplexing  for protein production with homogeneous N-glycan profiles Thomas Amann1*, Anders H. Hansen1, Nuša Pristovšek1, Ankita Singh1, Gyun M. Lee1,2, Mikael R. Andersen3, Helene F. Kildegaard1  References:   1. Xu, X. et al. Nat. Biotechnol. 29,735–741(2011) 2. Lewis, N.E. et al. Nat. Biotechnol. 31,759–765(2013) 3. Yang, Z. et al. Nat. Biotechnol. 33, 2014–2017 (2015) 4. Grav LM et al. Biotechnol. J. 10 (9), 1446-1456 (2015) 5. Walsh, G. Drug Discov. Today 15,773–780(2010)  4. Sequencing of pools and clones 1The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Lyngby, Denmark 2Korea Advanced Institute of Science and Technology, Department of Biological Sciences, Daejon, South Korea 3Institute for Biotechnology and Biomedicine, Technical University of Denmark, Lyngby, Denmark *Correspondance: thoam@biosustain.dtu.dk 3. Experimental Overview B. Rituximab quantification C. Secretome N-glycan analysis A. Clone Level Sequencing B. Pool Level Sequencing A. Background information B. Hypothesis Figure 1:  Post-translational N-glycan processing with nucleotide sugars and several N-glycosyltransferases anchored in the golgi membrane to be targeted for improved IgG N-glycan profile. Although CHO cells’ strength is the production of similar N-glycans to what is found on glycosylated human proteins5, non-engineered CHO cells display a broad variety of N-glycans which often includes N-glycan structures, that have an undesired effect on e.g. efficacy, antibody-dependent cell cytotoxicity (ADCC) or lectin-mediated clearance of the glycoprotein.  In this work, we investigate the limitations of targeting up to ten gene targets via multiplexing in a Rituximab producing CHO cell line. The targets include N-glycosyltransferases, enzymes involved in nucleotide sugar synthesis, N-glycosyltransferase modulation, apoptosis and glutamine synthesis. 1. KEY MESSAGE 2. Introduction: N-glycan engineering 5. Results: Growth, Rituximab titers and secretome N-glycosylation Bax Bak1 Target 4 B4GalT1 B4GalT2 B4GalT3 B4GalT4 B4GalT5 Target 10 GLUL Indel out of frame WT sequence Phenotype based GLUL-KO (no growth in glutamine-free medium) Indel partially in-frame A. Growth and Viability in Batch Experiment Golgi Cytosol Glycosyltransferases Glyco-engineering Improved ADCC Bulk Sorting of Cell Pools Characterization of sgRNA efficiency Figure 3:  Out of 94 screened clones, 20 clones harbor at least one genetic modification after multiplexing with 10 sgRNA´s (top 14 clones shown here).  Figure 2:  Target transcript levels and workflow of cell sorting after transfection to enrich transfected cells via GFP or generate single cell clones in 384 well format. Figure 6:  The three KO clones display hetero-geneous titers and productivity, where the 4x KO has the lowest and the 6x KO the highest titer. Within the control group, the two non-engineered clones reveal similar t i t e r s a n d s p e c i f i c Rituximab productivities.  Figure 7:  N-glycan profiles of the secretome from (i) a non-engineered clone ctr 1 and (ii) the 9x KO clone with indels in Bax, Bak1, GLUL, Target 4, B4GalT2, 3, 4 & 5 and target 10 (SA = sialic acid).  (ii) 9x KO Figure 4: Frequency of indel generation after pool level sequencing of the different target regions before (blue) and after (red) FACS sorting for GFP-positive cells representing Cas9_2A_GFP expressing populations. The frequency of indel generation increased at least one-fold for all sgRNA targets after FACS and range from 1-80%. Figure 5:  The three top-KO clones were grown in parallel to the parental producer cell line and two controls w i t h  n o  M i S e q - confirmed indels being present. Compared to the non-engineered controls, the KO clones show a slower drop in viability after 120h of cultivation (grey area), n=2. Combining the chinese hamster ovary (CHO) - K1 draft genome1,2, identified CHO glycosyl-transferases3 and the power of multiplexing gene knock-outs with CRISPR/Cas94 via co-transfection of Cas9 and one single guiding RNA (sgRNA) per target, we generated 20 Rituximab expressing CHO-S cell lines differing in amount and combination of insertions or deletions (indels) in the targeted genes. Clones harboring 9, 6 and 4 indels were further investigated for growth, Rituximab productivity and secretome N-glycosylation.  Single Cell Cloning  Characterization of: •  Genotype: Sequencing of target regions  •  Phenotype:  N-glycan profile, growth & viability in batch cultivation  Sialic acid Fucose Galactose Mannose N-Acetylglucosamine Bax	   	  Bak1	   GLUL	   Target	  4	   B4GalT1	   B4GalT2	   B4GalT3	   B4GalT4	   B4GalT5	   Target	  10	  before	  sort	   27.3	   15.7	   0.8	   38.0	   13.8	   22.1	   17.8	   23.7	   5.0	   8.4	  a<er	  sort	   75.9	   52.3	   1.6	   80.1	   32.0	   56.0	   55.2	   55.2	   20.0	   18.2	  0	  20	  40	  60	  80	  100	  Indel [%] 9x KO 6x KO 4x KO 5x A 4x A 4x B 3x A 3x B 3x C 2x A 2x B 2x C 2x D 1x A batch in 30 ml shake flask for growth, titer and N-glycan analysis Acknowledgement: Special thanks to Nachon C. Petersen for assistance with FACS experiments, Helle M. Petersen for Rituximab titer analysis & purification and Zulfiya Sukhova for preparing the MiSeq libraries. [FPKM] PO315 Cas9_2A_GFP [viable cells / ml] viability [%] 1st round 2nd round This resulted in clones with prolonged viabilites, no changes in N-glycan galactose contents but an increase of matured and sialylated N-glycan structures in the secretome. Additionally we point out, that multiplexing an increasing amount of genes most likely results in clones only revealing a few of all possible combinations of the targets and is highly driven by the sgRNA efficiency which can differ from each other by factor 4, even after FACS sorting. cultivation time [h] 11.68	  9.78	  0.05	  4.46	  6.21	  ctr	  1	   ctr	  2	   4x	  KO	   9x	  KO	   6x	  KO	  spec.	  prod.	  [pg/cell*day]	  56.21	   53.30	  0.44	  26.90	  46.62	  ctr	  1	   ctr	  2	   4x	  KO	   9x	  KO	   6x	  KO	  Iter	  day	  4	  [mg/L]	  N A 0	  10	  20	  30	  40	  50	  60	  70	  80	  90	  100	  CHO	  K1	   CHO	  DG44	   CHO	  S	  relative abundance relative abundance E:\DATA\...\20170424_ThoAm_TK_05 ThoAm - Ctrl A2 2.000000C:\Xcalibur\Methods\20170404_RapiFluor_Pos_NoSF_Ver5.meth BF4 4/24/2017 7:45:26 PMRT: 6.00 - 40.006 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40Time (min)-10-505101520253035404550556065707580859095100Relative AbsorbanceNL:8.15E6Emission_1  UV 20170424_ThoAm_TK_052x	  SA	  2x	  SA	  3x	  SA	  E:\DATA\...\20170424_ThoAm_TK_07 ThoAm - KO B4 2.000000C:\Xcalibur\Methods\20170404_RapiFluor_Pos_NoSF_Ver5.meth BF6 4/24/2017 9:20:15 PMRT: 6.00 - 40.006 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40Time (min)05101520253035404550556065707580859095100Relative AbsorbanceNL:3.50E7Emission_1  UV 20170424_ThoAm_TK_072x	  SA	  3x	  SA	  2x	  SA	  elution time [min] elution time [min] 1x	  SA	  1x	  SA	  (i) ctr 1 -­‐125	  -­‐100	  -­‐75	  -­‐50	  -­‐25	  0	  25	  50	  75	  100	  0.E+00	  2.E+06	  4.E+06	  6.E+06	  8.E+06	  1.E+07	  0	   24	   48	   72	   96	   120	   144	   168	  parental	   ctr	  1	   ctr	  2	   4x	  KO	   9x	  KO	   6x	  KO	  N-glycan sampling 

CHO glyco-engineering using CRISPR/Cas9 multiplexing for protein production with homogeneous N-glycan profiles

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CHO glyco-engineering using CRISPR/Cas9 multiplexing for protein production with homogeneous N-glycan profiles

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