{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/395871"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/395871","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Using high-throughput proteomics to identify novel engineering targets for enhanced rAAV production in HEK293 cells","abstract":"Gene therapies are a novel group of biotherapeutics based upon the delivery of nucleic acids to target cells. For delivery directly into a patient, termed in vivo gene therapy, recombinant adeno-associated virus (rAAV) vectors have taken centre stage. The most common method by which rAAV vectors are manufactured is through transient triple transfection of the necessary genetic components into HEK293 cells. Unfortunately, low yields during this process prevent rAAV production from meeting increasing demands. Optimisation of rAAV manufacturing through rational design will require an increased understanding of the HEK293 cell production system. This thesis details the characterisation of rAAV2, rAAV6.2 and rAAV8 production by transient triple transfection of HEK293-VP 2.0 cells. First, the effect of rAAV production on HEK293-VP 2.0 cell growth and viability was monitored by trypan blue exclusion assays, and the cell cycle status was evaluated using propidium iodide staining coupled with flow cytometry. High-throughput expression and subcellular spatial proteomics were then applied to profile the response of HEK293-VP 2.0 cells to rAAV production. The effect of rAAV production on global protein abundance was determined by quantifying HEK293-VP 2.0 cell proteins in mock transfected and rAAV2-, rAAV6.2- and rAAV8-producing cells at 48- and 96-hours post-transfection. A total of 7,469 and 7,180 cellular proteins were quantified by TMT shotgun proteomics at each timepoint, respectively. Next, the effect of rAAV production on global protein subcellular localisation was investigated using the Localisation of Organellar Proteins using Isobaric Tagging with Differential Centrifugation (LOPIT-DC) technique combined with downstream analysis using the Bayesian ANalysis of Differential Localisation Experiments (BANDLE) algorithm. Subcellular proteome maps were generated for mock transfected and rAAV6.2-producing HEK293-VP 2.0 cells at 96-hours post transfection. Interrogation of these datasets revealed the serotype-independent response of HEK293-VP 2.0 cells to rAAV production. This common response included the upregulation of ribosome biogenesis, translation initiation factors, and cell death, as well as proteins belonging to the nuclear pore complex, endoplasmic reticulum and peroxisome. Meanwhile, the DNA damage response, mitochondrial fatty acid β-oxidation, amino acid transport, histones and lysosomal proteins were downregulated. The HEK293-VP 2.0 cells were also arrested in S-phase upon rAAV production. Together, these data provide a powerful hypothesis-generating tool for the identification of proteins and pathways that could be modulated to enhance rAAV production. The potential of this data-guided approach was illustrated by selecting three targets, namely C11orf96, PKR and TFEB, for the rational design of an improved rAAV-producing HEK293-VP 2.0 cell line. Preliminary results showed that knockdown of C11orf96 or PKR could enhance rAAV6.2 yields by up to 100% and 50%, respectively.","abstract_html":"Gene therapies are a novel group of biotherapeutics based upon the delivery of nucleic acids to target cells. For delivery directly into a patient, termed in vivo gene therapy, recombinant adeno-associated virus (rAAV) vectors have taken centre stage. The most common method by which rAAV vectors are manufactured is through transient triple transfection of the necessary genetic components into HEK293 cells. Unfortunately, low yields during this process prevent rAAV production from meeting increasing demands. Optimisation of rAAV manufacturing through rational design will require an increased understanding of the HEK293 cell production system. This thesis details the characterisation of rAAV2, rAAV6.2 and rAAV8 production by transient triple transfection of HEK293-VP 2.0 cells. First, the effect of rAAV production on HEK293-VP 2.0 cell growth and viability was monitored by trypan blue exclusion assays, and the cell cycle status was evaluated using propidium iodide staining coupled with flow cytometry. High-throughput expression and subcellular spatial proteomics were then applied to profile the response of HEK293-VP 2.0 cells to rAAV production. The effect of rAAV production on global protein abundance was determined by quantifying HEK293-VP 2.0 cell proteins in mock transfected and rAAV2-, rAAV6.2- and rAAV8-producing cells at 48- and 96-hours post-transfection. A total of 7,469 and 7,180 cellular proteins were quantified by TMT shotgun proteomics at each timepoint, respectively. Next, the effect of rAAV production on global protein subcellular localisation was investigated using the Localisation of Organellar Proteins using Isobaric Tagging with Differential Centrifugation (LOPIT-DC) technique combined with downstream analysis using the Bayesian ANalysis of Differential Localisation Experiments (BANDLE) algorithm. Subcellular proteome maps were generated for mock transfected and rAAV6.2-producing HEK293-VP 2.0 cells at 96-hours post transfection. Interrogation of these datasets revealed the serotype-independent response of HEK293-VP 2.0 cells to rAAV production. This common response included the upregulation of ribosome biogenesis, translation initiation factors, and cell death, as well as proteins belonging to the nuclear pore complex, endoplasmic reticulum and peroxisome. Meanwhile, the DNA damage response, mitochondrial fatty acid β-oxidation, amino acid transport, histones and lysosomal proteins were downregulated. The HEK293-VP 2.0 cells were also arrested in S-phase upon rAAV production. Together, these data provide a powerful hypothesis-generating tool for the identification of proteins and pathways that could be modulated to enhance rAAV production. The potential of this data-guided approach was illustrated by selecting three targets, namely C11orf96, PKR and TFEB, for the rational design of an improved rAAV-producing HEK293-VP 2.0 cell line. Preliminary results showed that knockdown of C11orf96 or PKR could enhance rAAV6.2 yields by up to 100% and 50%, respectively.","abstract_has_math":false,"creators":["Hutchings, Charlotte"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Lilley, Kathryn"],"committee_chairs":[],"committee_members":[],"year":2025,"date_issued":"2025-09-12","date_published":"2025-09-12","updated_at":"2026-07-22T22:24:04Z","subjects":["Subcellular proteomics","Expression proteomics","Advanced therapeutics"],"languages":["eng"],"rights":[],"rights_urls":["https://www.repository.cam.ac.uk/bitstreams/531cac11-1499-4441-8c49-38e9f05d52f1/download","http://purl.org/NET/rdflicense/allrightsreserved"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.125234","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Lilley, Kathryn"]},{"key":"dc:contributor.sponsor","label":"Sponsor","values":["BBSRC CASE award with AstraZeneca (BB/W509929/1)"]},{"key":"dc:creator","label":"Author","values":["Hutchings, Charlotte"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2025-09-12"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/395871"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Subcellular proteomics","Expression proteomics","Advanced therapeutics"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://www.repository.cam.ac.uk/bitstreams/531cac11-1499-4441-8c49-38e9f05d52f1/download","http://purl.org/NET/rdflicense/allrightsreserved"]},{"key":"dc:rights.embargodate","label":"Dc Rights Embargodate","values":["2027-01-26"]},{"key":"dc:rights.embargotype","label":"Dc Rights Embargotype","values":["embargo"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.17863/CAM.125234"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://www.repository.cam.ac.uk/bitstreams/f3824234-2a52-4bec-a6f6-69d88c097fbc/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Gene therapies are a novel group of biotherapeutics based upon the delivery of nucleic acids to target cells. 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High-throughput expression and subcellular spatial proteomics were then applied to profile the response of HEK293-VP 2.0 cells to rAAV production. The effect of rAAV production on global protein abundance was determined by quantifying HEK293-VP 2.0 cell proteins in mock transfected and rAAV2-, rAAV6.2- and rAAV8-producing cells at 48- and 96-hours post-transfection. A total of 7,469 and 7,180 cellular proteins were quantified by TMT shotgun proteomics at each timepoint, respectively. Next, the effect of rAAV production on global protein subcellular localisation was investigated using the Localisation of Organellar Proteins using Isobaric Tagging with Differential Centrifugation (LOPIT-DC) technique combined with downstream analysis using the Bayesian ANalysis of Differential Localisation Experiments (BANDLE) algorithm. Subcellular proteome maps were generated for mock transfected and rAAV6.2-producing HEK293-VP 2.0 cells at 96-hours post transfection. Interrogation of these datasets revealed the serotype-independent response of HEK293-VP 2.0 cells to rAAV production. This common response included the upregulation of ribosome biogenesis, translation initiation factors, and cell death, as well as proteins belonging to the nuclear pore complex, endoplasmic reticulum and peroxisome. Meanwhile, the DNA damage response, mitochondrial fatty acid β-oxidation, amino acid transport, histones and lysosomal proteins were downregulated. The HEK293-VP 2.0 cells were also arrested in S-phase upon rAAV production. Together, these data provide a powerful hypothesis-generating tool for the identification of proteins and pathways that could be modulated to enhance rAAV production. The potential of this data-guided approach was illustrated by selecting three targets, namely C11orf96, PKR and TFEB, for the rational design of an improved rAAV-producing HEK293-VP 2.0 cell line. 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The effect of rAAV production on global protein abundance was determined by quantifying HEK293-VP 2.0 cell proteins in mock transfected and rAAV2-, rAAV6.2- and rAAV8-producing cells at 48- and 96-hours post-transfection. A total of 7,469 and 7,180 cellular proteins were quantified by TMT shotgun proteomics at each timepoint, respectively. Next, the effect of rAAV production on global protein subcellular localisation was investigated using the Localisation of Organellar Proteins using Isobaric Tagging with Differential Centrifugation (LOPIT-DC) technique combined with downstream analysis using the Bayesian ANalysis of Differential Localisation Experiments (BANDLE) algorithm. Subcellular proteome maps were generated for mock transfected and rAAV6.2-producing HEK293-VP 2.0 cells at 96-hours post transfection. Interrogation of these datasets revealed the serotype-independent response of HEK293-VP 2.0 cells to rAAV production. This common response included the upregulation of ribosome biogenesis, translation initiation factors, and cell death, as well as proteins belonging to the nuclear pore complex, endoplasmic reticulum and peroxisome. Meanwhile, the DNA damage response, mitochondrial fatty acid β-oxidation, amino acid transport, histones and lysosomal proteins were downregulated. The HEK293-VP 2.0 cells were also arrested in S-phase upon rAAV production. Together, these data provide a powerful hypothesis-generating tool for the identification of proteins and pathways that could be modulated to enhance rAAV production. The potential of this data-guided approach was illustrated by selecting three targets, namely C11orf96, PKR and TFEB, for the rational design of an improved rAAV-producing HEK293-VP 2.0 cell line. 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