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AAV production system proteomics

ABSTRACT: We performed absolute quantification (AQUA) of viral proteins by targeted quantitative proteomics and conducted tandem mass tag (TMT)-based proteomics on the three rAAV and wtAAV production systems to identify potential factors limiting rAAV productivity in HEK293 cells

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Kidney

DISEASE(S): Disease Free

SUBMITTER: Yu-Chieh Lin

LAB HEAD: Wei-Shou Hu

PROVIDER: PXD050484 | Pride | 2024-07-03

REPOSITORIES: Pride

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			Action	DRS
	wshu_lin00917_18977_20230226_FAIMS_TMT16_re.pdResult	Other
	wshu_lin00917_18978_20220208_PRM_S01.raw	Raw
	wshu_lin00917_18978_20220208_PRM_S02.raw	Raw
	wshu_lin00917_18978_20220208_PRM_S03.raw	Raw
	wshu_lin00917_18978_20220208_PRM_S04.raw	Raw

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Publications

Comparative transcriptomic and proteomic kinetic analysis of adeno-associated virus production systems.

Lin Yu-Chieh YC Lu Min M Cai Wen W Hu Wei-Shou WS

Applied microbiology and biotechnology 20240619 1

Recombinant adeno-associated virus (rAAV) is a major gene delivery vehicle. We have constructed a stable rAAV producer cell line by integrating essential rAAV genome, viral and helper genes into the genome of HEK293 cell under the control of inducible promoters. Upon induction, the cell line produces transducing rAAV. To gain insight into enhancing rAAV productivity and vector quality, we performed a comparative transcriptomic and proteomic analysis of our synthetic cell line GX2 and two wild-ty ...[more]

PMID: 38896252

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Proteome Analysis of AAV producing HEK293 cells

Project description:Adeno-associated viral (AAV) vectors are widely used for gene therapy, providing treatment for diseases caused by absent or defective genes. Despite the success of gene therapy, AAV-manufacturing is still challenging, with production yields being limited. With increased patient demand, improvements in host cell productivity through various engineering strategies will be necessary. Here, we study the host cell proteome of AAV5 producing HEK293 cells using reversed phase nano liquid chromatography and tandem mass spectrometry (LC-MS/MS). Rela-tive label-free quantitation (LFQ) was performed allowing a comparison of transfected vs. un-transfected cells. Gene ontology enrichment and pathway analysis revealed differential expres-sion of proteins involved in fundamental cellular processes such as metabolism, proliferation and cell death. Furthermore, changes in expression of proteins involved in endocytosis and lysosomal degradation were observed. Our data provides highly valuable insights into cellular mechanisms involved during recombinant AAV production by HEK293 cells thus potentially enabling further improvements of gene therapy product manufacturing.

2021-12-09 | PXD028154 | Pride

Transcriptomics-informed pharmacology identifies epigenetic and cell cycle regulators as enhancers of AAV production

Project description:Recombinant adeno-associated virus (rAAV) is a widely used viral vector for gene therapy. Despite its clinical efficacy, the manufacturing of rAAV faces challenges in productivity and quality, leading to limited availability. To address the growing demand, next-generation process development should be informed by a mechanistic understanding of the cellular response to rAAV. In this study, we performed transcriptomic analysis of 5 cell lines with variable capacities for rAAV production. Using an intersectional approach, we assessed the transcriptional response to rAAV production and compared transcriptional profiles between high and baseline producers to identify possible targets for enhancing production. Modulation of cell cycle and nucleosome components suggested a reduction of proliferative capacity and a shift toward DNA replication to support rAAV production. During rAAV production, we observed upregulation of several core functions including transcription, stress response, and Golgi and endoplasmic reticulum organization. Conversely, inhibitors of DNA-binding proteins and mitochondrial components were consistently downregulated during rAAV production. We next performed a drug connectivity analysis of these results and identified 5 classes of drugs predicted to enhance rAAV production. Validation studies confirmed the efficacy of HDAC and microtubule inhibitors. Our data uncover novel and previously identified pathways that may enhance rAAV productivity, potentially enabling a path to engineer improved processes and cell lines for higher yields and better quality rAAV production.

2024-08-29 | GSE269485 | GEO

Secretory pathway differences between HEK293 and CHO enable rescue of difficult to express human proteins

Project description:Biologics have emerged as leading blockbuster therapeutics, but many advanced recombinant protein moieties remain difficult to produce. Here we identify bottlenecks limiting expression of challenging recombinant human proteins through a systems biology analysis of the transcriptomes of CHO and HEK293. Surprisingly, one third of the challenging human proteins displayed an improved secretion upon host cell swapping from CHO to HEK293. While most components of the secretory machinery showed comparable expression levels in both expression hosts, a key set of genes with extreme expression variation was identified. Among these, ATF4 and SRP9 were validated as general productivity boosters in CHO. Further, we found that more heavily glycosylated products benefit more from the elevated activities of the N- and O-glycosyltransferases found in HEK293 cells. Collectively, our results demonstrate the utilization of HEK293 cells for expression rescue of human proteins and suggest a strategy for identification of secretory pathway components improving yield and quality in HEK293 and CHO.

2022-06-22 | GSE157729 | GEO

Selective Disruption of Core Regulatory Transcription [AQuA-HiChIP]

Project description:With emerging methods recently developed to capture protein-anchored 3D epigenome folding we herein report an experimental advance yielding a fundamental and systematic improvement in understanding the 3D genome: integrated normalization of orthologous chromatin for measurement of absolute changes in the landscape. With Absolute Quantification of chromatin Architecture (AQuA-HiChIP) global and local changes in the 3D epigenome can be measured, and the absolute differences in protein-anchored folding can be determined. These changes can be defined in a way that couples the relative occupancy of chromatin regulatory factors or histone marks to absolute quantification of 3D chromatin structure. While our method has intrinsic limitations, including restriction by the scope of available ChIP-grade antibodies with mouse/human cross-reactivity, the approach to measuring absolute components of chromatin folding will enable new insights into the topological determinants of transcriptional control and tissue-specific epigenetic memory.

2019-09-27 | GSE120770 | GEO

A Robust System for Production of Superabundant VP1 Recombinant AAV Vectors.

Project description:Recombinant adeno-associated viral (rAAV) vectors have been widely used in human gene therapy. One major impediment to its broad application is the inability to produce high-quality vectors in mass quantity. Here, an efficient and scalable suspension cell culture system for the production of rAAV vectors is described. In this system, the AAV trans factors, Rep78, Rep52, VP1, VP2, and VP3, were stably integrated into a single vaccinia virus carrier by maximizing the use of alternative codons between genes with identical amino acids, and the cis rAAV genome was carried by an E1/E3 gene-deleted adenovirus. Infection of improved, E1 integrated, suspension-cultured cells with these two viral vectors resulted in the robust production of rAAV vectors. The newly enhanced system can consistently produce ∼1 × 1015 genome containing rAAV vectors per liter of suspension cells. Moreover, the capsid composition of rAAV vectors produced by this system is markedly different from those produced using the traditional system in that the VP1 protein is more abundant than the VP2 protein (19:1 versus 1:1). The unique VP1 superabundant rAAV vectors produced in this new system exhibited improved transduction in vivo after intravitreal injection.

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Stoichiometry of Nucleosome Remodelling and Deacetylase (NuRD) complex

Project description:The Nucleosome Remodeling and Deacetylase (NuRD) complex is essential for development in complex animals but has been refractory to detailed analysis because of its low abundance and resistance to recombinant production. As part of a larger project, absolute quantification using isotopically-labelled AQUA peptides was performed on natively-purified NuRD and six recombinantly-expressed NuRD subcomplexes. This is the first time the NuRD complex has been quantified using the AQUA strategy.

2021-01-08 | PXD010110 | Pride

Chemically Defined, High-Density Insect Cell-Based Expression System for Scalable AAV Vector Production.

Project description:The recombinant adeno-associated virus (AAV) vector is one of the most utilized viral vectors in gene therapy due to its robust, long-term in vivo transgene expression and low toxicity. One major hurdle for clinical AAV applications is large-scale manufacturing. In this regard, the baculovirus-based AAV production system is highly attractive due to its scalability and predictable biosafety. Here, we describe a simple method to improve the baculovirus-based AAV production using the ExpiSf Baculovirus Expression System with a chemically defined medium for suspension culture of high-density ExpiSf9 cells. Baculovirus-infected ExpiSf9 cells produced up to 5 × 1011 genome copies of highly purified AAV vectors per 1 mL of suspension culture, which is up to a 19-fold higher yield than the titers we obtained from the conventional Sf9 cell-based system. When mice were administered the same dose of AAV vectors, we saw comparable transduction efficiency and biodistributions between the vectors made in ExpiSf9 and Sf9 cells. Thus, the ExpiSf Baculovirus Expression System would support facile and scalable AAV manufacturing amenable for preclinical and clinical applications.

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Extra-HEK293-AAV: Transcriptomics of modified HEK293 cell line for higher productivity of biologics (e.g. viral vectors, AAV)

Project description:The human EXT1 gene has been knockdown in HEK293 cells to improve their performance in producing viral vectors such as AAV. This work compares the gene expression for the wildtype and kd_EXT1 cells, in adherent and suspension cultures.

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Regulatory mechanism of diurnal fluctuated laminarin in diatom Phaeodactylum tricornutum

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