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

Abstract 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-type AAV (wtAAV) production systems, one by virus co-infection and the other by multi-plasmid transfection. The three systems had different kinetics in viral component synthesis but generated comparable copies of AAV genomes; however, the capsid titer of GX2 was an order of magnitude lower compared to those two wtAAV systems, indicating that its capsid production may be insufficient. The genome packaging efficiency was also lower in GX2 despite it produced higher levels of Rep52 proteins than either wtAAV systems, suggesting that Rep52 protein expression may not limit genome packaging. In the two wtAAV systems, VP were the most abundant AAV proteins and their levels continued to increase, while GX2 had high level of wasteful cargo gene expression. Furthermore, upregulated inflammation, innate immune responses, and MAPK signaling, as well as downregulated mitochondrial functions, were commonly observed in either rAAV or wtAAV systems. Overall, this comparative multi-omics study provided rich insights into host cell and viral factors that are potential targets for genetic and process intervention to enhance the productivity of synthetic rAAV producer cell lines. Key points • wtAAV infection was more efficient in producing full viral particles than the synthetic cell GX2. • Capsid protein synthesis, genome replication, and packaging may limit rAAV production in GX2. • wtAAV infection and rAAV production in GX2 elicited similar host cell responses. Supplementary Information The online version contains supplementary material available at 10.1007/s00253-024-13203-5.

transcripts were expressed at high levels in HEK293.A TPM level of about 10 3 was seen at 24 h in all three cases.
Ad5 co-infection provided additional E1A and E1B gene copies in Case B, but no major further increase of their transcripts was seen compared to other two cases.E1B transcript was more abundant than E1A transcript in all three    x-axis titles of (a) and (b).DUSP8 encodes dual specificity phosphatase 8, a key negative regulator of MAPK.
Overexpression of DUSP8 induces expression of the proapoptotic p53 upregulated modulator of apoptosis (PUMA), indicating the onset of programmed cell death (Lin et al. 2020).GPNMB encodes the transmembrane glycoprotein NMB, has been implicated in inflammation and neuroinflammation.Increased levels of GPNMB were commonly found in pro-inflammatory and pathological conditions (Saade et al. 2021).SH3RF2 encodes a SH3-domaincontaining RING finger protein, which is involved in promoting cell survival and apoptosis (Kim et al. 2014); UNC5CL, which encodes Unc-5 family C-terminal like protein, was identified as an inducer of a pro-inflammatory signaling cascade (Heinz et al. 2012).Across the transcript and protein levels, there were indications that functional classes related to inflammatory and immune responses were enriched (Cluster II in Fig. 4a and b).Together with the GX2 vs. wtAAV transfection GX2 vs. wtAAV-Ad5 co-infection a. b.
upregulation of these MAPK signaling genes, the evidence for the occurrence of inflammation is stronger in all three cases.
VP123-Rep52 denotes the transcript encoding VP1/2/3 and Rep52 in GX2.VP23A denotes the truncated transcript encoding VP2, VP3 and AAP.(m) Percentage of transcripts of different categories of genes in the four AAV production systems at 48 h.The y-axis indicates the percentage (%) of total transcripts per million (TPM)."Ad other" represents the transcripts generated by all adenoviral genes except helper genes.S1.Primers used in qPCR assay.

Supplemental
Fig. S1.Schematic diagram of (a) gene modules used for the construction of rAAV2 producer cell line GX2 construction and (b) pAV1 and pHelper plasmids.(a) The genome of HEK293 cells were integrated with three transposon vectors: Genome Module contains a LacSwitch promoter-driven cargo gene GFP flanked by AAV2 inverted terminal repeats (ITRs) with a repressor gene lacI linked to a puromycin resistance gene driven by a phosphoglycerate kinase promoter (PPGK).Replication Module contains an adenoviral helper E4orf6 and a DNA binding protein (DBP) CDS, and a destabilization domain (DD) and mCherry-tagged AAV2 Rep68 CDS under the control of inducible TetOn promoters with a reverse Tet transactivator gene rtTA3 linked to a hygromycin resistance gene driven by a PPGK.Packaging Modules contains an inducible CumateSwitch promoter-driven intronless AAV2 cap CDS (VP123) linked to a Rep52 CDS via an internal ribosome entry site (IRES).In addition, the Rep52 CDS is linked through P2A to a small ultra-red fluorescent protein (smURFP) CDS.This module also contains a Cym repressor gene CymR linked to a blasticidin resistance gene driven by a PPGK.Followed by antibiotic selection, single-cell cloning and productivity screening, clone GX2 was obtained.(b) pAV1 plasmid contains wtAAV2 genome, while pHelper plasmid contains adenoviral helper genes E2A, E4 and VA RNA.
Electron microscopic determination of full particle contents of viral particles produced in Case A (GX2) (a), Case B (wtAAV-Ad5 co-infection) (b) and Case C (wtAAV transfection) (c).AAV particles were produced by induced GX2, wtAAV-Ad5-infected or pAV1-pHelper-transfected HEK293 cells in multiple 150 mm cell culture dishes and followed by purification using affinity chromatography.The procedure of AAV purification, sample preparation and equipment for cryogenic electron microscopy (Cryo-EM) were as described (Lu et al. 2024).The captured Cryo-EM images underwent image processing and the full and empty were manually counted to determine the full particle contents.Transcripts of adenoviral helper E1A, E1B, E2A and E4 genes and their time dynamic during AAV production in three production systems.(a) The schematic diagram of the alternatively spliced transcripts and protein coding sequences of E1, E2 and E4 genes.(b, e): GX2 (Case A); (c, f, h, j): wtAAV-Ad5 coinfection (Case B); and (d, g, i, k): wtAAV transfection (Case C).E4orf6-DBP denotes the transcript encoding E4orf6 and DBP in GX2.The data are presented as the means±SDs (n=3).As shown in (b-d), E1A and E1B cases.The abundance and dynamics of different alternatively spliced transcripts generated from E1A and E1B gene as shown in (e-g).The abundance level of different transcripts was in the same range among the three cases, but the dynamics varied somewhat in three cases.E1B-22S, encoding E1B-55k protein, was the most abundant among all alternatively spliced transcript isoforms of E1A and E1B in all three cases.The E1B-55k protein plays a key role in regulation of host cell response, which blocks p53 from inhibiting the cell cycle and prevents p53 from inducing apoptosis(Debbas and White 1993).Adenoviral E2A and E4 are two additional helper genes commonly used in AAV production.They are both provided through co-infected Ad5 in Case B, and through the pHelper plasmids in Case C. In GX2, only E4orf6 CDS in E4 and DBP CDS in E2A were provided as a single transcript E4orf6-DBP.In GX2, E4orf6-DBP transcript reached 10 4 transcript per million (TPM) levels at 24 h and stayed at this high level as shown in (b).In Case C, E2A and E4 were also at a very high level as E4orf6-DBP transcript in GX2 as shown in (b) and (d).Surprisingly, the transcript expression of E2A and E4 was more moderate in Case B as shown in (c).At 48 h, TPM of both E2A and E4 were an order of magnitude lower than those in Case C. The lower E2A expression at the transcript level was also reflected at the protein level.At 24 h, DBP in Case B had a lower abundance than in Case A and C (Fig.3j-l).The adenoviral E2 and E4 genes in either Ad5 or pHelper plasmids have alternatively spliced transcripts of varying abundance, in contrast to GX2, which has only DBP and E4orf6 CDS integrated, as shown in (h-k).Notably, transcripts encoding E4orf6/7, E4orf3 and E4orf1 had relatively higher expression levels compared to other E4 alternatively spliced transcripts in either Case B and C. As reported, E4orf6/7 interacts with E2F transcription factors and plays a role in the regulation of DNA replication and cell cycle progression(Schaley et al. 2005).E4orf3 is involved in supporting viral replication and the inactivating cellular antiviral defenses(Araujo et al. 2005).E4orf1 encodes a protein that enhances host cell glucose uptake(Shastri et al. 2018).Taken together, it is likely that these E4-encoded proteins could provide additional helper functions to both wtAAV cases and enhance their AAV productivity.Supplemental Fig. S4.Western blot analysis of Rep protein.Samples were harvested at 24 and 48 h post induction, co-infection or transfection.The harvested cells were lysed using NP-40 cell lysis buffer containing protease inhibitors (Roche, Indianapolis, IN, USA).The obtained cell lysates were subsequently clarified via centrifugation and protein concentration was determined by bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific, Waltham, MA, USA). 10 g of denatured and reduced proteins was loaded to each lane of NuPAGE Bis-Tris gels (Thermo Fisher Scientific, Waltham, MA, USA) and subsequently transferred to the 0.45 μm nitrocellulose membrane (Bio-Rad, Hercules, CA, USA).The blotted membranes were then incubated with mouse anti-AAV Rep protein (Fitzgerald, Gardner, MA, USA) and anti-GAPDH monoclonal antibodies (mAbs) (Cell Signaling Technology, Danvers, MA, USA) and corresponding anti-mouse IgG conjugated with alkaline phosphatase mAb (Sigma-Aldrich, St. Louis, MO, USA).The chemiluminescent signal was detected using the Immun-StarTM AP Chemiluminescence Kits (Bio-Rad, Hercules, CA, USA).Case A: GX2; Case B: wtAAV-Ad5 co-infection; Case C: wtAAV transfection.AAV2 Rep68 protein in GX2 was tagged by a destabilization domain (DD) and mCherry.The DD-tagged protein was ubiquitinated and degraded and seen in multiple bands (80-110 kDa).AAV2 Rep52 protein in GX2 was linked with small ultra-red fluorescent protein (smURFP) by the self-cleaving peptide P2A, a 22 amino acid peptide.After cleavage, a segment of 21 extra amino acids from P2A was attached to the Rep52 protein.Therefore, bands of Rep52 with partial P2A were observed in the Western blotting membrane.Percentage of transcripts of different categories of genes in the three AAV systems.In the upper figures, different colors indicate different categories of genes, and the y-axis indicates the percentage (%)of total transcripts per million (TPM)."Ad other" represents the transcripts generated by all adenoviral genes except helper genes.The details of the percentage of TPM of each category of genes in the three AAV cases at each measured time point are listed in the table below.N/A: Not applicable.
Differential expression of transcripts in three AAV production systems.(a-c): rAAV production by synthetic cell line GX2 cells induced with 10 μg/mL doxycycline and 90 μg/mL cumate (10D90C))for 24, 48 or 72 h were compared to that of uninduced GX2 cells (0 h); (d, e): wtAAV production by co-infection of HEK293 cells with wtAAV and Ad5. 24, 48 hours post infection (hpi) samples were compared to that of uninfected cells (0 hpi); (f, g): wtAAV production by plasmid transfection of HEK293 cells.Samples of 24, 48 hours post transfection (hpt) were compared to that of un-transfected cells (0 hpt).For all the comparisons, a threshold of |Log2 fold change (FC)| > 1 and FDR p-value < 0.05 was used to identify differentially expressed genes (DEGs).In all three cases, about 20,000 transcripts were identified of which about 2600 to 3000 were differentially expressed at 48 h post induction, co-infection or transfection.The peak number of DEGs was seen at 72 h for Case A as shown in (c).
Differential expression of proteins upon switching to different AAV production cases.(a)For Case A of rAAV production, the proteome of 10D90C-induced GX2 cells (72 h) was compared to that of uninduced cells (0 h). (b, c) For Case B of wtAAV production, the proteome of wtAAV-Ad5 co-infected HEK293 cells (24, 48 hpi) was compared to that of uninfected cells (0 hpi).(d) For Case C of wtAAV production, the proteome of pAV1-pHelper co-transfected HEK293 cells (48 hpt) was compared to that of un-transfected cells (0 hpt).For all the comparisons, a threshold of unadjusted p-value < 0.05 was used to identify differentially expressed proteins (DEPs).In all three cases, about 5,000 proteins were identified.In each comparison, about 400 to 500 proteins were differentially expressed.Transcript fold change of genes in the term MAPK signaling (GO:0000165) in three AAV systems.The fold change of all the MAPK signaling related genes were plotted and compared among three AAV cases.The fold change of antiviral genes upon production of wtAAV by co-infection (log2 (Co-48 hpi/Co-0 hpi)) (Case B) (a) and transfection (log2 (TF-48 hpi/TF-0 hpi) (Case C) (b) was compared to that by induction of GX2 (log2 (GX2-72 hpi/GX2-0 hpi) (Case A).The dash line in (a) and (b) mark a twofold change (log2 FC = 1).All labeled MAPK signaling-related genes had a FDR p-value below 0.05 in each comparison shown in the y-axis and