Mechanistic model for production of recombinant adeno-associated virus via triple transfection of HEK293 cells

Abstract

Manufacturing of recombinant adeno-associated virus (rAAV) viral vectors remains challenging, with low yields and low full:empty capsid ratios in the harvest. To elucidate the dynamics of recombinant viral production, we develop a mechanistic model for the synthesis of rAAV viral vectors by triple plasmid transfection based on the underlying biological processes derived from wild-type AAV. The model covers major steps starting from exogenous DNA delivery to the reaction cascade that forms viral proteins and DNA, which subsequently result in filled capsids, and the complex functions of the Rep protein as a regulator of the packaging plasmid gene expression and a catalyst for viral DNA packaging. We estimate kinetic parameters using dynamic data from literature and in-house triple transient transfection experiments. Model predictions of productivity changes as a result of the varied input plasmid ratio are benchmarked against transfection data from the literature. Sensitivity analysis suggests that (1) the poorly coordinated timeline of capsid synthesis and viral DNA replication results in a low ratio of full virions in harvest, and (2) repressive function of the Rep protein could be impeding capsid production at a later phase. The analyses from the mathematical model provide testable hypotheses for evaluation and reveal potential process bottlenecks that can be investigated.

Keywords: AAV-based gene therapy; adeno-associated virus; biomanufacturing; gene therapy; gene-therapy manufacturing; mechanistic modeling; mechanistic understanding; transfection; viral vector; viral vector manufacturing.

Graphical abstract

Figure 1

PEI-mediated plasmid-delivery schematic for triple transfection (A) Triple transfection. (B) Model of plasmid delivery. Non-viral gene-delivery kinetics are assumed to be similar across all three different plasmids. The cellular uptake parameter (k_uptake) describes the combined process of the extracellular complex (ExComplex) binding to the cell membrane followed by its internalization into the cytosol in the form of endosomal complexes (EndosComplex). Endosomal escape (k_escape) then follows and releases the complexes into the cytosol (CytoComplex).PEI complexes in the cytosol can either migrate into the nucleus before unpacking to release plasmids, or they can unpack in the cytosol; all of the steps required to deliver the plasmids from inside of the cytosol complexes to inside of the nucleus are described by an effective kinetic event (k_{nuclear_entry}) , and the final product is naked plasmid in the nucleus (NuclearPlasmid). In addition, the degradation rates of all plasmid-equivalent species in the cytosol are described by one single parameter (k_{plasmid_degrade}).This framework was adapted from previous non-viral gene-delivery model^, and simplified to ensure model identifiability/observability with available measurement data.

Figure 2

Viral vector synthesis network Helper plasmid (pHelper) activates expression of the rep/cap gene on the packaging plasmid (pPackaging) and synthesis (k_{Cap_syn} and k_{Rep_syn}) of viral protein (VP) and Rep protein (RepProtein). Capsid proteins are assembled (k_assembly) into empty capsids in the nucleus (EmptyCapNuc), and each capsid particle consists of 60 protein subunits. With helper functions from the helper plasmid, the Rep protein replicates (k_{DNA_rep}) viral DNA (vDNA) from the transgene vector plasmid (pVector). Rep proteins dock on empty capsids (k_{Rep_bind_capsid}) to form intermediate complexes (CapRepcomplex), which then interact with viral DNA and encapsidate them inside capsids (k_{DNA_pack}) at a 1:1 ratio to form full virions inside the nucleus (RepRCcomplex). Regardless of their contents (empty or full), capsids can be secreted out of the nucleus (k_secrete) into the cytosol (EmptyCapCyto and FullCapCyto). Rep protein binding (k_{Rep_bind_plasmid}) forming bounded plasmids (RepRCcomplex) negatively regulates expression of the rep/cap gene on the packaging plasmid. Degradation of proteins (k_{Rep_protein_degrade} and k_{VP_degrade}) is possible during the process. Dotted lines imply that reactants do not get consumed in the reaction.

Figure 3

Dynamics of PEI-mediated gene delivery (A) Plasmid copies per cell over time. The model was fitted to single-transfection data for HEK293-EBNA1 cells from Carpentier et al. with initial plasmid input of 2.7 × 10⁵ plasmids/cell. (B) Plasmid content in the nucleus over time. The model prediction was generated using estimated parameters from the Carpentier et al. study and plotted against HeLa cell transfection data from Glover et al. The initial plasmid input was 2.2 × 10⁶ plasmids/cell.

Figure 4

Dynamics of viral DNA and rAAV5 capsid synthesis in in-house triple-transfection experiments for HEK293 cells (A) Dynamics of viral GFP DNA replication. (B) Dynamics of total and full rAAV capsid production. Viral production parameters were fitted to in-house triple-transfection experimental data. Measurements of total and full rAAV capsids are per-milliliter culture, harvested from both cell lysate and supernatant, normalized by the cell density at the sampling time. The dotted line indicates time point of media exchange. Initial plasmid input was 7.6 × 10⁴/cell for each of the three plasmids.

Figure 5

Quantity of full virions produced as a function of the molecular ratios of helper, packaging, and vector plasmids Data/model simulation was normalized to the maximum value reported/predicted in the four cases. The experimental data with error bars were taken from a study by Grieger et al.

Figure 6

Dynamic profiles of the full virion sensitivities (Equation 17) for all of the model parameters.

Figure 7

Model simulation of intermediate species. Dynamic trends of the (A) species concentrations and (B) reaction fluxes. The values are qualitative. The simulation parameters were according to the in-house experiments described in Materials and methods.