Comparison of different technologies for producing recombinant adeno-associated virus on a laboratory scale

Adeno-associated virus vectors are among the most promising ones for the delivery of transgenes to various organs and tissues. Recombinant adeno-associated virus (rAAV) is able to transduce both dividing and non-dividing cells, has low immunogenicity, and is able to provide long-term expression of transgenes. Modern technologies make it possible to obtain rAAV for in vivo use, but they are not without drawbacks associated with laboriousness, scalability difficulties, and high cost, therefore, improvement of technological schemes for obtaining rAAV is an urgent issue. The aim of the study was to compare different technological approaches to rAAV production based on different conditions of the transfected HEK293 cell line cultivation on a laboratory scale. Materials and methods: HEK293 cell culture, AAV-DJ Packaging System, PlasmidSelect Xtra Starter Kit were used in the study. The technologies were compared using a model rAAV vector with a single-domain antibody transgene fused to the Fc-fragment of IgG1 specific to botulinum toxin. HEK293 cells were transfected with supercoiled plasmid DNA isolated by three-step chromatographic purification. The identity of the rAAV preparation was determined by electrophoresis, immunoblotting, and real-time polymerase chain reaction. Results: the study demonstrated the efficiency of the chromatographic method for obtaining a supercoiled form of plasmid DNA that can be used for efficient transfection of cell culture in order to produce rAAV. The study compared the following processes of rAAV production: using transient transfection and cultivation of the transfected HEK293 cell suspension in Erlenmeyer flasks, adherent culture in T-flasks, and adherent culture in a BioBLU 5p bioreactor on a matrix of Fibra-Cel disks. Conclusions: the data obtained showed the possibility of using the described approaches to purification of plasmid DNA, cell transfection, and cultivation of the transfected cells under various conditions to obtain rAAV samples that expresses the antibody gene. The BioBLU 5p reactor with Fibra-Cel discs was used for the first time to produce preparative quantities of rAAV on a laboratory scale, which increased the adherent surface area during cell culture and transfection, and, as a result, increased the yield of the target product.

Handling Massive Proportion of Missing Labels in Multivariate Long-Term Time Series Forecasting

Training Deep Learning (DL) models with missing labels is a challenge in diverse engineering applications. Missing value imputation methods have been proposed to try to address this problem, but their performance is affected with Massive Proportion of Missing Labels (MPML). This paper presents a approach for handling MPML in Multivariate Long-Term Time Series Forecasting. It is an two-step process where interpolation (using Gaussian Processes Regression (GPR) and domain knowledge from experts) and prediction model are separated to enable the integration of prior domain knowledge. First, a set of samples of the possible interpolation of the missing outputs are generated by the GPR based on the domain knowledge. Second, the observed input sensor data and interpolated labels from GPR are used to train the prediction model. We evaluated our approach with the development of a soft-sensor with one real datasets to forecast the biomass during recombinant adeno-associated virus (rAAV) production in bioreactors. Our experimental results demonstrate the potential of the approach through quantitative evaluation of the generated forecasts in a case that would be extremely difficult to train a DL model due to MPML.

Monobac System–A Single Baculovirus for the Production of rAAV

Large-scale manufacturing of rAAV is a bottleneck for the development of genetic disease treatments. The baculovirus/Sf9 cell system underpins the first rAAV treatment approved by EMA and remains one of the most advanced platforms for rAAV manufacturing. Despite early successes, rAAV is still a complex biomaterial to produce. Efficient production of the recombinant viral vector requires that AAV replicase and capsid genes be co-located with the recombinant AAV genome. Here, we present the Monobac system, a singular, modified baculovirus genome that contains all of these functions. To assess the relative yields between the dual baculovirus and Monobac systems, we prepared each system with a transgene encoding γSGC and evaluated vectors’ potency in vivo. Our results show that rAAV production using the Monobac system not only yields higher titers of rAAV vector but also a lower amount of DNA contamination from baculovirus.

Improved Genome Packaging Efficiency of AAV Vectors Using Rep Hybrids

Recombinant Adeno-associated viruses (rAAVs) are one of the most commonly used vectors for a variety of gene therapy applications. In the last two decades research focused primarily on the characterization and isolation of new cap genes resulting in hundreds of natural and engineered AAV capsid variants while the rep gene, the other major AAV open reading frame, has been less studied. This is due to the fact that the rep gene from AAV serotype 2 (AAV2) enables the ssDNA packaging of recombinant genomes into most AAV serotype and engineered capsids. However, a major byproduct of all vector productions is empty AAV capsids, lacking the encapsidated vector genome, especially for non-AAV2 vectors. Despite the packaging process being considered the rate-limiting step for rAAV production, none of the rep genes from the other AAV serotypes have been characterized for their packaging efficiency. Thus, in this study AAV2 rep was replaced with the rep gene of a select number of AAV serotypes. However, this led to a lowering of capsid protein expression, relative to the standard AAV2- rep system. In further experiments the 3’end of the AAV2 rep gene was reintroduced to promote increased capsid expression and a series of chimeras between the different AAV Rep proteins were generated and characterized for their vector genome packaging ability. The utilization of these novel Rep hybrids increased the percentage of genome containing (full) capsids ∼2-4-fold for all of the non-AAV2 serotypes tested. Thus, these Rep chimeras could revolutionize rAAV production. Importance A major byproduct of all Adeno-associated virus (AAV) vector production systems are “empty” capsids, void of the desired therapeutic gene, and thus do not provide any curative benefit for the treatment of the targeted disease. In fact, empty capsids can potentially elicit additional immune responses in vivo gene therapies if not removed by additional purification steps. Thus, there is a need to increase the genome packaging efficiency and reduce the number of empty capsids from AAV biologics. The novel Rep hybrids from different AAV serotypes described in this study are capable of reducing the percentage of empty capsids in all tested AAV serotypes and improve overall yields of genome-containing AAV capsids at the same time. They can likely be integrated easily into existing AAV manufacturing protocols to optimize the production of the generated AAV gene therapy products.

Improved Genome Packaging Efficiency of AAV Vectors Using Rep Hybrids

Recombinant Adeno-associated viruses (rAAVs) are one of the most commonly used vectors for a variety of gene therapy applications. In the last two decades research focused primarily on the characterization and isolation of new cap genes resulting in hundreds of natural and engineered AAV capsid variants while the rep gene, the other major AAV open reading frame, has been less studied. This is due to the fact that the rep gene from AAV serotype 2 (AAV2) enables the ssDNA packaging of recombinant genomes into most AAV serotype and engineered capsids. However, a major byproduct of all vector productions is empty AAV capsids, lacking the encapsidated vector genome, especially for non-AAV2 vectors. Despite the packaging process being considered the rate-limiting step for rAAV production, none of the rep genes from the other AAV serotypes have been characterized for their packaging efficiency. Thus, in this study AAV2 rep was replaced with the rep gene of a select number of AAV serotypes. However, this led to a lowering of capsid protein expression, relative to the standard AAV2-rep system. In further experiments the 3’end of the AAV2 rep gene was reintroduced to promote increased capsid expression and a series of chimeras between the different AAV Rep proteins were generated and characterized for their vector genome packaging ability. The utilization of these novel Rep hybrids increased the percentage of genome containing (full) capsids ~2-4-fold for all of the non-AAV2 serotypes tested. Thus, these Rep chimeras could revolutionize rAAV production.

Popularizing recombinant baculovirus-derived OneBac system for laboratory production of all recombinant adeno-associated virus vector serotypes

Objective: On the basis of our previously established single recombinant baculovirus expression vector (BEV)-derived OneBac system, we have optimized the process and expanded the rAAV production range to the full range of serotypes rAAV1-13. Firstly, the AAV Cap gene was optimized to translate by ribosome leaky scanning and the gene of interest (GOI) was cloned into the pFD/Cap-(ITR-GOI)-Rep2 shutte plasmid. Following the classical Bac-to-Bac method, sufficient BEV stock containing all rAAV packaging elements can be quickly obtained. Finally, we can repeatedly scale up production of rAAVs in one week by using a single BEV to infect suspension-cultured Sf9 cells. The rAAV1-13 show relatively high yields ranging from 5×104 to 4×105 VG/cell. More than 1×1015 VG purified rAAVs can be easily obtained from 5 L suspension-cultured Sf9 cells. As expected, rAAV serotypes 1-13 show different potencies for in vitro transduction and celltype tropisms. Background: Recombinant adeno-associated virus (rAAV) has been widely used as an efficient transgenic vector in biomedical research, as well as gene therapy. Serotype-associated transduction efficiency, tissue- or cell-type tropism and immunological profile are major considerations in the various applications of rAAVs. There are increasing needs for different serotypes of rAAV, either naturally isolated or artificially engineered. However, affordable and scalable production of a desired serotype of rAAV remains very difficult, especially for researchers lacking relevant experience. Conclusion: In summary, the single BEV-derived OneBac system should prove popular for laboratory scaling-up production of any serotype of rAAV.

Dielectric Spectroscopy to Improve the Production of rAAV Used in Gene Therapy

The insect cell-baculovirus expression vector system is an established method for large scale recombinant adeno-associated virus (rAAV) production, largely due to its scalability and high volumetric productivities. During rAAV production it is critical to monitor process parameters such as Spodoptera frugiperda (Sf9) cell concentration, infection timing, and cell harvest viabilities since they can have a significant influence on rAAV productivity and product quality. Herein we developed the use of dielectric spectroscopy as a process analytical technology (PAT) tool used to continuously monitor the production of rAAV in 2 L stirred tank bioreactors, achieving enhanced control over the production process. This study resulted in improved manufacturing robustness through continuous monitoring of cell culture parameters, eliminating sampling needs, increasing the accuracy of infection timing, and reliably estimating the time of harvest. To increase the accuracy of baculovirus infection timing, the cell growth/permittivity model was coupled to a feedback loop with real-time monitoring. This system was able to predict baculovirus infection timing up to 24 h in advance for greatly improved accuracy of infection and ensuring consistent high rAAV productivities. Furthermore, predictive models were developed based on the dielectric measurements of the culture. These multiple linear regression-based models resulted in correlation coefficients (Q2) of 0.89 for viable cell concentration, 0.97 for viability, and 0.92 for cell diameter. Finally, models were developed to predict rAAV titer providing the capability to distinguish in real time between high and low titer production batches.

Subgenomic satellite particle generation in recombinant AAV vectors results from DNA lesion/breakage and non-homologous end joining

Recombinant AAV (rAAV) vectors have been developed for therapeutic treatment of genetic diseases. Nevertheless, current rAAV vectors administered to patients often contain non-vector related DNA contaminants. Here, we present a thorough molecular analysis of the configuration of non-standard AAV genomes generated during rAAV production. In addition to the sub-vector genomic size particles containing incomplete AAV genomes, our results found that rAAV preparations were contaminated with multiple categories of subgenomic particles with either snapback genomes or vector genomes with deletions in the mid regions. Through CRISPR and restriction enzyme-based in vivo and in vitro modeling, we identified that the main mechanism leading to the formation of non-canonical genome particles occurred through nonhomologous end joining of fragmented vector genomes caused by genome lesions or DNA breaks that were generated by the host cell/environment. The results of this study advance our understanding of AAV vectors and provide new clues on improving vector efficiency and safety profile for use in human gene therapy.

Wild type AAV, recombinant AAV, and Adenovirus super infection impact on AAV vector mobilization

Recombinant Adeno-associated viral vector (rAAV) mobilization is a largely theoretical process in which intact AAV vectors spread or “mobilize” from transduced cells and infect additional cells within, or external, of the initial host. This process can be replication independent (vector alone), or replication-dependent (de novo rAAV production facilitated by super-infection of both wild-type AAV (wtAAV) and Ad helper virus). Herein, rAAV production and mobilization with and without wtAAV were analyzed following plasmid transfection or viral transduction utilizing well established in vitro conditions and analytical measurements. During in vitro production, wtAAV produced the highest titer with rAAV-luc (4.1 Kb), rAAV-IDUA (3.7 Kb), and rAAV-NanoDysferlin (4.9 Kb) generating 2.5-, 5.9-, or 10.7-fold lower amounts, respectively. Surprisingly, cotransfection of a wtAAV and a rAAV plasmid resulted in a uniform decrease in production of wtAAV in all instances with a concomitant increase of rAAV such that wtAAV:rAAV titers were at a ratio of 1:1 for all constructs investigated. These results were shown to be independent of the rAAV transgenic sequence, size, transgene, or promoter choice and point to novel aspects of wtAAV complementation that enhance current vector production systems yet to be de fined. In a mobilization assay, a sizeable amount of rAAV recovered from infected 293 cell lysate remained intact and competent for a secondary round of infection (termed non-replicative mobilization). In rAAV infected cells co-infected with Ad5 and wtAAV, rAAV particle production was increased > 50-fold compared to non-replicative conditions. In addition, replicative dependent rAAV vectors mobilized and resulted in >1,000 -fold transduction upon a subsequent 2nd round infection, highlighting the reality of these theoretical safety concerns that can be manifested under various conditions. Overall, these studies document and signify the need for mobilization resistant vectors and the opportunity to derive better vector production systems.