GEN-How-to-Develop-AAV-Process-eBook-CY29204-03Jan23-EBHow to Develop a Scalable AAV Process, from Start to FinishCytiva Scientists Lend Their Insights from Two Years' WorkA SPONSORED PUBLIC ATION FR OMAcceleratingpath to new therapies isThe demands of innovation are evolving ever faster with eachnew discovery, each new molecule, each new insight. At Cytiva,we evolve with you, helping advance forward with flexible, modular solutions. We shorten the time to the next milestone and to market with automated manufacturing processes.Together, we'll accelerate brave science.How to Develop a Scalable AAV Process, from Start to FinishCytiva Scientists Lend Their Insights from Two Years' Work05 Meet the team06 Challenge 1: Choose a Host Cell and Growth Conditions Suitable for GMP08 Challenge 2: Optimize Transient Transfection for Scalability11 Challenge 3: Produce Virus in Single-Use Bioreactors14 Challenge 4: Produce Different Serotypes16 Challenge 5: Capture Virus Efficiently18 Challenge 6: Perform AAV Full and Empty Capsid Separation23 Challenge 7: Determine Titer Accurately25 What We Learned26 AAV Vectors Advance the Frontiers of Gene TherapyCytiva and the Drop logo are trademarks ofLife Sciences IP Holdings Corporation or an affiliate.©2021 CytivaFor local office contact information, visit cytiva.com/contactCY21470-18May21-AD© GEN Publishing Inc., December, 2022GENengnews.com|3IntroductionViral vectors are an integral delivery vehicle in modern medicine and are used in everythingMeet the TeamHere are some of the Cytiva scientists who have helped to develop this start-to-finish process, with input from adeno-associated virus (AAV) process developers and manufacturers.from oncolytic viruses to vaccines and gene therapies. With two gene therapies approved by regulatory agencies in 2021 and more in 2022, these therapies are delivering on their promise despite concerns on dosing and safety. Eyes were the first targets; there is growing interest in potential indications for targets in the central nervous system (CNS), liver, and muscles. Of the viral vectors used in gene therapy, adeno-associated virus (AAV) has emerged as the frontrunner.In 2021, there were more than 300 gene therapies in clinical trials, about half using AAV. This virus is a versatile tool, with serotypes that target specific tissue types. This fact, combined with the prevalence of engineered variants, creates an almost boundless array of choices. On the flip side of this versatility is the challenge: How do we find ways to produce the various serotypes with high efficiency, sufficient to generate many doses of therapy?Can we learn from monoclonal antibodies (mAbs), which use platform approaches to make grams of material? How do we achieve something similar for AAV? First off, we can't expect similar outputs for a burgeoning field. With mAbs, it took decades to get such high titers upstream that downstream became a bottleneck. And decades to achieve high recoveries following purification. But what we can do is learn more quickly than we did with mAbs to get better and better. This will take a combination of grit, perseverance, and innovation.AAV is not a mAb. It's five times larger and is packaged into capsids, which can contain all of the necessary genetic information, partial, or no genetic information. A main challenge upstream is getting a high percentage of full capsids in the harvest. Downstream, it's effectively separating the full capsids from empties that will reduce efficacy and potentially provoke an immune response.We don't have all the answers, but we have made progress both upstream and downstream. And we learned a lot of lessons along the way.We believe in the promise of gene therapy, and if you're reading this you do, too. We hope you find inspiration in the articles in this eBook, so you can bring your own therapy to life.Here we present highlights from a process that has been developed - and continues to be refined - with help from nearly two dozen Cytiva scientists. We frame these highlights around common challenges we hear from those who are developing and producing AAV.Ann-Christin MagnussonStaff Research Engineer, Protein and Viral Production, CytivaJoining Cytiva in 2008,Ann-Christin currently leads development of cell culture solutions for virus production supporting both vaccine manufacturing and genetherapy applications. Ann-Christin holds a degree in Biomedical Engineering from Uppsala University, Sweden and has over 30 years of hands-on experience in cell culture of different cell lines.Extended TeamDr. Åsa Hagner McWhirter, PhDPrincipal Scientist, Bioprocess applications R&D, CytivaÅsa joined Cytiva in 2003, currently leading developments in thedownstream process for viral vectors. She has focusedon customer collaborations to understand the specific needs for new dedicated products as well as to show use and performance of existing products in case studies for viral vector purification and analysis. Åsa holds a PhD in Medical Biochemistry from Uppsala University.Anna MobergStaff Research Engineer and Project Manager, CytivaAnna Moberg studied analytical chemistry at Uppsala University, Sweden. Since 1998 she has worked in R&D with system, application, andconsumables development for Biacore™ systems in Uppsala, Sweden. Currently she heads the Biacore™ Market Support team focusing on customer collaborations and generating marketing and training materials.Pictured above: Ann Lövgren, Josefin Thelander, Åsa Hagner McWhirter, Ann-Christin Magnusson, Lena Sandberg, Sebastian Persson, Anna Mattson, Åsa Lagerlöf, Elin Monie, Eva Blanck, Viktor Björklund, Robert MagnussonNot pictured: Albin Larsson, Camilla Estmer Nilsson, Daphne Areskoug, Anna Moberg, Marcus Kjellander, Linnea Nygren Babol, Mikaela Löfbacka, Hans Blom, Greta Hulting, Magnus Bergman, Christine Sund-Lundström, Jean-Luc Maloisel, Brigitta Németh, and Ola LindChallenge 1: Choose a Host Cell and Growth Conditions Suitable for GMPBackgroundFor research studies, adeno-associated virus(AAV) is often prepared in cells that adhere to the bottom of culture plates or flasks. Fetal bovine serum is a typical addition to spur cell growth.When it's time to increase the amount of virus for further studies, the number of containers is increased, or 'scaled out.' While this works for small quantities, there's a practical limit to the number of containers and hands-on time for larger quantities. Also, flask-based processes are prone to contamination.Best practice for biologic production in good manufacturing practices (GMP) environments is to close and automate processing to minimize the risk of contamination and human error.Avoiding the use of serum and other animal- derived components confers a regulatoryadvantage. These sources can carry adventitious viruses that will be challenging to remove later since their sizes are similar to that of the desired recombinant AAV (rAAV).So, what are the options for scalable cell growth?One possibility is to grow adherent cells in fixed-bed reactors. Another is to grow cells in suspension. We chose the latter option for our process. Furthermore, we directly adapted the cells into a serum-free medium for suspension culture that would be used for subsequent transfection, to avoid the need for a medium change.Experimental highlightsWe adapted adherent HEK293T cells to HyClone™ cell culture transfection medium, HyCell™ TransFx-H. In parallel, HEK293 cells (without theSV40 large T antigen) were also successfully adapted to the same medium using the same strategy. The illustration in Figure 1 provides an overview of the steps.As a starting point for adaptation, cells were grown in classicalHyClone™ DMEM mediumwith high glucose and 10% HyClone™ Fetal Bovine Serum (FBS). Then, they were transferred directly into the new transfection medium, which was supplemented with 4 mM of HyClone™L-glutamine and 0.1% nonionic surfactant. The cell growth and morphology of the HEK293T suspension cells were evaluated microscopically over 10 passages. To avoid aggregates and reduce shear stress, we used 0.1% surfactant from the beginning and kept the cell density below 2.5 × 106 cells/mL.Bottom lineCells adapted to HyClone™ medium met our set success criteria of < 10% aggregation (smallaggregates of < 10 cells) with robust cell growth.DetailsRead the full app note on AAV process development.Next step in start-to-finish process: Optimize the transfection conditions using a design of experiments (DoE) approach..."Before you move into clinical studies, it's important to be using a process that is robust, reproducible, and will scale up all the way to commercialization. Otherwise, you may be forced to switch, which can cause delays trying to convince regulators that AAV from your new process is comparable to what's produced in flasks."-Ann-Christin MagnussonFig 1. Illustration of cell culture adaptation: direct adaptation; detachment of adherent cells and subculture; seeding into shake-flask culture; monitoring of viable cell density, morphology, and aggregates; and creating a working cell bank.Challenge 2: Optimize Transient Transfection for ScalabilityBackgroundMaking AAV is a complex endeavor. AAV belongs to the parvovirus family and is dependenton co-infection with other viruses, mainly adenoviruses, in order to replicate in mammalian cells. The following genetic information is required for AAV production: AAV genes to make the viral capsid proteins; AAV helper Rep (Replication) genes for viral genome replication and packaging; and the gene of interest (GOI) for expression of the protein to elicit the desired genetic effect.The main options to produce recombinant AAV include incorporating all elements into a stable producer cell or delivering the genes on multiple plasmids by transient transfection. HEK293 cell lines are typically used.Triple-plasmid transfection is the mostcommon method, as it's quick to implement given the pressures to be first to market with any new gene therapy. However, transienttransfection is challenging and costly to scale.Because it's the most common and accessible method, we chose to perform triple-plasmid transfection using polyethyleneimine (PEI), as shown in Figure 2.Experimental HighlightsA series of DoE studies were performed to evaluate the optimal conditions for thetransfection. Five studies were performed with four being applied to rAAV2. During our process development, the goal was to achieve thefollowing criteria to meet the requirements for the downstream process: 1014 virus particles(VP)/L, 1013 viral genomes (VG)/L, and 10% or more full capsids in harvested material.We started to investigate the effects of plasmid DNA concentration, different cell densities, PEI:DNA ratio, transfection volume, and incubation time. We continued with fixed parameters from the previous DoE and added new parameters to the next DoE. Evaluation of temperature, as well as DNA plasmid ratio,were investigated and evaluated. DoE studies 1 through 4 showed that we could not achieve all these requirements for rAAV2, and consequently, the developed transfection protocol was tested for rAAV5. DoE5 was therefore designed as averification study that confirmed the optimal DNA concentration and DNA-PEI complex incubation time for rAAV5 production.Transfection efficiency and transduction assays detecting GFP-expressing cells were evaluated by flow cytometry. Viral genomes (VG, full capsids) were determined by qPCR (VG/L), and the total viral particle titer (VP/L) was determined by ELISA. The ratio of qPCR:ELISA was used to estimate the percentage of full capsids.In our DoE experiments, we found that ahigh concentration of DNA improved VG titer and transfection incubation time did notsignificantly affect the VG titer. However, when we looked at the percentage of full capsids, we saw the opposite effect; by reducing the DNA"Of course you want to have good productivity with a high viral titer. But at the same time, you want a high percentage of full capsids in the harvest material. It's important to find the balance in different parameters to create a robust production process."-Ann-Christin MagnussonFig 2. Transient transfection of adapted HEK293T cells using a triple plasmid transfection approach. PEI forms a complex with DNA, which is then introduced into the host cells.concentration and having a short incubation time, the percentage of full capsids increased.Bottom LineThe verification for rAAV5 production in the fifth DoE study showed that a reduced transfection incubation time positively affected the percentage of full capsids. From the responses in the DoE experiments, we conclude that during transfection, a DNA concentration of 0.75 µg/mL and an incubation time of 15 min is likely optimal under the conditions tested.We could summarize our DoE studies by doing a design space optimization based on a morerealistic scenario, calculating the percentage of full capsids we were aiming for. Figure 3 shows the green area, which is the operating space if we were aiming for 15% full capsids. The red x marks the conditions selected.DetailsRead the full app note on AAV process development.Next steps in start-to-finish process: Use the optimized process to produce virus in bioreactors. . .Challenge 3: Produce Virus in Single-Use BioreactorsBackgroundIt's one thing to make AAV in shake flasks; it's another to make it in vessels suitable for a good manufacturing practices (GMP) environment.Bioreactors are the preferred systems for clinical and commercial production of biologics. With the right bioreactor platform, a thoughtfully developed process can be scaled up in a straightforward manner as larger volumes are required. Single-use bioreactors are preferred for biologic production, including viral vectors, for several reasons. The risks for cross-contamination are minimized, and it's possible to increase the production capacity with shorter changeover procedures between batches compared witha flexible option for multi-product manufacturing.Transient transfection is known to be challenging to scale up; however, it remains the predominant method of introducing the viral genes, helper gene, and GOI into cells. During our process development work, we optimized the conditions to balance the resulting titer and percentage of full capsids with the goal of developing a robust protocol that would scale up well.Experimental highlightsUsing the protocol we optimized during process development, we expanded HEK293 or HEK293T suspension cells from small-volume cell cultureFig 3. Results from DoE5 to determine the operating space to achieve > 15% full rAAV5 capsids. The red x marks the conditions that were selected (DNA concentration and incubation time).stainless steel. Also, single-use bioreactors provide(20 mL) in shake flasks up to a 10 L culture inFor More Info...Develop and Refine Your AAVProduction Process...Help yourself to knowledge on strategies and solutions across the AAV vector workflowWAVE™ 25 bioreactor (right)Fig 4. Verification batches for rAAV5 at 10 L scale using Xcellerex™ XDR-10 (left) andconfirmed that criteria for rAAV5 production could be achieved with the developed transfection protocol.a benchtopXcellerex™ XDR-10 single-usebioreactor system. Also, we produced rAAV5 inReadyToProcess WAVE™ 25 bioreactor system, up to 25 L. Our goal with the study was to achieve 1010 viral genomes (VG)/mL (1013/L), and 1011 of viral particles (VP)/mL (1014/L) in crude harvest material. These criteria are based on levels described in the literature, which of course can be improved by further process development.We aimed for at least 10% full capsids in the harvest material to allow efficient AAV full and empty capsid separation during the purification process. The percentage of full capsids was calculated as a ratio between VG/mL and VP/mL.We performed verification batches to confirmthat these criteria could be achieved, which they were (Fig 4). Then, we performed three production runs in each bioreactor type.To confirm transfection efficiency in the XDR-10 bioreactor, we performed brightfield and fluorescent microscopic evaluation of cells in addition to flow cytometry analysis. Figure 5 shows GFP-positive fluorescing cells in a diluted sample of rAAV5 at harvest.Bottom lineWe confirmed that both the ReadyToProcess WAVE™ 25 and Xcellerex™ XDR-10 bioreactors are suitable for AAV production with similar productivity. While good results were observedfor both bioreactors, the XDR process offers scalability up to 2000 L with the Xcellerex™ XDR family. Three production batches inXDR-10 bioreactor gave a similar viral titer with approximately 1014 VP/L. All three production batches successfully yielded > 15% full capsids, as shown in Figure 6. The consistency between the three batches indicates that the process design is robust and suitable for manufacturing purposes.DetailsRead the full app note on AAV production in single-use bioreactors.Next step in start-to-finish process Determine whether the developed process works for other AAV serotypes. . .Fig 5. (A) Bright field and (B) fluorescent microscope images from diluted sample of rAAV5 at harvest from the XDR-10 bioreactor.Fig 6. Summary of the three production batches in XDR-10 bioreactors. All criteria were met with respect to viral genomes, viral particles, and percentage of full viral capsids for rAAV5 production."Our mindset is to create a 'simple' process which will improve reproducibility, robustness, and flexibility in our AAV production process." -Ann-Christin MagnussonChallenge 4: Produce Different SerotypesBackgroundResearch and development in the AAV field is highly active. Efforts are aimed at improving efficacy and tissue specificity as well as reducing some of the potential safety concerns. Numerous natural and synthetic or engineered serotypes are in various stages of development. It would be useful to have a scalable process that can produce a variety of serotypes.Experimental highlightsIn addition to rAAV5, we used our optimized protocol to produce rAAV2, rAAV8, and rAAV9. The same method was applied using shake flasksand the XDR-10 and WAVE™ 25 bioreactors. We achieved similar titers as for rAAV5.Bottom LineFigure 7 shows the average productivity from shake flask (20 mL to 2 L) and bioreactor (3 L to 25 L) demonstrating good scalability and productivity from shake flask to bioreactor for all four serotypes.We conclude from these findings that the transfection protocol we describe for bioreactor scale-up should allow production of different AAV serotypes at large scale.Next steps in start-to-finish process Harvest, clarify, concentrate, and capture the viruses. . .DetailsRead the full app note on AAV production in single-use bioreactors.View the WebinarWebinar: From cells to purified capsids: How to develop a scalable rAAV process...In this webinar you will learn:"We have heard from many people who want the flexibility to use different serotypes in their programs. When developing a scalable process, consider the serotypes you're using now - as well as the ones you may use in the future." -Ann-Christin MagnussonFig 7. Average of the titer for viral particles shows scalability of the transfection protocol for varioust AAV serotypes.Challenge 5: Capture Virus EfficientlyBackgroundAffinity capture is an efficient way to specifically bind and concentrate the target. At the same time, this step removes a large portion of impurities. However, affinity ligands don't discriminate between the full rAAV capsid (containing the VG and GOI) and empty rAAV capsids (product-related impurity lacking viral genomes).Experimental highlightsThe starting materials for all experiments were obtained from rAAV production by triple plasmid transfection in HEK-293T cells, followed by harvest using detergent lysis (Tween 20), clarification, and concentration and buffer exchange by tangential flow filtration using hollow fibers. We optimized the affinity capture step using Capto™ AVB resin.Capto™ AVB is an affinity chromatography resin designed for the purification of rAAV. The ligand of Capto™ AVB resin is a single domain antibody fragment that specifically binds rAAV 1, 2, 3, and 5 serotypes. The ligand also binds genetically engineered recombinant variants depending on the presence of the AVB binding epitope region.We applied concentrated and buffer-exchanged rAAV2 and rAAV5 samples toCapto™ AVB and eluted in 50 mM citrate at pH3.0 with gradient elution from 500 toFig 10. Separation of rAAV5 full and empty capsids using Capto™ Q ImpRes anion exchange chromatography resin. The optimized protocol included an 18 mM MgCl2 wash to elute the empty capsids followed by a NaCl linear gradient to elute the full capsids. The gray shaded area represents the pooled fractions containing the highest percentage of full capsids and sufficient viral genome recovery.122Fig 11. Separation of rAAV8 capsids on (A) Capto™ Q ImpRes (manufactured without dextran surface extenders) vs (B) Capto™ Q (manufactured with surface extenders) in the presence of 2 mM MgCl2. Separation performance of (C) Capto™ Q with 2 mM MgCl2 and (D) 18 mMMgCl2 is also shown. Elution was performed with an increasing NaCl linear gradient up to 400 mM.elution concentration for the full capsids in the prescreening run (Fig 12). The peak UV 260:280 ratios (~ 0.6-0.7 for empy capsids and ~ 1.1-1.25 for full capsids) were confirmed by qPCR and ELISA analysis. The VG recovery and % full capsids in peak 2 were above 80%. (Fig 12 Table).Bottom lineAffinity capture can require optimization depending on serotype to maximize recovery. Higher viral genome recovery and % full capsids can be obtained in the polishing step byusing Capto™ Q with surface extenders, whichCapto™ Q - Baseline Separation in 2 Stepssignificantly enhances the separation. We also learned that step elution and MgCl2 enhance the separation. To be able to achieve a protocol with good performance for AAV9, we reduced the conc. MgCl2 and changed to sodium acetate as elution salt. Our new protocol is compatiblewith AAV2, AAV5, AAV8, and AAV9. The developed process is suitable for large-scale clinical-grade production.DetailsRead the full app note on separation of empty and full rAAV capsids and the app note on capture and polishing.Fig 12. Separation of rAAV2, rAAV5, rAAV8, and rAAV9 full and empty capsids using Capto™ Q with dextran surface extenders. We used 20 mM BTP, pH 9.0 containing 2 mM MgCl2 without additives. Elution was performed with sodium acetate (250 mM as 100% buffer B). The % buffer B in step 1 and 2 was selected based on prescreening using 5% bufferB incremental steps. Step 1 was extended to 20 CV eluting the empty capsids, and step 2 was 5 CV, which led to elution of the full capsids. The mass balance based on total UV signal was 70% to 100%.Challenge 7: Determine Titer AccuratelyBackgroundDuring process development, final production, and purification, we monitor a range of critical quality attributes including infectious titer, viral genome titer, viral total particle, host cell protein (HCP), and DNA levels. Many of the techniques used for this are labor-intensive with low precision and a low degree of automation. In addition, the analyses are often time-consuming and costly. Simplified, high-precision analytics like surface plasmon resonance (SPR) can enhance today's workflows with a high degree of automation.Standard titer analysis uses ELISA, an assay that requires a lot of manual interaction and can come with challenges in precision and reproducibility.Experimental highlightsWe developed SPR assays for both rAAV2 and rAAV5. For rAAV2 we amine coupled an anti-AAV2 in one flow cell on Sensor Chip CM5 inBiacore™ T200. We also usedBiacore™ T200 for analysis of AAV5 virus titer.The latter assay is run using capture to Sensor Chip Protein A and doesn't require separate immobilization. We used a capture formatUnderpinning all this work: Analytics...Fig 13. Comparison between ELISA and Biacore™ assay determination of virus titer in different types of upstream and downstream AAV5 samples. We obtained slightly higher titerswith ELISA, which might be due to nonspecific binding to the ELISA plate.instead of covalent coupling because of the binding antibody for this assay.Also, we set up the rAAV2 assay on aBiacore™8K system, which significantly increases sample throughput. For a full 96-well plate of samples, the system reduces run time to less than 3 h, compared to 19 h on a single-needle system.We performed a Biacore™ analysis of a set of upstream and downstream AAV5 processsamples and compared the results with ELISA (Progen AAV5 Titration ELISA, art no. PRAAV5). Our results correlate well (Fig 13, on the previous page).For this sample set, the Biacore™ assay determined the average intra assay precision tobe 2%, while the ELISA determined it to be 15%.Bottom lineThe developed assays for both rAAV2 and rAAV5 provide repeatable results and correlate well with established total AAV capsid titer assays using ELISA. Compared to ELISA, Biacore™ assays havea higher degree of automation and precision and are compatible with process samples from harvest to final purified bulk. Their performance and ease-of-use make them very suitable as process development tools.DetailsRead the full app note on titer analysis using SPR.What we learnedWe learned that:for compatibility with different buffers, and include controls and references.The start-to-finish process we developed for recombinant AAV5 (rAAV5) is presented in Figure 14.For details on the work presented here, please visit our website to read the full application notes:"During production and purification it is crucial to monitor critical quality attributes such as viral genome titer, HCP, and DNA levels. This is often done with labor- intensive, low precision techniques. By using Biacore™ SPR systems for titer analysis, we get better precision compared to ELISA. We also save time since the assays are very easy to run and more highly automated." -Anna MobergFig 14. Summary of our start-to-finish process for rAAV, showing upstream, downstream, and analysis.A Report on the Present Science of Gene EditingAnis H. Khimani, PhD Senior Strategy Leader, PerkinElmer Life SciencesAdditionally, early-generation lentiviral vectors have shown clinical efficacy in more than 300 patients who received gene therapy for a number of diseases8-10; however, a recent gene therapy trial to treat cerebral adrenoleukodystrophy was halted after a participant inArun Srivastava, PhD Professor of Genetics, University of Florida College of Medicinein serious adverse events22 including the deaths of four patients.23Although gene therapy with AAV vectors continues tobe a promising treatment modality, it has also become increasingly clear that noneof the first generation of AAVthe study developed myelodysplastic syndrome, a bone marrow disorder that can lead to leukemia.11Meanwhile, recombinant AAV vectors, based on a nonpathogenic parvovirus, have been used or arecurrently in use in 264 Phase I/II/III clinical trials for diseasesvectors currently in use is ideal for the following reasons:Gene delivery vehicles have helped realizethe concept of treating human diseases by introducing normal alleles of genes into appropriate target cells. These gene delivery vehicles include recombinant andnonrecombinant lentiviral vectors and adeno- associated virus (AAV) vectors.Recombinant retroviral vectors have been used in clinical trials for nearly three decades. Initial results with retroviral vectors were encouraging,1,2 but the use of these vectors innonhuman primate studies was reported to leadto T-cell lymphoma.3 These vectors were also implicated in the development of T-cell leukemia in several children who received gene therapy for X-linked severe combined immunodeficiency in clinical trials.4,5Adenoviral vectors in gene therapy of cystic fibrosis were reported to lack efficacy.6 Also, an adenoviral vector was suspected of playing a role in the death of a patient in a trial for gene therapy of ornithine transcarbamylase deficiency.7acid decarboxylase deficiency,16 choroideremia,17 Leber hereditary optic neuropathy,18 hemophilia A,19 and spinal muscular atrophy (SMA),20 unexpected, remarkable clinical efficacy has also been achieved.Thus far, two AAV "drugs"-Luxturna for LCA and Zolgensma for SMA-have been approved by the FDA.21 However, in some cases, relatively large vector doses are needed to achieve clinical efficacy. The use of high doses has been shown to provoke host immune responses culminatingorgans.Next-generation AAVs come into focus These limitations have been addressed in various ways. First, scientists have developed capsid-modified next-generation (NextGen) AAVserotype vectors. (These vectors are up to 80-fold more efficacious at reduced doses26,27; they are also less immunogenic.28) Second, scientists developed genome-modified generation X (GenX) AAV vectors. (These vectors mediate upto eightfold enhanced transgene expression.29) Third, scientists have combined both strategies to produce optimized AAV serotype vectors. (These vectors are about 20-30-fold more efficientat further reduced doses.30) Fourth, scientists have identified AAV3 and AAV6, serotypes that possess remarkable tropisms for human liver and primary human hematopoietic stem cells, respectively.31-36In vivo evolution of large AAV libraries in combination with massive parallel screening of barcoded AAV vectors are now being actively employed to identify AAV vectors with further enhanced transduction properties and improved specificity.37,38but it is clear that attachment to putative cell surface receptors is the initial step for successful transduction. A wealth of information has been obtained from studies in mice, where different AAV serotype vectors have been shown to exhibit distinct tropism for various tissues and organs. The efficacy of some of the AAV serotype vectors has also been evaluated in other animals, small and large, such as rodents, canines, and nonhuman primates.40Innovative Tools to Advance DiscoveryFor therapeutic vector design, optimization of the size of the packaged therapeutic expression cassette can be achieved. Expression cassettes that are oversized (>5 kb) can result in theIn this schematic illustration, the recombinant AAV serotype vectors that have been used in various animal models and Phase I/II/III trials in humans are indicated. The animal models shown are the ones most commonly used to evaluate vector safety and efficacy. Also indicated are the organs that were targeted by the different vectors.AAV Serotype ConsiderationsMultiple AAV serotypes have been isolated from tissue culture stocks from humans and nonhuman primates.39 To date, 13 distinct AAV serotype vectors (AAV1-AAV13) have been described, and this number is likely to grow.Viral vector characterization is also an important application in gene therapy. It facilitates the evaluation of critical quality attributes (CQAs)such as identity, potency, purity, and stability.The precise mechanism of tissue tropism of AAV serotype vectors in vivo remains unknown,packaging of truncated genomes,41 whereas packaging of expression cassettes that are undersized can result in increased cross- packaging of plasmid-derived prokaryotic sequences that incorporate antibiotic resistance genes.42 Inclusion of large non-immunogenic inverted terminal repeat (ITR)-flanking spacer sequences significantly reduces unwantedpackaging of plasmid-derived prokaryotic sequences.The design, development, and production workflow for gene therapy vectors such as AAV is similar to the workflow for classical large- molecule biotherapeutics. Living producer systems that support an efficient, sustainable, and scalable growth environment for the vectors are desirable. Such systems can drive the development of the surrounding infrastructure for workflow management, automation, and regulatory compliance.A commonly used cell system for AAV transient transfection and production is HEK293. For other producer biological model systems, either mammalian or insect cell lines have been used to scale up AAV sequences containing thetransgene. For stable mammalian cell lines, either BHK cells or HeLa cells are used. With the insectsystem, Sf6 cells infected with recombinant baculovirus have been used as well to scale up recombinant AAV. With either of the above cell systems, production levels of 104 to 105 genome copies/L are obtained.Viral vector characterization is also an important application in gene therapy. It facilitates the evaluation of critical quality attributes (CQAs) such as identity, potency, purity, and stability.To evaluate CQAs and comply with regulatory guidelines, companies engaged in gene therapy need to adopt reproducible and robust methods of development and validation.A number of analytical techniques enable chemical and physical characterization. Viral structure and particle integrity and aggregation can be assessed with transmission electron microscopy (TEM), contributing to the quality control of viral vectors. TEM can also be used incombination with molecular techniques such as droplet digital PCR (ddPCR) or quantitative real time PCR (qPCR) to determine AAV identity and purity.Recent attention has been focused on analytical ultracentrifugation and mass spectrometryas well. However, some of these technologies have limited throughput. Higher throughput microfluidic capillary electrophoresis platforms, such as the LabChip GXII Touch with the ProteinEXact assay, can enable AAV capsid protein (VP1, VP2, VP3) analysis.Some of the above technologies are critical to the orthogonal validation of conventional or well- established methods such as electrophoresis, spectroscopy, and chromatography.Artificial intelligence and machine learning, along with analytical data management solutions, are significantly supporting analytical techniquesto achieve automation, predictability, and decision making along the vector development, characterization, scale up, and QA/QC workflows.Advanced tools, methods, applications, and services can facilitate the design, manufacture, and characterization of gene therapy vectors. End-to-end solutions can incorporate platforms for liquid handling and microfluidics; platforms for the analysis of macromolecules; platforms for cellular analysis and in vivo imaging; as well as platforms for bioinformatics applications. In addition, enhanced security software tools that enable 21 CFR Part 11 compliance to meet the regulatory requirements within the GMP lot release environment are critical.Therapeutic gene and cell therapy researchand development can progress more readily when advanced technologies and services for viral vector design and manufacturing areadopted. Such technologies, in combination with technologies for CRISPR-based gene editing, RNA interference, base editing, and prime editing, can move innovative therapeutics forward.Anis H. Khimani, PhD, is the senior strategy leader for pharmaceutical development at PerkinElmer Life Sciences. Christian Thirion, PhD, is the CEO of Sirion Biotech. Arun Srivastava, PhD, is the George H. Kitzman Professor of Genetics at the University of Florida College of Medicine.ReferencesThe only therapy we canblameon ourgenes.When you're striving to bring potential gene therapies to life,speed is critical. That's where we come in. With our workflowsolutions, you can make the viral vectors and plasmids you need, quickly. Because patients - and their genes - are waiting.It's in our genes atCytiva and the Drop logo are trademarks of Life Sciences IP Holdings Corp.or an affiliate doing business as Cytiva.©2022 CytivaFor local office contact information, visit cytiva.com/contactCY29175-18Apr22-AD
