suspension-culture-production-purification-adeno-associated-virus

Suspension Culture Production and Purification of Adeno-Associated Virus by Iodixanol Density Gradient Centrifugation for In Vivo Applications

This protocol describes recombinant adeno-associated virus (rAAV) production and purification by iodixanol density gradient centrifugation, a ...

University of Florida

The major drawback of the Baculovirus/Sf9 system for recombinant adeno-associated viral (rAAV) manufacturing is that most of the Bac-derived rAAV vector serotypes, with few exceptions, demonstrate altered capsid compositions and lower biological potencies. Here, we describe a new insect cell-based production platform utilizing attenuated Kozak sequence and a leaky ribosome scanning to achieve a serotype-specific modulation of AAV capsid proteins stoichiometry. By way of example, rAAV5 and rAAV9 were produced and comprehensively characterized side by side with HEK293-derived vectors. A mass spectrometry analysis documented a 3-fold increase in both viral protein (VP)1 and VP2 capsid protein content compared with human cell-derived vectors. Furthermore, we conducted an extensive analysis of encapsidated single-stranded viral DNA using next-generation sequencing and show a 6-fold reduction in collaterally packaged contaminating DNA for rAAV5 produced in insect cells. Consequently, the re-designed rAAVs demonstrated significantly higher biological potencies, even in a comparison with HEK293-manufactured rAAVs mediating, in the case of rAAV5, 4-fold higher transduction of brain tissues in mice. Thus, the described system yields rAAV vectors of superior infectivity and higher genetic identity providing a scalable platform for good manufacturing practice (GMP)-grade vector production.

Direct Head-to-Head Evaluation of Recombinant Adeno-associated Viral Vectors Manufactured in Human versus Insect Cells.

Despite numerous advancements in production protocols, manufacturing AAV to meet exceptionally high demand (10-10 viral genomes [VGs]) in late clinical stages and for eventual systemic delivery poses significant challenges. Here, we report an efficient, simple, scalable, robust AAV5 production process utilizing the most recent modification of the OneBac platform. An increase in volumetric yield of genomic particles by ∼6-fold and functional particles by ∼20-fold was achieved by operating a high-cell-density process in shake flasks and bioreactors that involves an Sf9-based  stable cell line grown at a density of about 10 million cells/mL infected with a single baculovirus. The overall volumetric yields of genomic (VG) and bioactive particles (enhanced transducing units [ETUs]) in representative fedbatch bioreactor runs ranged from 2.5 to 3.5 × 10 VG/L and from 1 to 2 × 10 ETU/L. Analytical ultracentrifugation analyses of affinity-purified AAV vector samples from side-by-side batch and fedbatch production runs showed vector preparations with a full and empty particle distribution of 20%-30% genomic and 70%-80% empty particles. Moreover, the stoichiometric analysis of capsid proteins from fedbatch production in shake flask and bioreactor run samples demonstrated the incorporation of higher VP1 subunits, resulting in better functionality.

Achieving High-Yield Production of Functional AAV5 Gene Delivery Vectors via Fedbatch in an Insect Cell-One Baculovirus System.

Endotoxin is the most common contaminant found in protein samples. Even a small amount of endotoxin can induce strong allergic reaction and death of a host organism. Endotoxin is also often detected in recombinant adeno-associated virus (rAAV) stocks prepared in research laboratories using off-the-shelf reagents; purifying rAAV stocks from endotoxin using commercial reagents sometimes results in significant titer loss. The problem is exacerbated due to the recently expanded diversity of rAAV serotypes and capsid variants, which, due to their variable capsid surface charge, display differential affinity toward endotoxin. In this paper, we describe a simple universal protocol of purifying vector stocks irrespective of AAV serotype. The protocol is based on subjecting endotoxin-contaminated rAAV to mild detergent treatment, followed by repeated buffer-exchange washing and concentrating viral stock by low-speed centrifugation. Multiple assays were employed to test the physical and biological equivalency of the viral stocks before and after purification. The described protocol has been routinely utilized to purify vector stocks contaminated at levels as high as >1,000 endotoxin units (EU)/mL to produce viral vectors with practically undetectable levels of endotoxin (<2.5 EU/mL), with the titer's recovery in the range of 50%-100%.

Removal of Endotoxin from rAAV Samples Using a Simple Detergent-Based Protocol.

Limitations to successful gene therapy with adeno-associated virus (AAV) can comprise pre-existing neutralizing antibodies to the vector capsid that can block cellular entry, or inefficient transduction of target cells that can lead to sub-optimal expression of the therapeutic transgene. Recombinant serotype 3 AAV (AAV3) is an emerging candidate for liver-directed gene therapy. In this study, we integrated rational design by using a combinatorial library derived from AAV3B capsids with directed evolution by  selection for liver-targeted AAV variants. The AAV3B-DE5 variant described herein was undetectable in the original viral library but gained a selective advantage upon  passaging in human hepatocarcinoma spheroid cultures. AAV3B-DE5 contains 24 capsid amino acid substitutions compared with AAV3B, distributed among all five variable regions, with strong selective pressure on VR-IV, VR-V, and VR-VII. , AAV3B-DE5 demonstrated improved human hepatocyte tropism in a liver chimeric mouse model. Importantly, this variant exhibited reduced seroreactivity to human intravenous immunoglobulin (i.v. Ig), as well as individual serum samples from 100 healthy human donors. Therefore, molecular evolution using a combinatorial library platform generated a viral capsid with high hepatocyte tropism and enhanced evasion of pre-existing AAV neutralizing antibodies.

Engineering and  Selection of a Novel AAV3B Variant with High Hepatocyte Tropism and Reduced Seroreactivity.

Machine learning (ML) can aid in novel discoveries in the field of viral gene therapy. Specifically, big data gathered through next-generation sequencing (NGS) of complex capsid libraries is an especially prominent source of lost potential in data analysis and prediction. Furthermore, adeno-associated virus (AAV)-based capsid libraries are becoming increasingly popular as a tool to select candidates for gene therapy vectors. These higher complexity AAV capsid libraries have previously been created and selected ; however,  analysis using ML computer algorithms may augment smarter and more robust libraries for selection. In this study, data of AAV capsid libraries gathered before and after viral assembly are used to train ML algorithms. We found that two ML computer algorithms, artificial neural networks (ANNs), and support vector machines (SVMs), can be trained to predict whether unknown capsid variants may assemble into viable virus-like structures. Using the most accurate models constructed, hypothetical mutation patterns in library construction were simulated to suggest the importance of N495, G546, and I554 in AAV2-derived capsids. Finally, two comparative libraries were generated using ML-derived data to biologically validate these findings and demonstrate the predictive power of ML in vector design.

Applying machine learning to predict viral assembly for adeno-associated virus capsid libraries.

Non-human primates (NHPs) are a preferred animal model for optimizing adeno-associated virus (AAV)-mediated CNS gene delivery protocols before clinical trials. In spite of its inherent appeal, it is challenging to compare different serotypes, delivery routes, and disease indications in a well-powered, comprehensive, multigroup NHP experiment. Here, a multiplex barcode recombinant AAV (rAAV) vector-tracing strategy has been applied to a systemic analysis of 29 distinct, wild-type (WT), AAV natural isolates and engineered capsids in the CNS of eight macaques. The report describes distribution of each capsid in 15 areas of the macaques' CNS after intraparenchymal (putamen) injection, or cerebrospinal fluid (CSF)-mediated administration routes (intracisternal, intrathecal, or intracerebroventricular). To trace the vector biodistribution (viral DNA) and targeted tissues transduction (viral mRNA) of each capsid in each of the analyzed CNS areas, quantitative next-generation sequencing analysis, assisted by the digital-droplet PCR technology, was used. The report describes the most efficient AAV capsid variants targeting specific CNS areas after each route of administration using the direct side-by-side comparison of WT AAV isolates and a new generation of rationally designed capsids. The newly developed bioinformatics and visualization algorithms, applicable to the comparative analysis of several mammalian brain models, have been developed and made available in the public domain.

A comprehensive study of a 29-capsid AAV library in a non-human primate central nervous system.

Capsid engineering of adeno-associated virus (AAV) can surmount current limitations to gene therapy such as broad tissue tropism, low transduction efficiency, or pre-existing neutralizing antibodies (NAb) that restrict patient eligibility. We previously generated an AAV3B combinatorial capsid library by integrating rational design and directed evolution with the aim of improving hepatotropism. A potential isolate, AAV3B-DE5, gained a selective proliferative advantage over five rounds of iterative selection in hepatocyte spheroid cultures. In this study, we reanalyzed our original dataset derived from the AAV3B combinatorial library and isolated variants from earlier (one to three) rounds of selection, with the assumption that variants with faster replication kinetics are not necessarily the most efficient transducers. We identified a potential candidate, AAV3B-V04, which demonstrated significantly enhanced transduction in mouse-passaged primary human hepatocytes as well as in humanized liver chimeric mice, compared to the parental AAV3B or the previously described isolate, AAV3B-DE5. Interestingly, the AAV3B-V04 capsid variant exhibited significantly reduced seroreactivity to pooled or individual human serum samples. Forty-four percent of serum samples with pre-existing NAbs to AAV3B had 5- to 20-fold lower reciprocal NAb titers to AAV3B-V04. AAV3B-V04 has only nine amino acid substitutions, clustered in variable region IV compared to AAV3B, indicating the importance of the loops at the top of the three-fold protrusions in determining both transduction efficiency and immunogenicity. This study highlights the effectiveness of rational design combined with targeted selection for enhanced AAV transduction via molecular evolution approaches. Our findings support the concept of limiting selection rounds to isolate the best transducing AAV3B variant without outgrowth of faster replicating candidates. We conclude that AAV3B-V04 provides advantages such as improved human hepatocyte tropism and immune evasion and propose its utility as a superior candidate for liver gene therapy.

Characterization of a Bioengineered AAV3B Capsid Variant with Enhanced Hepatocyte Tropism and Immune Evasion.

Adeno-associated virus (AAV) continues to be the gold standard vector for therapeutic gene delivery and has proven especially useful for treating ocular disease. Intravitreal injection (IVtI) is a promising delivery route because it increases accessibility of gene therapies to larger patient populations. However, data from clinical and non-human primate (NHP) studies utilizing currently available capsids indicate that anatomical barriers to AAV and pre-existing neutralizing antibodies can restrict gene expression to levels that are "sub-therapeutic" in a substantial proportion of patients. Here, we performed a combination of directed evolution in NHPs of an AAV2-based capsid library with simultaneous mutations across six surface-exposed variable regions and rational design to identify novel capsid variants with improved retinal transduction following IVtI. Following two rounds of screening in NHP, enriched variants were characterized in intravitreally injected mice and NHPs and shown to have increased transduction relative to AAV2. Lead capsid variant, P2-V1, demonstrated an increased ability to evade neutralizing antibodies in human vitreous samples relative to AAV2 and AAV2.7m8. Taken together, this study further contributed to our understanding of the selective pressures associated with retinal transduction via the vitreous and identified promising novel AAV capsid variants for clinical consideration.

Intravitreal injection of a rationally designed AAV capsid library in non-human primate identifies variants with enhanced retinal transduction and neutralizing antibody evasion.

The widespread successful use of recombinant Adeno-associated virus (rAAV) in gene therapy has driven the demand for scale-up manufacturing methods of vectors with optimized yield and transduction efficiency. The Baculovirus/Sf9 system is a promising platform for high yield production; however, a major drawback to using an invertebrate cell line compared to a mammalian system is a generally altered AAV capsid stoichiometry resulting in lower biological potency. Here, we introduce a term of the structural and biological "fitness" of an AAV capsid as a function of two interdependent parameters: (1) packaging efficiency (yield), and (2) transduction efficiency (infectivity). Both parameters are critically dependent on AAV capsid structural proteins VP1/2/3 stoichiometry. To identify an optimal AAV capsid composition, we developed a novel Directed Evolution (DE) protocol for assessing the structural and biological fitness of Sf9-manufactured rAAV for any given serotype. The approach involves the packaging of a combinatorial capsid library in insect Sf9 cells, followed by a library screening for high infectivity in human Cre-recombinase-expressing C12 cells. One single DE selection round, complemented by Next-Generation Sequencing (NGS) and guided by in silico analysis, identifies a small subset of VP1 translation initiation sites (known as Kozak sequence) encoding "fit" AAV capsids characterized by a high production yield and superior transduction efficiencies.

Article	Year
Direct Head-to-Head Evaluation of Recombinant Adeno-associated Viral Vectors Manufactured in Human versus Insect Cells. Molecular therapy : the journal of the American Society of Gene Therapy\| PubMed ID: 28890324	2017
Achieving High-Yield Production of Functional AAV5 Gene Delivery Vectors via Fedbatch in an Insect Cell-One Baculovirus System. Molecular therapy. Methods & clinical development\| PubMed ID: 30886878	2019
Removal of Endotoxin from rAAV Samples Using a Simple Detergent-Based Protocol. Molecular therapy. Methods & clinical development\| PubMed ID: 31649960	2019
Engineering and Selection of a Novel AAV3B Variant with High Hepatocyte Tropism and Reduced Seroreactivity. Molecular therapy. Methods & clinical development\| PubMed ID: 33145371	2020
Applying machine learning to predict viral assembly for adeno-associated virus capsid libraries. Molecular therapy. Methods & clinical development\| PubMed ID: 33511242	2021
A comprehensive study of a 29-capsid AAV library in a non-human primate central nervous system. Molecular therapy : the journal of the American Society of Gene Therapy\| PubMed ID: 34298128	2021
Characterization of a Bioengineered AAV3B Capsid Variant with Enhanced Hepatocyte Tropism and Immune Evasion. Human gene therapy\| PubMed ID: 36950804	2023
Intravitreal injection of a rationally designed AAV capsid library in non-human primate identifies variants with enhanced retinal transduction and neutralizing antibody evasion. Molecular therapy : the journal of the American Society of Gene Therapy\| PubMed ID: 37814449	2023
Exploring the Comprehensive Kozak Sequence Landscape for AAV Production in Sf9 System. Viruses\| PubMed ID: 37896760	2023

Oleksandr Kondratov

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