production of recombinant aav by transient transfection

Purification of AAV Vectors by Single-Step Heparin Affinity Chromatography

Adeno-associated virus (AAV) vector is conquering its way as one of the most promising delivery systems in current gene therapy studies. Initially identified in 19651, AAV is a small, non-enveloped virus, with an icosahedral protein capsid of about 25 nm in diameter, harboring a single-stranded DNA genome. AAVs belong to the Parvoviridae family and to the Dependoparvovirus genus owing to their unique dependence on co-infection with a helper virus, such as herpes simplex virus or, more frequently, adenovirus, to complete their lytic cycle2,3.
The 4.7 kilobase genome of AAVs consists of two open reading frames (ORFs) flanked by two inverted terminal repeats (ITRs) that form characteristic T-shaped hairpin ends4. ITRs are the only cis-acting elements critical for AAV packaging, replication, and integration, therefore being the only AAV sequences retained in recombinant AAV (rAAV) vectors. In this system, the genes necessary for vector production are supplied separately, in trans, allowing the gene of interest to be packaged inside the viral capsid5,6.
Each viral gene codifies different proteins through alternative splicing and start codons. Within the Rep ORF, four non-structural proteins (Rep40, Rep52, Rep68, and Rep78) are encoded, playing crucial roles in replication, site-specific integration, and encapsidation of viral DNA7,8. The Cap ORF serves as a template for the expression of three structural proteins differing from each other at their N-terminus, (VP1, VP2, and VP3), that assemble to form a 60-mer viral capsid at a ratio of 1:1:104,9. Additionally, an alternative ORF nested in the Cap gene with a nonconventional CUG start codon encodes an assembly-activating protein (AAP). This nuclear protein has been shown to interact with the newly synthesized capsid proteins VP1-3 and promote capsid assembly10,11.
Differences in the amino acid sequence of the capsid account for the 11 naturally occurring AAV serotypes and over 100 variants isolated from humans and non-human primate tissues7,12,13. Variations in the conformation of structurally variable regions govern the distinct antigenic properties and receptor-binding specificities of capsids from different strains. This results in distinct tissue tropisms and transduction efficiencies across different mammalian organs14.
Early production methods of rAAVs relied on adenovirus infection for helper purposes15,16,17,18,19. Despite being efficient and usually easy to produce on a large scale, several problems arise from this infection. Even after the purification and a heat-denaturing step for inactivation, adenoviral particles may still be present in AAV preparations, constituting an unwanted safety issue20. Moreover, the presence of denatured adenoviral proteins is unacceptable for clinical use. Other production strategies take advantage of recombinant herpes simplex virus strains engineered to bring the Rep/Cap and transgene into the target cells21 or of the baculovirus-insect cell system22. Although these systems offer advantages in terms of scalability and GMP compatibility, they still face similar problems.
The triple transfection method for rAAV production has been commonly adopted to easily overcome these issues. Briefly, the rAAV assembly relies on the transient transfection of cells with three plasmids encoding for: 1) the transgene expression cassette packed between the ITRs from the wild-type AAV2 genome (pITR); 2) the Rep/Cap sequences necessary for replication and virion assembly (pAAV-RC); and 3) the minimal adenoviral proteins (E1A, E1B, E4, and E2A) along with the adenovirus virus-associated RNAs required for the helper effect (pHelper)6,20,23. While plasmid transfection methods provide simplicity and flexibility for rAAV production in preclinical studies, these procedures have limitations in terms of scalability and reproducibility when applied to large-scale production. As an alternative approach, rAAV production can be achieved through the use of AAV producer cell lines (of both adherent and suspension growth), stably expressing either AAV Rep/Cap genes or Rep/Cap in combination with vector constructs. In these systems, the adenoviral helper genes are introduced through plasmid transfection. Even though this strategy improves the scalability of the cell culture process, it is technically complex and time-consuming21,24,25.
In either case, the producer cells are then lysed and subjected to one or multiple purification steps. Currently, the principal methods for purifying rAAVs include the use of cesium chloride (CsCl) or iodixanol ultra-high speed density gradient centrifugation followed, or not, by chromatography techniques26. The original purification scheme for viral precipitation used ammonium sulfate, followed by two or three rounds of ultracentrifugation through a CsCl gradient. The main advantages of this process include the possibility to purify all serotypes and the ability to physically separate full particles from empty capsids based on their different densities. This method, however, is elaborate, time-consuming, and has limited scalability, often resulting in poor yield and low sample quality27,28,29,30. Moreover, dialysis against a physiological buffer is often necessary before in vivo studies due to the toxic effects that CsCl can exert on mammals.
Iodixanol has also been used as an alternative iso-osmotic gradient medium to purify rAAV vectors, with advantages over CsCl from both safety and vector potency points of view. However, like CsCl, the iodixanol method presents some drawbacks related to the loading capacity of cell culture lysate (and thus the scalability of rAAV purification) and it remains a time-consuming and expensive method30,31.
To overcome these constraints, researchers turned their attention to chromatography techniques. In this regard, several purification approaches were developed that either incorporate affinity, hydrophobic, or ion-exchange chromatography methods. These methods rely on the biochemical properties of a particular serotype, including their natural receptors, or the charge characteristics of the viral particle32. For instance, the discovery that AAV2, AAV3, AAV6, and AAV13 preferably bind to heparan sulfate proteoglycans (HSPG), opened the possibility of using the closely related heparin in affinity chromatography purification. However, the binding sites to HSPG can differ among serotypes, mediating AAV attachment and infection of target cells in different ways2,33,34,35,36. On the other hand, AAV1, AAV5, and AAV6 bind to N-linked sialic acid (SA), while AAV4 uses O-linked SA2,14,34. Following the same rationale, a single-step affinity chromatography protocol for the purification of rAAV5 has also been developed based on the use of mucin, a mammalian protein highly enriched in SA37. Like heparin-based techniques, this purification is also dependent on the specific serotype being generated. Apart from heparin and mucin, other ligands have been explored for affinity chromatography, such as the A20 monoclonal antibody and camelid single-domain antibodies (AVB Sepharose and POROS CaptureSelect)22,23,38,39,40,41. Other innovative strategies to improve the previously existing purification methods involve the introduction of small modifications in the rAAV capsid to present specific binding epitopes. For instance, hexa-histidine-tagged or biotinylated rAAVs can be purified using ligands that target those epitopes (nickel nitrilotriacetic acid and avidin resins, respectively)42,43,44.
In an effort to broaden the desired characteristics of rAAVs, investigators have cross-dressed the virions by mixing their capsids. This is accomplished by supplying the capsid gene from two distinct AAV serotypes in equimolar or different ratios during production, giving rise to a capsid structure composed of a mixture of capsid subunits from different serotypes34,45,46,47,48,49,50. Previous studies provide physical evidence that co-expressing capsid proteins from AAV2 with AAV1 (1:1 ratio) and AAV2 with AAV9 (1:1 ratio) results in the generation of mosaic rAAV1/2 and rAAV2/9 vectors, respectively45,46,48. A major benefit of the generation of mosaic rAAVs is the capacity to integrate advantageous traits from different AAV serotypes, resulting in synergistic improvements in transgene expression and tropism, while maintaining other properties useful during rAAV production. Interestingly, certain mosaic variants even exhibit novel properties different from either parental virus46,47,49. By taking advantage of the heparin-binding ability of AAV2, mosaic rAAV vectors could potentially be generated and purified by mixing AAV2 with other natural or new AAV capsids generated by directed evolution and/or rational design. Nonetheless, previous studies have highlighted the importance of capsid subunit compatibility when attempting to assemble mosaic vectors. For instance, Rabinowitz and colleagues demonstrated that although the transcapsidation of AAV1, AAV2, and AAV3 led to an efficient co-assembly of mosaic capsids, the cross-dressing of these serotypes with AAV4 hindered the generation of stable virions34,45,47. Additionally, AAV1, AAV2, and AAV3 showed low compatibility with AAV5, given the reduced viral titers obtained when mixing these capsids at different ratios. Interestingly, mosaic rAAV2/5 showed decreased heparin-binding properties, while maintaining mucin-binding ability like parental AAV5. However, rAAV3/5 at a 3:1 ratio preserved the dual binding to heparin and mucin. Overall, the generation of new mosaic rAAVs with enhanced transduction, specific tropism, or low immunogenicity could greatly benefit from our understanding of capsid assembly and receptor interactions, with specific combinations still requiring thorough investigation and optimization.
In the present work, we describe a step-by-step protocol for the production and purification of rAAVs using an optimized heparin affinity chromatography method. rAAVs are produced by transient transfection and are purified using a fast protein liquid chromatography (FPLC) system. After the concentration of selected purified fractions, the resulting viral stocks are characterized in terms of titer, purity, intrinsic physical properties, and biological activity in vitro and in vivo. As a proof of concept, we demonstrate the improvements and applicability of this protocol for the generation of mosaic rAAV1/2 and rAAV2/9 vectors. The choice of each serotype was based on their strikingly different tropisms, potentially conferring their unique characteristics to the mosaic versions as well. AAV serotype 1, with an overall moderate tropism for the central nervous system (CNS), has the ability to transduce neurons and glia (to a lesser extent) and undergoes axonal transport in the anterograde and retrograde directions in vivo2,7,8. Additionally, AAV serotype 9 was chosen for its natural ability to cross the blood-brain barrier and target the CNS in both neonatal and adult mice51,52. Finally, AAV serotype 2 was selected given its ability to bind to heparin, allowing affinity chromatography33. The purified rAAV1/2 and rAAV2/9 particles combine the properties of both parental AAV serotypes and, therefore, constitute suitable vectors for the transduction of the CNS45,46,48,49.

Author Spotlight: Improved Method for Production and Purification of Adeno-Associated Viral Vectors

Isolation of Adeno-Associated Viral Vectors Through a Single-Step and Semi-Automated Heparin Affinity Chromatography Protocol

In this work, we intend to develop an improved protocol for the production, purification, and characterization of mosaic adeno-associated viral vectors that could prove their worth in preclinical studies. Despite efforts to establish stable producer cell lines, transient transfection in mammalian cells is still the predominant workflow for AAV production. Then the AAVs are purified by ultra-high speed density gradient centrifugation, or by chromatography techniques that rely on the biochemical properties of viral particles.The commonly used methods to purify AAVs make use of cesium chloride, or iodixanol density gradient ultracentrifugation. Despite their advantages, they have some limitations. They are time-consuming, they have limited scalability, and they are often giving results to some vectors with low purity.Now, to overcome these constraints, several researchers have turned their attention to chromatography techniques. We describe a step-by-step protocol for the generation of mosaic rAAVs based on the heparin-binding ability of AAV2. This method renders highly pure and biologically active rAAVs ready to use in six days, presenting itself as a semi-automated, scalable, and cost-effective strategy to generate rAAVs for preclinical studies.Having demonstrated the applicability of this method for the generation of mosaic rAAVs, the production system can now be fine-tuned to achieve a better scalability and cost effectiveness by establishing a producer cell line. Additionally, we aim to remove empty particles by including a second polishing purification step.

Production of Recombinant AAV by Transient Transfection

production-of-recombinant-aav-by-transient-transfection

Research

JoVE Journal

Bioengineering

405 次观看.  University of Coimbra. 首先，通过在 10 个直径为 15 厘米的处理过的培养皿中加入 22 毫升补充培养基来铺HEK293T细胞。孵育板 24 小时，达到 70% 至 80% 的汇合度。对于细胞转染，通过将试剂混合在微量离心管中来制备混合物，并通过敲击进行混合。在 50 毫升离心管中将转染混合物加入 4.56 毫升未添加的 DMEM 中，并通过敲击混合。然后，逐滴加入 1.37 毫升无菌聚乙烯亚胺溶液。通过敲击混合并在...

视频: 通过瞬时转染生产重组AAV

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. However, the commonly used methods to purify AAVs make use of cesium chloride or iodixanol density gradient ultracentrifugation. Despite their advantages, these methods are time-consuming, have limited scalability, and often result in vectors with low purity. To overcome these constraints, researchers are turning their attention to chromatography techniques. Here, we present an optimized heparin-based affinity chromatography protocol that serves as a universal capture step for the purification of AAVs.
This method relies on the intrinsic affinity of AAV serotype 2 (AAV2) for heparan sulfate proteoglycans. Specifically, the protocol entails the co-transfection of plasmids encoding the desired AAV capsid proteins with those of AAV2, yielding mosaic AAV vectors that combine the properties of both parental serotypes. Briefly, after the lysis of producer cells, a mixture containing AAV particles is directly purified following an optimized single-step heparin affinity chromatography protocol using a standard fast protein liquid chromatography (FPLC) system. Purified AAV particles are subsequently concentrated and subjected to comprehensive characterization in terms of purity and biological activity. This protocol offers a simplified and scalable approach that can be performed without the need for ultracentrifugation and gradients, yielding clean and high viral titers.

This manuscript describes a detailed protocol for the generation and purification of adeno-associated viral vectors using an optimized heparin-based affinity chromatography method. It presents a simple, scalable, and cost-effective approach, eliminating the need for ultracentrifugation. The resulting vectors exhibit high purity and biological activity, proving their value in preclinical studies.

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. ...

FPLC Purification and Characterization of Recombinant AAV

Production and Titering of Recombinant Adeno-associated Viral Vectors

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently being tested in human clinical trials. Wild-type AAV is a non-pathogenic member of the parvoviridae family and inherently replication-deficient. The broad transduction profile, low immune response as well as the strong and persistent transgene expression achieved with these vectors has made them a popular and versatile tool for in vitro and in vivo gene delivery. rAAVs can be easily and cheaply produced in the laboratory and, based on their favourable safety profile, are generally given a low safety classification. Here, we describe a method for the production and titering of chimeric rAAVs containing the capsid proteins of both AAV1 and AAV2. The use of these so-called chimeric vectors combines the benefits of both parental serotypes such as high titres stocks (AAV1) and purification by affinity chromatography (AAV2). These AAV serotypes are the best studied of all AAV serotypes, and individually have a broad infectivity pattern. The chimeric vectors described here should have the infectious properties of AAV1 and AAV2 and can thus be expected to infect a large range of tissues, including neurons, skeletal muscle, pancreas, kidney among others. The method described here uses heparin column purification, a method believed to give a higher viral titer and cleaner viral preparation than other purification methods, such as centrifugation through a caesium chloride gradient. Additionally, we describe how these vectors can be quickly and easily titered to give accurate reading of the number of infectious particles produced.

Recombinant adeno-associated virus (rAAVs) vectors are becoming increasingly valuable for in vivo studies in animals. We describe how rAAVs can be produced in the laboratory and how these vectors can be titered to give an accurate reading of the number of infectious particles produced.

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently ...

Immunology and Infection

An Efficient Method for Adenovirus Production

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types and organs. However, recombinant adenoviral technology is laborious, time-consuming, and expensive. Here, we present an improved protocol using the pAdEasy system to obtain purified adenoviral particles that can induce a strong green fluorescent protein (GFP) expression in transduced cells. The advantages of this improved method are faster preparation and decreased production cost compared to the original method developed by Bert Vogelstein. The main steps of the adenoviral technology are: (1) the recombination of pAdTrack-GFP with the pAdEasy-1 plasmid in BJ5183 bacteria; (2) the packaging of the adenoviral particles; (3) the amplification of the adenovirus in AD293 cells; (4) the purification of the adenoviral particles from cell lysate and culture medium; and (5) the viral titration and functional testing of the adenovirus. The improvements to the original method consist of (i) the recombination in BJ5183-containing pAdEasy-1 by chemical transformation of bacteria; (ii) the selection of recombinant clones by &#8220;negative&#8221; and &#8220;positive&#8221; PCR; (iii) the transfection of AD293 cells using the K2 transfection system for adenoviral packaging; (iv) the precipitation with ammonium sulfate of the viral particles released by AD293 cells in cell culture medium; and (v) the purification of the virus by one-step cesium chloride discontinuous gradient ultracentrifugation. A strong expression of the gene of interest (in this case, GFP) was obtained in different types of transduced cells (such as hepatocytes, endothelial cells) from various sources (human, bovine, murine). Adenoviral-mediated gene transfer represents one of the main tools for developing modern gene therapies.

Here, we present a protocol for adenovirus production using the pAdEasy system. The technology includes the recombination of the pAdTrack and pAdEasy-1 plasmids, the adenovirus packaging and amplification, the purification of the adenoviral particles from cell lysate and culture medium, the viral titration, and the functional testing of the adenovirus.

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types ...

Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), including gene therapy applications. AAV vectors are non-replicating, able to infect both dividing and non-dividing cells&#160;and provide long-term transgene expression. Importantly, some serotypes, such as the newly described PHP.B, can cross the blood-brain-barrier (BBB) in animal models, following systemic delivery. AAV vectors can be efficiently produced in the laboratory. However, robust and reproducible protocols are required to obtain AAV vectors with sufficient purity levels and titer values high enough for in vivo applications. This protocol describes an efficient and reproducible strategy for AAV vector production, based on an iodixanol gradient purification strategy. The iodixanol purification method is suitable for obtaining batches of high-titer AAV vectors of high purity, when compared to other purification methods. Furthermore, the protocol is generally faster than other methods currently described. In addition, a quantitative polymerase chain reaction (qPCR)-based strategy is described for a fast and accurate determination of the vector titer, as well as a silver staining method to determine the purity of the vector batch. Finally, representative results of gene delivery to the CNS, following systemic administration of AAV-PHP.B, are presented. Such results should be possible in all labs using the protocols described in this article.

Here, we describe an efficient and reproducible strategy to produce, titer, and quality-control batches of adeno-associated virus vectors. It allows the user to obtain a vector preparation with high-titer&#160;(&#8805;1 x 1013 vector genomes/mL) and a high purity, ready for in vitro or in vivo use.

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), ...

Neuroscience

Production of Adeno-Associated Virus Vectors in Cell Stacks for Preclinical Studies in Large Animal Models

Adeno-associated virus (AAV) vectors are among the most clinically advanced gene therapy vectors, with three AAV gene therapies approved for humans. Clinical advancement of novel applications for AAV involves transitioning from small animal models, such as mice, to larger animal models, including dogs, sheep, and nonhuman primates. One of the limitations of administering AAV to larger animals is the requirement for large quantities of high-titer virus. While suspension cell culture is a scalable method for AAV vector production, few research labs have the equipment (e.g., bioreactors) or know how to produce AAV in this manner. Moreover, AAV titers are often significantly lower when produced in suspension HEK 293 cells as compared to adherent HEK293 cells. Described here is a method for producing large quantities of high-titer AAV using cell stacks. A detailed protocol for titering AAV as well as methods for validating vector purity are also described. Finally, representative results of AAV-mediated transgene expression in a sheep model are presented. This optimized protocol for large-scale production of AAV vectors in adherent cells will enable molecular biology laboratories to advance the testing of their novel AAV therapies in larger animal models.

Here we provide a detailed procedure for large-scale production of research-grade AAV vectors using adherent HEK 293 cells grown in cell stacks and affinity chromatography purification. This protocol consistently yields &#62;1 x 1013 vector genomes/mL, providing vector quantities appropriate for large animal studies.

Adeno-associated virus (AAV) vectors are among the most clinically advanced gene therapy vectors, with three AAV gene therapies approved for humans. ...

Efficient Adeno-Associated Virus Vector Production Using a Cell Factory Platform

A Streamlined and Standardized Procedure for Generating High-Titer, High-Quality Adeno-Associated Virus Vectors Utilizing a Cell Factory Platform

Preclinical gene therapy research, particularly in rodent and large animal models, necessitates the production of AAV vectors with high yield and purity. Traditional approaches in research laboratories often involve extensive use of cell culture dishes to cultivate HEK293T cells, a process that can be both laborious and problematic. Here, a unique in-house method is presented, which simplifies this process with a specific cell factory (or cell stacks, CF10) platform. An integration of polyethylene glycol/aqueous two-phase partitioning with iodixanol gradient ultracentrifugation improves both the yield and purity of the generated AAV vectors. The purity of the AAV vectors is verified through SDS-PAGE and silver staining, while the ratio of full to empty particles is determined using transmission electron microscopy (TEM). This approach offers an efficient cell factory platform for the production of AAV vectors at high yields, coupled with an improved purification method to meet the quality demands for in vivo studies.

Author Spotlight: Advancing Gene Therapy Research with High-Titer Adeno-Associated Virus Vector Production

As the field of gene therapy continues to evolve, there is a growing need for innovative methods that can address these challenges. Here, a unique method is presented, which streamlines the process of generating high-yield and high-purity AAV vectors using a cell factory platform, meeting the quality standards for in vivo studies.

Preclinical gene therapy research, particularly in rodent and large animal models, necessitates the production of AAV vectors with high yield and purity. ...

Simplified and Efficient AAV Production Using HEK293 Suspension Culture

Production of High-Yield Adeno Associated Vector Batches Using HEK293 Suspension Cells

Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.eB, demonstrate extensive transduction of the CNS by intravenous injection in mice. To achieve comparable transduction, a 100-fold higher titer (minimally 1 x 1011 genome copies/mouse) is needed compared to direct injection in the CNS parenchyma. In our group, AAV production, including AAV.PHP.eB relies on adherent HEK293T cells and the triple transfection method. Achieving high yields of AAV with adherent cells entails a labor- and material-intensive process. This constraint prompted the development of a protocol for suspension-based cell culture in conical tubes. AAVs generated in adherent cells were compared to the suspension production method. Culture in suspension using transfection reagents Polyethylenimine or TransIt were compared. AAV vectors were purified by iodixanol gradient ultracentrifugation followed by buffer exchange and concentration using a centrifugal filter. With the adherent method, we achieved an average of 2.6 x 1012 genome copies (GC) total, whereas the suspension method and Polyethylenimine yielded 7.7 x 1012 GC in total, and TransIt yielded 2.4 x 1013 GC in total. There is no difference in in vivo transduction efficiency between vectors produced with adherent compared to the suspension cell system. In summary, a suspension HEK293 cell based AAV production protocol is introduced, resulting in a reduced amount of time and labor needed for vector production while achieving 3 to 9 times higher yields using components available from commercial vendors for research purposes.

Author Spotlight: Advancing Gene Therapy with High-Yield AAV Vectors Through HEK293 Suspension Cell Cultivation

Here, a suspension HEK293 cell-based AAV production protocol is presented, resulting in reduced time and labor needed for vector production using components that are available for research purposes from commercial vendors.

Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.eB, ...