Amira D. Rghei, Brenna A. Y. Stevens, Sylvia P. Thomas, and Jacob G. E. Yates were recipients of Ontario Veterinary College Student Stipends as well as Ontario Graduate Scholarships. Amira D. Rghei was the recipient of a Mitacs Accelerate Studentship. This work was funded by the Canadian Institutes for Health Research (CIHR) Project Grant (#66009) and a Collaborative Health Research Projects (NSERC partnered) grant (#433339) to SKW.

<ol>
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Sarah K. Wootton is an inventor on a US patent US10806802B2 for the AAV6.2FF capsid.

The production of recombinant AAV (rAAV) vectors described in this paper uses common materials, reagents, and equipment found in majority of the molecular biology research labs and facilities. This paper allows for high-quality in vitro and in vivo grade rAAV to be produced by the reader. Above all, this protocol for rAAV production, compared to more tedious protocols involving cesium chloride purification, is efficient and avoids the use of ultracentrifugation. Once HEK293 cells have been transfected, ...

Gene therapy utilizing adeno-associated virus (AAV) vectors has made huge strides over the past three decades1,2. Demonstrated improvements in a diverse range of genetic diseases, including congenital blindness, hemophilia, and diseases of the musculoskeletal and central nervous system, have brought AAV gene therapy to the forefront of clinical research3,4. In 2012, the European Medicines Agency (EMA) approved Glybera, an AAV1 vector expressing lipoprotein lipase (LPL) for the treatment of LPL deficiency, making it the first marketing authorization for a gene therapy treatment in either Europe or the United States5. Since then, two additional AAV gene therapies, Luxturna6 and Zolgensma7, have received FDA approval, and the market is expected to expand quickly over the next 5 years with as many as 10-20 gene therapies expected by 20258. Available clinical data indicate that AAV gene therapy is a safe, well-tolerated, and efficacious modality making it one of the most promising viral vectors, with above 244 clinical trials involving AAV registered with ClinicalTrials.gov. The increasing interest in clinical applications involving AAV vectors requires robust and scalable production methods to facilitate the evaluation of AAV therapies in large animal models, as this is a critical step in the translational pipeline9.
For AAV vector production, the two main requirements are the AAV genome and the capsid. The genome of wild-type (wt)-AAV is single-stranded DNA that is approximately 4.7 kb in length10. The wt-AAV genome comprises inverted terminal repeats (ITRs) found at both ends of the genome, which are important for packaging, and the rep and cap genes11. The rep and cap genes, necessary for genome replication, assembly of the viral capsid, and encapsulation of the genome into the viral capsid, are removed from the viral genome and provided in trans for AAV vector production12. The removal of these genes from the viral genome provides room for therapeutic transgenes and all the necessary regulatory elements, including the promoter and polyA signal. The ITRs remain in the vector genome to ensure proper genome replication and viral encapsulation13,14. To improve the kinetics of transgene expression, AAV vector genomes can be engineered to be self-complementary, which mitigates the need for conversion from single-stranded to double-stranded DNA conversion during AAV genome replication, but reduces the coding capacity to ~2.4 kb15.
Beyond AAV genome design, capsid serotype selection determines the tissue and cell tropism of the AAV vector in vivo2. In addition to tissue tropism, different AAV serotypes have been shown to display different gene expression kinetics16. For example, Zincarelli et al.17&#160;classified different AAV serotypes into low expression serotypes (AAV2, 3, 4, 5), moderate expression serotypes (AAV1, 6, 8), and high expression serotypes (AAV7 and 9). They also categorized AAV serotypes into slow-onset expression (AAV2, 3, 4, 5) or rapid-onset expression (AAV1, 6, 7, 8, and 9). These divergent tropisms and gene expression kinetics are due to amino acid variations in the capsid proteins, capsid protein formations, and interactions with host cell receptors/co-receptors18. Some AAV capsids have additional beneficial characteristics such as the ability to cross the blood-brain barrier following intravascular administration (AAV9) or reside in long-living muscle cells for durable transgene expression (AAV6, 6.2FF, 8, and 9)19,20.
This paper aims to detail a cost-effective method for producing high-purity, high-titer, research-grade AAV vectors for use in preclinical large animal models. Production of AAV using this protocol is achieved using dual-plasmid transfection into adherent human embryonic kidney (HEK)293 cells grown in cell stacks. Furthermore, the study describes a protocol for heparin sulfate affinity chromatography purification, which can be used for AAV serotypes that contain heparin-binding domains, including AAV2, 3, 6, 6.2FF, 13, and DJ21,22.
A number of packaging systems are available for the production of AAV vectors. Among these, the use of a two-plasmid co-transfection systems, in which the Rep and Cap genes and Ad helper genes (E1A, E1B55K, E2A, E4orf6, and VA RNA) are contained within one plasmid (pHelper), has some practical advantages over the common three-plasmid (triple) transfection method, including reduced cost for plasmid production23,24. The AAV genome plasmid containing the transgene expression cassette (pTransgene), must be flanked by ITRs, and must not exceed ~4.7 kb in length. Vector titer and purity can be affected by the transgene due to potential cytotoxic effects during transfection. Assessment of vector purity is described herein. Vectors produced using this method, which yield a 1 x 1013 vg/mL for each, were evaluated in mice, hamsters, and ovine animal models.
Table 1: Composition of required solutions. Necessary information, including percentages and volumes, of components needed for various solutions throughout the protocol. <a href="https://www.jove.com/files/ftp_upload/62727/Table 1- 62727_R2.xlsx" target="_blank">Please click here to download this Table.</a>

<table><tbody><tr><td>0.22 &amp;mu;m filter</td><td>Millipore Sigma</td><td>S2GPU05RE</td><td></td></tr><tr><td>0.25% Trypsin</td><td>Fisher Scientific</td><td>SM2001C</td><td></td></tr><tr><td>1-Butanol</td><td>Thermo Fisher Scientific</td><td>A399-4</td><td>CAUTION. Use under a laminar flow hood. Wear gloves</td></tr><tr><td>10 chamber cellstack</td><td>Corning</td><td>3271</td><td></td></tr><tr><td>1L PETG bottle</td><td>Thermo Fisher Scientific</td><td>2019-1000</td><td></td></tr><tr><td>30% Acrylamide/Bis Solution</td><td>Bio-Rad</td><td>1610158</td><td></td></tr><tr><td>96-well skirted plate</td><td>FroggaBio</td><td>FS-96</td><td></td></tr><tr><td>Adhesive plate seals</td><td>Thermo Fisher Scientific</td><td>08-408-240</td><td></td></tr><tr><td>Ammonium persulfate (APS)</td><td>Bio-Rad</td><td>161-0700</td><td>CAUTION. Use under a laminar flow hood. Wear gloves</td></tr><tr><td>Blood and Tissue Clean up Kit</td><td>Qiagen</td><td>69506</td><td>Use for DNA clean up in section 4.6 of protocol</td></tr><tr><td>Bromophenol blue</td><td>Fisher Scientific</td><td>B392-5</td><td>CAUTION. Use under a laminar flow hood. Wear gloves</td></tr><tr><td>Cell Culture Dishes</td><td>Greiner bio-one</td><td>7000232</td><td>15 cm plates</td></tr><tr><td>Culture Conical Tube</td><td>Thermo Fisher Scientific</td><td>339650</td><td>15 mL conical tube</td></tr><tr><td>Culture Conical Tube</td><td>Fisher Scientific</td><td>14955240</td><td>50 mL conical tube</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM) with 1000 mg/L D-glucose, L-glutamine</td><td>Cytiva Life Sciences</td><td>SH30022.01</td><td></td></tr><tr><td>ECL Western Blotting Substrate</td><td>Thermo Fisher Scientific</td><td>32209</td><td></td></tr><tr><td>Ethanol</td><td>Greenfield</td><td>P016EA95</td><td>Dilute ethyl alcohol(95% vol) to 20% for section 3.7.4 and 70% for section 6.1.1.1</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Thermo Fisher Scientific</td><td>SH30396.03</td><td></td></tr><tr><td>Glacial acetic acid</td><td>Fisher Scientific</td><td>A38-500</td><td>CAUTION. Use under a laminar flow hood. Wear gloves</td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>BP229-1</td><td></td></tr><tr><td>Glycine</td><td>Fisher Scientific</td><td>BP381-500</td><td></td></tr><tr><td>HBSS with Mg&lt;sup&gt;2+ &lt;/sup&gt;and Ca&lt;sup&gt;2+&lt;/sup&gt;</td><td>Thermo Fisher Scientific</td><td>SH302268.02</td><td></td></tr><tr><td>HBSS without Mg&lt;sup&gt;2+ &lt;/sup&gt;and Ca&lt;sup&gt;2+&lt;/sup&gt;</td><td>Thermo Fisher Scientific</td><td>SH30588.02</td><td></td></tr><tr><td>HEK293 cells</td><td>American Tissue Culture Collection</td><td>CRL-1573</td><td>Upon receipt, thaw the cells and culture as described in manufacturer&amp;rsquo;s protocol. Once cells have been minimally passaged and are growing well, freeze a subfraction for future in aliquots and store in liquid nitrogen. Always use cells below passage number 30. Once cultured cells have been passaged more than 30 times, it is recommended to restart a culture from the stored aliquots</td></tr><tr><td>HEK293 host cell protein ELISA kit</td><td>Cygnus Technologies</td><td>F650S</td><td>Follow manufacturer&amp;rsquo;s instructions</td></tr><tr><td>Heparin sulfate column</td><td>Cytiva Life Sciences</td><td>17040703</td><td></td></tr><tr><td>Kimwipe</td><td>Thermo Fisher Scientific</td><td>KC34120</td><td></td></tr><tr><td>&amp;nbsp;L-glutamine (200 mM)</td><td>Thermo Fisher Scientific</td><td>SH30034.02</td><td></td></tr><tr><td>Large Volume Centrifuge Tube Support Cushion</td><td>Corning</td><td>CLS431124</td><td>Support cushion must be used with large volume centrifuge tubes uless the centrifuge rotor has the approriate V-bottom cushions</td></tr><tr><td>Large Volume Centrifuge Tubes</td><td>Corning</td><td>CLS431123-6EA</td><td>500 mL centrifuge tubes</td></tr><tr><td>MgCl&lt;sub&gt;2&amp;nbsp;&lt;/sub&gt;</td><td>Thermo Fisher Scientific</td><td>7791-18-6</td><td></td></tr><tr><td>Microcentrifuge tube</td><td>Fisher Scientific</td><td>05-408-129</td><td>1.5 mL microcentrifuge tube, sterilize prior to use</td></tr><tr><td>Molecular Grade Water</td><td>Cytiva Life Sciences</td><td>SH30538.03</td><td></td></tr><tr><td>N-Lauroylsarcosine sodium salt</td><td>Sigma Aldrich</td><td>L5125</td><td>CAUTION. Wear gloves</td></tr><tr><td>NaCl</td><td>Thermo Fisher Scientific</td><td>BP35810</td><td></td></tr><tr><td>Optimem, reduced serum medium</td><td>Thermo Fisher Scientific</td><td>31985070</td><td></td></tr><tr><td>Pasteur pipets</td><td>Fisher Scientific</td><td>13-678-20D</td><td>Sterilize prior to use</td></tr><tr><td>PBS (10x)</td><td>Thermo Fisher Scientific</td><td>70011044</td><td>Dilute to 1x for use on cells</td></tr><tr><td>Penicillin-Streptomycin Solution</td><td>Cytiva Life Sciences</td><td>SV30010</td><td></td></tr><tr><td>pHelper plasmid</td><td>De novo design or obtained from plasmid repository</td><td>NA</td><td></td></tr><tr><td>Pipet basin</td><td>Thermo Fisher Scientific</td><td>13-681-502</td><td>Purchase sterile pipet basins</td></tr><tr><td>Polyethylene glycol tert-octylphenyl ether (Triton X-100)</td><td>Thermo Fisher Scientific</td><td>9002-93-1</td><td>CAUTION. Wear gloves</td></tr><tr><td>Polyethylenimine (PEI)</td><td>Polyscience</td><td>24765-1</td><td>Follow manufacturer&amp;rsquo;s instructions to produce a 1L solution. 0.22&amp;mu;m filter and store at 4&amp;deg;C</td></tr><tr><td>Polypropylene semi-skirted PCR Plate</td><td>FroggaBio</td><td>WS-96</td><td></td></tr><tr><td>Polysorbate 20 (Tween 20)</td><td>Thermo Fisher Scientific</td><td>BP337-100</td><td>CAUTION. Wear gloves</td></tr><tr><td>polyvinylidene difluoride (PVDF) membrane</td><td>Cytiva Life Sciences</td><td>10600023</td><td>Use forceps to manipulate. Wear gloves.</td></tr><tr><td>Primary antibody</td><td>Progen</td><td>65158</td><td></td></tr><tr><td>Protein Ladder</td><td>FroggaBio</td><td>PM008-0500</td><td></td></tr><tr><td>Proteinase K</td><td>Thermo Fisher Scientific</td><td>AM2546</td><td></td></tr><tr><td>pTrangene plasmid</td><td>De novo design or obtained from plasmid repository</td><td>NA</td><td>Must contain SV40 polyA in genome to be compatible with AAV titration in section 5.0</td></tr><tr><td>Pump tubing</td><td>Cole-Parmer</td><td>RK-96440-14</td><td>Optimize length of tubing and containment of virus in fractions E1-E5</td></tr><tr><td>RQ1 Dnase 10 Reaction Buffer</td><td>Promega</td><td>M6101</td><td>Use at 10x concentration in protocol from section 4.0</td></tr><tr><td>RQ1 Rnase-free Dnase</td><td>Promega</td><td>M6101</td><td></td></tr><tr><td>Sample dilutent</td><td>Cygnus Technologies</td><td>I700</td><td>Must be purchased separately for use with HEK293 host cell protein ELISA kit</td></tr><tr><td>Secondary antibody, HRP</td><td>Thermo Fisher Scientific</td><td>G-21040</td><td></td></tr><tr><td>Skim milk powder</td><td>Oxoid</td><td>LP0033B</td><td></td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>Thermo Fisher Scientific</td><td>28312</td><td>CAUTION. Use under a laminar flow hood. Wear gloves</td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Thermo Fisher Scientific</td><td>SS266-4</td><td></td></tr><tr><td>SV40 polyA primer probe</td><td>IDT</td><td></td><td>Use sequence in Table X for quote from IDT for synthesis</td></tr><tr><td>Tetramethylethylenediamine (TEMED)</td><td>Thermo Fisher Scientific</td><td>15524010</td><td>CAUTION. Use under a laminar flow hood. Wear gloves</td></tr><tr><td>Tris Base</td><td>Fisher Scientific</td><td>BP152-5</td><td></td></tr><tr><td>Trypan blue</td><td>Bio-Rad</td><td>1450021</td><td></td></tr><tr><td>Ultra-Filter</td><td>Millipore Sigma</td><td>UFC810024</td><td>Ultra-4 Centrifugal 10K device must be used, as it has a 10000 molecular weight cutoff</td></tr><tr><td>Universal Nuclease for cell lysis</td><td>Thermo Fisher Scientific</td><td>88702</td><td></td></tr><tr><td>Universal qPCR master mix</td><td>NEB</td><td>M3003L</td><td></td></tr><tr><td>Whatman Paper</td><td>Millipore Sigma</td><td>WHA1001325</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Fisher Scientific</td><td>21985023</td><td>CAUTION. Use under a laminar flow hood. Wear gloves</td></tr><tr><td>CAUTION:&amp;nbsp;Refer to the Materials Table for guidelines on the use of dangerous chemicals.</td><td></td><td></td><td></td></tr></tbody></table>

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.

Translation from small rodent models to larger animal models and eventual clinical application presents a significant challenge due to the large amount of AAV required to transduce larger animals and achieve therapeutic effects. To compare transduction efficiency of the rationally designed AAV6.2FF capsid, previously demonstrated a 101-fold increase in transduction efficiency in murine muscle cells compared to AAV63, mice, hamsters, and lambs were all administered AAV6.2FF expressing a human monoc...

Watch this Scientific Journal Video about Production of Adeno-Associated Virus Vectors in Cell Stacks for Preclinical Studies in Large Animal Models at JoVE.com

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

production-adeno-associated-virus-vectors-cell-stacks-for-preclinical

Research

JoVE Journal

Immunology and Infection

5.8K Views.  University of Guelph. 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 >1 x 1013 vector genomes/mL, providing vector quantities appropriate for large animal studies.

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

Estimating Virus Production Rates in Aquatic Systems

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative estimates of this process is critical as we can then develop better models of microbial community structure and function as well as advance our understanding of how viruses work to alter aquatic biogeochemical cycles. The virus reduction technique allows researchers to estimate the rate at which virus particles are released from the endemic microbial community. In brief, the abundance of free (extracellular) viruses is reduced in a sample while the microbial community is maintained at near ambient concentration. The microbial community is then incubated in the absence of free viruses and the rate at which viruses reoccur in the sample (through the lysis of already infected members of the community) can be quantified by epifluorescence microscopy or, in the case of specific viruses, quantitative PCR. These rates can then be used to estimate the rate of microbial mortality due to virus-mediated cell lysis.

The turnover rate of viruses in marine and freshwater systems can be estimated by a reduction and reoccurrence technique. The data allow researchers to infer rates of virus-mediated microbial mortality in aquatic systems.

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative ...

Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized animals. Before embarking on an examination of animals for parasites, two things should be considered. One: what use will be made of the information collected, and two: which test is the most appropriate. Knowing that animals are parasitized may be something that the facility accepts, but there is often a need to treat animals and then to determine the efficacy of treatment. Parasites may be detected in animals through various techniques, including samples taken from live or euthanized animals. Historically, the tests with the greatest diagnostic sensitivity required euthanasia of the animal, although PCR has allowed high-sensitivity testing for several types of parasite. This article demonstrates procedures for the detection of endo- and ectoparasites in mice and rats. The same procedures are applicable to other rodents, although the species of parasites found will differ.

This article describes various procedures for screening rats and mice to detect endo- or ectoparasitism. Several diagnostic assays will be demonstrated, both those suitable for use on live animals and those used after euthanasia of the animal. Photographs to aid in identification of rat and mouse parasites will be included.

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized ...

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 ...

Engineering and Evolution of Synthetic Adeno-Associated Virus (AAV) Gene Therapy Vectors via DNA Family Shuffling

Adeno-associated viral (AAV) vectors represent some of the most potent and promising vehicles for therapeutic human gene transfer due to a unique combination of beneficial properties1. These include the apathogenicity of the underlying wildtype viruses and the highly advanced methodologies for production of high-titer, high-purity and clinical-grade recombinant vectors2. A further particular advantage of the AAV system over other viruses is the availability of a wealth of naturally occurring serotypes which differ in essential properties yet can all be easily engineered as vectors using a common protocol1,2. Moreover, a number of groups including our own have recently devised strategies to use these natural viruses as templates for the creation of synthetic vectors which either combine the assets of multiple input serotypes, or which enhance the properties of a single isolate. The respective technologies to achieve these goals are either DNA family shuffling3, i.e. fragmentation of various AAV capsid genes followed by their re-assembly based on partial homologies (typically &gt;80% for most AAV serotypes), or peptide display4,5, i.e. insertion of usually seven amino acids into an exposed loop of the viral capsid where the peptide ideally mediates re-targeting to a desired cell type. For maximum success, both methods are applied in a high-throughput fashion whereby the protocols are up-scaled to yield libraries of around one million distinct capsid variants. Each clone is then comprised of a unique combination of numerous parental viruses (DNA shuffling approach) or contains a distinctive peptide within the same viral backbone (peptide display approach). The subsequent final step is iterative selection of such a library on target cells in order to enrich for individual capsids fulfilling most or ideally all requirements of the selection process. The latter preferably combines positive pressure, such as growth on a certain cell type of interest, with negative selection, for instance elimination of all capsids reacting with anti-AAV antibodies. This combination increases chances that synthetic capsids surviving the selection match the needs of the given application in a manner that would probably not have been found in any naturally occurring AAV isolate. Here, we focus on the DNA family shuffling method as the theoretically and experimentally more challenging of the two technologies. We describe and demonstrate all essential steps for the generation and selection of shuffled AAV libraries (Fig. 1), and then discuss the pitfalls and critical aspects of the protocols that one needs to be aware of in order to succeed with molecular AAV evolution.

Engineering and Evolution of Synthetic Adeno-Associated Virus AAV Gene Therapy Vectors via DNA Family Shuffling

We demonstrate the basic technique to molecularly engineer and evolve synthetic Adeno-associated viral (AAV) gene therapy vectors via DNA family shuffling. Moreover, we provide general guidelines and representative examples for selection and analysis of individual chimeric capsids with enhanced properties on target cells in culture or in mice.

Adeno-associated viral (AAV) vectors represent some of the most potent and promising vehicles for therapeutic human gene transfer due to a unique ...

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high packaging capacity (up to 35 kb), low immunogenicity, and low toxicity. However, for many laboratories the use of HCAdV is hampered by the complicated procedure for vector genome construction and virus production. Here, a detailed protocol for efficient cloning and production of HCAdV based on the plasmid pAdFTC containing the HCAdV genome is described. The construction of HCAdV genomes is based on a cloning vector system utilizing homing endonucleases (I-CeuI and PI-SceI). Any gene of interest of up to 14 kb can be subcloned into the shuttle vector pHM5, which contains a multiple cloning site flanked by I-CeuI and PI-SceI. After I-CeuI and PI-SceI-mediated release of the transgene from the shuttle vector the transgene can be inserted into the HCAdV cloning vector pAdFTC. Because of the large size of the pAdFTC plasmid and the long recognition sites of the used enzymes associated with strong DNA binding, careful handling of the cloning fragments is needed. For virus production, the HCAdV genome is released by NotI digest and transfected into a HEK293 based producer cell line stably expressing Cre recombinase. To provide all adenoviral genes for adenovirus amplification, co-infection with a helper virus containing a packing signal flanked by loxP sites is required. Pre-amplification of the vector is performed in producer cells grown on surfaces and large-scale amplification of the vector is conducted in spinner flasks with producer cells grown in suspension. For virus purification, two ultracentrifugation steps based on cesium chloride gradients are performed followed by dialysis. Here tips, tricks, and shortcuts developed over the past years working with this HCAdV vector system are presented.

A protocol for generation of high-capacity adenoviral vectors lacking all viral coding sequences is presented. Cloning of transgenes contained in the vector genome is based on homing endonucleases. Virus amplification in producer cells grown as adherent cells and in suspension relies on a helper virus providing viral genes in trans.

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high ...

Bioengineering

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

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. ...

Subconjunctival Administration of Adeno-associated Virus Vectors in Small Animal Models

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ease of accessibility via multiple administration routes. Subconjunctival (SCJ) injections offer advantages over other intraocular administration routes as they are simple, safe, and usually performed in an outpatient setting. SCJ injections in small animals usually require the assistance of an operating microscope due to the size of the eye. Previous work has demonstrated that SCJ injection of specific adeno-associated virus (AAV) serotypes is a valid gene delivery strategy for targeted transduction of the ocular surface, eye muscle, cornea, and optic nerve, providing a potential approach for the treatment of many ocular diseases.
Herein, a detailed protocol is presented for SCJ injections in a mouse model using an injection system consisting of a programmable infusion/withdrawal syringe pump (which allows for consistent and precise injection speed and pressure) and a gastight removable syringe coupled with microinjection needles. The injection system is also adaptable for other intraocular administration routes such as intrastromal, intracameral, intravitreal, and subretinal injections in small animals. Although the delivery of adeno-associated viral vectors for ocular gene therapy studies is described, the protocol herein can also be adapted for a variety of ophthalmic solutions in small animal models. The key practical steps in the administration route, setup for the injection platform, preparation of the injection, and tips from direct experience will be discussed in detail. In addition, common validation techniques for AAV delivery confirmation to the desired tissues will also be briefly discussed.

In this manuscript, subconjunctival injection is demonstrated as a valid vector delivery method for ocular tissues in mice using an injection system consisting of an infusion/withdrawal syringe pump and a gastight removable syringe coupled with microinjection needles. This injection system is also adaptable for other intraocular administration routes.

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ...

Medicine

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, ...

Process Development for the Production and Purification of Adeno-Associated Virus (AAV)2 Vector using Baculovirus-Insect Cell Culture System

Adeno-associated viruses (AAV) are promising vectors for gene therapy applications. Here, the AAV2 vector is produced by co-culture of Spodoptera frugiperda (Sf9) cells with Sf9 cells infected with baculovirus (BV)-AAV2-GFP (or therapeutic gene) and BV-AAV2-rep-cap in serum-free suspension culture. Cells are cultured in a flask in an orbital shaker or Wave bioreactor. To release the AAV particles, producer cells are lysed in buffer containing detergent, which is subsequently clarified by low-speed centrifugation and filtration. AAV particles are purified from the cell lysate using AVB Sepharose column chromatography, which binds AAV particles. Bound particles are washed with PBS to remove contaminants and eluted from the resin using sodium citrate buffer at pH 3.0. The acidic eluate is neutralized with alkaline Tris-HCl buffer (pH 8.0), diluted with phosphate-buffered saline (PBS), and further concentrated with tangential flow filtration (TFF). The protocol describes small-scale pre-clinical vector production compatible with scale-up to large-scale clinical-grade AAV manufacturing for human gene therapy applications.

Process Development for the Production and Purification of Adeno-Associated Virus AAV2 Vector using Baculovirus-Insect Cell Culture System

In this protocol, AAV2 vector is produced by co-culturing Spodoptera frugiperda (Sf9) insect cells with baculovirus (BV)-AAV2-green fluorescent protein (GFP) or therapeutic gene and BV-AAV2-rep-cap infected Sf9 cells in suspension culture. AAV particles are released from the cells using detergent, clarified, purified by affinity column chromatography, and concentrated by tangential flow filtration.