Viral Vector Production

Genome Editing Core provides facilities and training for affordable high-titer viral vector preparation for research use; we cater to customers engaged in both in vitro and in vivo genome editing projects. Please, do not hesitate to contact us for consulting vector and project design. Our main focus is on training scientists to prepare their viral vectors, but we can also help in virus production.

BSL2 laboratory for working with replication-incompetent viruses

Our facility is a restricted access area in the Biocity building at the Turku Bioscience Centre. In the laboratory, the users have access to general cell culture equipment in an environment with biosafety level 2 standards. Before conducting to work, users are introduced to safe working practices and get acquinted with the instrumentation.

Lentiviruses are a group of retroviruses that includes such viruses as human (HIV), simian (SIV), and feline (FIV) immunodeficiency viruses. These viruses are characterised by their long incubation period (lente, Latin for “slow”) and a single-stranded RNA genome packaged inside an enveloped protein capsid. Replication-deficient lentiviral vectors are often derived from the HIV virus and particles are produced by an in trans complementation system. This system consists of required packaging-related components, envelope proteins, and transgenes of interest divided into three or more plasmids to prevent the generation of replication-competent viruses through recombination. When simultaneously transfected into suitable production cells, these plasmids produce packaged viral particles that are released into the culture medium from which they can be harvested. What really sets these vectors apart from preceding retroviral vectors is their ability to integrate the transgene into the genomes of both dividing and non-dividing cells. Additionally, the transduction efficiency is usually very high, especially when working with smaller inserts. The maximum packaging capacity is ca. 8-9 kb, though insertions of up to 11 kb have been reported. Lentiviral particles are often the vector of choice for in vivo use due to their low immunogenicity, and wide tropism through pseudotyping.

Production

Vector preparations are available as either non-concentrated culture medium, highly concentrated ultracentrifuged stock suspended in culture medium (DMEM) or double ultracentrifuged stocks suspended in any buffer of choice. Our lentiviral vectors are often pseudotyped with vesicular stomatitis virus G-protein (VSVG) to achieve high titres. Particles with this envelope proteins are also more stable during ultracentrifugation and cryopreservation. Titration of preparations and testing for replication competent virus is possible by a qPCR-based test, or ELISA (provided by the University of Helsinki) depending on the vectors used and your preference.

Most (non-lenti) retroviral vectors were derived from two gammaretroviruses, Moloney murine leukaemia virus (MMLV) and murine stem cell virus (MSCV). They are large, enveloped viruses whose genome is composed of two identical single-strand RNA molecules. Similar to lentivectors, retroviral particles are produced by transfecting producer cells with three or more plasmids which contain components required for replication and integration, capsid proteins and transgenes of interest. These components are separated into different plasmids to prevent the recombination of these units into a self-replicating virus. Unlike lentiviral vectors, retroviruses can only transduce actively dividing cells due to the large size of their integrase complex. Though the insert is transduced into host cells relatively efficiently, and the maximum packaging capacity is similar to lentiviruses (ca. 8-9 kb). Retroviral vectors can also be slightly more immunogenic than lentiviruses but are still widely used both in vitro and in vivo.

Production

Vector preparations are available as either non-concentrated filtered culture medium, highly concentrated ultracentrifuged stock suspended in culture medium (DMEM) or double ultracentrifuged stocks suspended in any buffer of choice. Please, consult Genome Editing Core staff if your project requires changes to standard procedures (e.g. a specific envelope protein or suspension buffer) for your project so that we can optimise production parameters accordingly.

Adeno-associated virus (AAV) is a small (20-25 nm in diameter) non-enveloped single-strand DNA virus in the family parvoviridae. They were discovered as a contaminant in adenovirus preparations and it soon became clear that these viruses could only complete their infection cycle in the presence of simultaneous adenovirus or herpesvirus infection: over the course of its evolution, this virus had prioritised small size over having a fully functional replication machinery of its own. AAV genome remains in host cells as a circular episome and has a small chance to integrate into host genome in a site-dependent manner, though this rarely occurs when using recombinant AAV vectors. Therefore they are considered more of a transient method of transduction as the episomes are diluted out of actively dividing cell populations over time. These viruses are known for their low immunogenicity and thus have been used extensively in in vivo models. Host and cell specificity are highly modifiable by using various capsids isolated from different serotypes.

Production

We are currently attempting to optimise the production of various AAV serotypes at our facility. Ask us whether your project can be a part of this endeavor! We offer AAV preparations as non-concentrated, filtered lysate supernatant or purified stocks concentrated by ultracentrifugation. Concentration by affinity chromatography may be available for certain serotypes.

Quick reference

	Lentivirus	Retrovirus	AAV
Insert packaging capacity	8 Kb	8 Kb	4.5 Kb
Virion diameter	80-130 nm	80-130 nm	18-26 nm
Expression	Transient or stable	Stable	Transient (or stable)
Infects dividing cells	Yes	Yes	Yes
Infects non-dividing cells	Yes	No	Yes
Integrates into cell genome	Yes or no	Yes	No (WT yes)
Immunogenicity	Low	Moderate	Very low
Average titre	High	Moderate	High
Transduction efficiency	High	Moderate	High