Vectorology Facility
Harnessing the power of recombinant viral vectors to target the genome
About
Recombinant viral vectors harness the natural ability of viruses to transduce living cells and are powerful tools for transgene delivery. The Vectorology Facility is an innovative academic scientific core facility aimed at supporting basic and translational research in vector-based gene editing and therapy to target genetic diseases. The Vectorology Facility focuses on the packaging of lentiviral, adenoviral, and AAV vectors and offers a range of services to assist with basic and translational research.
Our Services
We enable basic and translational research in vector-based gene therapy by providing access to the latest technologies, consultation, technical support, and study design.
- Customised vector design and preparation
- AAV production and purification
- Screen kit with 51 different AAV variants
Techniques and Technologies
Viral Vector Systems
Lentiviral and adenoviral vectors have been used in research for many years, but the immense potential of adeno-associated viruses (AAVs) has recently taken the scientific community by storm.
Widespread and virtually innocuous, AAVs are naturally found in a variety of serotypes, each with its own specificity for different cell types. The AAV capsid has proven to be highly malleable, such that new and better serotypes are constantly being developed. In addition, AAVs are promising candidates for gene therapy, due to their low immunogenicity and cytotoxicity, as well as the fact that they can be delivered systemically, but act locally on cells and tissues of interest.
AAV Purification Techniques
We offer a range of purification scales to suit a variety of budgets and applications. These methods range from direct harvesting of vectors to deep purification using gradient ultracentrifugation and liquid chromatography. While so-called ‘crude preps’ are suitable for quick experiments, additional purification steps are recommended for more high-end applications requiring higher purity and vector titres. Ultracentrifugation over an iodixanol or caesium chloride (CsCl) gradient eliminates contaminating protein, broken or empty capsids and produces a cleaner and more concentrated product.
AAV Serotypes and Tropism
The tissue selectivity and transduction efficiency of AAVs varies widely between the various serotypes, and is the subject of extensive study and experimentation, both in vitro and in vivo. The vectors themselves can be highly promiscuous (e.g. AAV 8, 9 10) or highly selective (AAV 3, 4). Conversely, certain cell types and tissues, such as the central nervous system, can only be targeted by very few serotypes while others (e.g. liver cells) are accessible to many different variants. Some AAVs are best suited to in vitro applications (e.g. AAV-DJ), while others transduce much more efficiently in vivo (e.g. AAV-DJ/8, 9, 10).
The following table* summarizes the tropisms of a few common AAV serotypes based their relative expression in a given tissue or cell type.
ND = Not detected, “–” = Some detectable expression, ”+”, “++”, ”+++” = Low, Medium and High Expression
* Information adapted from Westhaus et al, HumGeneTher. 2020 Feb 26. doi: 10.1089/hum.2019.264
Barcoded AAV Vector Screening Kit
We recognize that it can sometimes be difficult to decide which AAV vector to use. With the large and constantly growing number of available vectors, it is often best to perform a screen to identify the optimal vector serotype for a particular application. To assist investigators in this endeavor, we offer “AAV Vector Screening Kits”, which contain iodixanol-purified barcoded GFP expression cassettes packaged in a variety of AAV variants. The barcode is unique for each AAV variant and can easily be detected by NGS sequencing at the DNA and RNA level. This provides a prediction of the relative efficiency of the different serotypes in a given cell type or tissue.
Currently, VGEF offers a kit containing 51 individual AAVs, each one identifiable by two unique barcodes for greater experimental confidence. The amount provided is sufficient for most in vitro studies or small-scale in vivo screens. The “AAV vector screening kit” is constantly growing as new variants become available, so please visit our website for up-to-date info.
If you are interested in utilizing such a kit, contact us for a free consultation and quote.
Don’t see what you need? Not to worry! Contact us to discuss an AAV solution that’s right for you.
Just contact us for a consultation.
Lentivirus Production
Lentiviral vectors typically yield lower titres compared to AAVs, but can be used at much lower MOIs. Lentiviral particles are pelleted through a 20% sucrose cushion. The pellet is washed and re-pelleted in PBS before finally being finally re-solubilized. Lentiviral particles are extremely sensitive to freezing and thawing, thus all vector preps are pre-aliquoted before being frozen down.
Please Read the Below Guidelines Before Submitting Plasmids
Guidelines for working with AAV plasmids/the best conditions for maintaining ITR integrity
Bacteria should not be incubated for more than 16 hours at any stage including:
- Bacterial plate streaking
- Bacterial culture for Miniprep
- Bacterial culture for Maxiprep
These steps should all be started last thing in the afternoon and removed from incubation immediately the following morning.
DNA Requirements – AAV
Plasmid provided must be purified using an Endotoxin-free plasmid DNA kit (<0.1 EU/µg).
AAV constructs require AhdI, MscI and XmaI digest (3 separate digests) to verify ITR integrity. Excessive amounts of linearized full-length plasmid indicate recombination has occurred. The Vectorlogy Facility will not be liable for a low titre if these digests are not carried out.
Restriction digest guidelines can be found on this page. Gel images of each digest must be provided electronically to the Vectorology Facility Scientific Lead, as well as plasmid map (snapgene file preferred) prior to submitting DNA.
Gel imaging guidelines can be found on this page. DNA concentration must be measured preferably using a Nanodrop system prior to sample submission. Plasmid DNA should be checked for purity and have an A260/280 ratio of >1.8.
- A minimum of 50 µg DNA per 5 plates is required for Iodixanol purification production. If multiple sets of plates (multiples of 5) are required, additional DNA will be required.
- A minimum of 500 µg DNA is required for Cesium purification production.
DNA must arrive at the Vectorology Facility by 12PM on Wednesdays if to be included in the current weeks schedule. DNA delivered after this time will be included in the following weeks production schedule.
DNA Requirements – Lentivirus
Plasmid provided must be purified using an Endotoxin-free plasmid DNA kit (<0.1 EU/µg).
Lentivirus constructs require 3 individual restriction digests that each generate 3 sufficiently separated bands. CMRI’s Vectorlogy Facility will not be liable for a low titre if these digests are not carried out.
Restriction digest guidelines can be found on this page. Gel images of each digest must be provided electronically to the Vectorology Facility Scientific Lead, as well as plasmid map (snapgene file preferred) prior to submitting DNA.
Gel imaging guidelines can be found on this page. DNA concentration must be measured preferably using a Nanodrop system prior to sample submission. Plasmid DNA should be checked for purity and have an A260/280 ratio of >1.8.
- A minimum of 30 µg DNA is required for crude prep production. If multiple sets of plates (multiples of 5) are required, additional DNA will be required.
- A minimum of 320 µg DNA is required for purified Lentivrus production
DNA must arrive at the Vectorology Facility no later than 12PM on Friday if to be included following weeks schedule (Tuesday transfection). DNA delivered after this time will be included in the following weeks production schedule.
Transfection Workflow – AAV
Transfection Workflow – Lentivirus
Restriction Digest Guidelines
For a reaction volume of 10 µl use:
- 1 µl restriction enzyme buffer,
- 0.5 µl of restriction enzyme and
- 500-750 ng of plasmid DNA
Incubate 2 hours at 37°C
Run on a 1% agarose gel at 100V for 1 hour or until bands are sufficiently separated. An uncut control should also be run on the gel next to the digested DNA.
Gel Imaging Guidelines
The following information must be provided to CMRI’s Vectorology Facility Scientific Lead prior to AAV and/or Lentivirus production:
- A gel image. Gels should be run on a 1% agarose gel at 100V for 1 hour or until bands are sufficiently separated. An uncut control should also be run on the gel next to the digested DNA.
- An image of the simulated gel (if possible).
- Table containing the expected band size for each of the restriction enzymes, as well as sequence file and plasmid map (Snapgene file preferred).
- Information on the size of the DNA ladder used on the gel.
- DNA ladder must cover the range of band sizes expected.
Trusted Expertise
Dr Betty Kao
Vectorology Facility Lead
Dr Betty Kao is a molecular biologist with over 10 years’ experience in gene therapy. Her passion for gene therapy led her to earn a PhD from the University of Melbourne investigating gene therapy for beta-thalassaemia, followed by two postdoctoral fellowships researching gene therapy for Duchenne Muscular Dystrophy at Royal Holloway University of London and University College London. Through her PhD and postdoctoral fellowships, Betty gained expertise in design, production, quality testing and transduction of retrovirus, lentivirus and AAV in both in vitro and in vivo models. Betty joined the Vectorology Facility at Children’s Medical Research Institute in December 2021 and is responsible for viral vector production, the day-to-day operation of the facility, supervision of the team, as well as advising researchers on the most appropriate gene delivery vectors for their specific needs. Please reach out for questions, consultation, or to discuss using the facility to support your project design and implementation.