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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 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
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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.
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. For additional purification, the vector mixture can be separated by FPLC/HPLC to produce ultra-high purity vectors.
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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 HBSS 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.