Utilities of AAV vectors in in vivo phenotypic screening and functional studies

KEYWORDS:

• AAV
• phenotypic screening
• target validation
• drug discovery

Target identification and validation is an essential part of drug discovery process. Large scale in vivo phenotypic screening has been pursued to accelerate therapeutic target identification and validation [1]. Such an approach requires the development of highly efficient in vivo gene delivery method. Hydrodynamic tail vein injection (HTV)​ as an in vivo gene delivery method for direct phenotypic screening in animal models for the purpose of target identification and validation has been described previously [1]. While HTV has the advantages of low cost and high throughput, it also has a number of limitations. For example, HTV usually results in a relatively short duration of expression; for about one-third of the constructs tested, target of interest (TOI) expression dropped significantly 2 weeks after injection​. Other limitations include low expression levels for some targets, liver targeting only with unknown percentage of hepatocytes receiving plasmid DNA​, and acute injury to the liver [1]. These limitations may result in a significant number of false negatives. They also make it difficult to study membrane proteins, especially when it is desired to target tissues other than liver.

In contrast, adeno-associated virus (AAV) vector-mediated gene delivery may overcome some of these limitations associated with HTV. AAV is a small single-stranded DNA virus. Vectors based on AAV have been used as gene delivery vehicle in both preclinical and clinical studies [2]. Multiple AAV serotypes have been isolated from human and nonhuman primates. To date, 13 distinct AAV serotypes have been developed that show different tissue tropisms in animal models. More selective tissue delivery could be further achieved by a combination of AAV serotypes, promoter​ selected to drive TOI expression, miRNA-mediated regulation of TOI expression [3], and route of administration. In order to utilize AAV as a large-scale in vivo phenotypic screening gene delivery method, high-throughput production of AAV is highly desirable. However, most AAV production is carried out by transient transfection into adherent human embryonic kidney (HEK) 293T cells and its scalability is limited. Recently, AAV production in scalable suspension-adapted HEK293T cells has been described [4]. Purification of AAV is also a bottleneck for scalable manufacturing. This is especially true for the AAV vectors discovered in recent years, whose ligands are either unknown or not available for affinity purification. Combinations of ion-exchange and molecular sieve columns have been employed to overcome these limitations, but they require multiple chromatography steps with buffer exchanges in between [5]. Therefore, we also developed scalable automated affinity column chromatography purification methods for several AAV serotypes [6]. The combination of AAV production in suspension-adapted HEK293T followed by automated affinity chromatography purification enabled the utility of AAV as a gene delivery method for in vivo phenotypic screening and target validation at a large scale​.

While the generation of AAV is still more resource demanding than HTV and the packing capacity of AAV vector also limits the size of the gene that can be delivered, overall, AAV vector-mediated gene delivery complements many of the limitations associated with HTV. Some of its advantages include:

1. The duration of expression is sustained (we have observed expression that lasts at least a year, unpublished observations). This not only allows the assessment of efficacy of the TOI, but also allows assessment of any potential undesirable effects of the TOI, providing valuable safety readouts of novel targets early in the drug development process. In most applications, AAV can also replace the need for transgenic animals.

2. High levels of TOI expression can be achieved, and the expression levels are more precisely titratable than with HTV by injecting different amounts of AAV particles [7].

3. The high transduction efficiency in liver and in multiple tissues, allowing greater than 95% of parenchymal cells to express TOI, expands the ability to study not only secreted proteins but also transmembrane proteins.

4. AAV-mediated sustained​ TOI expression in mouse or rat allows creation of animal models that can be used for screening of therapeutic candidates.

We believe AAV adds significant value to the previously proposed HTV-based in vivo phenotypic screening strategy [1]. Therefore, we propose the following modified screening workflow: HTV remains the primary screening approach given the advantages of low cost and high throughput; negatives from the HTV screen due to low expression or short duration of expression can be made into AAV for a second round of screening to eliminate false negatives. Furthermore, AAV can be used for evaluation of target efficacy and safety due to sustained TOI expression in mouse and rat. In addition, animals expressing​ TOI to mimic human disease can be developed for screening of therapeutic candidates. We believe this new workflow takes advantages of both approaches and significantly enhances the quality, the confidence, and the value of the in vivo phenotypic screening paradigm.

Declaration of interest

K. J. Lee and Y. Li are employees of Amgen Inc. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Additional information

Funding

This work is funded by Amgen Inc.