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Applications of rAAV

Adeno-Associate Virus (AAVs) are an effective tool for the delivery of genetic payloads for discovery science experiments through applied Gene and Cell Therapy (GCT). Importantly, AAV infection does not trigger a severe immune response in mammalian hosts, and therefore offers a much safer alternative to other virus-based genetic payload delivery systems such as adenovirus and lentivirus. The efficacy and safty profile of AAVs has led to their widespread application as a both a research tool and as a tool for the delivery of clinical therapeutics.

rAAV as Research Tool

In vivo Over-expression

The first experimental use of rAAV delivered a neomycin-resistance gene into mammalian cells, thus demonstrating that AAV can be used to drive exogenous protein expression in mammalian cells. Since early proof of concept work rAAV have continuously been used as a gene transfer tool for biological research.

Over-expressing a gene of interest in mammalian cells via rAAV has a several unique advantages over alternative viral systems. For example, rAAV infection does not result in a significant host immune response that is associated with alternative virus based genetic payload delivery systems including adenovirus and lentivirus. rAAV can achieve also long-term expression without genome integration in vivo, whereas the use of lentiviruses may raise concerns associated with genome integration. Furthermore, rAAV serotypes exhibit varying degrees of tissue tropism that provide a mechanism for tissue specific infection that cannot be achieved using a lentiviral approach. This tissue tropism not only provides a mean of specificity, but also allows AAV to transfer target genes into almost all animal tissues with considerations of different serotypes and promoter-specificity.

Under normal conditions the over expression of a transgene by rAAV occurs through a multistage process in which (1) single-stranded the AAV vector genome enters the host cell nucleus, (2) the AAV genome is converted to a double stranded circular episome via second strand synthesis, and (3) episomes are converted to high molecular weight concatemers that provide an substrate for long-term expression of the transgene. It was found that synthesis of a second strand is the rate limiting step in this process, and in an effort to bypass this step McCarty and colleagues developed the double-stranded (self-complementary) AAV (dsAAV). dsAAV house a modified ITR that cannot be cut by the Rep protein, and thus the self-complementary single stranded DNA can form the double-stranded DNA [1]. Based on this advancement, we generated “K” serial ssAAV entry vectors with 14 kinds of promoters for targeting universal or specific organs or cell types, and “KD” serial dsAAV (scAAV) with 12 kinds of promoter to drive customized genes.

In vivo Knock-Out

rAAVs have recently been paired with,with CRISPR technology to drive in vivo gene knock-outs. Zhang and his colleague injected rAAV8 carrying liver-specific promoter-driven SaCas9 targeting PCSK9 gene into C57BL/6 mice and achieved 50% knock-out efficiency and 95% decline in serum Pcsk9, while three other groups successfully used rAAV9 to deliver SpCas9/SaCas9 system to skip up to 40% of the negligible mutated exon 23 of DMD mouse. Since these early studies, AAV-based CRISPR knockout strategies have become well adopted, and have been successfully employed in large number of studies. PackGene has designed and developed several AAV vectors to support AAV-based CRISPR knockout strategies. PackGene’s A/B/C/D/F/G/H vector lines drive expression of one 7 CRISPR/Cas and variants including: SpCas9, SpCas9HF, SaCas9, SaCas9HF, NmCas9, AsCpf1 and LbCpf1. PackGene Entry vectors are equipped with optimized gRNA scaffold and mammalian or human-codon-optimized CRISPR/Cas.

In vivo Activation

rAAV can also be used to drive gene activation with CRISPR based technologies. The CRISPR system has be modified to act as a gene-activation system in two ways. First, a cutting deficient mutant of Cas9 called dCas9 can be used in place of Cas9. dCas9 is capable of binding to the host genome by the same binding mechanism as Cas9, but is not capable of cutting the DNA and instead remains bound. dCas9 can be fused with a transcription activator, like VP64, or coupled with a modified sgRNA that forms sgRNA loops capable of recruiting transcriptions factors such as MS2-P65-HSF1. Recruitment of transcription elements can thereby drive transcription of a target gene. Alternatively, short gRNAs that prevent uncoupling of Cas9/dCas9 from cutting or uncoupling from the host DNA after binding the target sequence. Modified MS2-binding sgRNA loops then recruit the MS2-P65-HSF1 activation component, which will make it possible to apply wild-type Cas9 for simultaneous activation and knock-out by delivering both a short and a standard length gRNA that are targeted at different genes. PackGene offers our E series SpCas9-specific activation vector for gene activation including when paired with our A series SpCas9 vector or a SpCas9 mouse. In addition, vector lines for SaCas9 gene activation are coming soon and will provide more promoter choices for both SaCas9 and MS2-P65-HSF1.

In vivo Knock-Down

RNA interference (RNAi) is a highly specific tool for protein knockdown in which via small non-coding RNA bind to target a proteins mRNA to drive mRNA degradation and therefore reduce protein translation. RNAi mechanisms are endogenously present in most eukaryotes, and have been widely adopted as a molecular tool for gene function studies, drug discovery, and gene silencing therapy. rAAV can be used to drive the expression of RNAi, and PackGene’s M series of vectors serve as an exceptional resource for driving RNAi expression. Our M series of vector are designed to express short-hairpin RNAi under the H1 or U6 promoters whole simultaneously expressing an EGFP, mCherry, or Firefly-Luciferase reporter protein for easyt identification of transfected cells.

rAAV as Clinical Trials Vector for Gene Therapy

Flotte et al. from the University of Florida were the first to use rAAV in clinical trials to correct cystic fibrosis, a genetic disease [2]. Loss of the cystic fibrosis gene coding for a chloride ion channel causes chronic lung infection, and emphysema, and reduces life span. Because of the relatively small capacity of rAAV, they counted on the weak promoter activity of ITR and put a promoterless CFTR gene into rAAV. The vectors were used to transduce nasal or lung airway epithelial cells. This first trial demonstrated the safety of the vector and showed varied transduction efficacy depending on the target tissues.

The first rAAV clinical success came from several groups that used rAAV-rpe65 to cure Leber congenital amaurosis (LCA) caused by homozygous recessive rpe65 deficiency [3]. After the rAAV-RPE65 was injected subretinally into one eye of each patient, significant recovery of vision was observed in some of them.

Currently, world-widely more than 200 clinical trials are completed or ongoing.
For a complete list of rAAV clinical trials, please click the below weblink


1. Douglas McCarty. Molecular Therapy 16, 1648-1656 (2008)
2. Terence R Flotte, et al., Human gene therapy 7 (9), 1145-1159 (1996)
3. WW Hauswirth et al., Human gene therapy 19 (10), 979-990 (2008)