Applications of rAAV

Adeno-Associate Virus (AAV) is an effective tool for delivery of genetic payloads in discovery science experiments through applied Gene and Cell Therapies (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 including adenovirus and lentivirus. The efficacy and safty profile of AAVs has led to their widespread application as 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 recombinant AAV (rAAV) delivered a neomycin-resistance gene into mammalian cells, and thus demonstrated that AAV can be used to drive exogenous protein expression in mammalian cells. Since this early proof of concept work rAAVs have been continuously 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 virus based genetic payload delivery systems. For example, rAAV infection does not produce a significant host immune response whereas both adenovirus and lentivirus do. Infection with rAAV also results in long-term expression of transgenes without genome integration in vivo while 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 degree of specificity, but also allows AAV to transfer target genes into most animal tissues.

Under normal conditions the over expression of a transgene by rAAV occurs through a multistage process in which (1) the single-stranded 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 a substrate for long-term expression of transgenes. It was found that synthesis of the 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). Stable dsAAV vectors were achieved through modifications of the ITR that functionally inhibit Rep protein’s ability to cut the circular AAV dsDNA vector [1]. Based on these two available technologies we first generated our “K” series of ssAAV entry vectors with 14 different promoters available, and our “KD” series of dsAAV vectors with 12 different promoters available. Available promoters can functionally drive ubiquitous gene expression across many tissues and cell types or may promote targeted gene expression in specific tissue or cell types.

In vivo Knock-Out

Recently, rAAVs have been paired with CRISPR technology to drive in vivo gene knock-outs. Zhang and colleague injected rAAV8 carrying liver-specific promoter-driven SaCas9 and guide RNA targeting the PCSK9 gene into C57BL/6 mice to achieve 50% knock-out efficiency and 95% decline in serum Pcsk9 protein. Since these early studies, AAV-based CRISPR knockout strategies have been well adopted and 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 of 7 CRISPR/Cas and variants including: SpCas9, SpCas9HF, SaCas9, SaCas9HF, NmCas9, AsCpf1 and LbCpf1. PackGene Entry vectors are equipped with an optimized gRNA scaffold and mammalian or human-codon-optimized CRISPR/Cas genes.

In vivo Activation

Gene activation can also be driven with rAAV using 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 traditional 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, traditional Cas9 can be paired with short gRNAs that prevent both the DNA cutting and the uncoupling of Cas9 from host DNA after binding the target sequence. Modified MS2-binding sgRNA loops then recruit the MS2-P65-HSF1 activation component and drive target gene transcription. By this mechanism it is possible to use wild-type Cas9 for simultaneous activation and knock-out of different genes. This is achieved through Cas9 expression with both a short modified gRNA that drives expression of one gene and a standard length gRNA that produces knockout of an alternate gene. PackGene offers our E series SpCas9-specific activation vector for gene activation 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. This technology uses a small non-coding RNA designed to bind to a target proteins mRNA. RNAi binding in turn drives target mRNA degradation and reduces 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 therapies. 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 vectors 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 easy identification of transfected cells.

rAAV as Clinical Trials Vector for Gene Therapy

In a pioneering set of studies Flotte et al. used rAAV in clinical trials experiments to treat the genetic disease cystic fibrosis [2]. Loss of the cystic fibrosis gene coding for a chloride ion channel leads to chronic lung infections, emphysema, and reduces patient life span. In these studies rAAV, were used to transduce nasal or lung airway epithelial cells. This first trial demonstrated the safety of rAAV for use in gene and cell therapies, and provided evidence for varied transduction efficacy in target human tissues.

The first rAAV clinical successes came from several groups that used rAAV-rpe65 to treat Leber congenital amaurosis (LCA), a disease caused by homozygous recessive rpe65 deficiency that causes vision impairments [3]. Significant vision recovery was observed in a subset of patients following subretinal rAAV-RPE65 injections into one eye of each patient. These studies not only demonstrate that rAAV-based treatments may be safe for use in human patients, but may also be efficacious.

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)