Adeno-Associated Virus (AAV) is one of the safest gene-delivering vectors for mammals as it requires helper viruses to propagate and generates relatively mild innate and adaptive immune responses compared to an adenovirus, which may cause a serious immune response. Recombinant AAV (rAAV) currently used in research has a clinical-safety profile since it contains AAV packaging signal sequences (Inverted terminal repeats, ITR) at both ends of the sequence of interest in addition to the absence of genes coding AAV proteins Rep and Cap. It forms an episomal concatemer in infected cells, which is almost free of integration into the host genome. In 1984, the first rAAV vector was proved to be capable of delivering foreign genes into mammalian cells. Since then, the rAAV has been used as a gene transfer vehicle in biological research and later clinical gene therapy.
rAAV as Research Tool
In vivo Over-expression
rAAV was first used to deliver a neomycin resistant gene into mammalian cells. Afterward, rAAV has been used in research as a gene transfer tool. Over-expression of a target gene in mammalian cells via rAAV has a unique advantage. For example, rAAV can obtain long-term expression without genome-integration in vivo, whereas lentivirus has limited tissue tropism and raises the concern of genome-integration, though it can express genes long term. Adenoviruses are transient expression vectors but it often causes severe immune response, which may affect or hide the correct phenotype of the transgene. In contrast, rAAV can transfer target genes into almost all tissues in animals depending on different serotypes and promoter-specificity. After the realization that synthesis of complemented second-strand of AAV is the bottle-neck for in vivo transduction efficiency, McCarty and colleagues invented a double-stranded (self-complementary) AAV, which contains a modified ITR unable to be cut by Rep protein; in turn the self-complementary single stranded DNA can form the double-stranded DNA [ 1 ]. 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
rAAV, in conjunction with CRISPR, has been recently used for in vivo gene knock-outs in several cases. 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. Three groups successfully used rAAV9 delivering SpCas9/SaCas9 system to skip up to 40% of the negligible mutated exon 23 of DMD mouse, and significantly recovered muscle function to a therapeutic level. Moreover, researchers from Temple University for the first time demonstrated the in vivo eradication of HIV-1 DNA in mouse genome by rAAV9 delivering CRISPR/Cas9 and sgRNA sequence in multiple organs, including blood cells, brain, heart, kidney, lung, spleen and liver. Serial “A/B/C/D/F/G/H” of PackGene Entry vectors contain 7 types of CRISPR/Cas and variants, including SpCas9, SpCas9HF, SaCas9, SaCas9HF, NmCas9, AsCpf1 and LbCpf1 for KO. All PackGene Entry vectors are equipped with optimized gRNA scaffold and mammalian or human-codon-optimized CRISPR/Cas.
In vivo Activation
CRISPR system can be adapted into a gene-activation system in two ways: 1) The mutant cutting-deficient dCas9 binds the target sequence but does not cut-and-release. Thus it can be fused with an activating protein, like VP64, or coupled with the recruitment of activating proteins like MS2-P65-HSF1 through modified MS2-binding sgRNA loops, thereby activating nearby gene transcription. 2) Short gRNA (14-15 nt) forbids either wild-type Cas9 or dCas9 from releasing 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 short and normal length gRNAs. In our Entry vectors portfolio, “E” SpCas9-specific activation serial plus “A” SpCas9 serial or the SpCas9 mouse will exert the activation function. Many more serials for short version SaCas9 activation are coming soon, and will allow you more choices of promoter for both SaCas9 and MS2-P65-HSF1, with which you will have the maximal possibilities for your experimental design.
In vivo Knock-Down
Driven by U6 or H1 promoters, short-hairpin RNA for silencing a specific gene can be expressed in vivo via rAAV vectors. Simultaneously expressing EGFP, mCherry, or Firefly-Luciferase can easily mark transduced cells or organs. We have the “M” serial customizable entry vectors for this unique purpose.
rAAV as Clinical trials vector
Flotte et al. from University of Florida were the first to use rAAV in clinical trials to correct cystic fibrosis, a genetic disease . Loss of the cystic fibrosis gene coding for a chloride ion channel causes chronic lung infection, 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 that 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 . After the rAAV-RPE65 was injected sub-retinally into one eye of each patient, significant recovery of vision was observed in some of them.
Currently, world-widely more than 165 clinical-trials are completed or ongoing. For a complete list of rAAV clinical trials, you may go http://www.genetherapynet.com/clinical-trials.html.
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)