Recombinantadeno-associated virus (rAAV) based genome engineering is a
genome editing platform centered on the use of
recombinant AAV vectors that enables insertion, deletion or substitution of
DNA sequences into the genomes of live mammalian cells. The technique builds on
Mario Capecchi and
Oliver Smithies'
Nobel Prize–winning discovery that
homologous recombination (HR), a natural hi-fidelity DNA repair mechanism, can be harnessed to perform precise genome alterations in mice. rAAV mediated genome-editing improves the efficiency of this technique to permit
genome engineering in any pre-established and differentiated human cell line, which, in contrast to mouse ES cells, have low rates of HR.
The technique has been widely adopted for use in engineering human cell lines to generate
isogenic human disease models. It has also been used to optimize bioproducer cell lines for the biomanufacturing of protein vaccines and therapeutics. In addition, due to the non-pathogenic nature of rAAV, it has emerged as a desirable vector for performing gene therapy in live patients.
rAAV Vector
The rAAV genome is built of single-stranded deoxyribonucleic acid (ssDNA), either positive- or negative-sensed, which is about 4.7 kilobases long. These single-stranded DNA viral vectors have high
transduction rates and have a unique property of stimulating endogenous HR without causing double strand DNA breaks in the genome, which is typical of other
homing endonuclease mediated genome editing methods.
Capabilities
Users can design a rAAV vector to any target genomic locus and perform both gross and subtle endogenous gene alterations in mammalian somatic cell-types. These include gene knock-outs for functional genomics, or the ‘knock-in’ of protein tag insertions to track translocation events at physiological levels in live cells. Most importantly, rAAV targets a single allele at a time and does not result in any off-target genomic alterations.[2] Because of this, it is able to routinely and accurately model genetic diseases caused by subtle
SNPs or point mutations that are increasingly the targets of novel drug discovery programs.[2]
Applications
To date, the use of rAAV mediated genome engineering has been published in over 2100 peer reviewed scientific journals.[3]
Another emerging application of rAAV based genome editing is for gene therapy in patients, due to the accuracy and lack of off-target recombination events afforded by the approach.
Bardelli A, Parsons DW, Silliman N, et al. (May 2003). "Mutational analysis of the tyrosine kinome in colorectal cancers". Science. 300 (5621): 949.
doi:
10.1126/science.1082596.
PMID12738854.
S2CID85934154.
Endogenous Expression of Oncogenic PI3K Mutation Leads to Activated PI3K Signaling and an Invasive Phenotype Poster Presented at AACR/EORTC Molecular Targets and Cancer Therapeutics, Boston, USA, Nov. 2009
Endogenous Expression of Oncogenic PI3K Mutation Leads to accumulation of anti-apoptotic proteins in mitochondria Poster Presented at AACR 2010, Washington, D.C., USA, April. 2010
The use of ‘X-MAN’ isogenic cell lines to define PI3-kinase inhibitor activity profiles Poster Presented at AACR 2010, Washington, D.C., USA, April. 2010
The use of ‘X-MAN’ mutant PI3CA increases the expression of individual tubulin isoforms and promoted resistance to anti-mitotic chemotherapy drugs Poster Presented at AACR 2010, Washington, D.C., USA, April. 2010
Kandavelou K, Chandrasegaran S (2008). "Plasmids for Gene Therapy". In Lipps, Georg (ed.). Plasmids: Current Research and Future Trends. Caister Academic Press.
ISBN978-1-904455-35-6.
Gao H, Smith J, Yang M, et al. (January 2010). "Heritable targeted mutagenesis in maize using a designed endonuclease". Plant J. 61 (1): 176–87.
doi:
10.1111/j.1365-313X.2009.04041.x.
PMID19811621.
Sussman D, Chadsey M, Fauce S, et al. (September 2004). "Isolation and characterization of new homing endonuclease specificities at individual target site positions". J. Mol. Biol. 342 (1): 31–41.
doi:
10.1016/j.jmb.2004.07.031.
PMID15313605.
Arnould S, Chames P, Perez C, et al. (January 2006). "Engineering of large numbers of highly specific homing endonucleases that induce recombination on novel DNA targets". J. Mol. Biol. 355 (3): 443–58.
doi:
10.1016/j.jmb.2005.10.065.
PMID16310802.