Recombinant AAV mediated genome engineering
Encyclopedia
Recombinant adeno-associated virus (rAAV) based genome engineering is a genome editing platform centered around the use of rAAV vectors that enables insertion, deletion or substitiution of DNA sequences into the genomes of live mammalian cells. The technique builds on Capecchi and Smithies’ Nobel Prize winning discovery that homologous recombination
(HR), a natural DNA repair mechanism, can be harnessed to perform precise genome alterations in mice. rAAV mediated genome 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.
mediated genome editing methods.
. These are precisely matched pairs of cell lines, where one harbours a disease-associated mutation in an endogenous gene, just as it occurs in real patients, while the other is a genetically identical cell line carrying a normal version of that gene.
Derived from this disease model panel, the company is also launching a range of genetically-defined DNA reference materials. These reagents represent several improvements over existing controls, including the ability to titrate mixtures of mutant and normal gDNA to accurately test assay sensitivity, as well as the way in which they represent the isogenic nature of patient tumour samples. Further work is underway by Horizon to apply rAAV genome editing in other translational areas, such as enhancing the yield of therapeutic biologic agents from ‘bioproducer’ mammalian cell lines by knocking-out the key apoptic genes responsible and/or disrupting the function of genes or catalytic binding domains responsible for the secretion of inactivating binding proteins.
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. There are a number of clinical trials currently in progress.
rAAV vs ZFNs
ZFN off-target effects are a potential drawback for functional genomics, although for rapid bi-allelic KOs, especially if there are more than 2-alleles or several genes that need to be performed in parallel, ZFNs could be preferable. For disease modeling, random integration of donor plasmid is a concern, although no measurable increase in the rate of random plasmid integration was observed in human cells efficiently edited with ZFNs that target a composite 24 bp recognition site. dsDNA breaks are also repaired more often by NHEJ than HR and ZFN's will commonly cut both alleles, making the modeling of patient relevant SNPs/mutations difficult. More typically ZFN knock-ins involve targeting the AAVS1 on Chromosome 19 site with high specificity by creating a double strand break. The cell then uses the pZDonor plasmid, with homology arms to the AAVS1 genomic region, in homology-directed repair to replace the natural AAVS1 locus. The knocked-in gene remains under the control of the endogenous AAVS1 promoter a fact that means the patient context of the inserted gene or mutation is not that observed in a real patient. For diagnostics, clones derived by ZFN treatment could also be less isogenic due to off-target effects. For reporter cell lines the presence of off-targets is less concerning as they may not overtly affect relevance of reporter function. For stem cells the only 2 reports of KI's using ZFNs having been performed in ES cells (1 in human, 1 in mouse), and absolute targeting frequencies are 1-2% in mouse ES cells (unselected) and 10-90% in human ES or IPs cells . rAAV vectors have been used in ES cell targeting on numerous occasions.
rAAV vs Homing Endonucleases
Natural homing endonucleases can be highly accurate, although the specificity of engineering homing endonuclease has not been well characterized. The potential accuracy of engineered homing endonuclease mediated knock-out comes at the price of extensive lead-time for vector design and high associated financial cost. This further limits the appropriateness of homing endonucleases for fundamental academic research into functional genomics. The potential accuracy of homing endonuclease mediated gene-knockout makes it a prime method for engineering bioproducer plant cells. Plant cell genome reparation does not involve homologous recombination which limits the applicability of rAAV to this field. However, the lower relative cost and precision nature of rAAV makes it a prime method for optimizing bioproducer mammalian cell lines.
Homologous recombination
Homologous recombination is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. It is most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks...
(HR), a natural DNA repair mechanism, can be harnessed to perform precise genome alterations in mice. rAAV mediated genome improves the efficiency of this technique to permit genome engineering
Genome engineering
Genome engineering refers to the strategies and techniques developed in recent years for the targeted, specific modification of the genetic information – or genome – of living organisms....
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
Isogenic human disease models
Isogenic human disease models are a family of cells that are selected or engineered to accurately model the genetics of a specific patient population, in vitro . They are provided with a genetically matched ‘normal cell’ to provide an isogenic system to research disease biology and novel...
. 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 kilobase 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 endonucleaseHoming endonuclease
The homing endonucleases are a type of restriction enzymes typically encoded by introns or inteins. They act on the cellular DNA of the cells that synthesize them, in the opposite alleles of the genes that encode them.- Origin and mechanism :...
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. 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.Applications
To date, the use of rAAV mediated genome engineering has been published in over 160 peer reviewed scientific journals. Scientists at the English biotechnology company, Horizon Discovery, have employed the method to generate genetically defined isogenic human disease modelsIsogenic human disease models
Isogenic human disease models are a family of cells that are selected or engineered to accurately model the genetics of a specific patient population, in vitro . They are provided with a genetically matched ‘normal cell’ to provide an isogenic system to research disease biology and novel...
. These are precisely matched pairs of cell lines, where one harbours a disease-associated mutation in an endogenous gene, just as it occurs in real patients, while the other is a genetically identical cell line carrying a normal version of that gene.
Derived from this disease model panel, the company is also launching a range of genetically-defined DNA reference materials. These reagents represent several improvements over existing controls, including the ability to titrate mixtures of mutant and normal gDNA to accurately test assay sensitivity, as well as the way in which they represent the isogenic nature of patient tumour samples. Further work is underway by Horizon to apply rAAV genome editing in other translational areas, such as enhancing the yield of therapeutic biologic agents from ‘bioproducer’ mammalian cell lines by knocking-out the key apoptic genes responsible and/or disrupting the function of genes or catalytic binding domains responsible for the secretion of inactivating binding proteins.
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. There are a number of clinical trials currently in progress.
Comparison to other genome engineering techniques
rAAV based genome engineering differs from other techniques in several respects:- Zinc finger nucleases are a class of restriction enzymes which can be used to engineer gene knock-outs by causing double strand DNA breaks. ZFNs are extremely efficient, but there are concerns about the accuracy of ZFNs and their tendency to result in off-target effects
- Homing endonucleases are analogous to the functioning of ZFNs yet with a much higher degree of specificity to the DNA targets to which they are designed.
rAAV vs ZFNs
ZFN off-target effects are a potential drawback for functional genomics, although for rapid bi-allelic KOs, especially if there are more than 2-alleles or several genes that need to be performed in parallel, ZFNs could be preferable. For disease modeling, random integration of donor plasmid is a concern, although no measurable increase in the rate of random plasmid integration was observed in human cells efficiently edited with ZFNs that target a composite 24 bp recognition site. dsDNA breaks are also repaired more often by NHEJ than HR and ZFN's will commonly cut both alleles, making the modeling of patient relevant SNPs/mutations difficult. More typically ZFN knock-ins involve targeting the AAVS1 on Chromosome 19 site with high specificity by creating a double strand break. The cell then uses the pZDonor plasmid, with homology arms to the AAVS1 genomic region, in homology-directed repair to replace the natural AAVS1 locus. The knocked-in gene remains under the control of the endogenous AAVS1 promoter a fact that means the patient context of the inserted gene or mutation is not that observed in a real patient. For diagnostics, clones derived by ZFN treatment could also be less isogenic due to off-target effects. For reporter cell lines the presence of off-targets is less concerning as they may not overtly affect relevance of reporter function. For stem cells the only 2 reports of KI's using ZFNs having been performed in ES cells (1 in human, 1 in mouse), and absolute targeting frequencies are 1-2% in mouse ES cells (unselected) and 10-90% in human ES or IPs cells . rAAV vectors have been used in ES cell targeting on numerous occasions.
rAAV vs Homing Endonucleases
Natural homing endonucleases can be highly accurate, although the specificity of engineering homing endonuclease has not been well characterized. The potential accuracy of engineered homing endonuclease mediated knock-out comes at the price of extensive lead-time for vector design and high associated financial cost. This further limits the appropriateness of homing endonucleases for fundamental academic research into functional genomics. The potential accuracy of homing endonuclease mediated gene-knockout makes it a prime method for engineering bioproducer plant cells. Plant cell genome reparation does not involve homologous recombination which limits the applicability of rAAV to this field. However, the lower relative cost and precision nature of rAAV makes it a prime method for optimizing bioproducer mammalian cell lines.
See also
- Biological engineeringBiological engineeringBiological engineering, biotechnological engineering or bioengineering is the application of concepts and methods of biology to solve problems in life sciences, using engineering's own analytical and synthetic methodologies and also its traditional...
- Genome engineeringGenome engineeringGenome engineering refers to the strategies and techniques developed in recent years for the targeted, specific modification of the genetic information – or genome – of living organisms....
- Homing endonucleaseHoming endonucleaseThe homing endonucleases are a type of restriction enzymes typically encoded by introns or inteins. They act on the cellular DNA of the cells that synthesize them, in the opposite alleles of the genes that encode them.- Origin and mechanism :...
- Homologous recombinationHomologous recombinationHomologous recombination is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. It is most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks...
- Meganuclease
- Zinc finger nucleaseZinc finger nucleaseZinc-finger nucleases are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target desired DNA sequences and this enables zinc-finger nucleases to target unique sequences within complex genomes...
- Isogenic human disease modelsIsogenic human disease modelsIsogenic human disease models are a family of cells that are selected or engineered to accurately model the genetics of a specific patient population, in vitro . They are provided with a genetically matched ‘normal cell’ to provide an isogenic system to research disease biology and novel...
Sources
- 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