Isogenic human disease models - AbsoluteAstronomy.com

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 ("within glass"; or, commonly, "in the lab", in an artificial environment). They are provided with a genetically matched ‘normal cell’ to provide an isogenic system to research disease biology and novel therapeutic agents. They can be used to model any disease with a genetic foundation. Cancer is one such disease for which isogenic human disease models have been widely used.

Historical models

Human isogenic disease models have been likened to ‘patients in a test-tube’, since they incorporate the latest research into human genetic diseases and do so without the difficulties and limitations involved in using non-human models.

Historically, cells obtained from animals, typically mice, have been used to model cancer related pathways. However, there are obvious limitations inherent in using animals for modelling genetically determined diseases in humans. Despite a large proportion of genetic conservation between humans and mice, there are significant differences between the biology of mice and humans that are important to cancer research. For example, major differences in telomere

Telomere

A telomere is a region of repetitive DNA sequences at the end of a chromosome, which protects the end of the chromosome from deterioration or from fusion with neighboring chromosomes. Its name is derived from the Greek nouns telos "end" and merοs "part"...

regulation enable murine cells to bypass the requirement for telomerase

Telomerase

Telomerase is an enzyme that adds DNA sequence repeats to the 3' end of DNA strands in the telomere regions, which are found at the ends of eukaryotic chromosomes. This region of repeated nucleotide called telomeres contains non-coding DNA material and prevents constant loss of important DNA from...

upregulation, which is a rate-limiting step in human cancer formation. As another example, certain ligand-receptor interactions are incompatible between mice and humans. Additionally, experiments have demonstrated important and significant differences in the ability to transform cells, compared with cells of murine origin. For these reasons, it remains essential to develop models of cancer that employ human cells.

Targeting vectors

Isogenic cell lines are created via a process called homologous gene-targeting. Targeting vectors that utilize homologous recombination are the tools or techniques that are used to knock-in or knock-out the desired disease causing mutation or SNP (single nucleotide polymorphism

Single nucleotide polymorphism

A single-nucleotide polymorphism is a DNA sequence variation occurring when a single nucleotide — A, T, C or G — in the genome differs between members of a biological species or paired chromosomes in an individual...

) to be studied. Although disease mutations can be harvested directly from cancer patients, these cells usually contain many background mutations in addition to the specific mutation of interest, and a matched normal cell line is typically not obtained. Subsequently, targeting vectors are used to ‘knock-in

Gene knockin

In molecular cloning and biology, a Knock-in refers to a genetic engineering method that involves the insertion of a protein coding cDNA sequence at a particular locus in an organism's chromosome. Typically, this is done in mice since the technology for this process is more refined, and because...

’ or ‘knock out

Gene knockout

A gene knockout is a genetic technique in which one of an organism's genes is made inoperative . Also known as knockout organisms or simply knockouts, they are used in learning about a gene that has been sequenced, but which has an unknown or incompletely known function...

’ gene mutations enabling a switch in both directions; from a normal to cancer genotype; or vice versa; in characterized human cancer cell lines such as HCT116 or Nalm6.

There are several gene targeting technologies used to engineer the desired mutation, the most prevalent of which are briefly described, including key advantages and limitations, in the summary table below.

Technique	Gene Knock-In	Gene Knock-out
rAAV (recombinant adeno-associated virus vectors)	The correct gene-regulation mechanisms; and Accurately reflect the disease events found in real patients. rAAV can introduce subtle point mutations, SNPs as well as small insertions with high efficiency. Moreover, many peer reviewed studies have shown that rAAV does not introduce any confounding off target genomic events. Appears to be the preferred method being adopted in academia, Biotech and Pharma on a precision versus time versus cost basis.>	Generate a heterozygous KO Generate a bi-allelic knockout by targeting the second allele. This process can therefore generate 3 genotypes (+/+; -/+ and -/-); enabling therefore the analysis of haplo-insufficient gene function. Current limitation is the need to sequentially target single alleles making generation of knock-out cell lines a two-step process.>
Plasmid based homologous recombination	Insertion is at the endogenous locus and has all the above benefits, but it is very inefficient. It also requires a promoterless drug selection strategy entailing bespoke construct generation. A large historical bank of cell-lines has been generated using this method which has been displaced by other methods since the mid 1990s.	Deletion is at endogenous locus and has all the above benefits, but it is inefficient. It also requires a promoterless drug selection strategy that entails bespoke construct generation
Flip-in	This is an efficient technique that allows the directed insertion of ‘ectopic’ transgenes at a single pre-defined genomic locus (integration via a FLP recombinase FLP-FRT Recombination In genetics, FLP-FRT recombination is a site-directed recombination technology used to manipulate an organism's DNA under controlled conditions in vivo. It is analogous to Cre-Lox recombination... site). This is not a technique for modifying an endogenous locus. Transgenes will usually be under the control of an exogenous promoter, or a partially defined promoter-unit in the incorrect genomic location. Their expression will therefore not be under the same genomic and epigenetic regulation as the endogenous loci, which limits the utility of these systems for studying gene-function. They are however, good for eliciting rapid and stable exogenous gene expression.	Not applicable
Zinc-Finger Nucleases Zinc finger nuclease Zinc-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... (ZFNs)	ZFNs have been reported to achieve high rates of genetic knock-outs within a target endogenous gene. If ZFNs are co-delivered with a transgene construct homologous to the target gene, genetic knock-in's or insertions can also be achieved. One potential drawback is that any off-target double strand breaks could lead to random off-target gene insertions, deletions and wider genomic instability; confounding the resulting genotype. However, 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	ZFNs are sequence-directed endonucleases which enable the rapid and highly efficient (up to 90% in a bulk cell population) disruption of both alleles of a target gene, although user- defined or patient relevant loss of-function alterations have not been reported at similar frequencies. Off target deletions or insertions elsewhere in the genome are a significant concern. The speed advantage of obtaining a biallelic KO in one step is also partially mitigated if one still needs to derive a clonal cell-line to study gene function in a homogenous cell-population.
Meganucleases	Meganucleases are operationally analogous to ZFN's. There are limitations inherent in their use such as the meganuclease vector design which can take up to 9 months and cost tens of thousands of dollars. This makes meganucleases more attractive in high-value applications such as gene therapy, agrobiotechnology and engineering of bioproducer lines.	\|

Homologous recombination in cancer cell disease models

Homologous recombination (HR) is a kind of genetic recombination in which genetic sequences are exchanged between two similar segments of DNA. HR plays a major role in eukaryotic cell division, promoting genetic diversity through the exchange between corresponding segments of DNA to create new, and potentially beneficial combinations of genes.

HR performs a second vital role in DNA repair, enabling the repair of double-strand breaks in DNA which is a common occurrence during a cell's lifecycle. It is this process which is artificially triggered by the above technologies, and bootstrapped in order to engender ‘knock-ins’ or ‘knockouts’ in specific genes5, 7.

A recent key advance was discovered using AAV-homologous recombination vectors, which increases the low natural rates of HR in differentiated human cells when combined with gene-targeting vectors-sequences.

Commercialization

Factors leading to the recent commercialization of isogenic human cancer cell disease models for the pharmaceutical industry and research laboratories are twofold.

Firstly, successful patenting of enhanced targeting vector technology has provided a basis for commercialization of the cell-models which eventuate from the application of these technologies.

Secondly, the trend of relatively low success rates in pharmaceutical RnD and the enormous costs have created a real need for new research tools that illicit how patient sub-groups will respond positively or be resistant to targeted cancer therapeutics based upon their individual genetic profile.

There are several companies working to address this need, a list of the key players and their technology offering is provided below.

News

http://web.mit.edu/piyush/www/diseasemodels.pdf
http://www.genengnews.com/gen-news-highlights/gsk-to-use-horizon-discovery-s-cell-lines-for-cancer-related-metabolomics-research/78565157/
http://www.genomeweb.com/biotechtransferweek/horizon-discoverys-umb-cell-line-deal-latest-example-its-academic-collaboration-
http://www.genomeweb.com/dxpgx/tgen-horizon-discovery-set-pgx-pact
http://www.tgen.org/news/index.cfm?pageid=57&newsid=1764 TD2
http://www.businessweekly.co.uk/life-sciences-archive/horizon-hooks-up-with-genentech.html
http://www.horizondiscovery.com/uploads/horizon-downloads/horizon-xman-genesis-faqs.pdf /
http://www.cellectis.com/genome-engineering/meganucleases/engineered-meganucleases/meganuclease-technologies/
http://www.sigmaaldrich.com/life-science/zinc-finger-nuclease-technology/custom-zfn.html
http://tools.invitrogen.com/content.cfm?pageid=3375

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