REPORTER CELL TECHNOLOGY

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Xactagen has developed technology that makes reporter cells a powerful tool for understanding gene expression in biological research and drug discovery.

We accomplish this by inserting the reporter into endogenous genes.  This is important because our reporter cells retain complete gene regulation including normal promoters, enhancers, suppressors, and DNA methylation.  We have further engineered the mRNA to retain the normal 3’ and 5’ un-translated regions of the gene, so that mRNA stability and direct responses to siRNAs and micro-RNAs may be retained.  Finally, we have engineered the vectors for the exceptional sensitivity required for high throughput screening (HTS).  Our reporter cells include known genes, predicted genes, and previously unidentified gene transcripts.

We provide reporter cells, libraries of reporter cells, assay reagents, and protocols for research use, as well as contract research and custom cell production for our customers.

 

I.  Overview of Xactagen Technology

A.  Xactagen Gene Trapping Technology:  A proprietary gene trap vector is inserted into an endogenous gene to retain normal transcriptional regulation.  The integrated vector encodes a fusion mRNA comprised of 5’ gene sequences, vector sequences, and 3’ gene sequences.  An internal ribosome entry site is used to translate the fusion mRNA into luciferase (reporter) and Neo (selectable marker).  Elements of the vector have been optimized for reporter expression, which far exceeds other reporter cell capabilities.  Vectors have also been engineered for cell production capabilities.  Tens of thousands of reporter cells can be produced.  Because the mRNA for reporter expression retains 5’ and 3’ exon sequences (including 5’ and 3’ UTRs), they report mRNA stability, such as that observed for micro-RNAs.

Xactagen reporters currently utilize Gaussia luciferase.  Xactagen has engineered Gaussia luciferase for ease of use.  Luciferase is monitored either by addition of coelenterazine (substrate) which emits light or by binding fluorescent antibodies directed against Gaussia luciferase (in progress).

Case Study of an Early Library in HCT116 Cells

(not commercially available)

Trapped Genes

Known or reseq genes	6,000
Predicted genes	7,000
Unknown transcripts	12,000
Total:	25,000

 

Wide Representations

Integrations were found on each chromosome.  811/862 (94%) of chromosomal loci (bands) represented.

 

 

B.  Xactagen Gaussia Luciferase Assay:  Xactagen has engineered Gaussia luciferase for ease of use, including HTS.  Half-life of the luciferase is ~ 5 hours so that effects of compounds, siRNAs, and other cellular stimulants on suppression of gene expression may be monitored.  Enhancers of gene expression may also be monitored.

Luciferase activity is monitored by addition of coelenterazine (substrate) prepared in Xactagen Assay Buffer.  For flash luciferase assays, Xactagen has prepared Assay Buffer that provides more activity (relative to background) and more consistent data with lower CV values.

With Xactagen’s Gaussia luciferase, there is no need to wash cells or change medium on cells to perform screening assays.  Simply add compounds, siRNAs, miRNAs or biologics (e.g. growth factors, cytokines, antibodies) and read luminescence.  The simplicity of the assay makes it ideal for both High Throughput Screening (HTS) and for basic research in academic laboratories.

              

C. Xactagen Reporter Cell Sensitivity:  Xactagen has prepared numerous reporter cell vectors.  The following data was attained with an earlier reporter cell vector.  The study was also performed using commercially available substrates that are inferior to those now used by Xactagen (see section B).  Similar studies with the current vector system and substrates are ongoing.

Xactagen’s reporter cell vector was introduced into HCT116 colon cancer cells.  A panel of randomly selected G418 resistant cells was cloned into a 96-well plate.  Gaussia luciferase activity was measured with a SpectraMax L plate reader (Molecular Devices).  Data is represented relative to luciferase activity in parental HCT116 cells (lacking Gaussia luciferase).  Data was sorted from lowest expressing clones to highest expressing clones.

 Eighty eight out of the ninety-six clones had expression levels greater than 5 fold over background.  Median activity was 30-fold over background.  The highest level observed was 1,263 fold over background.

II.  Utilization of Xactagen Technology

 A. Whole Genome Drug Discovery:  Expression of many genes (biomarkers) has been associated with human disease by DNA arrays and other genomics technologies.  Accordingly, compounds that affect key regulatory genes for the disease-associated genes represent potential drug candidates.  Xactagen reporter cells offer an efficient means to identify these potential drug candidates.  Screening disease-relevant reporter cells with libraries of “drug like” compounds identifies drug candidates that target regulatory genes for these disease-associated genes.  Targets for drug intervention include G-protein coupled receptors (GPCRs), growth factors and receptors, angiogenic factors, receptor and non-receptor kinases, protein and lipid phosphatases, small guanine nucleotide binding proteins, lipid metabolizing enzymes, cell cycle proteins, cytoskeletal proteins, DNA repair enzymes, proteins involved in DNA methylation, transcription factors, and many other potential targets.

 

Example of Drug Discovery:  Xactagen reporter cells for insulin response substrate 1 (IRS1) were plated into 384-well cell culture plates (white wall and bottom).  Cells were incubated compounds from a chemical diversity library (20uM) for ~36 hours.  Gaussia luciferase activity (flash) was determined using a SpectraMax L luminescence plate reader (Molecular Devices).  Data is plotted relative to control cells not receiving compound.   For another example of an early drug discovery study (using prior Xactagen vectors that utilized b-galactosidase reporter) please see our poster presented in 2007 (AACR meeting presentation).

                

B.  Discovery of Regulatory Networks and Drug Targets:  DNA arrays and other genomics technologies have associated expression of specific sets of genes with various cellular stimulants (e.g., growth factors, cytokines, or drug products).  DNA arrays have also associated abnormal expression of many genes with human disease.  The regulatory networks for these genes are often unknown.  Screening Xactagen reporter cells with libraries of siRNAs identifies these regulatory genes and pathways.  Should the reporter cell correspond to a disease-associated gene, identified regulatory genes represent potential drug targets.  Included are G-protein coupled receptors (GPCRs), growth factors and receptors, angiogenic factors, receptor and non-receptor kinases, protein and lipid phosphatases, small guanine nucleotide binding proteins, lipid metabolizing enzymes, cell cycle proteins, cytoskeletal proteins, DNA repair enzymes, proteins involved in DNA methylation, transcription factors, and many other possible targets.  For an example of early drug discovery study (using prior Xactagen vectors that utilized b-galactosidase reporter) please see our poster presented in 2007 (AACR meeting presentation).

C. Biomarker Studies for Discovery of Personalized Medicines:  DNA arrays and other genomics technologies have associated abnormal expression of many biomarkers with human disease.  Key to future drug discovery is the ability to utilize biomarker data to identify patients most likely (and least likely) to respond to a drug candidate.  Xactagen reporter cells offer an efficient means to associate biomarkers with drug candidate efficacy.  First, reporter cells responsive to drug candidates are identified.  Genes responsive to the drug candidate are typically identified by DNA arrays or may be identified using Xactagen reporter cells and whole genome drug discovery.  Second, responsive reporter cells are screened with the drug candidate alone, a siRNA library alone, or a combination of the drug candidate and a siRNA library.  SiRNAs that accentuate the activity of the drug candidate disclose genes, pathways, and biomarkers that potentiate the activity of the drug candidate (e.g., as shown above).  SiRNAs that abrogate activity of the drug candidate disclose genes, pathways, and biomarkers that suppress the activity of the drug candidate.  Implications for restricting patient enrollment in clinical trials and reducing timelines and costs for clinical trials while enhancing probabilities for success are evident.

D. SiRNA and miRNA Drug Discovery:  Small interfering RNAs (siRNAs) and micro-RNAs (miRNAs) represent a new class of potential drug products that share sequence identity (target sequences) with mRNAs for disease-relevant genes.  Xactagen reporter cells provide highly sensitive and gene-specific assays for:

1.  Identifying the most active siRNA/miRNA target sequences and

2.  Monitoring siRNA/miRNA delivery in vitro and in vivo.

Example of Control siRNA Suppression of Reporter Activity:  Xactagen reporter cells for IRS1 (moderately expressed gene) and protocadherin 7 (relatively low expressing gene) were plated into 384-well cell culture plates (white wall and bottom; 1,500 cell per well) containing siRNA directed against Gaussia luciferase (e.g., reverse transfection).  Cells were incubated for 24 hours.  Gaussia luciferase activity (flash) was determined using a SpectraMax L luminescence plate reader (Molecular Devices). 

E.  In vivo Imaging with Gaussia luciferase in mice:

See Molecular Therapy (2005) 11, 435–443; doi: 10.1016/j.ymthe.2004.10.016.  Codon-optimized Gaussia Luciferase cDNA for Mammalian Gene Expression in Culture and in Vivo, Tannous, B.A. et. al., http://www.nature.com/mt/journal/v11/n3/full/mt200555a.htmlMonitoring Activation of Transcription Factors, DNA Methylation, siRNA, and miRNA Activity

Note:  This did not use Xactagen cells, but our cell libraries are currently being produced in tumor cell lines used as a standard in xenograft studies.

III.  Production

Xactagen’s vectors have not only been designed for superior signal to noise, but also contain elements designed to enhance production capabilities.  Production is scalable.  Thousands to tens of thousands of reporter cells may be produced.

The sensitivity of Xactagen’s vector system is reflected in our ability to capture up to 4-5 time more genes than standard vectors.  We estimate that from 70-80  percent of known human genes may be attained for human cell lines.  This observation was at first surprising and suggests that most genes in the human genome are expressed in cancer cell lines and that our vectors possess the sensitivity to detect them.

The following is an example of gene trapping.  The ability of Xactagen vectors to generate reporter cells was compared to the ability of a standard gene trap vector.

Interpretation:

·        Only a small fraction of standard vectors that insert into genomic DNA are capable of producing a gene expression reporter cell.

·        In contrast, Xactagen’s early vectors could trap ~ 2 fold more genes while Xactagen’s current vectors trap 4- to 5-fold more genes.

Our production capacity is currently scalable.  Tens of thousands of reporter cells corresponding to an identified gene/transcription unit may now be produced.  Each is represented as an individual clone in the Xactagen reporter cell library.

For examples of a gene expression reporter cell library, please see section A. 

IV.  Products and Services:

·        Off-the-shelf reporter cells.

·        Custom-produced libraries of reporter cells.

·        Gaussia luciferase assay kits.

·        Contract research:  Drug discovery (small molecule inhibitors).

·        Contract research:  Drug discovery (siRNAs).

·        Contract research:  Personalized medicine.

·        Contract research:  Discovery of regulatory genes, pathways, and potential drug targets.

Copyright Xactagen 2008