MONTREAL--Procrea Biosciences, a reproductive medicine company known for offering paternity testing, genetic counseling, and infertility treatment at its clinic here, is venturing into functional genomics.
According to Patrice Hugo, vice president, research and development, Procrea intends to address the bottleneck that biotechnology and pharmaceutical companies face in identifying which gene is related to which disease.
Most current bioinformatics tools designed to find candidate genes for human diseases compare genes expressed in a diseased cell versus a healthy one to find anywhere from 500 to 5,000 differentially expressed genes. But Hugo said, “The hurdle for most companies is to determine which of these genes is the real McCoy.” What big pharmaceutical companies want is a smaller cluster of candidate genes to test as potential drugs, he added.
With a license from Montreal University for a method developed by geneticist Pierre Chartrand, Procrea is tackling these bottlenecks by taking a different approach to narrow the search.
In the early 1990s, Chartrand developed Rapid Access Mapping. RAM is based on a ten-year-old concept that states that chromosomes in a nucleus of any given cell are tightly compacted with interacting regions called chromosomal domains. Genes on chromosome 1, for example, can physically touch genes on chromosomes 3, 6, and 9 at the periphery of these domains. The organization of these domains is also thought to be highly dependent on cell and tissue type.
These domains involve genes from distant chromosomes made up of chromosomal loops that bring the genes together physically in the same space called factories. The theory behind RAM Genomics assumes first that these genes are regulated through a common mechanism such as mutual transcription factors, and second, that they have common functions in shared or complementary pathways. Hugo emphasized that the concept, which has been accepted by several people in genomics, “is cutting-edge science.”
Between 1995 and 1997 Chartrand filed for European international patent rights for RAM. Subsequently, Procrea received exclusive worldwide rights from Montreal University. Chartrand then left academia to further develop his technology at the company, where he is now director of genomics.
To identify the genes in a factory, RAM Genomics needs at least one gene that is known to be involved in a disease, for example BRCA1 for breast cancer. Indeed, this is Procrea’s first test target. “This gene is now the bait to identify all the partners of BRCA1’s factory,” Hugo said. “Requiring this first gene as a starting point is the limiting factor for RAM.”
From this gene scientists build a vector that contains sequences similar to BRCA1, with which they have transfected mammary gland tissue. The RAM probe then finds its way to the BRCA1 gene in the tissue, guided by its homologous BRCA1 sequence. The key event is that the RAM vector, following its interaction with BRCA1, does not integrate randomly into the genome.
Procrea researchers found that when a RAM vector integrates it might do so on the same chromosome as the starting gene or a different one; however it does combine preferentially with genes that are within the assumed factory of the initial targeted gene. The RAM technology, Chartrand explained, deals with far fewer numbers of genes compared to those derived from expression methods. For this reason, RAM does not require complicated bioinformatics software to discriminate patterns of interactions of genes on different chromosomes.
Using FISH analysis, they also found that a probe for the RAM vector and a probe for the initial targeted gene were situated side-by-side in the nucleus. Procrea scientists visualize these gene clusters by looking at the fluorescing patterns in the nuclei of cultured cells under specially equipped microscopes. They capture the images on computers and analyze the data with ISIS-C imaging software. However, adds Chartrand, because of the small numbers of genes in question, this analysis can also be done by eye: “From here we use almost standard karyotyping techniques to determine the exact position of the signal on which band on which chromosome.”
“Now we can fish out from the genome the flanking sequences near the RAM vector,” noted Hugo. “We just have to type in a short sequence of nucleotides--a flanking region that was harvested using the RAM vector--which then tells us, for example, we’re on chromosome 16 near a gene that’s involved in DNA repair and here is the protein it encodes.” For this part of the process Procrea must rely on public or proprietary genomic databanks.
All of the flanking genes contained in the chromosomal factories are potential drug targets. Hugo said current genomics research indicates that there are somewhere between 5 and 50 genes within a given factory. Although Hugo declined to say what genes Procrea is working with other than BRCA1, he acknowledged that the company is in the process of identifying the genes flanking BRCA1.
Procrea employs some 100 people--about half in the fertility clinic. The company also maintains a diagnostic lab that performs such services as paternity tests, forensic DNA fingerprinting, amniocentesis, and other fertility tests. About 30 are employed in R&D, including 10 PhDs, mostly with backgrounds in molecular biology, genetics, and immunology. The evolving genomics group is comprised of about 10 people. Alan Wolffe, senior vice-president and chief scientific officer of Sangamo BioSciences known for his work on the chromatin structure and its role in the regulation of gene expression, recently joined Procrea’s scientific advisory board to offer consultation to the RAM genomics group.
One of this group’s main efforts is to identify one target gene involved in endometriosis, which will be used as the bait for RAM Genomics to find other genes involved in this common condition.
It is this approach to narrowing the search for viable disease gene targets that motivated Procrea to label its technology “the next generation of functional genomics.”
--Karen Kreeger