Cells are the building blocks of life. Within all creatures and plants, collectively called "organisms", there are large numbers of cells that work together to allow the organism to develop and function. In humans, it is thought that there may be as many as 100 trillion (100,000,000,000,000) cells. This implies that cells must be small. In fact, about 60,000 human cells can fit on the head of a pin.
To perform the complex functions of the organism, some cells must take on specialized roles. In mammals, for example, liver cells perform different roles than brain cells, and heart cells perform different roles than skin cells. But, even within tissues, there are specialized cells. For example, in human blood, there are red cells, T cells, B cells, plasma cells, and so on. All of these cells have important roles in normal development and health. The overall control of how cells become specialized as organisms develop is not well understood, but this control must in general function reliably to avoid disease and ensure the ability to reproduce.
In general, all of the cells within sexually reproducing organisms receive one complete set of genetic instructions from each parent. This complete set of genetic instructions is called the genome, and the scientific approach to studying the genome is called genomics. The genome is composed of DNA and contains the code for genes. It is known that cells within an organism become specialized, and hence different from each other, as a consequence of turning certain genes on or off. Genes that are turned on are said to be "expressed", and this expression can be measured by looking for the presence of another molecule called RNA, which is produced from a gene when it is turned on. Disease can result if the wrong genes are expressed or have their expression turned off.
Many scientists have used the tools of genomics to discover which genes are expressed within normal and diseased tissues as a way to identify the genes that are mistakenly turned on or off in diseased tissues. Some of these studies have eventually resulted in drugs which are designed to interfere with genes when they are mistakenly turned on. Gleevec, which is used to treat leukemia, is an example of such a drug.
One critical problem that scientists face in identifying the genes whose mis-expression results in disease is the very small size of individual cells. This small size means that very little RNA is present, much less than can be studied using the current tools of genomics. Another critical problem is that within a tissue there are typically many different types of cells, only one of which may be relevant to the disease process. For example, many cancer tissues contain confusing mixtures of normal and diseased cells, and scientists studying gene expression in such cancers are most likely mis-led by this confusing mixture. In fact, discovery of the most important mis-expressed genes in such tissues is usually impossible.
Our project, which involves leading experts in genomics and engineering, proposes to solve these critical problems by developing a new approach that will allow scientists to apply the tools of genomics to many single cells at once. By studying single cells we hope to avoid the confusion that accompanies the study of complex mixtures of cells, and more accurately measure the RNA that is present within each cell. Broad availability and application of our approach will allow scientists to accurately measure RNA in single cells, which will in turn lead to a dramatically improved ability to identify mis-expressed genes in diseased tissues. This will eventually produce high quality gene targets for the design of new drugs. We foresee that our approach has the potential to revolutionize many areas of research in addition to health care, including examination of genomic differences in "normal" non-diseased tissues, and in studying the genomes of un-culturable microbes inhabiting interesting ecological and environmental niches.
The research undertaken has allowed continued success in responding to RFAs and writing research grant applications. Overall, since the inception of this project the researchers have been successful in garnering over $3.7 million in research funding, specifically involving single cell microfluidic technologies and genomics. These awards are listed in the leveraged funding section of this report (following pages). This funding will in turn create new research training opportunities, related IP, and potential commercial ventures within Canada, thereby providing a certain return on Genome Canada’s investment in the Towards Single Cell Genomics technology development project.
It is anticipated that translation of the single cell technologies to the scientific, biotechnology and pharmaceutical industries could have profound consequences for several fields including biomarker discovery, drug development, environmental genomics, metagenomics and personalized medicine. In particular, the Single Cell RT-qPCR device developed by the project which is a microfluidic device for performing hundreds of simultaneous single cell gene expression measurements using RT-qPCR. This technology along with future improvements to increase multiplexing will find immediate application in biomedical research and is particularly well suited to developmental biology, stem cell science, and cancer biology. In addition we anticipate that this technology may ultimately be applied to allow new modalities of diagnosis based on single cell transcription with possible applications in cancer monitoring and prognosis as well as in reproductive medicine. New protocols and bioinformatics analysis tools developed through the present work are being refined and made available through the Michael Smith Genome Science Center and will open new avenues of research within BC and Canada. The impact of this is already evidenced by recent funding applications that contain elements of single cell genetic analysis. Finally, in addition to microfluidic devices this work will lead to new assay designs that may be transferred to industry. These include methods for accurate measurements of sequence abundance by binning reads to unique molecular tags, and assay designs for the targeting of sequence regions by PCR.
In the near term the project’s interactions with Fluidigm Corporation through the in-kind contribution of the BioMark™ and the Collaborative Research Agreement between Dr. Hansen and Fluidigm Corp has had considerable impact. See Appendix 4 for an article which recently came out in Genetic Engineering and Biotechnology News highlighting Fluidigm and Dr. Hansen’s lab about the potential for gene expression profiling using single cells. We anticipate that the technologies developed here for single cell gene expression measurements by RTqPCR will be rapidly commercialized and widely adopted. The work developed in this proposal has been the basis for a newly negotiated collaborative research agreement that is aimed at further development and commercialization of technologies for single cell analysis, sample preparation for sequencing, and genetic diagnostics.
Our PCR-Based Target Enrichment technology we have developed for performing multiplexed target amplification from spotted oligos also has potential to be commercialized as a platform for sequence enrichment prior to genomic analysis. This technology would find adoption in genomics research and would be particularly well-suited to large cohort studies in which a panel of hundreds to thousands of mutations are being correlated with experimental or phenotypic observations. Ultimately we envision that this technology would find a broad market as a sample preparation tool for cancer diagnosis based on tumour sequencing.
The proposed research program has helped meet the growing need for biotechnology personnel in Canada by training highly skilled and multidisciplinary personnel. In addition to the training opportunities directly funded through this project, this work will undoubtedly generate new funding through grants and economic ventures that ultimately will attract and train a larger base of researchers and highly skilled technicians. In summary, this research has created new technologies with significant potential to improve health care and generate new economic opportunities in Canada. Through a commitment to cutting edge research and the training of highly qualified personnel Canada will be positioned to maximize the academic and economic awards that must come from taking an internationally leading role in technology and science.