Missing Genes Point to Possible Drug Targets

Human knockout projectEvery person’s genetic blueprint, or genome, is unique because of variations that occasionally occur in our DNA sequences. Most of those are passed on to us from our parents. But not all variations are inherited—each of us carries 60 to 100 “new mutations” that happened for the first time in us. Some of those variations can knock out the function of a gene in ways that lead to disease or other serious health problems, particularly in people unlucky enough to have two malfunctioning copies of the same gene. Recently, scientists have begun to identify rare individuals who have loss-of-function variations that actually seem to improve their health—extraordinary discoveries that may help us understand how genes work as well as yield promising new drug targets that may benefit everyone.

In a study published in the journal Nature, a team partially funded by NIH sequenced all 18,000 protein-coding genes in more than 10,500 adults living in Pakistan [1]. After finding that more than 17 percent of the participants had at least one gene completely “knocked out,” researchers could set about analyzing what consequences—good, bad, or neutral—those loss-of-function variations had on their health and well-being.

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Find and Replace: DNA Editing Tool Shows Gene Therapy Promise

Neutrophil being edited with CRISPR/Cas9

Caption: This image represents an infection-fighting cell called a neutrophil. In this artist’s rendering,  the cell’s DNA is being “edited” to help restore its ability to fight bacterial invaders.
Credit: NIAID, NIH

For gene therapy research, the perennial challenge has been devising a reliable way to insert safely a working copy of a gene into relevant cells that can take over for a faulty one. But with the recent discovery of powerful gene editing tools, the landscape of opportunity is starting to change. Instead of threading the needle through the cell membrane with a bulky gene, researchers are starting to design ways to apply these tools in the nucleus—to edit out the disease-causing error in a gene and allow it to work correctly.

While the research is just getting under way, progress is already being made for a rare inherited immunodeficiency called chronic granulomatous disease (CGD). As published recently in Science Translational Medicine, a team of NIH researchers has shown with the help of the latest CRISPR/Cas9 gene-editing tools, they can correct a mutation in human blood-forming adult stem cells that triggers a common form of CGD. What’s more, they can do it without introducing any new and potentially disease-causing errors to the surrounding DNA sequence [1].

When those edited human cells were transplanted into mice, the cells correctly took up residence in the bone marrow and began producing fully functional white blood cells. The corrected cells persisted in the animal’s bone marrow and bloodstream for up to five months, providing proof of principle that this lifelong genetic condition and others like it could one day be cured without the risks and limitations of our current treatments.

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Creative Minds: The Worm Tissue-ome Teaches Developmental Biology for Us All

C. elegans

Caption: An adult Caenorhabditis elegans, 5 days
Credit: Coleen Murphy, Princeton University, Princeton, NJ

In the nearly 40 years since Nobel Prize-winning scientist Sydney Brenner proposed using a tiny, transparent soil worm called Caenorhabditis elegans as a model organism for biomedical research, C. elegans has become one of the most-studied organisms on the planet. Researchers have determined that C. elegans has exactly 959 cells, 302 of which are neurons. They have sequenced and annotated its genome, developed an impressive array of tools to study its DNA, and characterized the development of many of its tissues.

But what researchers still don’t know is exactly how all of these parts work together to coordinate this little worm’s response to changes in nutrition, environment, health status, and even the aging process. To learn more, 2015 NIH Director’s Pioneer Award winner Coleen Murphy of Princeton University, Princeton, NJ, has set out to analyze which genes are active, or transcribed, in each of the major tissues of adult C. elegans, building the framework for what’s been dubbed the C. elegans “tissue-ome.”

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Creative Minds: Building the RNA Toolbox


Caption: Genetically identical mice. The Agouti gene is active in the yellow mouse and inactive in the brown mouse.
Credit: Dana Dolinoy, University of Michigan, Ann Arbor, and Randy Jirtle, Duke University, Durham, NC

Step inside the lab of Dana Dolinoy at the University of Michigan, Ann Arbor, and you’re sure to hear conversations that include the rather strange word “agouti” (uh-goo-tee). In this context, it’s a name given to a strain of laboratory mice that arose decades ago from a random mutation in the Agouti gene, which is normally expressed only transiently in hair follicles. The mutation causes the gene to be turned on, or expressed, continuously in all cell types, producing mice that are yellow, obese, and unusually prone to developing diabetes and cancer. As it turns out, these mutant mice and the gene they have pointed to are more valuable than ever today because they offer Dolinoy and other researchers an excellent model for studying the rapidly emerging field of epigenomics.

The genome of the mouse, just as for the human, is the complete DNA instruction book; it contains the coding information for building the proteins that carry out a variety of functions in a cell. But modifications to the DNA determine its function, and these are collectively referred to as the epigenome. The epigenome is made up of chemical tags and proteins that can attach to the DNA and direct such actions as turning genes on or off, thereby controlling the production of proteins in particular cells. These tags have different patterns in each cell type, helping to explain, for example, why a kidney and a skin cell can behave so differently when they share the same DNA.

Some types of genes, including Agouti, are particularly vulnerable to epigenomic effects. In fact, Dolinoy has discovered that exposing normal, wild-type (brown) mice to certain chemicals and dietary factors during pregnancy can switch on the Agouti gene in their developing offspring, turning their coats yellow and their health poor. Dolinoy says these experiments raise much larger questions: If researchers discover populations of humans that have been exposed to lifestyle or environmental factors that modify their epigenomes in ways that may possibly contribute to risk for certain diseases, can the modification be passed on to their children and grandchildren (referred to as transgenerational epigenetic inheritance, a controversial topic)? If so, how can we develop the high-precision tools needed to better understand and perhaps even reduce such risks? The University of Michigan researcher received a 2015 NIH Director’s Transformative Research Award to undertake that challenge.

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Of Mice and Men: Study Pinpoints Genes Essential for Life

Many people probably think of mice as unwanted household pests. But over more than a century, mice have proven to be incredibly valuable in medical research. One of many examples is how studies in mice are now helping researchers understand how mammalian genomes work, including the human genome. Scientists have spent decades inactivating, or “knocking out,” individual genes in laboratory mice to learn which tissues or organs are affected when a specific gene is out of order, providing valuable clues about its function.

More than a decade ago, NIH initiated a project called KOMP—the Knockout Mouse Project [1]. The goal was to use homologous recombination (exchange of similar or identical DNA) in embryonic stem cells from a standard mouse strain to knock out all of the mouse protein-coding genes. That work has led to wide availability of such cell lines to investigators with interest in specific genes, saving time and money. But it’s one thing to have a cell line with the gene knocked out, it’s even more interesting (and challenging) to determine the phenotype, or observable characteristics, of each knockout. To speed up that process in a scientifically rigorous and systematic manner, NIH and other research funding agencies teamed to launch an international research consortium to turn those embryonic stem cells into mice, and ultimately to catalogue the functions of the roughly 20,000 genes that mice and humans share. The consortium has just released an analysis of the phenotypes of the first 1,751 new lines of unique “knockout mice” with much more to come in the months ahead. This initial work confirms that about a third of all protein-coding genes are essential for live birth, helping to fill in a major gap in our understanding of the genome.

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