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Sinai Health | Magazine

The New Power Tools of Medical Research

Peek inside the toolkit of researchers at the LTRI to learn how today’s most exciting biomedical technologies — organoids, optogenetics, CRISPR — are powering research

3D art by Julia Seo Artist Julia Seo used layers of card stock and Korean hanji paper to create a 3-D representation of an intestinal organoid used in Dr. Jeff Wrana’s lab to study the progression of cancer.

By Patricia Hluchy

In the wake of game-changing developments — such as the Human Genome Project, which identified and mapped more than 20,000 individual genes in humans, and next-generation DNA sequencing, technology which dramatically improved the efficiency and lowered the cost of genome mapping — the field of biomedical research has undergone staggering growth over the past two decades.

Scientists at the Lunenfeld-Tanebaum Research Institute (LTRI), part of Sinai Health System, are leveraging a revolution in biomedical technologies that is yielding an ever more intricate understanding of how the human body, and its diseases, work. Three of the latest technologies that are transforming research are organoids, optogenetics and CRISPR — if you haven’t heard of them yet, you certainly will in the months and years to come.

Here, we talk with four world-class LTRI researchers who are using these hugely exciting innovations to answer crucial questions including: What happens when cancer starts and grows? How do brain disorders develop? And how can we regenerate tissues or develop drugs tailored to a specific patient’s disease?

ORGANOIDS: MINI ORGANS IN A DISH

multi color organoid Image 1 - An intestinal organoid from the lab of Dr. Jeff Wrana. Dr. Wrana’s team uses organoids like these to study how organs regenerate after injury and how colorectal cancer initiates and grows.

Organoids are cells grown from human or mouse organ tissues to create simplified, three-dimensional mini organs that possess some of the same structures and functions as their real counterparts. The Scientist Magazine named organoids one of the biggest scientific advances of 2013, and since then the field has snowballed rapidly. Organoids help researchers understand tissue development and study disease directly by using a patient’s own tissues, which allows for the possibility of personalized medicine.

Dr. Jeff Wrana, CIBC Breast Cancer Research Scientist and the Mary Janigan Research Chair in Molecular Cancer Therapeutics at the LTRI, aims to expose the communication networks that control how these complex structures form and how disturbances cause disease. He creates organoids to understand the progression of cancer and how to regenerate tissue.

TELL US ABOUT YOUR WORK WITH ORGANOIDS.

Dr. Wrana: With sequencing of the human genome, you basically went from a sailing ship trying to find new land to a satellite that showed you the entire Earth. And then the challenge became, OK, what do all the genes do and how do they all interact with each other and make a human, which led to the discovery of stem cells. And what goes wrong with cells globally and not just on a single-gene level when people get a disease? We started to study how proteins, which are the functional product of our genome, communicate between cells and instruct them on how to become a liver cell or a kidney cell. This work provided a toolkit that has driven an explosive area of biomedical research, where we can take stem cells and get them to make different kinds of organoids.

Watch our cover image come to life

WHICH BIG QUESTIONS ARE YOU USING ORGANOIDS TO EXPLORE?

Dr. Wrana: One is how cancer initiates. The second is, how do tissues regenerate? The intestine has a tremendous turnover capacity: We turn over our intestinal lining every two to three weeks. The kidney has a limited regenerative capacity. So we’re interested in trying to compare and contrast those two tissues. We’re applying some remarkable new technologies that allow us to profile, basically, all individual cells in a tissue, and this gives us remarkable insight into how organs respond to injury.

And because the organoid is in a dish, it’s much easier to do those experiments than if you were trying to do it in the context of a whole animal, like a mouse. And what we discovered, actually, is taking some cells out of a tissue and then making them grow for a couple of weeks in a dish — that’s basically regeneration.

Also, you can grow an organoid in a dish and knock out a gene in the dish and see what happens in terms of tumour initiation and regenerative capacity. What are the signals that are controlling these processes, and how can we exploit them to identify potential targets for new therapies?

In our recent work, we discovered a new cell type that arises when we damage the intestine, and it seems to be critical to repair damaged intestine. Now we’re really interested in whether this cell type is also present in tissues with limited regenerative capacity, like the kidney, and whether we might be able to activate it to improve repair.

HOW MIGHT ORGANOIDS ONE DAY IMPROVE PATIENT CARE?

Dr. Wrana: One example of what we’re working on is glioblastoma, the devastating tumour that Gord Downie, lead singer of The Tragically Hip, recently died from. So now we’re taking human cerebral organoids — mini brains — and growing glioblastomas in them so we can understand how the glioblastoma and the normal human brain tissue are interacting with each other. This could be a really important model to screen for new therapeutics.

ARE THERE ETHICAL CONCERNS AROUND THE USE OF ORGANOIDS?

Dr. Wrana: With cerebral organoids, there is considerable discussion with respect to ethics. At what point does brain tissue become truly human brain-like? At what point is there consciousness, or pain? But it’s important to remember that at this point in the technology, these are really tiny pieces of tissue in a dish — they’re only a millimetre in diameter and have nowhere near the complexity of even a mouse brain, let alone a human brain.

WHAT’S MOST EXCITING FOR YOU ABOUT THIS RESEARCH?

Dr. Wrana: The discovery of a new cell type critical for regeneration. I gave you the analogy of going up into a satellite. Now it’s like being above the solar system, looking at this exquisite global view of tissues but also seeing it at the single-cell level. It’s blowing my mind.

OPTOGENETICS: CONTROLLING BRAIN MOLECULES WITH LIGHT

blue and green brain slice Image 2 - A microscope image from Dr. Kenichi Okamoto’s lab of a brain slice showing expression of a light-activated protein in green. The expression of this protein was targeted to the dentate gyrus because this area of the brain is crucial for memory formation.

The most complicated structure in the known universe, as it’s often described, the human brain is one of science’s final frontiers. Researchers still have limited understanding of the workings of its tens of billions of cells — called neurons — and the ways they communicate with each other through synapses, or of its diseases and disorders. Historically, scientists stimulated the brain with electrodes to study its activity, but that proved to be a crude tool. Now, with optogenetics, everything has changed. The technology makes targeted neurons light-sensitive and then activates those cells with light. And it has ushered in a whole new era of brain study.

An Investigator at the LTRI, Dr. Kenichi Okamoto has propelled optogenetics to the microscopic level, using infrared lasers to trigger target molecules critical for brain functions such as learning and memory. He hopes this work will lead to greater insight into a variety of neurological disorders.

TELL US ABOUT HOW YOU ARE USING OPTOGENETICS TO UNDERSTAND MEMORY.

Dr. Okamoto: With optogenetics, we’re able to directly study memory function in mice at the level of neurons and synapses, which are critical for memory. In particular, we target signalling pathways, which are molecules in synapses that act as messengers between neurons. These signalling pathways are hot drug-discovery targets for brain disease.

Optogenetics allows us to control the function of a target molecule with light, which means you can immediately turn critical molecules in the brain on or off, much as you would with a light switch.

To do this, we inject live mice with a nontoxic virus that generates a light-sensitive molecule in the target neurons. Then we insert two optical fibres with LED lights at the tip into each hemisphere of the mouse’s brain. We do some mouse learning tests to see what happens when you break or activate signalling pathways for a specific time in the target neurons of the brain, to find out how these pathways are critical for memory. We use a unique-to-us custom microscope equipped with infrared laser light to trigger target molecules in synapses in deep regions of the brain.

WHICH BIG RESEARCH QUESTIONS IS OPTOGENETICS HELPING YOU EXPLORE?<,/p>

Dr. Okamoto: Using our unique optogenetics approaches, we are trying to address the molecular mechanisms that encode learning and memory in the brains of mice and, by extension, humans. We revealed the inter-synaptic functions of these signalling molecules for the first time and are currently studying how these mechanisms function for learning and memory compared to other systems, such as computers. We hope to find out what role these molecules might have in Alzheimer’s disease, autism and schizophrenia.

HOW WILL OPTOGENETICS INFLUENCE CARE FOR BRAIN DISEASES AND DISORDERS IN THE FUTURE?

Dr. Okamoto: Drugs that activate the signalling pathway that we are studying do exist, and they are now being used to treat heart failure. It is a critical pathway for brain function too, but the drugs don’t work well in the brain. So we are hoping to use optogenetics as a new therapeutic tool because it enables us to precisely control the timing and duration of the signaling effect. With optogenetics, we will be able to maintain the effects for a very long time compared to the more traditional chemical, pharmaceutical approach.

ARE YOU WORKING ON ANYTHING ELSE IN THE REALM OF LEARNING AND MEMORY?

Dr. Okamoto: We are also trying to establish sensors to monitor target molecules in the living brains of freely moving animals. In combination with a tiny micro-camera system, we will be able to watch the effect of optogenetics to precisely determine the effective light strength or duration of the photoactivation for learning, memory and related therapeutic functions.

CRISPR: “ESSENTIALLY A PAIR OF SCISSORS”

cell division Image 3 - Image of a human cell in culture in the process of cell division. Dr. Daniel Durocher and his team routinely use tissue culture cell in CRISPR experiments to study processes that influence cancer.

CRISPR is a powerful gene-editing tool that can change the genome in any species, including mice and humans. Science magazine’s 2015 Breakthrough of the Year, CRISPR — an acronym for the process whereby bacteria use proteins to break down the DNA of attacking viruses — allows scientists to quickly, cheaply and very precisely target and induce mutations in any of our 20,000 genes. Which means it holds the promise of treating an array of health conditions and diseases caused by genetic mutations.

Dr. Daniel Durocher, Thomas Kierans Research Chair in Mechanisms of Cancer Development and Assistant Director of the LTRI and the Canada Research Chair in Molecular Genetics of the DNA Damage Response, studies how cells detect and repair damage to their DNA. He aims to understand how normal cells become cancerous.

Dr. Daniel Schramek, Kierans/Janigan Cancer Research Scientist at the LTRI, focuses on the mutations and genes responsible for the development of cancerous tumours and their progression through metastasis. He has developed a new technique that uses CRISPR to weed out the random genetic bystander mutations and identify the ones critical for cancer.

HOW ARE YOU USING CRISPR IN YOUR RESEARCH?

Dr. Durocher: My team is trying to disrupt the function of each gene in the human genome, one by one, and ask, “If we remove this gene, are we impacting the response to DNA damage, DNA repair and DNA damage signaling events?” We use CRISPR to do this in a cell culture dish, and we use next-generation sequencing technologies. CRISPR is essentially a pair of molecular scissors that allows you to cut DNA at will. And we’ve been able to design a strategy to improve CRISPR.

Dr. Schramek: What we’re doing is similar to what Dr. Durocher is doing, but we do it in mice rather than in cell culture. While we are limited in the number of genes we can interrogate in a mouse, the animal gives you more of a physiologic context compared to a cell culture dish. When a tumour grows in an organism such as a mouse or a human, you’ve got 20 to 30 different cell types, including immune cells, blood cells, lymph cells. So when we start to model tumours in vivo, the advantage is that we are looking at the interactions of all these cell types together.

mouse Image 4 - This photo from Dr. Daniel Schramek’s lab shows a mouse infected with CRISPR viruses. The coloured patches represent clones where specific genes have been destroyed. This allows Dr. Schramek’s team to identify which genes may play a critical role in cancer development.

When a patient comes to the clinic with a tumour, and the tumour is excised and sequenced to see which mutations it harbours, it turns out that a normal patient’s tumour carries about 200 gene mutations. But it’s not clear which handful of these 200 mutations are actually the driver genes of this tumour and which are just passenger mutations which randomly accumulate as the tumour grows. We can take these 200 genes and destroy them one by one in an organ of a mouse and see which of them give rise to a tumour. CRISPR makes it much, much faster and cheaper to do this.,/p>

Now, our team has built a system where we take a pregnant mouse and inject the embryonic sac with a virus that carries the components of the CRISPR machinery. Then we reinsert the embryo, and when the baby is born it now carries these mutated genes and the mouse develops a tumour. We can do this for about 500 genes in a single mouse.

WHICH BIG SCIENTIFIC QUESTIONS ARE YOU USING CRISPR TO EXPLORE?

Dr. Durocher: I’m trying to understand how cells protect their genetic information, which is essentially written on the DNA — how cells deal with broken DNA, how they repair it and how they tell the cell that there’s broken DNA. In oncology this is very important, first because you can boil down cancer to a disease of DNA that has been broken and mutated and where there have been errors in putting it back together. And second, it’s relevant because a lot of cancer therapies work by damaging the DNA.,/p>

We ask a simple but important question: Which genes and gene products — proteins — are involved in repairing and signalling DNA damage? We will only understand how to defeat cancer when we understand how cells — normal cells or cancer cells — take care of their DNA because cancer is caused by changes in the makeup of the DNA. In our research we have discovered the function of proteins that are key to the efficient repair of DNA damage.

Dr. Schramek: My lab is looking at which genes in breast, brain and head and neck cancers are actually responsible for these tumours developing and metastasizing. We know that no two cases of, say, head and neck cancer have the same mutations. But we’ve discovered in 60 per cent of instances of head and neck cancers the mutations affect the same pathway, which is a sort of communication network within cells.

HOW DO YOU THINK CRISPR WILL IMPROVE PATIENT CARE?

Dr. Durocher: As we uncover more and more knowledge about the making of cells and how cancer exploits this biology, there’s the possibility of new cancer treatments tailored to the exact genetic makeup of a particular tumour. Two years ago I co-founded a company, Repare Therapeutics, that is based on that premise. We think we will have products ready for trials in 2020.

WHAT ARE THE ETHICAL IMPLICATIONS OF THIS TECHNOLOGY?

Dr. Durocher: This kind of precision will require essentially reading the genetic makeup — the DNA — of individuals. So, it will be costly, which raises the issue of accessibility to such treatment. Then there are considerations related to the privacy of genetic information used for tailoring a treatment. I certainly wouldn’t want my genetic information to get into the hands of anyone, like marketers.

Dr. Schramek: Since we are applying the technology in mouse models rather than cell cultures, the ethics are more complex. The truth is that without animal research, we will simply not have any new cures; we wouldn’t be able to test a new drug or therapeutic strategy before going into humans. But equally important is that we conduct this research according to the highest ethical standards, which means things like minimizing the number of animals we use and avoiding any pain or suffering to them.

Also, and I think everybody in Canada and the U.S. is very clear on this, CRISPR should not become a tool for creating designer babies, which scientifically we do not know how to do — yet.

ANYTHING ELSE YOU WANT OUR READERS TO KNOW ABOUT CRISPR?

Dr. Durocher: I read a futuristic science fiction book called Seveneves last summer. They were doing gene editing in the novel, but we’re already way past the pretty primitive way the book described it. This field is moving so fast that science fiction has problems keeping up.

Dr. Schramek: Well, CRISPR technology actually comes from milk industry research into yogurt, which is a reminder that most scientific discovery comes from the unexpected.


Image credits: Image 1 Dr. Masahiro Narimatsu, Image 2 Jelena Borovac, Image 3 Dr. Nicole Hustedt, Image 4 Ellen Langille

Sinai Health Magazine

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