Tag Archives: mouse

Of Mice and Mammaries, Part 2: Breast cancer in a dish?

In light of Breast Cancer Awareness Month, Justin Varholick traces how mice have helped breast cancer research over the past century. In the second post of this 4-part series, we look at advances made from 1960 to 1975 when scientists were studying a virus in the milk.

Last week, in Part 1 of this series, we discussed how scientists from the early 1900s studied the growth and spread of mammary tumors in mice. We also walked through an experiment discovering how the breast milk from mother mice carried “something” that was more responsible for breast cancer than the genes the mother had passed down to her pups. This week we learn how scientists determined that this new “something” was a virus, and this virus could only be found in mice – it was never found in humans.

1940 to 1960 — What’s in the milk?

After Dr. John Bittner found out that there was “something” in the milk, many scientists wanted to figure out what it was. A first thought was to filter the milk and see if it still showed the same effects as Dr. Bittner’s experiments. Scientists took very special filters used to filter out bacteria, and ran mouse milk from “high tumor” mothers through these filters. Despite filtering, the results remained the same – filtered milk from “high tumor” mothers still led to tumors in pups fed the milk.

Then in the early 1950s scientists started using electron microscopes to compare “high-tumor” mouse breast milk with normal “low tumor” mouse breast milk. The electron microscopes gave scientists the power to magnify the milk 2,000,000x the normal size – magnification beyond what the bacteria filters could filter out. After comparing the types of milk, Dr. Leon Dmochowski found many small particles in the “high tumor” milk and very few of these particles in the “low tumor” milk. After other scientists repeated these experiments, they concluded that these particles were the “something” in the milk that may be responsible for tumors in the pups. Many scientists looked for these particles in human breast tissue and milk, but could never find it – only mice have this “something” in the milk. It was interesting, however, that the particles were in both “high tumor” mothers and “low tumor” mothers, albeit in different amounts, which indicated that both types of mothers were at risk to have the mammary tumors – “high tumor” mothers just had a higher risk.

While all this science with electron microscopy was going on, scientists studying mammary tumor cells in mice determined that the something in the milk acted very much like a virus and gave it the name mouse mammary tumor virus, or MMTV for short.

Cover of “Immunity against the mouse mammary tumor virus” by Paula Creemers. Electron microscope image of MMTV with a sketch of two mice with mammary tumors.

1960 to 1975 — Can we grow the milk virus in a dish?

Now that scientists had established that a virus in the milk was responsible for mammary tumors in mice, they wanted to see if they could grow the virus in a petri dish. Scientists had already grown viruses in a dish that were responsible for cancer in chickens, and other viruses that were responsible for skin cancer in humans, so they believed they would be able to grow MMTV in the dish. Unfortunately, growing the milk virus dish proved very difficult.

In the 1970s many scientists tried to grow the virus in a dish and were unsuccessful. One group of scientists at the Cancer Research Genetics Laboratory (CRGL) of the University of California, Berkeley showed that the virus could be grown in a dish, but it was too cumbersome for many scientists to use for research. Because MMTV could not be easily grown in a dish, scientists interested in mammary tumors in mice knew they had to find a new method if they wanted to continue using mice to understand more about breast cancer.

To be continued…

Tune in next week, to learn how scientists started using new methods with stem cells to make genetically engineered mice and how they validated that the mouse could be used as a model for humans!

Justin Varholick

References:

  1. Cardiff R, Kenney N. (2011). A compendium of the mouse mammary tumor biologist: From the initial observations in the house mouse to the development of genetically engineered mice. Cold Spring Harb Perspect Biol. 3(6).

 

Of Mice and Mammaries, Part 1: There’s something in the milk

In light of Breast Cancer Awareness Month, Justin Varholick traces how mice have helped breast cancer research over the past century. In the first post this 4-part series, we look at advances made from 1854 to 1940, including the understanding of the role of breast milk in causing certain types of tumors. 

Image credit: Jackson Labs

Breast cancer is one of the most serious forms of cancer facing women. Each year, over 300,000 women in the U.S. will be diagnosed with breast cancer, and it is estimated that 40,610 women will die from it in 2017 alone. Thankfully, death rates from breast cancer have dropped almost 40% from 1989 to 2015, and there are over 3.1 million breast cancer survivors living in the U.S. today.

Breast cancer grows and spreads through many stages, and can start in different parts of the breast. Some types of breast cancer cause lumps, others form no lumps. Some forms of it spread very quickly throughout the body, while others spread more slowly. Because breast cancer spreads and forms at different rates and in different areas of the breast, treating it is no easy task. It is also unlikely that we will one day have a “cure” for breast cancer — one size cannot fit all.

Despite the complicated nature of breast cancer, scientists feel a responsibility to understand it as much as possible in efforts to find new treatment methods and forms of a cure. Over the years they have made great strides in their research by studying mice. These mice serve as an essential step between early research on mammary cells and clinical trials in humans.

Over the course of this month, I will highlight some of the key findings scientists have discovered about breast cancer through their studies in mice.

1854 to 1903 — The first mouse mammary tumors

The first discovery of a mammary tumor in a mouse was in 1854. In these early days, scientists were able to find tumors spontaneously growing in female mice kept as pets and in the wild. Although they were able to detect and describe these tumors, it was difficult to understand where they came from, and how they grew and possibly spread or metastasized.

Thankfully in 1903, Dr. Carl Jensen developed a line of “high tumor” mice that readily grew mammary tumors, which could be easily transplanted to other mice. By transplanting the tumors in other mice, they could measure how and where the tumors spread, in otherwise healthy mice.

During this time in history, 1.2* women per 1,000 died from breast cancer in the U.S. Today it is around 0.13 women per 1,000. (*at this time we only had reports on the number of white women in the U.S.).

1933 to 1940 — There’s something in the milk

After bringing mice into the laboratory and thoroughly studying their biology, a great discovery was made — there was something in the milk. This discovery was made by Scientists at Jackson Laboratories, in Bar Harbor, Maine. They bred “high tumor” mice with “low tumor”* mice and found that offspring were more likely to get mammary tumors if they had a “high tumor” mother. Although some scientists were able to replicate this finding in other labs, very few were convinced there was something in the milk — they believed it was passed down through the genes. (* an extremely low number of “low tumor” mice were found with mammary tumors; because of this scientists could not call them “no tumor” mice.)

To answer whether there was either something in the genes or the milk, Dr. John J. Bittner did a more complex study 3 years later. In this key study, Bittner cross-fostered mouse pups from “high tumor” and “low tumor” mice to opposite mothers (see diagram). This method allowed him to determine whether the parent’s genes or the foster mother’s milk lead to mammary tumors. If it was the genes then “high tumor” offspring would have tumors whether they had “high tumor” or “low tumor” foster parents. If it was the milk then any offspring nursed by “high tumor” mothers would get tumors.

Through this experiment, Bittner found out that milk was a key factor. “Low tumor” pups cross-fostered to “high tumor” mothers had many mammary tumors, while “high tumor” pups cross-fostered to “low tumor” mothers had very few tumors. “High tumor” pups nursed by their own mothers, however, had the highest rates of tumor growth. It didn’t always matter who the parents were, it also mattered who nursed the pups. This verified that indeed there was something in the milk. This something was labeled as the Mouse Mammary Tumor Virus (MMTV).

Dr. Bittner was often heard stating that he only studied the milk because nobody else wanted it — they all wanted to study the genes.

To be continued…

Tune in next week to read what we learned about the milk virus, MMTV, and what we did with this new power!

Justin Varholick

References

  1. Cardiff R, Kenney N. (2011). A compendium of the mouse mammary tumor biologist: From the initial observations in the house mouse to the development of genetically engineered mice. Cold Spring Harb Perspect Biol. 3(6).
  2. Holen I, Speirs V, Morrissey B, Blyth K. (2017). In vivo models in breast cancer research: progress, challenges and future directions. Dis Model Mech. 10(4).
  3. Tarone RE, Chu KC. (1992). Implications of birth cohort patterns in interpreting trends in breast cancer rates. J Natl Cancer Inst. 84(18).

Research Roundup: Lupus protein identified, vaccine for Type 1 diabetes, new chronic pain treatment, and more!

Welcome to this week’s Research Roundup. These Friday posts aim to inform our readers about the many stories that relate to animal research each week. Do you have an animal research story we should include in next week’s Research Roundup? You can send it to us via our Facebook page or through the contact form on the website.

  • A protein that may cause Lupus has been identified.Lupus is a chronic inflammatory disease that occurs when your body’s immune system attacks its own tissues and organs. An estimated 1.5 million Americans, and at least 5 million people worldwide, have a form of lupus. Previous research has implicated the gene PRDM1 as a risk factor for lupus. Scientists looking at Blimp-1, a protein that is encoded by the PRDM1 gene, have found in mice thatthat a low level of or no Blimp-1 in a particular cell type led to an increase in the protein cathepsin S (CTSS) which caused the immune system to identify healthy cells as something to attack — particularly in females.” These results are particularly striking as women have an increased risk for lupus compared to men. While this work needs to be replicated and validated, this research provides some valuable insight into the etiology and treatment of lupus. This research was published in the journal Nature Immunology.
lupus mice

Mice from the Lupus study. Source: AJP Renal Physiology.

  • Vaccine for virus induced Type 1 diabetes successful in mice. Coxsackie B viruses are the most common enteroviruses and are believed to be associated with the development of Type 1 diabetes. Type 1 diabetes is  a common human disease defined by a decrease in the production of insulin, which is a hormone that allows blood glucose (sugar) to enter energy producing cells. Thus, without insulin your body cannot effectively produce energy. This week, a team of Finnish researchers published a preclinical evaluation of a Coxsackie B1 vaccine using mice and found that the vaccine successfully protected the mouse after administering the Coxsackie B1 virus. Pre-clinical trials in humans are the next logical step for this vaccine, and the researchers believe that this research will aid in the development of vaccines for other disease caused by enteroviruses such as; hand-foot-and-mouth disease, meningitis, and myocarditis. This research was published in the journal Vaccine.
  • High iron levels in brain linked to progression of Alzheimer’s. Alzheimer’s is a degenerative neurological disease that causes dementia in humans. Previous research has linked Alzheimer’s to the buildup of amyloid protein in the brain, but research on drugs that reduce amyloid levels have not successfully slowed the progression of the disease. New research from the University of Melbourne however, discovered that humans with high levels of iron and amyloid were suffering from rapid dementia, while those with just high levels of amyloid protein were stable. This finding will fuel a five year trial on whether an anti-iron drug can slow the progression of the disease. This research was published the journal Brain.
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Brain MRI using Quantitative Susceptibility Mapping (QSM). Source: University of Melbourne.

  • Compound protects macaques from simian HIV. Research being presented this week at the National AIDS Treatment Advocacy Project (NATAP) Conference on HIV Pathogenesis Treatment and Prevention in Paris shows that weekly administration of a compound called MK-8591 repeatedly protected 8 of 8 macaques from simian HIV (SHIV) 6 days after treatment. Researchers from the Aaron Diamond AIDS Research Center in New York, Merck, and the Tulane National Primate Research Center studies 16 male macaques, 8 of which received weekly treatments of MK-8591 for up to 14 weeks, the other 8 of which received a placebo. MK-8591 is a nucleoside reverse transcriptase translocation inhibitor (NRTTI) that thwarts HIV. All 8 monkeys treated with MK-8591 remained SHIV-free even after 12 challenges with SHIV, to the end of the 168-day study. In contrast, the monkeys not treated with the drug all became infected with SHIV. The researchers noted that protective intracellular active MK-8591 concentrations can be attained in humans at low drug doses. These new findings support the potential use of MK-8591 as a prophylactic treatment for high-risk individuals.
  • New drug acting at two opioid receptors shows promise to treat chronic pain without the adverse effects of morphine. An epidemic of opioid abuse is killing people by the hundred of thousands in the United States, becoming the main cause of death for people under 40. The epidemic has been traced to the prescription of new opioid analgesics like Oxycontin over the last years, which has led people to become addicted to it and then to harder drugs like heroin. A group of scientist followed the strategy of creating drugs that bind not only to the receptor for morphine in the brain, the mu-opioid receptor, but also to a new opioid receptor called the nociceptin FQ receptor. The new drug, BU08028, was shown to reduce pain responses in rats as effectively as morphine. Now the drug is being tested in rhesus monkeys, where it also decreased pain. Importantly, the monkeys showed no desire to self-administer BU08028, an indication that the drug is not addictive.
nhp_c2a9kathyweststudios-40

Male rhesus macaque. Source: Kathy West.

  • Computer-designed opioid drug may decrease pain without producing respiratory arrest. People dying during the current opioid epidemic do so because the drugs inhibit the centers in the brain that drive breathing, leading to suffocation. Scientists proposed that the pain-relieving and breathing-suppressive effects of the opioids depend on different interactions of these drugs with the mu-opioid receptor, and set up to produce a compound that would suppress pain but not breathing. To do that, they modeled the drug binding site at the receptor using computers. After testing millions of compounds in the computer, they found a drug, PZM21, that showed promise. Then they tested PZM21 in mice and found that it suppressed pain but not breathing. They also found that PZM21 did not show the rewarding effect that typically lead to addiction. This demonstrates how even when new drugs are designed using computer models, animal studies are still needed to evaluate their effects.

Research Roundup: Malaria vaccine, mouse sperm in space, animal welfare prizes, and more!

Welcome to this week’s Research Roundup. These Friday posts aim to inform our readers about the many stories that relate to animal research each week. Do you have an animal research story we should include in next week’s Research Roundup? You can send it to us via our Facebook page or through the contact form on the website.

  • New study finds that mouse sperm stored in space still functions on Earth. Increasingly in the news we read about the upcoming reality of commercial space travel (for example, here and here). Of course, with such advances there is caution with respect to feasibility — and of course imagination with respect to possibilities (e.g., colonizing Mars). With such goals on the horizons, these researchers investigated whether sperm that had been freeze dried, and transported to the International Space Station (ISS) and then back to Earth would be able to produce viable offspring. To accomplish this they used freeze dried mouse spermatozoa — which provided a unique advantage, as the addition of water — maintains the viability of the sperm to fertilize an egg and allows for the sperm to be stored at room temperature. Other sperm when freeze dried do not survive. Microinjection  of these “space” sperm into an egg on Earth — produced healthy viable  “space offspring”. Moreover, these offspring all grew to healthy adults and were able to produce offspring of their own. This study was published in the Proceedings of the National Academy of Sciences of the USA.

Space mouse and pups. Source: PNAS

Laboratory frogs. Source: University of Portsmouth

  • Modified experimental vaccine protects monkeys from deadly malaria. Researchers at the National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health, discovered that a modified version of an experimental malaria vaccine completely protected 4 of 8 monkeys from a malaria parasite, and delayed the first appearance of the parasites in 3 more monkeys. Scientists modified an existing malaria vaccine by including a particular protein, RON2L, so that it closely mimicked the protein complex used by the parasite to infect blood cells. Vaccination with the modified vaccine resulted in more neutralizing antibody, indicating a better quality response to parasitic infection. Additionally, the modified vaccine seemed to protect against other parasite strains that differed from those used to create the vaccine, suggesting that this new modified vaccine may protect against multiple parasite strains. This research will pave the way toward eventual human trials. The study was published in NPJ Vaccines.

A female Aedes mosquito. Source: NIAID.

 

 

 

 

 

 

 

 

 

 

 

  • Researchers at the University of Helsinki has found the lymphatic vessels extend into the brain – overturning 300 years of accepted wisdom. By genetically altering mice using the luminescent GFP gene, so that lymphatic vessels glowed under light, Aleksanteri Aspelund found that there were lymphatic vessels in the brain. The research was repeated by Karl Alitalo with the same results.  Other researchers have found evidence linking problems with the lymphatic and glymphatic systems to Alzheimer’s; one study in mice showed it could lead to the buildup of amyloid beta in the brain – a key sign of the Alzheimer’s. The study was published in the Journal of Experimental Medicine.

    Red fluorescence of the membrane protein aquaporin-4 in an individual with Alzheimer’s (left) and a healthy individual (right). Source: OHSU

  • Mice help researchers identify genes responsible for a severe congenital heart defect.  Congenital heart disease affect up to 1 percent of all live births. Hypoplastic Left Heart Syndrome (HLHS) is a rare congenital heart disease resulting in an inability to effectively pump blood  throughout the body.  Current treatment involves multiple complex surgeries during the first few years of a child’s life. For some, the surgical interventions improve heart function.  For others, it does not,  leading to heart failure and the need for heart transplants. It has been known that genetic risk factors play a role in HLHS but specific genes have been hard to identify.  Researchers at the University of Pittsburgh Schools of the Health Sciences used fetal ultrasound imaging to look for structural heart defects in genetically modified mice to identify HLHS.  Then by comparing the genomes of affected and non affected mice, and confirming using CRISPR technology they found that mutations in two specific genes that interact were required for HLHS.,   Dr. Cecilia Lo, a professor and the F. Sargent Cheever Chair in Developmental Biology at Pitt says, “Studying diseases with complex genetics is extremely challenging…By understanding the genetics and biology of HLHS, this can facilitate development of new therapies to improve the prognosis for these patients.” This study was published in the journal Nature Genetics.
  • The University of Bristol has awarded prizes in its first Animal welfare and 3Rs Symposium. The 3Rs, developed by Russel and Burch in 1954, have advanced the humane treatment of animals used in research by advocating for replacement (aiming to replace animals where possible, with alternatives), to reduce the number of animals used to the minimum required to answer and experimental question and and to refine their experiments to minimise any adverse effects experienced by the animals.These awards went to three research projects that have advanced the 3Rs in their various lines of research.

“The research project that won first prize has developed a refined method for producing aortic aneurysms in mice.  An aortic aneurysm is a bulge in a section of the aorta, which is the body’s main artery, and if the bulge ruptures it can cause sudden death. The research team has also developed a new human aortic aneurysm model in the laboratory, potentially replacing the need for animal models, using arteries taken from the discarded umbilical cord of newly born babies.

The second prize was awarded to a research team who has developed a method for giving oral drugs using solutions that mice and rats both like and which avoids the need for restraint and reduces stress in the animals. The research team found that liquid foods such as condensed milk, milkshake and fruit puree baby food are good solutions to use for giving a wide range of drugs.
The final prize was awarded to a research team who has developed photographic techniques that can be used in conscious animals.  This new technique has revolutionised preclinical eye research and has markedly reduced the number of animals needed for research studies.”

The 3Rs. Source: Bayer

SR-Research Roundup

July 21st-July 28th

Welcome to this week’s Research Roundup. These Friday posts aim to inform our readers about the many stories that relate to animal research each week. Do you have an animal research story we should include in next week’s Research Roundup? You can send it to us via our Facebook page or through the contact form on the website.

  • A protein that may cause Lupus has been identified. Lupus is a chronic inflammatory disease that occurs when your body’s immune system attacks its own tissues and organs. An estimated 1.5 million Americans, and at least 5 million people worldwide, have a form of lupus. Previous research has implicated the gene PRDM1 as a risk factor for lupus. Scientists looking at Blimp-1, a protein that is encoded by the PRDM1 gene, have found in mice thatthat a low level of or no Blimp-1 in a particular cell type led to an increase in the protein cathepsin S (CTSS) which caused the immune system to identify healthy cells as something to attack — particularly in females.” These results are particularly striking as women have an increased risk for lupus compared to men. While this work needs to be replicated and validated, this research provides some valuable insight into the etiology and treatment of lupus. This research was published in the journal Nature Immunology.

Source: http://ajprenal.physiology.org/content/308/10/F1146

  • Vaccine for virus induced Type 1 diabetes successful in mice. Coxsackie B viruses are the most common enteroviruses and are believed to be associated with the development of Type 1 diabetes. Type 1 diabetes is a common human disease defined by a decrease in the production of insulin, which is a hormone that allows blood glucose (sugar) to enter energy producing cells. Thus, without insulin your body cannot effectively produce energy. This week, a team of Finnish researchers published a preclinical evaluation of a Coxsackie B1 vaccine using mice and found that the vaccine successfully protected the mouse after administering the Coxsackie B1 virus. Pre-clinical trials in humans are the next logical step for this vaccine, and the researchers believe that this research will aid in the development of vaccines for other disease caused by enteroviruses such as; hand-foot-and-mouth disease, meningitis, and myocarditis. This research was published in the journal Vaccine.
  • High iron levels in brain linked to progression of Alzheimer’s. Alzheimer’s is a degenerative neurological disease that causes dementia in humans. Previous research has linked Alzheimer’s to the buildup of amyloid protein in the brain, but research on drugs that reduce amyloid levels have not successfully slowed the progression of the disease. New research from the University of Melbourne however, discovered that humans with high levels of iron and amyloid were suffering from rapid dementia, while those with just high levels of amyloid protein were stable. This finding will fuel a five year trial on whether an anti-iron drug can slow the progression of the disease. This research was published the journal Brain.

Source:https://pursuit.unimelb.edu.au/articles/rusty-brains-linked-to-alzheimer-s?utm_source=twiter&utm_medium=social&utm_content=story

  • Compound protects macaques from simian HIV. Research being presented this week at the National AIDS Treatment Advocacy Project (NATAP) Conference on HIV Pathogenesis Treatment and Prevention in Paris shows that weekly administration of a compound called MK-8591 repeatedly protected 8 of 8 macaques from simian HIV (SHIV) 6 days after treatment. Researchers from the Aaron Diamond AIDS Research Center in New York, Merck, and the Tulane National Primate Research Center studies 16 male macaques, 8 of which received weekly treatments of MK-8591 for up to 14 weeks, the other 8 of which received a placebo. MK-8591 is a nucleoside reverse transcriptase translocation inhibitor (NRTTI) that thwarts HIV. All 8 monkeys treated with MK-8591 remained SHIV-free even after 12 challenges with SHIV, to the end of the 168-day study. In contrast, the monkeys not treated with the drug all became infected with SHIV. The researchers noted that protective intracellular active MK-8591 concentrations can be attained in humans at low drug doses. These new findings support the potential use of MK-8591 as a prophylactic treatment for high-risk individuals.
  • New drug acting at two opioid receptors shows promise to treat chronic pain without the adverse effects of morphine. An epidemic of opioid abuse is killing people by the hundred of thousands in the United States, becoming the main cause of death for people under 40. The epidemic has been traced to the prescription of new opioid analgesics like Oxycontin over the last years, which has led people to become addicted to it and then to harder drugs like heroin. A group of scientist followed the strategy of creating drugs that bind not only to the receptor for morphine in the brain, the mu-opioid receptor, but also to a new opioid receptor called the nociceptin FQ receptor. The new drug, BU08028, was shown to reduce pain responses in rats as effectively as morphine. Now the drug is being tested in rhesus monkeys, where it also decreased pain. Importantly, the monkeys showed no desire to self-administer BU08028, an indication that the drug is not addictive.
  • Computer-designed opioid drug may decrease pain without producing respiratory arrest. People dying during the current opioid epidemic do so because the drugs inhibit the centers in the brain that drive breathing, leading to suffocation. Scientists proposed that the pain-relieving and breathing-suppressive effects of the opioids depend on different interactions of these drugs with the mu-opioid receptor, and set up to produce a compound that would suppress pain but not breathing. To do that, they modelled the drug binding site at the receptor using computers. After testing millions of compounds in the computer, they found a drug, PZM21, that showed promise. Then they tested PZM21 in mice and found that it suppressed pain but not breathing. They also found that PZM21 did not show the rewarding effect that typically lead to addiction. This demonstrates how even when new drugs are designed using computer models, animal studies are still needed to evaluate their effects.

Nobel Prize 2016 – how yeast and mouse studies uncovered autophagy

Congratulations to Professor Yoshinori Ohsumi Tokyo Institute of Technology on being awarded the 2016 Nobel Prize in Physiology or Medicine for “for his discoveries of mechanisms for autophagy“!

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Yoshinori Ohsumi. Image: Tokyo Institute of Technology

The process of autophagy is hardly one familiar to most people, but is is absolutely crucial to all complex life on out planet, including ourselves. The name autophagy comes from the Greek words for “self” and “eating” and describes the ordered process through which cells break down and recycle unnecessary or damaged structures or proteins, and allows the cell to reach an equilibrium between the synthesis and degradation of proteins.

The discovery of autophagy

The process itself was identified through studies in tissues of mice and rats back in the 1950’s and 1960’s, by scientists including Christian de Duve, who was subsequently awarded the Nobel Prize in Physiology or Medicine in 1974 for this and other work. They first discovered that mammalian cells contain a compartment which they termed the lysosome where proteins are broken down, and then that proteins and other molecules that were to be degraded were first isolated from the rest of the cell by the formation of a membrane sac around the protein in question  (later called the autophagosome). The process through which the autophagosome fused with the lysosome to deliver its protein cargo for degradation was given the name autophagy by Christian de Duve.

Image: NobelPrize.org

Image: NobelPrize.org

Progress in understanding how autophagy worked was slow, as at the time the genes or proteins involved in regulating the process had been identified. With the research methods available at the time it was difficult to measure autophagy as it happened in mammalian cells, and hence difficult to determine how altering different components affected the overall process, a key step towards understanding their role. It may have seemed an unpromising field to join, but Yoshinori Ohsumi had a different career philosophy to most researchers, which he described in an interview given in 2012:

I am not very competitive, so I always look for a new subject to study, even if it is not so popular. If you start from some sort of basic, new observation, you will have plenty to work on.

From cells to genes

What was needed was a simple experimental system in which to study the process, and the bakers yeast Saccharomyces cerevisiae  – a simple single celled organism separated from us by hundreds of millions of years of evolution, but sharing many of our key biological processes – was one candidate. Yoshinori Ohsumi had worked with yeast, and in particular had identified many proteins in a subcellular component of the yeast cell known as the vacuole, which was important as there was evidence that the vacuole performed the same role in yeast cells as the lysosome in mammalian cells. Still, as the Nobel Prize website highlights there were still hurdles to overcome as he began his study of autophagy in yeast at the end of the 1980’s:

But Ohsumi faced a major challenge; yeast cells are small and their inner structures are not easily distinguished under the microscope and thus he was uncertain whether autophagy even existed in this organism. Ohsumi reasoned that if he could disrupt the degradation process in the vacuole while the process of autophagy was active, then autophagosomes should accumulate within the vacuole and become visible under the microscope. He therefore cultured mutated yeast lacking vacuolar degradation enzymes and simultaneously stimulated autophagy by starving the cells. The results were striking! Within hours, the vacuoles were filled with small vesicles that had not been degraded (Figure 2). The vesicles were autophagosomes and Ohsumi’s experiment proved that authophagy exists in yeast cells. But even more importantly, he now had a method to identify and characterize key genes involved this process.

With an experimental system available Yoshinori Ohsumi and his team studied the process of autophagy in thousands of mutant strains of yeast, and identified 15 individual genes (most of them of previously unknown function) that are essential for the process in yeast, tho order in which the key events in autophagy take place, and the roles of the individual genes in them. This was the work for which he was awarded the Nobel Prize.

From yeast genes to us!

But it is not the end of the story! Identifying the genes essential for autophagy in yeast, and their roles in the process, was a major breakthrough, but what about humans and other mammals?

It turns out that that in humans and other mammals there are counterparts to almost all the yeast autophagy genes, though the situation is made a lot more complicated by the face that mammals have more than one copy for each of the genes…starting with yeast was a wise move! Professor Noboru Mizushima of the University of Tokyo made an important advance when, working with Yoshinori Ohsumi,  he developed a transgenic mouse in which a protein called LC3 that is found in the autophagosome membrane is fused to Green Fluorescent Protein (GFP – see Nobel Prize for Chemistry 2008) which allowed him and his colleagues to observe and monitor the process of autophage in vivo in mice for the first time.

Laboratory Mice are the most common species used in research

This LC3-GFP transgenic mouse proved to be a very powerful research tool for studying mammalian autophagy, allowing not only the role of indicudual genes in the process to be determined, but also the role of autophagy itself in processes as diverse as early embryonic development, tumor suppression, nerve cell survival and function, and protection against infection.

This research is still at a relatively early stage, but techniques such as the LC3-GFP system in mice – and others used in organisms such as fruit flies, are showing us how defects in autophagy contribute to many diseases, including neurodegenerative disorders such as Parkinson’s Disease, and metabolic disorders such as type 2 Diabetes. While the development of specific therapies to correct these defects in autophagy is still some way off, it is already clear that understanding autophagy has the potential to improve the treatment of a wide range of illnesses.

What the work of Yoshinori Ohsumi demonstrates once again is the crucial contribution of basic biological research in model organisms that may at first glance appear to share little with us to the advancement of medicine.

Speaking of Research

 

 

Clinical trial success for Cystic Fibrosis gene therapy: built on animal research

This morning the Cystic Fibrosis Gene Therapy Consortium (GTC) announced the results of clinical trial in 140 patients with cystic fibrosis, which demonstrate the potential for gene therapy to slow – and potentially halt – the decline of lung function in people with the disorder. It is a success that is built on 25 years of research, in which studies in animals have played a crucial role.

Cystic fibrosis is one of the most commonly inherited diseases, affecting about one in every four thousand children born in the USA, and is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The CFTR gene produces a channel that allows the transport of chloride ions across membranes in the body, and the many mutations identified in cystic fibrosis sufferers either reduce the activity of the channel or eliminate it entirely. This defect in chloride ion transport leads to defects in several major organs including the lungs, digestive system, pancreas, and liver. While the severity of the disease and the number of organs affected varies considerably, cystic fibrosis patients often ultimately require lung transplant s, and too many still die early in their 20’s and 30’s as the disease progresses.

In a paper published in Lancet Respiratory Medicine today (1), the GTG members led by Professor Eric Alton of Imperial College London compared monthly delivery to the airway of a non-viral plasmid vector containing the CFTR gene in the liposome complex pGM160/GL67A using a nebuliser with a placebo group who received saline solution via the nebuliser. They reported stabilisation of lung function in the pGM169/GL67A group compared with a decline in the placebo group after a year. This is the first time that gene therapy has been shown to safely stabilise the disease, and while the difference between the treated and control group was modest, and the therapy is not yet ready to go into clinical use, it provides a sound bases for further development and improvement.

Blausen_0286_CysticFibrosis

The Chief Executive of the Cystic Fibrosis Trust, which is one of the main funders of the GTC, has welcomed the results, saying:

Further clinical studies are needed before we can say that gene therapy is a viable clinical treatment. But this is an encouraging development which demonstrates proof of concept.

“We continue to support the GTC’s ground-breaking work as well as research in other areas of transformational activity as part of our mission to fight for a life unlimited by cystic fibrosis.”

So how did animal research pave the way for this trial?

Following the identification of the CFTR gene in 1989 scientists sought to create animal models of cystic fibrosis with which to study the disease, and since the early 1990’s more than a dozen mouse models of cystic fibrosis have been created. In some of these the CFTR gene has been “knocked out”, in other words completely removed, but in others the mutations found in human cystic fibrosis that result in a defective channel have been introduced. These mouse models show many of the defects seen in human cystic fibrosis patients and over the past few years have yielded important new information about cystic fibrosis, and in 1993 Professor Alton and colleagues demonstrated that it is possible to deliver a working copy of the CFTR gene using liposomes to the lungs of CFTR knockout mice and correct some of the deficiencies observed.

To get a working copy of the CFTR gene to the lungs of cystic fibrosis patients Professor Alton and colleagues needed three things:

• A DNA vector containing the working CFTR gene that is safe and  can express sufficient amounts of the CFTR channel protein in the lungs to correct the disease

• A lipid-like carrier that can form a fatty sphere around the DNA vector to so that it can cross the lipid membrane of cells in the lung, as “naked” DNA will not do this efficiently.

• A nebuliser device that produces an aerosol of the gene transfer agent so that it can be inhaled into the lungs of the patient.

Several early attempts to use gene therapy using viral vectors to deliver the working copy of the CFTR gene to patients failed because the immune response rapidly neutralised the adenoviral vector (see this post for more information on challenges using adenoviral vectors), and while attempts to use non-viral vectors were more promising, it was found that they caused a mild inflammation in most patients, which would make then unsuitable for long term use. As reported in a paper published in 2008 the GTC members developed and assessed in mice a series of non-viral DNA vectors, repeatedly modifying them and testing their ability to both drive CFTR gene expression in the lungs and avoid inducing inflammation. They finally hit on a vector – named pGM169 – which fulfilled both key criteria.

Earlier the consortium had undertaken a study to determine which carrier molecule to use in their non-viral gene transfer agent (GTA). To do this they assessed 3 GTA’s, each consisting of a lipid like molecule that could form a sphere around the non-viral DNA vector; either the 25 kDa-branched polyethyleneimine (PEI), the cationic liposome GL67A, or as a compacted DNA nanoparticle formulated with polyethylene glycol-substituted lysine 30-mer. Because there are significant differences in airway physiology between mouse and human they carried out this study in sheep, whose lung physiology more closely matches that of humans. The study identified the cationic liposome GL67A as the most promising candidate, resulting in robust expression of the CFTR transgene in the sheep lungs.

Studies in sheep play a key role in the development of gene therapy for cystic fibrosis

Studies in sheep play a key role in the development of gene therapy for cystic fibrosis

It now remained to bring the DNA vector and carrier together. In a 2013 publication the consortium reported that repeated aerosol doses of pGM169/GL67A to sheep over a 32 week period were safe and induced expression of the CFTR transgene in the sheep lungs, although the level of expression varied between individuals (this variation was also observed in human CF patients in the clinical trial reported today). A final study, this time in mice, assessed the suitability of the Trudell AeroEclipse II nebuliser as a device to create stable pGM169/GL67A aerosols, finding that it did so in a reproducible fashion. When aerosolized to the mouse lung, the new pGM169/GL67A formulation was capable of directing persistent CFTR transgene expression for at least 2 months, with minimal inflammation. These studies provided the evidence to support the gene delivery system and dosage strategy used in the clinical trial reported today.

The trial results announced today are an important accomplishment, but they mark a beginning rather than the end for Cystic Fibrosis gene therapy. It will be necessary to improve the efficiency of the therapy before it can enter widespread clinical use. Animal research will certainly play an important part in this work, notably the observation that the efficiency of CFTR gene delivery using this strategy was varied between individuals in both sheep and humans indicates that sheep are a good model in which to assess changes to improve the consistency and effectiveness of the gene therapy.

If you would like to know more about this cystic fibrosis gene therapy clinical trial you can watch two videos recorded at a meeting for cystic fibrosis patients at ICL on the  Cystic Fibrosis Trust website.

Paul Browne

1) Alton E.W.F.W. et al. “Repeated nebulisation of non-viral CFTR gene therapy in patients with cystic fibrosis: a randomised, double-blind, placebo-controlled, phase 2b trial” Lancet Respiratory Medicine Published online July 3, 2015