Tag Archives: Genetic modification

ERV blogs on GMO Herpes vs severe cancer pain

As gene therapy emerges as one of the hottest areas of medical research, one thing that is striking is how it employs viruses – sometimes very nasty viruses – to deliver the gene to where it is needed in the human body.

Yesterday virologist Abbie Smith discussed another excellent example of this on the ERV blog in a post entitled “GMO Herpes vs. severs cancer pain”, describing how scientists at the Universities of Michigen and Pittsburgh have used a genetically modified herpes virus to deliver the preproenkephalin gene – which produced a precursor to pain-killing opiates – to the nerve cells of terminal cancer patients who were suffering from severe pain.

Abbie remarks that “This was one of the most depressing, yet hopeful, papers I have ever read.”. It’s difficult to disagree, after all most of the patients participating in the trial died within 3 months of it starting. But to focus on this sobering statistic would miss the reason for this study, namely that the pain-relief available to patients with severe chronic pain is often inadequate, as the drugs are not specific enough and cause unacceptable side effects when used at the high doses often required for prolonged periods of time. By targeting the opiate molecules to the nerve ccells themselves these side effects can be avoided, and more effective pain relief provided.

The paper “Gene Therapy for Pain: Results of a Phase I Clinical Trial” is available for anyone to read in PubMed Central and makes it very clear that this is a therapy that was discovered, evaluated and refined in animal models of different types of pain before entering this first clinical trial. The first two paragraphs of the introduction noting that:

A significant limitation to the development of analgesic drugs is that off-target effects at doses below the maximal analgesic threshold restrict the ability to selectively interrupt nociceptive neurotransmission1. To address this limitation, we developed a series of replication defective HSV-based vectors to deliver gene expression cassettes directly to DRG neurons from skin inoculation 2, 3. The anatomically defined projection of DRG axons allows targeting of specific ganglia by injection into selected dermatomes. In preclinical studies, the release of anti-nociceptive peptides or inhibitory neurotransmitters in spinal dorsal horn from the central terminals of transduced DRG neurons effectively reduced pain-related behaviors in rodent models of inflammatory pain, neuropathic pain, and pain caused by cancer4-9.

The human PENK gene encodes for preproenkephalin, a precursor protein proteolytically cleaved to produce the endogenous opioid peptides met- and leu-enkephalin. In the spinal cord, enkephalin peptides inhibit pain signaling through actions at presynaptic opioid receptors located on central terminals of primary afferent nociceptors and postsynaptic opioid receptors on second order neurons involved in nociceptive neurotransmission10. HSV vectors expressing opioid peptides appear to be particularly effective in animal models of inflammatory and cancer pain4, 5, 8.”

And in the conclusion:

In preclinical animal studies, skin inoculation of HSV vectors expressing PENK reduce acute hyperalgesic responses27, and reduce pain-related behaviors in models of arthritis28, formalin injection4, peripheral nerve damage6 and bone cancer5. Because this was the first human trial employing HSV vectors to achieve gene transfer, we elected to carry out the phase 1 clinical trial for safety and dose-finding in patients with pain caused by cancer…This Phase I clinical trial primarily addressed the question of whether intradermal delivery of NP2 to skin would prove to be safe and well tolerated by subjects. The small number of patients and the absence of placebo controls warrant circumspect interpretation of the secondary outcome measures. But the observation that subjects in the low dose cohort had little change in the NRS or SF-MPQ while subjects in the higher dose cohorts reported substantial reduction in NRS and improvement in SF-MPQ is encouraging.”

Encouraging is possibly an understatement, seeing clear evidence of therapeutic benefits in a Phase I trial like this is very promising, or as Abbie puts it “A trial turning out this successful is a great starting point for optimizing this kind of therapy.”.

Paul Browne

p.s. Those interested in a more detailed account of the research that led to this clinical trial can find it in this review published in 2008 and available to read online for free.

Natural Antibiotic Casts a Net Against Bacteria

A “natural antibiotic” protects the body against bacteria by tangling them in a net, not poking holes in them, UC Davis researchers have found. Experiments with genetically-modified, or transgenic mice were crucial to the discovery, along with cell cultures, biochemistry and sophisticated studies of how small proteins assemble together.

It’s an entirely new mechanism of action for defensins, a group of molecules with natural antibiotic activity found in the gut, on the skin and in white blood cells. Most defensins studied so far work by punching holes in the membranes around bacterial cells. The discovery, published June 22 in the journal Science, also points to possible causes of Crohn’s disease and other inflammatory bowel diseases.

The researchers led by Hiutung Chu and Charles Bevins at the UC Davis School of Medicine were studying human defensin 6 (HD6), one of six related small proteins made by humans. HD6 is secreted by cells deep in the folds of the small intestine and seems to help keep the gut microbes in balance.

Other defensins kill bacteria in a lab dish, but early research studies repeatedly showed that HD6 just does not kill bacteria in culture.

However, when the researchers used transgenic mice that make HD6 in their intestines, they found that the HD6 mice could not be infected with bacterium Salmonella entericum through the gut. In control mice, the infection spread to other organs. This result indicated that a novel anti-microbial mechanism was at work.

The authors tested if HD6 blocked infection by interfering with proteins that form the mechanism through which S.entericum invades cells, but when they found that the transgenic mice expressing HD6 were also protected against invasion by the bacterium  Yersinia enterocolitica – which uses a different mechanism to invade cells – it became clear that HD6 must block infection by a different method.

Through a series of other experiments in vivo and using the transgenic HD6 mice, the team found that when HD6 encounters a bacteria like Salmonella, the small proteins rapidly link up to form a net or web that tangles the bacteria. This forms a barrier lining that stops hostile bacteria from crossing the gut lining and infecting the rest of the body.

In order to determint what part of the bacteria HD6 interacted with, the Bäumler laboratory created bacterial mutants lacking surface structures known as flagella and type I fimbriae. When those structures were removed HD6 was unable to form the fibrils on the bacterial surface, indicating that these structures act as anchoring points that trigger HD6 nanonet formation.

Put together, the molecular, cell culture, and transgenic mouse experiments build a case to show how HD6 works and protects the gut from infections.

Indeed in a commentary in this week’s issue of Science Professor Andre Ouelette  and Professor Michael Selsted of the University of Southern California note that this study highlights the need to undertake in vivo animal studies to complement and understand the relevance of in vitro observations.

“Aside from delineating the role of HD6 and its unusual mechanism of action, the report by Chu et al. should give us pause for reflection. HD6 has no evident activity in in vitro assays, yet it affects mucosal immunity profoundly. Thus, in vitro bactericidal assays, although useful for comparing peptides in structure-activity studies, may not predict or even hint at the potential impact of a peptide in vivo. Given the diversity of in vitro biological functions that have been associated with defensins and other hostdefense peptides, the challenge is to establish the physiological relevance of those activities.”

Inflammatory bowel disease

People with Crohn’s Disease have unusually large amounts of bacteria in the crypts of the small intestine, causing chronic inflammation. It may be that the gut defensins HD5 and HD6 are defective in these people, so the bacteria can invade and colonize the gut folds more easily. Understanding how these defenses work could lead to insights and new treatments for Crohn’s and similar conditions. No doubt animal models such as transgenic mice will play an important role in that research, too, along with other techniques.

Read more about this study at the UC Davis Health system Newsroom

Regards

Andy

Speaking of Research welcomes 2011 UK statistics for animal use in scientific research

The UK Home Office has released the animal research statistics for 2011, which show a 2% increase overall in procedures compared to 2010 figures. A summary of the report is available here, while the full report can be downloaded here.  Once again basic research and breeding of GM animals accounted for the lions share – 88% – of the total number of procedures, with mice being the most commonly used animal, bring used in 71 % of procedures.

Some other interesting trends, the number of procedures involving fish increased again, reflecting a trend over the past decade as the importance of the Zebrafish in scientific research increases. The number of procedures involving non-human primates – both new world and old world – fell substantially in 2011 compared to 2010, but the number of procedures using non-human primates tends to vary quite a lot from year to year because of the small number used overall, so it’s too early to say whether or not this is part of an overall trend.

Another interesting fact to note is that over 7o% of procedures did not require anaesthesia, and as Understanding Animal Research point out in their commentary a procedure under the Home Office regulations can be as mild as a blood draw, or indeed breeding in the case of GM animals.

There’s some useful commentary in the Guardian, and the Association of Medical Research Charities blog also provides some insightful analysis of what the figures mean.

Speaking of Research welcomes these figures as evidence that the biomedical research in the UK remains a vibrant despite the tough economic climate, and as evidence that scientists in the UK continue to adopt the most up-to-date animal research techniques as they further understanding of biological systems and develop new therapies for human and animal disease.

Speaking of Research

Millenium Technology Prize 2012: Mouse stem cell research heralds a new era in biomedical research

Every year since 2004 the Technology Academy Finland has awarded the prestigious Millennium Technology Prize as a tribute to life-enhancing technological innovations, and the list of past winners includes some of the world’s leading technological innovators. This year the Grand Prize is for the first time being shared between two innovators, Linus Torvalds, who created the open-source Linux operating system, and Shinya Yamanaka, who created the first induced pluripotent stem (iPS) cells.

The discovery by Professor Shinya Yamanaka of Kyoto University that it was possible to transform adult cells into stem cells that had the ability to then develop into any cell type – an ability only previously observed in embryonic stem cells – by inserting 4 genes associated with embryonic development, has electrified the scientific community, with many groups around the world now working to improve the technique and apply it to medical research and medicine. The Millenium prize notes that Prof. Yamanake first identified the genes required to transform skin cells to pluripotent stem cells through studies in mice.

In 2006 his research team successfully generated mouse induced pluripotent stem cells (iPS) cells with self-renewal and pluripotency (the ability to develop into different types of cell), which are almost equivalent to ES cells. The following year they established human iPS cells, by transducing the same four genes used in their earlier breakthrough, in human adult skin cells.”

The work briefly described above was a technological tour-de-force where Prof. Yamanaka and his colleagues selected 24 genes which had previously been identified as having key roles in mouse embryonic stem cells, and developed a screening method using skin fibroblast cells derived from mice that had be genetically modified with an antibiotic resistance gene that was only expressed in embryonic cells, so that only cells that were in an embryonic state would survive in a culture containing the antibiotic. Different combinations of these 24 genes were screened for their ability to induce to the production of colonies of embryonic -like cells from adult fibroblasts.  They eventually identified just 4 genes – Oct3/, Sox2, Klf4 and c-Myc – that together could reprogram adult mouse fibroblast cells to a pluripotent embryonic-like state (1), and subsequently demonstrated that these iPS cells could give rise to a wide variety of  tissue types when incorporated into mice, either by subcutaneous injection into adult mice or incorporation into early mouse embryos. By modifying their method slightly to also include expression of an important developmental gene named Nanog  they were then able to generate chimeric mice (mice whose tissues are made up of a mixture of cells derived from their own embryonic stem cells, and cells derived from iPS cells) which were capable of transmitting the iPS cells to the next generation of mice (2).

Soon after this Prof. Yamanaka succeeded in generating iPS cells from human fibroblasts, using the same techniques used for the mouse cells, and a whole new and exciting field of biomedical research was born.

Paul Browne

1)      Takahashi K, Yamanaka S. “Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.” Cell 2006 Vol. 126(4):663-76. PubMed: 16904174

2)      Okita K., Ichisaka T., Yamanaka S. “Generation of germline-competent induced pluripotent stem cells.” Nature Vol. 448:313-317 (2007). PubMed:17554338

The 21st Century Scientist

Earlier today we discussed some of the characteristics of the animal rights crank, so it’s perhaps appropriate that an award announced earlier this week has highlighted the best qualities of the scientists who are really shaping 21st century medicine.

The Grete Lundbeck European Brain Research Foundation has awarded its 2nd €1-million Brain Prize to Professor Karen Steel of Cambridge University, founder of the Mouse Genetics Programme at the Wellcome Trust Sanger Institute, and Professor Christine Petit of the College de France, head of the Genetics and Physiology of Hearing laboratory at the Institut Pasteur in Paris, for:

their unique, world-leading contributions to our understanding of the genetic regulation of the development and functioning of the ear, and for elucidating the causes of many of the hundreds of inherited forms of deafness”

Continue reading

Mice, rats, and the secrets of the genome.

It’s just over a decade since the completion of the first working draft of the human genome was announced, and seven years since the publication of the complete sequence, but in that short time the impact of this new knowledge on all areas medical research has been immense. Sequencing the human genome was a huge achievement, but having got the sequence an even greater task confronts scientists – working out what it all means.  To do this scientists have studied the natural variations that exist between individuals, and have also sequenced the genomes of a wide variety of species, some closely related to us, others separated from us by hundreds of millions of years of evolution. Scientists can analyze the similarities and differences between the genes of different species, and examine how changes to the structure or regulation of these genes are reflected in physiology.  In many cases it is also possible to use genetic modification to study the function of conserved genes in other species in ways that are just not possible, for technical and/or ethical reasons, in humans. A study published a couple of weeks ago in the scientific journal Nature provides an excellent example of how animal research contributes to our understanding of the human genome.

Blood flowing through the heart, but the cholesterol building up in an artery can cause a heart attack. Image courtesy of the British Heart Foundation

As the cost of technology such as DNA microarrays has fallen genome-wide association studies (GWAS) have become an increasingly popular way of examining the relationship between genetic differences between individuals and particular diseases. In a GWAS the whole genome of many individuals is screened for variations, and then any association between those variations and particular phenotypes or diseases is determined.  Tanya M. Teslovich and colleagues (1)  analysed the genomes of over 100,000 people who had been enrolled in 46 separate clinical studies, and identified 95 genes that have variants associated with increases in blood lipid (fat and cholesterol) levels.  One of the problems with GWAS studies is that while they are often good at identifying genes that are associated with a disease, they are not so good at identifying which genetic variations actually cause disease, or explaining how the genetic variations contribute to disease.  This is where Tanya Teslovich and colleagues scored highly; they were able to show that 14 of the 95 lipid-associated genes were also associated with the development of coronary artery disease, supporting the proposition that elevated blood lipids contribute to coronary artery disease. They also found that overall the effect of the variants was additive, the more risk variants of these 95 genes you have the greater your chance of having elevated blood lipids.

So that established that the gene variants were associated with elevated blood lipids, but to use that information to develop new treatments you need to know how the particular gene affects lipid levels. As you might expect many of the 95 genes identified were already known from previous studies to be involved in the regulation of blood lipids, and in several cases their precise role has been thoroughly studied. However, several of the genes had not been implicated in regulating blood lipids before, and the team decided to use genetically modified mice to investigate their function.  They injected viral vectors into the liver of the mice that contained either an extra copy of the gene being studied, to increase expression of the gene, or a short-hairpin RNA, to target the gene for knockdown via RNAi. This allowed them to discover that one gene, GALNT2, decreases levels of high-density lipoprotein cholesterol (HDLC), the so-called “good cholesterol”, while two other genes, Ttc39b and Ppp1r3b, increase  HDLC.

Another associated paper (2) in the same issue of Nature takes the analysis even further. Several studies, including the GWAS performed by Tanya M. Teslovich and colleagues, had demonstrated that variations in a particular region of chromosome 1 known as 1p13 were associated with high levels of Low-density lipoprotein “bad” cholesterol (LDLC) in the blood and heart disease, but that these variations were not within the coding sequence of any genes, so they would not affect the structure of any proteins. They first show through genetic studies of human subjects and human liver tissue culture that variations at 1p13 affect the expression of several genes –  and hence the amount of protein produced by those genes – and that one particular variation creates a binding site for the transcription factor C/EBP. Transcription factors are proteins that regulate the expression of genes, and this particular site altered the levels of a gene named SORT1. But what does SORT1 do? To answer this they again turned to GM mice, using virus vectors that specifically reduced or increased the levels of SORT1 in the mouse liver. Reducing or eliminating SORT1 expression in the mouse liver led to a reduction in the levels of LDLC in the blood, and that this was found to be due to SORT1 regulating the production of very-low-density lipoprotein (VLDL), a precursor to low-density lipoprotein, in the liver. As a result of this work a whole new pathway for the regulation of blood lipids has been uncovered, one that may offer new opportunities to scientists developing treatments for hypercholesterolemia.

As a BBC news report indicates, the identification of these genes and the elucidation of their function may aid the development of better diagnostic tools to identify those at risk of heart disease, and ultimately the development of better treatments.

These studies illustrate how important animal models, particularly GM mice, are to efforts to decode the human genome. As the biosciences move towards a more systems based approach to biology, one where knowledge of how networks of genes interact to produce a particular physiological or clinical outcome is applied to areas such as toxicology, the information that studies of GM animals can yield will become increasingly important. This importance has not gone unrecognized by the wider scientific community, the 2007 Nobel Prize in Medicine was awarded to Mario Capecchi, Sir Martin  Evans, and Oliver Smithies for their discoveries of  “ principles for introducing specific gene modifications in mice by the use of embryonic stem cells”.

With this in mind let’s turn briefly to another GM animal that’s been in the news lately – the rat.  While GM mice have become a mainstay of modern scientific research the rat has lagged behind, which is a shame since the larger size, longer lifespan, and more complex behavior of rats make them more effective animal models than mice for studying many human diseases, particularly neurological conditions. The lack of GM rats was due to the difficulty in growing rat embryonic stem cells (ESCs) in culture, a necessary first step in the most common methods of producing GM animals. Last year Matthew Evans wrote an article for the Pro-Test blog discussing how scientists at the University of Cambridge and the University of Southern California had developed a method for growing rat ESCs in culture, and how this achievement paved the way for the production of transgenic rats. Last week the same group of scientists announced that they had employed this method to produce GM rats whose p53 gene, a key tumor suppressor that is defective in several cancers, was deleted.

The humble lab rat, now available in GM. Image courtesy of Understanding Animal Research.

This is not the first time GM rats have been produced, as for the past few years scientists have been able to use zinc finger nucleases to knock-out rat genes. Zinc fingers, so called because one or more zinc ions stabilizes the finger like structure, are found in many proteins, allowing them to bind specifically to a structure within a cell, such as a particular DNA sequence. Scientists found that they could produce artificial zinc fingers that recognize particular genes, and then join a nuclease to that zinc finger so that it cuts out the target gene. This method, discussed in more detail in this excellent article by Elie Dolgin, allows scientists to knock-out genes in rat embryos. The downside of the zinc finger nuclease technique can only be used to knock-out genes, whereas the ESC method is more flexible – it can also be used to add extra copies of a gene, or to delete genes in specific tissues or stages of development.

It is now clear that the rat is joining the mouse at the forefront of the GM revolution in medicine, and that has to be great news for medical science and the patients that depend on it.

Paul Browne

1)      Teslovich T.M. et al. “Biological, clinical and population relevance of 95 loci for blood lipids” Nature Volume 466, Pages 707-713 (2010) DOI:10.1038/nature09270

2)      Musunuru K. et al. “From noncoding variant to phenotype via SORT1 at the 1p13 cholesterol locus” Nature Volume 466, Pages 714–719 (2010) DOI:10.1038/nature09266