National Academies Press: OpenBook

The Markey Scholars Conference: Proceedings (2004)

Chapter: Papers Presented by Markey Scholars5 Functional Genomics

« Previous: 4 New Directions in Genomic Research
Suggested Citation:"Papers Presented by Markey Scholars5 Functional Genomics." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
×

Papers Presented by Markey Scholars

Suggested Citation:"Papers Presented by Markey Scholars5 Functional Genomics." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
×

This page intentionally left blank.

Suggested Citation:"Papers Presented by Markey Scholars5 Functional Genomics." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
×

Functional Genomics

George D. Yancopoulos, M.D., Ph.D.

Regeneron Pharmaceuticals, Inc.

Let me begin by telling you the story about not only getting my Markey award but also why I believe I was the first recipient actually to give it back. I have to give you a little background for this. My parents were Greek immigrants whose lives were interrupted by World War II and the subsequent civil war against communism in Greece. They never got a chance to finish school and were forced to leave Greece. Like many other immigrants, they worked very hard to give their kids a better chance than they had. Toward this end they stressed education. To them education was a means to an end, and that end was a high-paying job as a doctor, lawyer, or something similar.

Needless to say, when they saw that their only son—to whom they had stressed all this education—was not achieving this end, and, in fact, had decided to become a scientist and slave away in a lab for 24 hours a day and make only $10 thousand a year, they were really disappointed.

When I received the Markey award, I thought that I might finally be vindicated, at least in my Dad’s eyes. I went home to tell him about this great award, and the first thing he asked me was, “Well, how much money are you going to make?” I tried to explain to him about how hard it was to fund a lab. I explained how over the course of the 7 years, the funds would add up to lots of money for my lab and so forth and so on. He cut me right off, and he said, “Well, but exactly how much money are you going to make?” When I told him that my income was going to be about $30 thousand a year, he basically started choking up. And then when he recovered, he said that without any education whatsoever he would still

Suggested Citation:"Papers Presented by Markey Scholars5 Functional Genomics." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
×

be making more than his son who had been going to school for more than 25 years and had both an M.D. and a Ph.D. degree.

I tried to argue that what I was doing could end up being up really important. It might even lead to new cures for important diseases. Then he said something which I’ve never forgotten. He said, “Well, if what you are doing is really so important, then maybe you should think about the fact you are in the greatest country in the world … and there are some incredible opportunities here. If what you are doing is so important, then people will pay for it.

I have been in biotech with my partner of about 13 or 14 years now, Leonard Schleifer. The Markey contribution does not end with the award. It turns out that three of the founding board members of this company—one of them, Eric Shooter, is here—were actually on the Selection Committee for the Markey Trust. And so they were three of the strongest advocates for me leaving academia and getting involved in what was then this fly-by-night operation.

I have been at Regeneron for over 13 years, and hopefully I can convince you that things have not worked out so badly. Our initial focus at Regeneron was to identify master regulatory genes involving biological processes with potential therapeutic value, such as neurotrophic growth factors, cytokines, peptide hormones, and their receptors. From the beginning what we were doing is what is now called “functional genomics.” We also do subtractive hybridization, or what is now called “differential display and microarrays.

We realized from the beginning that these sorts of platforms produced a lot of candidates. The hardest thing to do once you have these candidates identified is to understand what they are doing in vivo. There is really no algorithm or chip that can do that for you. It requires a lot of custom work and sophisticated biologists. From the very beginning we believed in genetic approaches, and one of the most powerful ways to understand what a gene did was to knock it out or do these reporter knock-ins where you exchange the gene of interest or do transgenics.

What we do is not that much different from what a lot of people always try to do, but there is an order and science to it. A lot of it involves developing new approaches and new technologies for each step, which we all know can go wrong at any given point. This has been a primary focus at Regeneron from its early days, whether it was to pioneer or greatly simplify techniques.

I would like to spend a moment on what I think is one of the most powerful recent approaches that we have developed that has produced a lot of biology (see Figure 1). This has to do with genetic approaches, knockouts, and reporter knockins in transgenics in particular, a technology that we have been using for only a couple of years at Regeneron. We have not published the technology yet, only the fruits of the technology.

Suggested Citation:"Papers Presented by Markey Scholars5 Functional Genomics." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
×

FIGURE 1 Traditional timing from a knockout service company.

This is the first truly industrialized and high precision automated way to produce knockouts and knockins in transgenics which we call “Velocigene.

We make knockout constructs overnight, and within two to four weeks we have knockout mice. It’s a completely automated process. What is the value of this? Well, we have hundreds, if not thousands of interesting genes we want to play with, and the thing we want to do is functionalize them. By using this Velocigene technology we can deal with things in the quantities of hundreds or thousands, which rivals what you can do in cells, worms, or fish.

The basic advantage to the bottom line is not only have we compressed the timeframe about 10-fold, but also that we can do hundreds in a period where we used to be able to do only one. I want to quickly describe some of the fruits of this technology, and show you how we can get rather dramatic, exciting data. Our goal is therapeutically interesting targets that we can eventually address to human diseases, and do translational research.

Suggested Citation:"Papers Presented by Markey Scholars5 Functional Genomics." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
×

FIGURE 2 Knockouts with substitution of reporter reveal genes site of expression—unparalleled with whole body resolution, yielding clues into gene function.

We have knocked out many unknown genes or genes that have defined sequences. I want to describe some dramatic examples. We substitute reporter genes at the initiation code of the gene of interest. We have found that it almost always replicates accurately the expression pattern of the gene of interest. Our favorite method is still starting with unknown genes. We look for one that has interesting expression patterns. In Figure 2 one gene is only expressed in the blood vessels of the eye. Another gene is expressed more generally in the blood vessels. A third is expressed only in the cartilage and the growth plate or on the surface of the bone. A fourth gene has an incredibly restricted expression pattern in a single layer of neurons in the dorsal part of the spinal cord. These are neurons that sense pain, a perfect place you might want for a pain target.

We produce hundreds of these patterns, and look for the most interesting. Here in Figure 3 is a gene that is only expressed in the nerves of the teeth. Another gene is expressed in association with hair follicles and particularly with the little muscle that makes your hair stand on end. This gene is expressed widely in muscle. Another gene is in the fat that coats the mesenteric vessels, so it is a gene expressed specifically in fat. Both of these Peyer’s patches are in the intestine. These are immunological structures, similar to a lymph node. And one of these genes is expressed on the outside, on the periphery of the little circular node-type structures that

Suggested Citation:"Papers Presented by Markey Scholars5 Functional Genomics." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
×

FIGURE 3 Additional gene expressions that yield clues into gene function.

you have in a Peyer’s patch. This other gene is expressed actually within the central part itself, so here we have two secreted proteins that have reciprocal patterns within the same structure.

We have many genes that we have discovered that are expressed specifically in cartilage. Of the genes that are expressed in the pancreas, we have many specifically expressed secreted proteins in the pancreas. The power of this technology is that we can simultaneously be screening hundreds, if not thousands, of genes and looking for the ones that fit into specified types of patterns.

This technology is amenable to grouping genes within their site of expression, and indicating why they might be interesting and important. The data can provide important clues, and also it is incredibly pretty and dramatic as well.

We have started to put genes into classes. If we have 5 or 10 secreted proteins or their receptors that are all expressed in cartilage, we can determine if they are only expressed in cartilage or if they are actually critical master regulators of the process in which they are seemingly expressing their specificity. Not only do we do reporter knockins, but those reporter knockins we can, in a few simple steps, turn into either knockouts or into transgenics. For example, we have a growth factor receptor gene. Not only is it expressed in cartilage, but obviously it must be a critical master regulator of cartilage growth because when you knock it out, the mouse is totally devoid of cartilage. So in this manner, you define not only genes

Suggested Citation:"Papers Presented by Markey Scholars5 Functional Genomics." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
×

that are specific to the process that you want, but obviously the critical regulators of the process.

And why is something like this important? These are the exact cell types that degenerate, have problems, get worn out and that are not replaced in disease of cartilage, such as in osteoarthritis. We are looking for clinically relevant targets.

We can determine that a gene, whose discovery and function was revealed by the genetic approaches in mice that I just described to you, is linked to a human disease. It turns out that in this case we were able to identify the mutation in the growth factor system that lead to cartilage growth defects in tragically involved patients. So I think it is very nice to link the gene to a patient problem, and hopefully ultimately to approaches that could help the patients.

OVER-EXPRESSION

I have shown you many genes that are expressed in blood vessels. I will now describe the discovery of a family of angiogenic factors, not only necessary but also absolutely critical for normal blood vessel formation. Instead of getting normal blood vessels patterning, when you knockout these genes you get this rather nondescript homogenous pattern.

You read about a lot of angiogenic regulators that are supposed proangiogenic and anti-angiogenic factors. It turns out that, in terms of genetic confirmation, only the Vascular Endothelial Growth Factor (VEGF) family and the family of factors known as the angiopiotiens has been genetically validated as true master regulators of blood vessel formation. Not only can we knockout these genes, but our technology is amenable to over-expressing them as well. When you over-express the same gene that in a knockout disrupts normal blood vessels, you actually get red mice. The reason the mice are red is that they have much higher densities of blood vessels. So clearly these approaches show you not only genes that are expressed in important biological processes, but that you can mediate them and that hopefully once again, these studies will have important therapeutic implications. There are a lot of diseases where the patient suffers from ischemia, low densities of blood vessels, or other settings where you want to stop blood vessel growth. I should point out this is the first genetically altered mouse by use of a growth factor that leads to stable and apparently functional and benign hyper-vascularization.

LYMPHATIC VESSEL FORMATION

Other members of this very same family that I have shown you are not necessarily involved in the same processes—they are not expressed

Suggested Citation:"Papers Presented by Markey Scholars5 Functional Genomics." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
×

by blood vessels. They are expressed by a different kind of vessel, lymphatic vessels. Lymphatic vessels coat the intestine, and the lacteals that dive into the villi of the intestine absorb the lipids and fats that you consume in your diet.

This gene has a very interesting expression pattern, and not only does it have an interesting expression pattern, but the knockout of this gene then ends up revealing that it is a master regulator of the process in which, once again, it is specifically expressing. So it is not a regulator of blood vessel growth, but of lymphatic vessel growth. And how do we know it? The belly of the knockout is totally filled with a milky fluid known as chyle. The knockout mouse develops huge ascites and lymphedema because it cannot absorb milk and lipids from its diet.

And why is that? This growth factor is required for the normal growth of the lymphatic vessel that allows you to absorb fat from the diet. Total disruption of the pattern, particularly of these central lacteal and lymphatics, results in a lack of absorption of this milky fluid.

Once again, genetics allows one to identify and to discover a therapeutically interesting target because if a knockout can disrupt lymphatic vessel growth, perhaps we can harness the gene to grow lymphatics in settings just like these that develop in patients where you get lymphedema.

MUSCLE FORMATION

Another biologically and therapeutically interesting area that we are interested in is muscle atrophy. We identify a lot of genes by other approaches that we thought might be specific to atrophying muscle, particularly things known as ubiquitin ligases that we thought could be involved in the atrophy process, because these are components of systems that degrades protein. And we thought, “Hey, these would be perfect candidates as mediators of muscle atrophy and decay since their job is to cause degradation of protein.” The first thing that this Velocigene approach allows you to do is validate these other techniques, which are purported to identify specific genes that are activated in atrophying muscles.

The reporter knockins confirm the specificity of the muscle and their induction during atrophy. But the most important point, of course, is once again the function.

On the far left panel of Figure 4 are normal muscle fibers. In the center panel is an example of the muscle fiber size after you have induced an atrophy process such as denervation. When you knockout one of two genes, individually, you can partially rescue the atrophy. But when you do double knockouts, you almost completely prevent the atrophy (far right panel). In the double knockout you completely maintain the fiber size, showing that indeed using these approaches we can validate that we

Suggested Citation:"Papers Presented by Markey Scholars5 Functional Genomics." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
×

FIGURE 4 Muscle atrophy: knockouts of ubiquitin liga components proves they mediate muscle throphy.

SOURCE: Bodine et al. Science 294:1702; Glass et al., to be submitted.

have discovered the key-signaling components, the potential drug targets for an important process that is detrimental in a variety of human conditions.

We identified what we thought might be a key growth factor pathway that might mediate muscle growth, but how do you prove it? In this case, we did a transgenic analysis. We created a transgenic mouse that we refer to as a “mighty mouse”—all of its muscles are two to three times the normal size. We have identified genes that are involved in muscle atrophy. We knocked them out and we proved that they can prevent muscle atrophy. And we transgenically over-expressed this growth factor pathway, and we get super hypermuscularized mice.

LEAN MICE

Another example of this process is found in a transgenic of an orphan glycoprotein hormone. The epidermal fat pads in these transgenics are much smaller than normal animals. It turns out that these mice are lean. They are of normal weight, but much less of their total body weight is contributed to by fat. The thing that really is the killer for all of us is that we are exposed to a modern-day diet, which is defined as a high-fat diet. A huge percentage of the human population, and also the mouse popula-

Suggested Citation:"Papers Presented by Markey Scholars5 Functional Genomics." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
×

tion will get fat when fed a high-fat diet. It turns out that these lean mice, which are constituently lean on normal chow or on normal food, actually are resistant to gaining weight on high-fat diets. So on high-fat diets, control animals can gain up to 50 percent more weight. These transgenic animals essentially gained no more weight on a high-fat diet.

G-PROTEIN COUPLED RECEPTOR

This next story shows the power of the technology and how we could never even imagine going after a therapeutic target such as this, but the power of genetics reveals them to you.

We had cloned an orphan G-protein coupled receptor (GPCR), and we could never figure out where it was expressed in the body. It turns out that when we did these reporter knockins using Velocigene we found out they were expressed only in one spot in the entire body. Before birth in mammals the testes are actually not located in the scrotum, but in the mid part of the body. There is a ligamentous-type structure called the gubernaculum that is attached to the testes. The most common congenital abnormality in males is actually failure of descent of the testes because this gubernaculum has not contracted. Now, I have to admit I did not even know what a gubernaculum was when we did this Velocigene knockout, but when we saw this expression pattern in something only expressed in one spot, what we realized was it was staining only the gubernaculum. Maybe we have discovered a gene involved in this process. That was the case because when we knocked out this gene, we got failure of descent of the testes. In knockout mice, the testes remain halfway up the body where they originally started because this GPCR is the peptide receptor that regulates gubernaculum contraction and descent of the testes. Once again, we got a totally unexpected potential therapeutic target.

We use powerful approaches and exploit them intentionally to identify and functionalize interesting gene products, and reveal therapeutic opportunities.

AXOKINE

We have taken things to the next step. The types of approaches that we have been at now for more than a decade have actually yielded some very exciting drugs that are in our pipeline. One drug known as Axokine, for obesity and diabetes, is in the final stages of testing in patients.

Many of you have heard about leptin. It turns out that Axokine works via a leptin-like receptor and signaling pathway, but it works even in so-called leptin-resistant mice. Leptin travels from the fat in the body to a key brain region known as the arcuate nucleus, and acts as a physiological

Suggested Citation:"Papers Presented by Markey Scholars5 Functional Genomics." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
×

regulator of body weight. When you have mice or humans that are deficient in leptin they become grotesquely obese. Giving leptin to these genetically leptin-deficient mice and humans causes dramatic weight loss. The only problem is that most obesity from which at least western man suffers from is diet-induced, not genetic in nature. All of this diet-induced obesity is leptin-resistant. So when you give leptin to animals or humans who are suffering from diet-induced obesity, it has almost no effect.

We started studying Axokine for a totally different reason. Axokine is a reengineered version of a naturally occurring neurotrophic factor known as CNTF. We started studying it as a neurotrophic factor, but found rather accidentally that it was causing weight loss when given to animals and to humans. So we did a lot more work on Axokine—including cloning its receptor and understanding the signaling pathway by which it worked.

We realized that Axokine is a distant leptin relative. It uses the receptor that is a close homologue of the leptin receptor, and this receptor is expressed in the same key brain region, the arcuate nucleus, as is the leptin receptor. Because the receptor is so similar, it activates the same exact intracellular signaling pathways—the STAT3 pathway that we initially characterized for the CNTF system. Axokine is really a leptin surrogate, but its big advantage is that it superactivates this brain region. It activates the leptin-like STAT3 response in studies of leptin resistance causing known activation, but most importantly, weight loss.

Genetics, once again, provide some of the strongest validation for this in terms of the knockouts and the transgenics of both Axokine and leptin. When you give patients or animals a protein, the arcuate nucleus is about the only part of the brain that the proteins can actually access, because almost the rest of the brain is behind a blood-brain barrier. This is where these types of proteins get in, and this is the part of the brain that seems to sample the periphery and respond to weight stimuli and so forth. Both leptin and Axokine share the ability to activate the arcuate nucleus. In the arcuate nucleus these two hormones do almost exactly the same thing in the same part of the brain.

In the leptin-resistant, diet-induced models, leptin does not do anything to this part of the brain. In the leptin-resistant study where you just give the mouse a high-fat diet, the equivalent of putting him on a McDonald’s-like diet, leptin has no effect. Axokine does induce weight loss, just as we predicted based on the biochemistry that I just showed you. Once again, the most important thing to us is the translation of this to humans. Everything that we have discovered and found out about how Axokine works, and what it does on animals, is starting to be confirmed and replicated in human patients in a phase-II trial in obesity.

Obesity is rapidly becoming the biggest and most dangerous epidemic facing humankind. Obesity is becoming the leading cause of preventable

Suggested Citation:"Papers Presented by Markey Scholars5 Functional Genomics." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
×

death in the United States today. There is essentially no satisfactory medication for it. No way to cause long-term, profound, and permanent weight loss. During treatment, we can get continuous weight loss while you are on the drug, but more importantly when you stop taking the drug, you maintain your weight loss due to Axokine. This has not been seen with any other treatment before.

After only a 3-month treatment, at the end of a year, you are more than 15 pounds below the placebo group, and the hope is that you can continue to lose even more weight if you stay on the drug longer, and we are now in the midst of the phase-III trial.

SUMMARY

I have given you a brief summary of how we have been going about doing things, and how we think this approach has led to exciting findings that might indeed have an impact on human disease. But the most important thing that I have learned after all these years is that no matter what, you still can’t make your Dad happy.

Suggested Citation:"Papers Presented by Markey Scholars5 Functional Genomics." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
×
Page 29
Suggested Citation:"Papers Presented by Markey Scholars5 Functional Genomics." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
×
Page 30
Suggested Citation:"Papers Presented by Markey Scholars5 Functional Genomics." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
×
Page 31
Suggested Citation:"Papers Presented by Markey Scholars5 Functional Genomics." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
×
Page 32
Suggested Citation:"Papers Presented by Markey Scholars5 Functional Genomics." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
×
Page 33
Suggested Citation:"Papers Presented by Markey Scholars5 Functional Genomics." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
×
Page 34
Suggested Citation:"Papers Presented by Markey Scholars5 Functional Genomics." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
×
Page 35
Suggested Citation:"Papers Presented by Markey Scholars5 Functional Genomics." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
×
Page 36
Suggested Citation:"Papers Presented by Markey Scholars5 Functional Genomics." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
×
Page 37
Suggested Citation:"Papers Presented by Markey Scholars5 Functional Genomics." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
×
Page 38
Suggested Citation:"Papers Presented by Markey Scholars5 Functional Genomics." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
×
Page 39
Suggested Citation:"Papers Presented by Markey Scholars5 Functional Genomics." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
×
Page 40
Suggested Citation:"Papers Presented by Markey Scholars5 Functional Genomics." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
×
Page 41
Next: 6 Electrophysiology of Synapses »
The Markey Scholars Conference: Proceedings Get This Book
×
Buy Paperback | $45.00 Buy Ebook | $35.99
MyNAP members save 10% online.
Login or Register to save!
Download Free PDF

This is the second of five reports to emerge from the evaluation of the Markey Trust. As part of this assessment, the NRC hosted a scientific conference for Markey Scholars and Visiting Fellows in Rio Grande, Puerto Rico on June 28-30, 2002. The purpose of the conference was to enable the Scholars and Fellows to share their research experiences, just as they did at the annual Scholars Conferences previously conducted by the Markey Trust. All of the attending Scholars and Fellows submitted abstracts of their poster sessions. Six scholars, along with other experts in the biomedical sciences, made formal presentations. These proceedings consist of shortened versions of the individual presentations and the poster session abstracts.

  1. ×

    Welcome to OpenBook!

    You're looking at OpenBook, NAP.edu's online reading room since 1999. Based on feedback from you, our users, we've made some improvements that make it easier than ever to read thousands of publications on our website.

    Do you want to take a quick tour of the OpenBook's features?

    No Thanks Take a Tour »
  2. ×

    Show this book's table of contents, where you can jump to any chapter by name.

    « Back Next »
  3. ×

    ...or use these buttons to go back to the previous chapter or skip to the next one.

    « Back Next »
  4. ×

    Jump up to the previous page or down to the next one. Also, you can type in a page number and press Enter to go directly to that page in the book.

    « Back Next »
  5. ×

    Switch between the Original Pages, where you can read the report as it appeared in print, and Text Pages for the web version, where you can highlight and search the text.

    « Back Next »
  6. ×

    To search the entire text of this book, type in your search term here and press Enter.

    « Back Next »
  7. ×

    Share a link to this book page on your preferred social network or via email.

    « Back Next »
  8. ×

    View our suggested citation for this chapter.

    « Back Next »
  9. ×

    Ready to take your reading offline? Click here to buy this book in print or download it as a free PDF, if available.

    « Back Next »
Stay Connected!