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Reprogramming Stars #6: A Venture Based in Cellular Reprogramming—An Interview with Dr. Cristiana Pires

2022; Mary Ann Liebert, Inc.; Volume: 24; Issue: 2 Linguagem: Inglês

10.1089/cell.2022.29061.cp

2152-4998

Cristiana F. Pires, Carlos‐Filipe Pereira,

CAR-T cell therapy research

Cellular ReprogrammingVol. 24, No. 2 InterviewOpen AccessCreative Commons licenseReprogramming Stars #6: A Venture Based in Cellular Reprogramming—An Interview with Dr. Cristiana PiresCristiana F. Pires and Carlos-Filipe PereiraCristiana F. PiresAddress correspondence to: Cristiana F. Pires, Asgard Therapeutics, Medicon Village, 223 81 Lund, Sweden E-mail Address: cristiana.pires@asgardthx.comAsgard Therapeutics, Medicon Village, Lund, Sweden.Molecular Medicine and Gene Therapy, Lund Stem Cell Centre, Wallenberg Centre for Molecular Medicine, Lund University, Lund, Sweden.Search for more papers by this author and Carlos-Filipe PereiraMolecular Medicine and Gene Therapy, Lund Stem Cell Centre, Wallenberg Centre for Molecular Medicine, Lund University, Lund, Sweden.Search for more papers by this authorPublished Online:4 Apr 2022https://doi.org/10.1089/cell.2022.29061.cpAboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail Dr. Cristiana F. PiresReprogramming star:Dr. Cristiana F. Piresis CEO and co-founder of Asgard Therapeutics. Asgard Therapeutics is a Swedish biotech company focusing on the development of groundbreaking cancer immunotherapies based on direct cell reprogramming technologies. Formed as a spin-off from Lund University, the company is pioneering a cancer gene therapy approach based on its proprietary TrojanDC technology. This is based on the expression of three transcription factors that convert somatic cells into dendritic cells. When reprogramming is applied to cancer cells, it induces efficient presentation of cancer own (neo-)antigens to the immune system, eliciting potent anticancer immune responses. Designed as an off-the-shelf gene therapy, Asgard's approach overcomes many of the logistic and manufacturing hurdles of conventional cell-based therapies.Introduction by Dr. Carlos-Filipe Pereira (Editor-in-Chief, CELLULAR REPROGRAMMING)Dr. Pereira: Good morning. My name is Filipe Pereira, Associate Professor at Lund University and Editor-in-Chief of Cellular Reprogramming. I'm very happy to bring you a new episode of Reprogramming Stars, our flagship series capturing the findings, projects, and ideas of the leaders in cellular reprogramming. Today we have a special episode: It's our first episode focusing on a company developing products based on cellular reprogramming. So, I am very happy to have Dr. Cristiana Pires, CEO and Co-Founder of Asgard Therapeutics in Lund, Sweden.Asgard Therapeutics is a spin-off from Lund University, developing cancer immunotherapies based in cellular reprogramming. Cristiana is a former trainee from my lab, the Pereira lab, where she performed her postdoctoral studies working in dendritic cell (DC) reprogramming strategies to modulate immune responses, which then led to the creation of Asgard Therapeutics. Before that, Cristiana trained in pharmaceutical sciences and performed her PhD studies in Miguel Seabra's Laboratory at Nova Medical School in Lisbon, Portugal. During her PhD, she explored direct reprogramming to generate retinal pigment epithelial cells for cell replacement therapy applications. Now as CEO, Cristiana has attracted a seed round of investment of €6 million, and more than €1.5 million in nondilutive funding, including the Horizon 2020 SME instrument and Eurostars grants. Dr. Pires, thanks so much for joining me today, it's a pleasure to have you featured as a reprogramming star.Dr. Pires: Thank you, Filipe, it's my pleasure to be here, and I'm honored to participate in the Reprogramming Stars interview series.Dr. Pereira: Can you tell us how you became CEO of Asgard Therapeutics?Dr. Pires: It has been an exciting challenge! It was never my goal and not even a dream to become a CEO of a spin-off company. I was always passionate about understanding diseases and how we could develop new treatments. That's what motivated me to study pharmaceutical sciences. But I also liked research and the prospects of contributing to new knowledge. So, I decided to do a PhD. At the time, I was looking at the gene therapy field, and I approached Miguel Seabra's Laboratory (CEDOC—Chronic Diseases Research Center, NOVA Medical School, Lisbon, Portugal) because they were developing a gene therapy for choroideremia, an X-linked retinal degenerative disorder that affects males at a young age. That line of research was already well advanced in preclinical development and led to a gene therapy product currently in Phase III clinical trials.And so, Miguel challenged me: why not explore cell reprogramming to generate cells of the retina that could be used for transplantation in patients with well-advanced degeneration of the retina and not eligible for gene therapy. I had been following with interest the concepts of the cell reprogramming field and the prospects with induced pluripotent stem cell (iPSC) technology. I decided this was a good challenge, and my PhD project came to life. As I joined the lab, the first paper I presented at journal club was Marius Wernig's paper on direct reprogramming on the induction of neuronal cells. My project was then centered in generating retinal pigment epithelium cells not only by differentiation of iPSC but also by direct reprogramming.At the end of my PhD, I took some time to reflect on what I should do next. I was on the edge between moving to industry and exploring my pharmaceutical background or staying in science. I found my sweet spot somewhere in the middle: still doing science, which is what excites me, but thinking on translational perspectives. I joined your group, Filipe, and together we started to develop direct reprogramming strategies to generate cells of the immune system. So, the aim is no longer to generate cells for transplantation, but rather to use cell reprogramming to modulate and kickstart immune responses (Pires et al., 2019).This is the work we've been developing since 2016, and it focuses on specific cells of the immune system. DCs are antigen-presenting cells—key players in driving immune responses and activating effector cells, such as T cells, against specific antigens. We thus developed a technology to induce DC fate, published in 2018 in Science Immunology (Rosa et al., 2018). This study opened several translational opportunities to develop new treatments exploring the DC reprogramming approach. And then, it kind of evolved naturally! As we wanted to develop our technology to the point where we could bring it to patients, we decided to create a company to give us the tools to achieve this goal in an efficient way. I stepped up as a CEO of Asgard Therapeutics, which was created in 2018 as a spin-off of Lund University, and I've progressively transitioned from the academic setting to the company.Dr. Pereira: Thank you very much for the insight. Your career path illustrates quite well how the cellular programming field has broadened in scope during the last years. How was it going from a lab where you focused on a particular disease, and cellular reprogramming providing a tool to generate cells, to a lab where you mainly focus on reprogramming problems and then focus on their potential?Dr. Pires: That's a good point. During my PhD, the lab where I was working focused on intracellular trafficking and the machinery regulating those processes. I was a bit of an “alien”—no one else was working in cell reprogramming. I was trying to generate the cells we're interested in at the lab for disease modeling and therapeutic purposes. But in the end, cell reprogramming was what really excited me—how does this mechanistically happen, how do you regulate cell identity? Then, for the postdoc, I was looking for a lab where I could focus on the process and mechanisms governing the conversion of one cell identity into the other. We started studying DCs, how they develop, and what transcription factors are involved in their specification. And only after we developed the reprogramming method did we start to think about the therapeutic application in detail. So, for me, it worked best starting from the process and then focusing on developing it further, but I believe both ways are complementary.Dr. Pereira: I know that Asgard combines cellular reprogramming and cancer immunotherapy, but tell us a bit more about the aims and long-term vision of the company. What is Asgard aiming to achieve?Dr. Pires: Well, we are ambitious … Asgard's vision is to become a world leader in cellular reprogramming, with a specific focus on DCs and antigen-presenting cells. For now, we are focusing on cancer, given the crucial role of DCs in inducing antitumor immunity. In the future, we will expand to other applications such as infectious diseases and autoimmunity where patients could also benefit from DCs or antigen-presenting cells. The overall idea is to start from good foundational science and develop the technology until we bring it to the patients who might benefit from these therapies.Dr. Pereira: So, what's the pillar technology of Asgard? What's the scientific foundation?Dr. Pires: It all started when Fábio Rosa and I identified a combination of three transcription factors—PU.1, IRF8, and BATF3—which, once expressed in a somatic cell, can rewire its identity to an antigen-presenting DC (Rosa et al., 2018). We screened more than 30 different transcription factors using mouse embryonic fibroblasts carrying a Clec9a reporter system. We used Clec9a because in hematopoietic cells, it is specifically expressed in the DC lineage.Recently, we have evolved these studies to make it efficient from human cells (Rosa et al., 2022). We have optimized how we overexpress the transcription factors and supplemented the process with other signaling molecules to improve the efficiency of DC reprogramming. Moreover, we dissected the mechanisms of action, looking into how the transcription factors engage chromatin and to which sites. We observed that the three factors cooperate and engage chromatin with PU.1 binding first and then recruiting IRF8 and BATF3 to its targets, inducing downregulation of fibroblast genes and upregulation of the DC genes.Thinking about how we could translate these findings, one option is to use this process to generate DCs ex vivo from accessible cell sources from patients, such as skin fibroblasts or mesenchymal stromal cells. Then, induced cells can be reinfused back to the patient after loading with tumor antigens, for example. This would be an attractive alternative to current sources of DC vaccines, which have been explored during the last 20 years in the clinic, such as DCs generated in vitro by differentiation of progenitors or monocytes. This process generates a mixture of different DC subsets with conflicting functions or cells that are inefficient in antigen cross-presentation and induction of CD8+ T-cell responses. In contrast, in our reprogramming approaches, when we overexpress PU.1, IRF8, and BATF3 we do not induce a mixture of cells. Rather, we specifically induce a defined subset of DCs, which are called conventional DCs type 1 (cDC1) that are efficient in the cross-presentation of antigens.Dr. Pereira: So, this really could enable cDC1-centric DC vaccines?Dr. Pires: Yes, recent data points out that the cDC1 subset is the most attractive subset for induction of antitumor immunity (Zhang et al., 2021). cDC1 are rare cells and thus cannot be isolated from peripheral blood in sufficient amounts, especially from cancer patients. Differentiation protocols also fail to give rise to a pure population of this specific subset. Thus, using cell reprogramming to generate the cDC1 subset can be a game changing for the field of DC vaccines. In this case, it would be an autologous cell-based therapy, which is associated with logistical challenges and high manufacturing costs because each patient needs to have a batch of cells. This is a common problem in the field of cell reprogramming and cell-based therapies, with allogeneic sources or cell banks being explored as alternatives.At Asgard, we decided to focus on using the transcription factors for in vivo reprogramming. For this, we are developing a gene therapy approach: a viral vector encoding the three reprogramming factors to co-express them in vivo in cancer cells. This will force them to be reprogrammed into immunogenic cDC1 cells. We are reprogramming the actual tumor to present their own tumor neoantigens and, by doing this, inducing an immune response against the neoantigens of that specific patient. This is an off-the-shelf gene therapy approach, always the same product, but the induced immune response is dependent on the tumor antigens that are presented by each tumor—thus, it will be personalized. This is a unique feature of TrojanDC: off-the-shelf and at the same time personalized.Dr. Pereira: So, turning cancer against itself with cell reprogramming?Dr. Pires: Yes, that's exactly how we explain it and why we named the concept TrojanDC, inspired by the Trojan Horse mythology.Dr. Pereira: And why Asgard? Where does the name come from?Dr. Pires: It's also a mythology-inspired name. In Norse mythology, the Trojans, after being defeated by the Spartans, migrated north. They became gods and created a fortified city resembling Troy. This city was named Asgard. And that's why we named the company Asgard. It's also a funny resemblance to the fact that the three founders are Portuguese who migrated to the north, to Sweden. In a sense, we are telling the world that we are embracing this Nordic identity now.Dr. Pereira: Can you tell us more about the current stage of development? What is Asgard doing now in terms of projects and operations?Dr. Pires: So, the company was created in 2018 as a spin-off from Lund University, just after our paper in Science Immunology. For a long time, the company existed in a semi-virtual mode. We were doing scientific work in the academic setting, but the company already existed and started attracting grants and nondilutive funding to develop the concept and business idea, including European H2020 SME instrument and Eurostars grants. We also received support from Novo Nordisk Foundation with exploratory and preseed grants to develop the scientific project until a point that was interesting to present to investors.In November 2021, we closed a €6 million seed round of investment, co-led by three major investors: Novo Holdings, as one of the investment arms from the Novo Nordisk Foundation, Boehringer Ingelheim Venture Fund, a corporate VC, and the Swedish investor Industrifonden. The aim of this funding round is to develop the in vivo reprogramming platform. We have proof-of-principle data showing that cancer cells can be reprogrammed into antigen-presenting DCs, but we need to select the optimal vectors further to guarantee an efficient reprogramming of cancer cells in vivo.One of the objectives is also to build the company. Now, we are located at SmiLe Incubator, which is part of Medicon Village in Lund, surrounded by life sciences companies and service providers. We are also growing the scientific and executive teams and attracting key opinion leaders and advisors to the board of directors and scientific advisory board. At the end of these two years, we will be ready to progress the IND-enabling studies and start producing GMP batches of the vector in preparation for clinical trials.Dr. Pereira: What are the key milestones that Asgard needs to accomplish for TrojanDC to be tested in humans?Dr. Pires: There's a lot of work to be done when you're developing a new cancer immunotherapy. One of the crucial ones is to define the ideal vector platform to overexpress the transcription factors in vivo. As this is not a typical gene therapy approach, we need to make sure that we induce the reprogramming process in the most efficient way. And then of course, we also need to dissect the antitumor immune response that is induced after reprogramming. In addition, to enter clinical trials, there are several regulatory requirements.This includes, for example, CMC studies—how we are manufacturing the viral vector at GMP grade and what the quality attributes are of the product that will be tested in the clinic. We will also perform safety studies in a controlled way, which are called the GLP tox studies to show the regulatory agencies that the approach is safe to be tested in the patients. Overall, I would expect the process to last three to four years before we reach clinical stage.Dr. Pereira: That's an exciting prospect, and I wish you the best of luck. As this is the first corporate Reprogramming Stars, it'll be interesting for the audience to understand how cellular reprogramming is now being perceived by investors. Are there other companies exploring cellular reprogramming paradigms right now, and how are investors in life sciences responding?Dr. Pires: After Yamanaka's discovery in 2006, investors understood the huge potential but were perhaps a bit skeptical in investing straight away. Then, I think gradually, with clinical trials in which retinal cells differentiated from iPSC have been started to be used in the clinic, confidence in the cell reprogramming field started growing (Yamanaka 2020). The number of treated patients was still reduced. It was a proof of concept—a stepping-stone in the right direction. Then, other companies started to explore the approach. Fate Therapeutics, for example, has a clinical program using natural killer cells from differentiation of iPSCs.More recently, there are some companies really focusing on cell reprogramming, for example Mogrify and Bit.bio in the United Kingdom. Mogrify focuses on identifying transcription factor combinations for direct reprogramming and exploring induced cells for ophthalmology for example. Bit.bio explores forward reprogramming, starting from iPSCs and forcing differentiation into the right cell subset using transcription factors. Even more recently, there's been this huge interest in Altos Lab, which received something in the region of $260 million in funding and is exploring cell reprogramming for rejuvenation.All in all, I think investors are looking into cell programming strategies, and there's a growing excitement in the field. I believe that with the right investment, we will see cell reprogramming coming to fruition. It's my expectation that in the next 5–10 years, we can really develop it into several clinical programs.Dr. Pereira: As Editor-in-Chief of Cellular Reprogramming, it's great to see excitement from the investors' point of view. It has been a fast-moving field in the last 20 years, but it looks like it will continue to be just as exciting! With major developments on both the academic and corporate sides, how do you look at collaboration? Is collaboration critical for a startup to succeed, and which type of collaborations are you establishing?Dr. Pires: That's a good point. I think, in general, no one is an expert in everything. I believe that it's better to look out for the right expertise to complement our own. At Asgard, we nurture different types. In the case of Eurostars, it's a consortium project in collaboration with an academic group but also with contract research organizations from other countries in Europe, which have expertise in in vivo and 3D organoid models that are interesting for us to test the direct reprogramming approach. We also have other grants and collaborations with academic and clinical groups, in Sweden and Denmark, to provide and test the DC reprogramming (our lead program TrojanDC) in primary cells from cancer patients. For other exploratory concepts that contribute to the pipeline of the company, we usually prefer to collaborate with academic groups. I think there's a bit more flexibility in how you approach problems on the academic side.In spirit, collaborations work similar to the academic setting. Of course, there needs to be a bit more caution in certain aspects if you are collaborating as a company. It's important always to have paper track and formal collaboration agreements in place to make sure that it doesn't compromise future investment. We must agree with the partners on how results, and potential intellectual property (IP), is regulated and owned by the different parties.Dr. Pereira: That's a good way of putting it. Thinking about the postdocs who are finishing their projects and want to become CEOs as well, what does it take to raise a €6 million round of investment in Europe?Dr. Pires: Well, it takes a lot of work, and it's important to be persistent with your goals. I have to say that I'm the CEO—in a sense, I'm the face of the company—but this is the result of many brilliant people working at the Pereira lab. So, the first thing of course: it's the team. I was really lucky because I had Fabio Rosa, currently Head of Research of the company, always supporting me, but also my mentor, Filipe Pereira, helping on the process. Having a good network is also critical. We connected with everyone around us who had been involved in any way in the process of taking a scientific technology into a company or even into the clinical setting. We are lucky to be here in Lund and South Sweden.There is also a good network around across the bridge in Copenhagen. For example, we enrolled in a Nordic entrepreneurship mentoring program (NOME), in which I have regular meetings with experienced mentors, with whom I can discuss my current challenges and get their input in how to take the next steps. Then, you need to build the business idea and business concept and start talking with investors. It's a trial-and-error process—a constant iteration.I would say that we approached 40–50 different investors and potential pharma partners. We need to build a relationship with them—frequent meetings with them to show progress. I have “pitched” the first time and received feedback, and honestly sometimes the feedback was not super positive at the beginning. And then, I have reflected on it and tried to improve it—I did it again until I got better and better at understanding what they want to see and how to explain it. When you get to the point that they are really interested, we discuss the scientific and business plans, but also then the legal investment terms. And that perhaps is not so fun if you're a scientist, but it comes with the game. Now, after we have closed the round, we are finally doing the experimental activities we planned for with a larger team and additional resources.Dr. Pereira: For the younger reprogramming scientists starting their PhD now, ambitious to follow your career path, do you have any specific advice?Dr. Pires: Well, if your vision is to become CEO of a company based in science, focus on the science. If you're doing your PhD, I wouldn't think too much about the company at this time. I would just focus on the science and make sure you develop something that is really robust—a really exciting finding. Then, once you've nailed that and you have your publication to finish the PhD, you can start looking at whether your findings can be explored from a commercial perspective—for a new therapy or new diagnostic tool, for example. I think it's always important to focus on the science and not the other way around, but this is perhaps my personal take on it.Dr. Pereira: But it's also important to focus on a scientific problem that will be exciting in 5–10 years, right? In a way, predicting the future!Dr. Pires: Yes, which is quite hard. If you focus on solving a specific problem, when you get to the answer, someone might have solved it already. So, I would focus more on the scientific question, as you are mentioning, and then try to see what it can be used for. I would just stress one crucial aspect for having an investment in the end: make sure that you have your IP protected before you present the results. I think it's important to bring this awareness around IP rights to the scientific community. It doesn't mean you cannot publish; it just means that you need to file a priority application before you publish the paper or before you disclose important details of your process, in scientific conferences for example.Dr. Pereira: So, at the end of the day, it's also about bringing something new to the table that could be exciting to be tested in humans, and there is enough supporting science for people to commit to see the results, right?Dr. Pires: Yes. And of course, many of the ideas and projects are not successful in the end. But I think we need to be passionate about the process as well. And of course, if you try once and it doesn't work, you can always go back and try to improve it. So, I think that's the overall process of it.Dr. Pereira: Thanks so much! I would like to close the interview with some questions that are not strictly related to science so that the audience can get to know you better. So, the first question would be: What's the best piece of advice that you've ever been given? This could be professional or not.Dr. Pires: Well, the best piece of advice is a bit hard to pinpoint. I have received a lot of advice over the years. More personally, my father always says that “First, you need to work, and then you can party.” So, I think this shaped who I am—for not being frightened by hard work but also dreaming big. And then, I had a mentor who always said, “We need to be looking for Eureka moments!” When you get really excited with the prospects of discovering something new that no one has ever seen. And I think it is important when you're doing science that a lot of the work needs to be repeated in a methodical and sometimes a bit tedious way. Then there are these rare occasions in which one critical experiment shows us that it is really working! We can use that energy to serve as an inspiration for the long months ahead of developing it further.Dr. Pereira: What is the biggest misconception about science or a science-based startup company that you would like to see resolved?Dr. Pires: I participated in a course for master's and PhD students at Lund University about entrepreneurship. Some of those students thought that the motivation to embark in such a challenge was monetary … or that you must be really crazy because there's no safety in the process. I have to say that money is not the reason. We have been working to attract funding, but it's for the company—to fulfill the dream as scientists of contributing to developing a new treatment. So it's all about having money to do the right science that can have an impact on society later on. That's the whole vision, and that's what motivates us.And the other one is about the risk. Well, of course, if you look at the statistics of startups that failed, it's daunting. There's a really small percentage of companies that are successful. But personally, I see it as a learning opportunity. I'm learning how to do science, but I'm also growing as a CEO and developing other capabilities of managing a team and the company, learning more about IP, regulatory processes, and so on. After this process, I'll be very prepared for several jobs in industry, whether this is in a new startup, pharma company, an IP firm, a VC investment firm—there's plenty of new opportunities that can arise.But the critical misconception I would say is that when you start doing entrepreneurship courses and developing business ideas, we learn that we need to explain what the problem is we are solving, what the market is, how many patients can be treated, and what will be the cost. And I'd say it's important—it's like a checklist to show investors that you understand the process. But at the end of the day, what they are really interested in is the science. And I've had more detailed scientific discussions with investors than I've ever had with the academic community. Even if you go, for example, to present at a scientific conference, you never get the same detailed and thorough scientific discussions…Dr. Pereira: A much higher level of scrutiny, right?Dr. Pires: Yes, much, much higher I would say. So that's why I keep stressing that in the end, focus on your science. If the science is good, if it's exciting, then you are on the right track to have some investment come out of it.Dr. Pereira: Dr. Pires, thanks so much for joining me today, and thank you for all your insight. I think this will be really useful for the cellular reprogramming audience and for the careers of the cellular reprogramming scientists in industry. So, thank you so much!Dr. Pires: Thank you. It was my pleasure.ReferencesPires , C.F., Rosa , F.F., Kurochkin , I., and Pereira , C.-F. (2019). Understanding and modulating immunity with cell reprogramming. Front. Immunol. 10, 2809. Crossref, Medline, Google ScholarRosa , F.F., Pires , C.F., Kurochkin , I., Ferreira , A.G., Gomes , A.M., Palma , L.G., Shaiv , K., Solanas , L., Azenha , C., Papatsenko , D., Schulz , O, Reis e Sousa , C, Pereira C-F. (2018). Direct reprogramming of fibroblasts into antigen-presenting dendritic cells. Sci. Immunol. 3, eaau4292. Crossref, Medline, Google ScholarRosa , F., Pires , C.F., Kurochkin , I., Halitzki , E., Zahan , T., Arh , N., Zimmermannová , O., Ferreira , A.G., Li , H., Karlsson , S., and others. (2022). Single cell transcriptional profiling informs efficient reprogramming of human somatic cells to cross-presenting dendritic cells. Sci. Immunol. 7, eabg5539. Crossref, Medline, Google ScholarYamanaka , S. (2020). Pluripotent stem cell–based cell therapy—promise and challenges. Cell Stem Cell 27, 523–531. Crossref, Medline, Google ScholarZhang , S., Chopin , M., and Nutt , S.L. (2021). Type 1 conventional dendritic cells: ontogeny, function, and emerging roles in cancer immunotherapy. Trends Immunol. 42, 1113–1127. Crossref, Medline, Google ScholarFiguresReferencesRelatedDetails Volume 24Issue 2Apr 2022 Information© Cristiana F. Pires and Carlos-Filipe Pereira, 2022. Published by Mary Ann Liebert, Inc.To cite this article:Cristiana F. Pires and Carlos-Filipe Pereira.Reprogramming Stars #6: A Venture Based in Cellular Reprogramming—An Interview with Dr. Cristiana Pires.Cellular Reprogramming.Apr 2022.57-62.http://doi.org/10.1089/cell.2022.29061.cpcreative commons licensePublished in Volume: 24 Issue 2: April 4, 2022Open accessThis Open Access article is distributed under the terms of the Creative Commons Attribution Noncommercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.PDF download