#sciencekaleidoscope

CRISPR/Cas9

Faster, cheaper and more accurate than any other previously developed method of engineering DNA. CRISPR/Cas9, based on the mechanisms of bacterial defence system, enables scientists to modify and replace genes. The technology was awarded the Breakthrough Prize of 2015 by the Science Magazine and sparked heated public debate on numerous occasions. Here’s why #CRISPR is still trending in media. CRISPR was used to edit human embryos The two papers, both published by Chinese scientists, reporting the use of CRISPR/Cas9 for genetic modification of human embryos stirred controversy around the world. The first group to cross this scientific line was led by Junjiu Huang and published their results in the online journal Protein & Cell (1). Although the authors used non‑viable human embryos (so-called 3PN embryos) to minimise ethical concerns, the paper was still rejected by top journals such as Science or Nature partly due to ethical objections. The team attempted to edit the gene responsible for β-thalassaemia, but reported low efficiency and many off-target mutations (called indels). The second, very recent publication, reported a number of similar technical problems associated with the use of CRISPR/Cas9 (2). Interestingly, this time the gene-editing technology was used to modify the form of CCR5 gene allele to reduce human susceptibility to HIV. The method, however, yielded only 5-15% efficiency and a number of unwanted mutations of CCR5 gene. As much as the idea of a human race that is completely resistant to HIV infection and free from any genetic disorders may appeal to some of the readers, CRISPR/Cas9 technology is still not ready to be used for clinical purposes. Low efficiency and the risk of unwanted off‑target mutations would result in a significant loss of viable embryos and other unpredictable developmental consequences that will be passed on future generations. This adds a scientific issue to the bigger view on human gene editing focused mainly on ethical concerns about designer babies. Generation of genetically modified humans remains strictly prohibited, and there is no indication that this will change any soon. If anyone is particularly interested in this idea, a book GMO Sapiens: The Life-Changing Science of Designer Babies seems to be a good read. Scientists in China are not the only ones interested in genetic modification of human embryos. Kathy Niakan, a stem cell scientist from London’s Francis Crick Institute, was given a license to carry out similar experiments. However, the aim of the Niakan’s investigation is purely for research, rather than clinical purposes. The group proposes to study the first 7 days of a fertilized egg to deepen the understanding of how the human embryo develops. Such experiments are expected to have an overwhelming scientific impact that may lead to the development of greater ways for prevention and treatment of developmental disorders and an improvement of IVF technology. Gene editing to produce modified human embryos, even if only in laboratory environment, is nonetheless very controversial from the view of general public. Will the next researchers take a step further and try to deploy CRISPR/Cas9 modification in the clinics for reproductive purposes? Who owns CRISPR/Cas9? Although some insist that CRISPR technology should not be patented at all, the patent dispute between Jennifer Doudna (University of California) and Feng Zheng ( Massachusetts Institute of Technology ) has been the subject of major attention. Patents go to the first to invent something and MIT paid for an accelerated review of its patent application. Zheng was then awarded the first patent for CRISPR/Cas9, which UC later claimed. The case is still awaiting a resolution with the most probable scenario being one of the sides to provide the earliest lab records for the discovery of CRISPR. This patent litigation is considered to be challenging and complex even for the most experienced patent practitioners. By bringing the attention of the attorneys and biotechnology industry interested in the invention, the popularity of CRISPR/Cas9 is spreading far beyond research institutes. Ideas.ted.com The lesson coming from all CRISPR/Cas9 stories is the importance of patience, in-depth consideration, but also critical thinking towards the breakthrough biomedical discoveries that can directly affect the human health. To avoid misuse and disappointment, the clinical application has to be approached step by step while supported by strong scientific evidence. In the case of CRISPR/Cas9, we are still far from seeing its full medical potential. Magdalena Plotczyk References 1. Liang P, et al. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell. 2015;6(5):363–72. 2. Kang X, et al . Introducing precise genetic modifications into human 3PN embryos by CRISPR/Cas‑mediated genome editing. J Assist Reprod Genet. 2016

Amniotic Membrane Transplantation in the Human Eye

Corneal disease is a major cause of blindness worldwide, second only to cataract, and its epidemiology involves an extensive variety of infectious and inflammatory eye diseases that can also be highly dependent on the geographical area under study. To place the disease into perspective, according to a report by the WHO, there were 45 million individuals worldwide in 2001 who were bilaterally blind and another 135 million that had severely impaired vision in both eyes. As a response to this, there are several treatments that range from the use of topic anti-inflammatory drugs or antibiotics, to corneal transplantation –more specifically, amniotic membrane transplantation –, which is the subject of this brief discussion. The amnion is the sac surrounding and protecting the developing embryo. Ever since 1910, when a surgeon first reported the use of fetal membranes as surgical material in skin, the use of amniotic membrane in surgery has been revised. In the following decades, numerous researchers reported the advantageous role of the amniotic membrane for the treatment of a variety of ocular surface disorders, without a clear or approved medical application, resulting in the abandonment of the technique. Nevertheless, recently, it has been making its way back into ophthalmological practice. The relevance of the subject is the scope that the application of amniotic membrane transplantation (AMT) in the management of ocular surface disorders offers. There are a number of applications for AMT given its transparency, low cost, availability as a surgical spare tissue, non-immunogenicity in a cryopreserved state, anti-inflammatory properties, and the natural antimicrobials present in its epithelial layer. Also, amniotic membrane contains growth factors and expresses anti-inflammatory cytokines that accelerate the healing of the tissue. Among the indications in ophthalmology are the persistent epithelial defects unresponsive to medical treatment, and its use as an alternative to conjunctival flaps, botulinum toxin injection, or tarsorrhaphy; with a very high success ratio. Regarding tissue engineering methods, AMT has a potential use in combination with limbal transplantation in patients with diffuse limbal stem cell deficiency and in diseases such as Stevens–Johnson syndrome or chemical and thermal burns. Generally, for the preparation of the membrane, after harvesting human placenta from a healthy woman during a cesarean section, the amnion is isolated from the chorion, washed in sterile physiological solution, put on nitrocellulose membrane fragments, and cryopreserved. The amnion as a scaffold with an intact epithelial layer has been proposed as an optimal choice for clinical usage since it has been proven to accelerate epithelialization of eye defects by promoting the migration of epithelial cells, their adherence to the basement membrane, and differentiation; along with the prevention of apoptosis. It also induces cell proliferation by secreting molecules with immunoregulatory and angiogenic properties. However, as every technique, amniotic membrane transplantation in the human eye also has its limitations. One of them is a shown failure in the case of its use when impending or recent corneal perforation is present. Another example would be the higher rate of recurrence – almost five times higher – of pterygium when compared to an autologous conjunctival graft during the removal of pterygia (bearing in mind that it could be used as an alternative in some specific cases). Moreover, the beneficial effect of amniotic membrane in the management of ocular surface disorders is still in need of well-designed clinical trials to obtain more solid information. Therefore, clinicians should be judicious when considering AMT applications. By Tania Choreño Machain

Why the ‘Game of Thrones’ in medical science?!

—Sparshita Nag Can you imagine the video game giant Nintendo being as successful as it has been, without its dedicated team of game developers? On the flipside, do you think Mario would’ve become a household name without the phenomenal execution of marketing strategies by Nintendo? I’m sure everybody agrees that this company or its team of R&D would not have existed at all had it not been for a steady base of pint-sized consumers going crazy over how to help Mario rescue his princess! In crude economics terminology, the source/inventor, mediator and benefactor are inter-dependent on each other constituting a universal system so as to be able to maintain equilibrium in the demand-supply curve. Now let’s shift the frame to something more familiar. How about Medicine? Sounds good? Well….In a world full of morbidities and fatalities, putting myself in the shoes of a patient honestly makes me bow down in gratitude before God for creating Doctors. Really, what would a Malaria patient in Africa or a RTA (Road traffic accident) victim with fracture shaft femur (a broken thigh bone) or a cancer patient anywhere in the world do without doctors? Boy this does feel good to write (read ‘it bloats my ego like never before!’) given I am an MBBS myself….conducting normal deliveries, resuscitating a newborn with respiratory distress, diagnosing an acute myocardial infarction and thrombolysing the patient to alleviate pain, assisting in immobilizing that heck of a broken arm or even suturing that gaping wound, I have done it all! I am also proud to have colleagues all around the world working in the harshest of conditions away from home and family to relieve the world of suffering. We save the world! Applause! Nevermind that there is still a significant number of conditions that we ‘cannot cure’ simply because the treatment is ‘Not Available’ (Read that again). Some wise saintly friend (my Conscience) once commented, “What if you did not have pharmaceutical drugs, diagnostic equipment or any surgical tools? Would you still be able to call yourself an independent medical practitioner or cure any of the conditions stated above? In essence, will you be able to deliver health to an ailing patient as easily as you do now?” This really got me thinking…Well, my conscience was right. After all, the biomedical engineers design surgical tools/diagnostic equipment, pharmacology researchers develop drugs which follows leads from what basic scientists discover after toiling for hours in their laboratories about the physiology and pathology of the organ system under study. I agree that we doctors do relieve a patient’s suffering and have an inhuman work pattern at times but at the end of the day, we are the mediators of what is being discovered/invented in the labs. Now this again makes me bow down with gratitude before God…only this time it is for Doctors as well as a wee bit for Medical Researchers too……(A tight slap on my face from my Conscience followed!! Whatt?? Why??)…oooookay….a 70-30 gratitude share for docs and researchers….that should do it? (A persisting venomous look from my Conscience)….ugh 60-40?! (Conscience progresses to a Parkinsonism-like masked facies…probably saw a slight lateralisation of lips too)…..alright…alright…50-50. And that’s it! I do acknowledge that we depend on the research fraternity for what we deliver, but even they depend on us for translating their research…so equal gratitude fits best here…(my Conscience finally smiles brightly!)…..time for a secret here…I still am as happy as I was before, even after pulling down the gratitude levels for docs….Reason? I am currently a medical researcher in-the-making!! Surprise!! As insensitive as this may sound, a word of gratitude to the patients for falling sick. After all it’s them that keep the medical profession alive and kicking and fuel medical research. May God bless one and all with good health, but after a bout of treatable sickness. No offence intended! Guess what? My elation continues…..because I happen to be a patient too! Anyone can be! (Don’t worry I won’t bow down before God this time! I don’t want a broken back now) So what is the take home message then? We all are part of a system in our professions with varied roles as an inventor, an executor/mediator or a benefactor. We are dependent on each other to serve a greater cause, which in case of medicine is fighting the disease. Like one of my friends summed it up, medical research is like catching that specific fish from the high seas while medical practice is akin to the fish-market both catering to fish-lovers!! Funny but undeniably true! So rather than getting sucked into the unfortunate reality of fierce professional rivalry over who gets more patients and perks (Doctors, are you listening?) or who has more journal publications/H-index to their credit (Attention Researchers!!) or even waging a war between these two professions to establish dominance like our animal brethren do, life would be easier if we all learn the art of mutual co-operation and knowledge transfer in addition to doing our bit to contribute towards the greater cause of eliminating sickness from the face of the world. We all are a team. Let’s keep it that way. Let’s just do away with this ‘Game of Thrones’ now! However if competing is in your nature, why not do it with yourself? As for the fact that we may very well be out of work in absence of sickness, let us not underestimate the efficient adaptive mechanisms of the creepy-crawly microbial beauties! Rest assured, cuz no amount of Dr. Mario would probably ever be enough to stop them forever! ———————x———————-x———————