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V O LVolume U M E 4 . 4. I S SIssue U E 2 .2.F February E B R U A R Y 2017 2017

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Gene Therapy as a Potential Pathway to Reverse Autism

CRISPR: From Food Industry to Human Gene Therapy

SLENDR: the CRISPR add-on for the brain

Victor Lunar Quintero

Shagun Gupta

Carol Chen











Table of Contents


SLENDR: the CRISPR add-on for the brain Shagun Gupta


Gene Therapy: A Promising Weapon in the Fight Against Cancer Denitsa Vasileva


Could Eye Drops be the Solution to an Effective Non-Invasive Administration of Gene Therapy? Abeer Wasim


Gene Therapy in Psychiatric Disorder Menghan Wang


Fate-Altering Therapies Aquila Akingbade An interview with Dr. Tod Tiele: How do neuroscientists use CRISPR to answer questions about the brain? Parandis Kazemi



Triple Trouble: Ethical Issues with Three-Parent Children Terese Mason Pierre




CRISPR: From Food Industry to Human Gene Therapy Victor Lunar Quintero

Gene Therapy as a Potential Pathway to Reverse Autism Carol Chen



Gene Therapy in Neurodegenerative Diseases Vanessa Gomes


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The age of the genome opened our eyes to the sea of genes and proteins involved in producing and sustaining life. The potential to discover genetic causes of diseases led many scientists to pursue genome-wide association studies, also referred to as GWAS. As technology improved, we could sequence an individual’s DNA much faster, and at a lower cost. The last step was to make sense of all the information that was being generated. At this point, researchers have made countless correlations between genes and diseases. Many of these candidate genes are being pursued in labs across the world. However, to study their exact role in a disease, we need to generate models. Models that overexpress genes. Models that lack genes. Models that carry single nucleotide polymorphisms (or SNPs). To create these models, science has turned to gene editing. For years, science has made use of known mutagens such as UV radiation and chemicals to try to change the sequence of DNA. Site-directed mutagenesis and recombination techniques were employed to introduce more selective mutations, but neither were very efficient. Slowly, we developed our knowledge of DNA repair mechanisms and made use of nucleases to cut and paste smaller fragments into genes. Eventually, we honed our skills and created more specific ways to edit DNA, including: zing finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALENs), and probably the most talked-about, CRISPR-Cas. In this issue, we look at the potential for gene therapies to treat a variety of conditions ranging from Parkinson’s Disease, to cancer. We also explore the discovery of the CRISPR-Cas system and hope to explain why researchers are excited by the technology. In addition to gathering student opinions, we were also fortunate enough to sit down with Dr. Tod Thiele, an active neuroscience at the University of Toronto Scarborough, to hear his opinion on the matter. Our submissions editors and contributors have put a lot of work into this issue and we hope that each of you can learn about how gene editing is being used. You might even be inspired to incorporate some of these techniques in your own research in the future! Priscilla and Ann Editors-in-Chief

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...the PGC1-alpha gene was inserted and injected directly into the aforementioned locations in the brain of a mouse.

Gene therapy is a novel form of remedy, only recently being applied to the many common diseases that plague our society, like cancer and Alzheimer’s Disease. With the rise of an aging population, neurodegenerative diseases, such as A l z h e i m e r ’s D i s e a s e ( A D ) , P a r k i n s o n ’s D i s e a s e ( P D ) , Amyotrophic Lateral Sclerosis (ALS), are becoming increasingly common. In recent attempts to alleviate the symptoms of t hese d iseases, research has turned to the capabilities of gene therapy as a tool for treatment [1]. The treatment of neurodegenerative diseases such as AD, PD, and ALS differs from that of other diseases in that an extra consideration needs to be made when choosing the appropriate vector [1]. Such a vector must be able to access the central nervous


system (CNS), and as such must pass through the blood brain barrier (BBB). Various applications of gene therapy have been undertaken for AD and PD respectively. Hallmarks of AD include loss of healthy neurons, the buildup of amyloid plaques, and neurofibrillary tangles [2]. The accumulation of amyloid plaques is hypothesized to cause the demise of brain cells. AD is associated with irregularity and damage to t he hippocampus (responsible for short-term memory, orientation) and the cortex (responsible for long-term memory, reasoning, mood) [3]. In an effort to alleviate symptoms of AD, a study conducted at the Imperial College London identified a gene called PGC1-alpha as a

candidate for proliferating gene therapy [3]. The project used a lentivirus within which the PGC1-alpha gene was inserted and injected directly into the aforementioned locations in the brain of a mouse [3]. To their delight, 4 months after the trial, mice that received the lentivirus had developed far fewer plaques, and had far less damage to brain cells in the hippocampus. While this seems like an absolute success story, one drawback is that the lentivirus must be administered directly into the brain [3]. This is a dangerous technique with lots of room for error. A safer method of delivery involves introducing the lentivirus into the bloodstream. However, the plasmids containing the gene of interest may not be able to cross the blood-brain barrier, and so the trial would remain ineffective.


Another study, from 2001, demonstrated that the injection of nerve growth factor (NGF) facilitated repair of damaged neurons in AD [4]. Again, NGF was injected directly into the brain because the large protein is unable to cross the BBB. The presence of NGF in the bloodstream also has adverse effects [4]. An important finding of this study was that degenerating neurons still maintain the ability to respond to growth factors, opening up a whole class of proteins that could be used as potential AD treatments. Parkinson’s Disease (PD) is an example of another progressive neurodegenerative disease. As a result of the damage to dopamine-releasing neurons of the midbrain, there is a depleted amount of dopamine in the striatum of the brain [1]. Symptoms of this dopamine reduction include tremors, rigidity, and bradykinesia. Familial PD occurs in about 5% of cases. A study published in Cell by a lab at the University of Copenhagen expressed a novel finding pertaining to the other 95% of PD cases – non-familial PD [5].

Professor Shohreh Issazadeh-Navikas established that the gene IFN-beta is responsible for keeping neurons healthy by ridding the brain of waste proteins. Dysfunction of this gene leads to a buildup of waste proteins into masses called Lewy bodies which are responsible for premature neuronal death [5]. When IFN-beta gene therapy was attempted, there was significantly less neuronal death and as a result, disease progression was halted [5]. This development has yet to be tested in humans. However, findings from mice studies appear promising and provide a path forward for further exploration into Parkinson’s Disease. Neurodegenerative diseases, and their growing incidence, demonstrate our harrowing

vulnerability. Humans are a unique species. Our lives are composed of experiences that play a role in determining the paths we take. It is imperative that we are able to recollect, and remember those experiences. Our past guides our present. Alzheimer’s patients do not live with that privilege. Our autonomy is another aspect of our personhood that we give great value to. This is not a privilege that Parkinson’s patients are afforded. In order to help the afflicted, an understanding of these diseases, and how to treat them, is key. With our continued humanity and curiosity, we are able to progress towards the ultimate goal of using gene therapy to treat diseases like Alzheimer’s and Parkinson’s.

References [1] O’Connor, D. M., & Boulis, N. M. (2015). Gene therapy for neurodegenerative diseases. Trends in Molecular Medicine, 21(8), 504-512. [2] Gomes, V. (2015). Genetic Markers for Alzheimer's Disease. Journal of Student Science and Technology, 8(2). [3] Katsouri, L., Lim, Y. M., Blondrath, K., Eleftheriadou, I., Lombardero, L., Birch, A. M., ... & Sastre, M. (2016). PPARγ-coactivator-1α gene transfer reduces neuronal loss and amyloid-β generation by reducing βsecretase in an Alzheimer’s disease model. Proceedings of the National Academy of Sciences, 113(43), 12292-12297. [4] Tuszynski, M. H., Yang, J. H., Barba, D., Hoi-Sang, U., Bakay, R. A., Pay, M. M., ... & Nagahara, A. H. (2015). Nerve growth factor gene therapy: activation of neuronal responses in Alzheimer disease. JAMA neurology, 72(10), 1139-1147. [5] Ejlerskov, P., Hultberg, J. G., Wang, J., Carlsson, R., Ambjørn, M., Kuss, M., ... & Ruscher, K. (2015). Lack of neuronal IFN-β-IFNAR causes lewy body-and parkinson’s disease-like dementia. Cell, 163(2), 324-339.



Over the past 10 years, scientists have identified hundreds of genes that are linked to the cause of autism [1]. While many of these mutations associated with autism come from multiple genes, there are a few rare cases where a single genetic mutation can cause autism [1]. This past year, in 2016, a group of neuroscientists from the Massachusetts Institute of Technology (MIT) decided to investigate one of these rare cases: the deletion of the Shank3 gene. By using gene therapy, they found that turning the Shank3 gene back on actually reversed autistic symptoms in mice!


VOLUME 4. ISSUE 2. FEBRUARY 2017 The Shank3 gene encodes for a major scaffolding protein at the excitatory synapse that recruits postsynaptic signalling molecules. The deletion of Shank3 is found to disrupt protein composition at t h e p o s t s y n a p t i c d e n s i t y, r e d u c i n g neurotransmission efficiency and causing anxiety, social interaction problems and repetitive behaviour, which are common symptoms in autism [2]. In their experiment, the MIT researchers used a gene therapy technique called the “Cre-dependent genetic switch (FLEx) strategy” to turn off Shank3 in the embryos of mice. This site-specific recombinase technology involves deleting, inverting and translocating specific sites to turn the expression of a gene off while another gene is simultaneously turned on. These Cre recombinases, or CreER recombinases, consist of Cre fused to mutated hormone-binding domains of the estrogen receptor [3]. These mice that had Shank3 turned off displayed typical autistic symptoms such as social interaction problems and repetitive behaviour. When the mice were older, they turned Shank3 back on by adding tamoxifen, an estrogen-blocking chemical, to their diet. Tamoxifen activates the inactive CreER recombinases by the synthetic estrogen receptor ligand 4-hydroxytamoxifen (OHT) [3]. The surprising result was that the mice did not display any of the common autistic symptoms [4]! In addition, the researchers found that it was more difficult to reverse all of the autistic symptoms as the mice aged. When the gene was turned on in mice a couple months after birth, they were able to eliminate repetitive behaviour and avoidance of social interaction, but they could not eliminate anxiety or deficiency in motor skills. However, when

they turned on the gene in mice only 20 days after birth, they were able to eliminate almost all autistic symptoms, including anxiety and motor deficiency [5]. Currently, the MIT researchers are studying the best time to intervene with Shank3 for the optimal reversal of autistic symptoms. One drawback of this gene-editing technique is that it can only be fully effective for an extremely small proportion of autistic people, as only 1% of autistic people are missing Shank3 [6]. However, this method could be used to guide more approaches to repair other brain connections, and lead the way to more research in other disorders related to autism. Researchers are attempting to identify the circuits that control certain behaviours to aid people who have the same defects in the same circuits (even with a different genetic mutation). For instance, re-expression of the gene in schizophrenic patients may reverse symptoms, since Shank3 mutations have also been implicated in schizophrenia [7]. There is still more to discover about fully reversing the effects of autism in humans. However, this novel gene therapy method, where turning on a single gene can reverse autism, has already paved a brighter pathway for completely curing autism in the future. R e f e r e n c e s
 [1] C., Gregorle. (2016, February 19). Some Autism Symptoms May Be Reversed By Gene Editing, Scientists Suggest. Retrieved January 17, 2017, from e 4 0 9 ? u t m _ h p _ r e f = b r a i n
 [2] Y., Mei, P., Monterio, Y., Zhou, J., Kim, X., Gao, Z., Fu, & G., Feng. (2016, February 25). Adult restoration of Shank3 expression rescues selective autistic-like phenotypes. Retrieved January 17, 2017, from
 [3] S., Feil, N., Valtcheva, & R., Feil. (n.d.). Inducible Cre Mice. In Methods in Molecular Biology ( V o l . 5 3 0 , p p . 3 4 3 - 3 6 3 ) . P u b m e d . d o i : 1 0 . 1 0 0 7 / 9 7 8 - 1 - 5 9 7 4 5 - 4 7 1 - 1 _ 1 8
 [4] Fixing disabled Shank3 gene eases autism-like behaviors in adult mice. (2016, February 18). Retrieved January 18, 2017, from e h a v i o r s - a d u l t - m i c e
 [5] A., Trafton. (2016, February 17). Neuroscientists reverse autism symptoms. Retrieved January 18, 2017, from
 [6] H., Osborne. (2016, February 18). Autism behaviours reversed in mice by switching Shank3 gene on in adulthood. Retrieved January 18, 2017, from - 1 5 4 4 6 6 1
 [7] A., Trafton . (2015, December 10). How one gene contributes to two diseases. Retrieved January 18, 2017, from

These mice that had Shank3 turned off displayed typical autistic symptoms such as social interaction problems and repetitive behaviour.

Artwork by Parandis Kazemi


CRISPR: From Food Industry to

Human Gene Therapy Author: Victor Lunar Quintero Timeline & Illustrations: Priscilla Chan One of the earliest practical applications of CRISPR as a biotechnology in use today pertains to the cultivation of bacteria in dairy products. Dr. Phillipe Horvath, a recipient of the Canada Gairdner International Award in 2016, works with this technology on a regular basis. His work focused on elucidated the function of CRISPR as a mechanism of bacteria's adaptive immune system. Horvath and his colleagues at Danisco (now known as DuPont) represent the turning point of CRISPR from theory to practicality, showing its potential as a gene editing tool. To produce yogurt, for example, S. thermophilus  bacteria must be allowed to grow in processed milk such that they convert the sugar in milk (lactose) to lactic acid. One of the difficulties in producing yogurt or cheese is preventing other bacteria or predatory phages from attacking the culture. Dr. Horvath earned his Gairdner Award  for


his research and application of gene editing to produce strains of S. thermophilus that were resistant to this harmful bacteria, therefore increasing the efficiency of food production. As more labs have begun to research CRISPR for applications in the medical field. When asked about the future of the technology at his recent talk in the University of Toronto, Dr. Horvath acknowledged that while there are ethical questions that must be addressed on the way, it would be unethical to not pursue this line of research in an attempt to save lives. The current direction of CRISPR gene therapy research echoes this sentiment, as scientists around the world come up with possible treatments for diseases like cancer or AIDS. Research is currently focused on genetically 'training' our immune cells to better fight disease as well as disrupting the genetic mechanisms of diseased cells. While the future of these treatments is still unclear, it is correct to assert that CRISPR and its associated DNA binding proteins constitute one of the greatest scientific breakthrough of the 21st century.




CRISPR first coined; Cas genes defined2 Jansen et al.

Experimental evidence of adaptive immunity4 Barrangou et al.

CRIS geno Cong



CRISPR repeats first observed1 Ishino et al.

Proposed adaptive immune function3 Mojica et al.


Characterized Cas9-mediate cleavage5 Jinek et al.


REFERENCES 1. Ishino, Y., Shinagawa, H., Makino, K., Amemura, M., & Nakata, A. (1987). Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. Journal of bacteriology, 169(12), 5429-5433. 2. Jansen, R., Embden, J., Gaastra, W., & Schouls, L. (2002). Identification of genes that are associated with DNA repeats in prokaryotes. Molecular microbiology, 43(6), 1565-1575. 3. Mojica, F.J.M., D ez-Villase or, C.S., Garcia-Martinez, J.S., and Soria, E. (2005). Intervening Sequences of Regularly Spaced Prokaryotic Repeats Derive from Foreign Genetic Elements. J Mol Evol 60, 174–182. 4. Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., Romero, D.A., and Horvath, P. (2007). CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709–1712. 5. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A., and Charpentier, E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821. 6. Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W., Marraffini, L.A., et al. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823. 7. Reardon, S. (2016). First CRISPR clinical trial gets green light from US panel. Retrieved January 29, 2017, from


ISPR-Cas9 for nome editing6 ng et al.

2017 CRISPR clinical trials7

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GENE THERAPY: A PROMISING WEAPON IN THE FIGHT AGAINST CANCER DENITSA VASILEVA Gene therapy is a promising new weapon in the battle against cancer (Cross, 2006). Broadly, there are three different types of gene therapy: immunotherapy, oncolytic virotherapy and gene transfer (Cross, 2006). Immunotherapy relies on genetically modified cells and viral particles to stimulate the immune system to fight the cancer cells (Cross, 2006). Oncolytic virotherapy programs specialized viral particles to replicate inside cancer cells to eventually kill them (Cross, 2006). This technique has shown encouraging results, particularly in patients with metastatic cancers (Cross, 2006). Lastly, gene transfer is a technique that includes introducing new genes into a cancer cell with the aim to slow down the growth of the cancer cell or outright kill it (Cross, 2006). A varied range of genes and vectors can be used in this technique (Cross, 2006).

Excitement about the potential of gene editing in the treatment of cancer has grown over the last several years, particularly with the advent of the CRISPR-Cas9 gene editing technology which is used in all three types of gene therapy (Cyranoski, 2016). CRISPR-Cas9 provides a much easier and more efficient way to edit genes into the cancer cells which makes gene editing for cancer treatment even more promising (Cyranoski, 2016). In fact, scientists in China have already used the CRISPR-Cas9 technique to inject modified cells into a patient with lung cancer (Cyranoski, 2016). Researchers took immune cells from the patient’s blood and used the CRISPR-Cas9 gene editing technique to disable a protein in the cancer cells, which usually inhibits the body’s immune response and allows the cancer cells to proliferate (Cyranoski, 2016). The edited cancer cells were then allowed to divide and were subsequently injected into the patient (Cyranoski, 2016).


Gene therapy is still a relatively new tool in the fight against cancer and only time will be able to determine its efficacy (Cyranoski, 2016). Moreover,

given the novelty of this procedure, its safety, side effects and long-term consequences for the patient will not be soon known. However, multiple clinical trials using the CRISPR technique to treat common cancers such as bladder, prostate and renal cancers are expected to begin shortly in China (Cyranoski, 2016). It is widely believed that with the introduction of CRISPR technology, gene editing has become the so-called new frontier of science and medicine, intensifying the competition between international powers ( Cyranoski, 2016). Many scientists believe that just like the space race of the 20th century, the competition to develop a safe and effective gene-editing therapy for cancer patients will define science in the early part of the 21st century (Cyranoski, 2016).

R e f e r e n c e s
 Cross, D. et al. 2006. Gene therapy for cancer treatment: past, present and future. Clin Med R e s . 4 ( 3 ) : 2 1 8 - 2 7 .
 Cyranoski, D. 2016. Chinese scientists to pioneer first human CRISPR trial. Nature. 535: 4 7 6 - 4 7 7 .
 Cyranoski, D. 2016. CRISPR gene-editing tested in a person for the first time. Nature. 539: 479.


TRIPLE TROUBLE ETHICAL ISSUES WITH THREE-PARENT CHILDREN TERESE MASON PIERRE Advancements in reproductive technologies are burgeoning year by year, and this is great news for geneticists and expectant parents. But with every step made by scientists, bioethicists can hold a hand up and say, “Wait!” While it’s easy to see the ethical issues arising from in vitro fertilization or surrogacy, the case of three-parent children is slightly more difficult to parse. Three-parent children arise from mitochondrial DNA donation, a process which occurs in the following way: if the mitochondria from a mother’s egg cell is abnormal or damaged, the nucleus is removed and placed inside a donor egg cell with healthy mitochondria (whose nucleus has also been removed to make way for this separate nucleus). This new egg cell is fertilized with the father’s sperm, although nucleus removal can occur from the abnormal-mitochondria egg cell before or after fertilization. The resultant child will have three sets of DNA: the nuclear DNA from the mother (who had abnormal mitochondria), the mitochondrial DNA from an egg donor (a “second mother” with healthy mitochondria) and DNA from the father [1]. Britain is the first country to legalize mitochondrial DNA donation for mothers with abnormal mitochondria [2]. Prima facie, this technique seems encouraging. Mitochondrial DNA donation arose from the desire to prevent genetic abnormalities in the mitochondria that could lead to loss of muscle coordination, dementia and others. However, as with various other

gene therapy techniques, the ethical issue of disability avoidance crops up. By undergoing techniques to reduce or eliminate the risk of some genetic disorders in vitro, what value assessments are we making about people who already have those disorders? Some—especially those who identify with their disability—can claim that it’s a tacit acknowledgement by society of their worthlessness, that it’s inherently bad and wrong to have a genetic disorder, and even more wrong to take steps to reduce instances of that disorder in the population. Other ethical mires that may come up, in particular when the child is growing, are issues of personal identity and donor anonymity. As the resultant child will have the DNA of three people, one can ask how the child will personally relate to the parents they live with, as well as how the child will see him/herself as an individual. Genetics is a large part of our humanness, not only because genes usually dictate how we look and act—which affects how others see and treat us—but also what sorts of diseases or illnesses we’re susceptible to.


Introducing a third parent—especially an anonymous donor, muddies the waters. Speaking of anonymous donors, questions about whether the child will have the right to know who their “second mother” is may be raised. A child may feel that they are being denied a part of themselves by not knowing who this third parent is [3]—a donor might wish to keep their identity a secret: what about their autonomy and privacy? How can this be solved? Interestingly, in Britain, egg and sperm donation aren’t anonymous, and if a person is involved in the creation of a child, they are effectively another parent [4]. A more practical ethical problem comes up during the process of mitochondrial DNA donation. A large number of eggs will need to be donated from women to further research this technique [5]—it’s not perfect, of course—and questions about whether such an invasive procedure is beneficial are worth considering. Wouldn’t scientists be better off studying ways to cure these mitochondrial diseases instead of “freaking people out” by altering genes? In one of the techniques, DNA is extracted from two cells to help form a third one, and since they won’t survive the transfer, in short, two embryos will be created to be destroyed [6]. One can ask: isn’t there something off about creating life merely to be used and tossed aside, eliminating any potentiality?

There’s also the larger, general ethical question of why we want to alter embryonic genes in the first place, and what right we have to do so, both legally and as a species. Many people, in many societies, often steer clear of altering the characteristics of children, fearing the famous “designer babies” outcome (see also the disability avoidance issue above). Not only does this sound like some weird dystopia—think: the film “Gattaca”—but, as humans, since we’re still susceptible to the forces of evolution, we don’t know how such a technique will alter the natural variation of humans, altering our adaptation and survival, now and in the future [7]. In conclusion, while mitochondrial DNA donation is a groundbreaking technique in gene therapy, with the potential to help many lives, it, like many medical procedures, carries a host of ethical issues, such as problems with identity, destruction of embryos and disability avoidance. Many of these ethical issues arise because of the way we view human life: as sacred, wholly important and inherently valuable—to be left unaltered as a way to preserve its purity. To use Marxist terms, we’ve commodified everything, and our lives and characteristics are the last holdout. If we sacrifice this, what do we have as people? What separates us from mere objects to pick and choose from? How quickly will our humanity disappear?

References [1] Stanley, T. (2017). Three parent babies: unethical, scary and wrong. London: The Telegraph. Retrieved from g.html [2] Rush, J. (2015).  'Three-parent babies': What is mitochondrial donation and what are the t e c h n i q u e s i n v o l v e d ?  L o n d o n : I n d e p e n d e n t . R e t r i e v e d f r o m -is-mitochondrial-donation-and-what-are-the-techniques-involved-10021567.html [3] Nazir-Ali, M. (2015).  Three-parent babies: an ethical boundary is being crossed. London: The Te l e g r a p h . R e t r i e v e d f r o m ing-crossed.html [4] Stanley, T. (2017).  Three parent babies: unethical, scary and wrong. London: The Telegraph. Retrieved from g.html [5] Nazir-Ali, M. (2015).  Three-parent babies: an ethical boundary is being crossed. London: The Te l e g r a p h . R e t r i e v e d f r o m ing-crossed.html [6] Stanley, T. (2017).  Three parent babies: unethical, scary and wrong. London: The Telegraph. Retrieved from g.html [7] Ibid



COULD EYE DROPS BE THE SOLUTION TO AN EFFECTIVE NON-INVASIVE ADMINISTRATION OF GENE THERAPY? ABEER WASIM Gene therapy is an experimental technique that uses genetic engineering to inactivate or introduce certain genes to treat or prevent diseases. Although it is a promising treatment for some diseases, it remains risky and impractical for a number of reasons, one being due to its invasive nature of administration and ineffective monitoring of therapeutics, especially in disorders of the brain [1]. Recently, a team of researchers from the Department of Radiology in Massachusetts General Hospital and Harvard Medical School worked together to create a noninvasive delivery and tracking mechanism of a gene transcript in mice following cerebral ischemia [2]. In the study, the group inserted G-CSF (granulocyte colonystimulating factor - a gene responsible for stimulating the production of new cells) in an adenovirus (delivery vector) that is safe for humans and known to infect brain cells [3]. This therapy was administered to the mice using eye drops at various time points post induced ischemia [3]. The results showed a significant decrease in mortality, cerebral atrophy and neurological deficits [3]. Notably, the researchers were also able to use Magnetic Resonance Imaging (MRI) to confirm the successful delivery and expression of G-CSF [2]. They were able to visualize the metabolic activity of tissue treated with and without the

growth factor. Mice treated with G-CSF were able to retain normal metabolic activity levels and close to normal striatum volume verses the significant decrease in metabolic activity and more than 3-fold reduction in striatum volume in un-treated mice [3]. Each year there are over 1.7 million new cases of brain injuries at all levels of severity in North America and 11% of those injuries result in death while almost all other result in some form of life-long disability [4]. This quick-touse method of delivering a therapeutic agent to the brain could lead to highly effective management of brain injuries during emergency situations, for example, following a stroke or heart attack where treatment within a few hours is critical [3]. With the technique set to move onwards to clinical trials, it is clear that this mechanism holds great translational qualities in delivering therapeutics to other diseases of the brain such as Alzheimer’s Disease and Parkinson’s Disease as well as delivering corrected genes for genetic brain disorders such as Tay-Sachs and Wilson disease. Who knows? Maybe one day, recovery after a stroke could be as simple as making sure you get your eye drops in time.


SLENDR: THE CRISPR ADD-ON FOR THE BRAIN SHAGUN GUPTA Precisely mapping the subcellular localization of a protein is essential for determining its effect on physiological function. In the brain, aberrant protein localization has been linked to various neurodegenerative disorders, speech-language diseases and the Fragile X syndrome [1]. Currently, only a fraction of proteins expressed within brain tissue have been localized. Traditional methods of examining protein localization have significant limitations including lack of antibody specificity, low contrast or resulting in altered protein function. To tackle these problems, Mikuni et al. at the Max Planck Florida Institute for Neuroscience developed a simple method that enables in vivo single-cell labeling of endogenous proteins by introducing an epitope or fluorescent tag via CRISPR-Cas9 mediated HDR (homology directed repair) [2]. The technique, called SLENDR (single-cell labeling of endogenous proteins by CRISPR-Cas9-mediated homology-directed repair), allows for subcellular localization of endogenous proteins and live imaging of protein dynamics in the mammalian brain with micrometer to nanometer resolution.

Although genes in the brain can be easily knocked out using CRISPR-Cas9, targeted insertion of a sequence using has been relatively difficult owing to the lack of HDR activity in the adult brain. HDR primarily occurs during the S and G2 phases of the cell cycle, while mature neuronal cells are frozen in the G0. To overcome this challenge, SLENDR introduces the CRISPR-Cas9 machinery into actively dividing neural progenitor cells via in-utero electroporation. Thus when the neuronal cells mature, they already carry the tagged protein. Additionally, by varying the electroporation angle and timing, researchers are able to target distinct neural progenitors in different regions of the brain, providing a developmental map of protein localization. Further harnessing the ability of CRISPR-Cas9, researchers have shown that SLENDR can be used in multiplex labelling of proteins by simultaneously labeling CaMKIIa and CaMKIIb (major subunits of Calcium/calmodulin-dependent protein kinase II) with different tags, making it a useful tool in co-localization assays. SLENDR also allows for mosaic analysis by combining protein labelling with single-cell knockout, providing researchers insight into disease-related changes in protein localization. SLENDR’s greatest strength lies in its ability to label most proteins with little optimization. Its simplicity combined with throughput cost-effectiveness compared to previous techniques have the potential to make it the go-to technique for single-cell labelling of protein within the brain. References [1] Mien-Chie Hung, Wolfgang Link. Protein localization in disease and therapy. J Cell Sci 2011 124: 3381-3392; doi: 10.1242/jcs.089110 [2] Mikuni, Takayasu et al.High-Throughput, High-Resolution Mapping of Protein Localization in Mammalian Brain by In  Vivo Genome Editing. Cell. 2016 165:1803-1817 doi: 10.1016/j.cell.2016.04.044 Source for CaMKII: Lisman et al. The molecular basis of CaMKII function in synaptic and behavioural memory. Nature Reviews Neuroscience. 202. 3:175-190 doi:10.1038/nrn75



GENE THERAPY IN PSYCHIATRIC DISORDER MENGHAN WANG Mental health has become an increasingly important component in determining one’s quality of life, and psychiatric disorders no doubt contribute negatively. According to the World Health Organization, there are about 350 million people of all ages in the world who suffer from depression. Depression has surpassed all other agents to become the leading cause of disability and disease (World Health Organization, 2010). Traditional treatments for psychiatric disorders, including depression, are mainly psychopharmacology and psychotherapy. Although these treatments are effective to some extent, we can hardly say that they are perfect. For example, antidepressants have a long list of side effects, and the effective size has been questioned a lot as well. In a recent antidepressant versus placebo study done by Dr. Rutherford, the mean placebo response was 31%, while the antidepressant response mean was 50% (Rutherford & Roose, 2013). It is hard to say that antidepressants are much more effective than placebos from a statistical point of view. Furthermore, the efficacy of psychotherapy is too subjective to

References World Health Organization. (2010). Depression (Fact sheet). Retrieved from ets/fs369/en/ Mei, L., & Xiong, W. C. (2008). Neuregulin 1 in neural development, synaptic plasticity and schizophrenia. Nature Reviews Neuroscience, 9(6), 437-452. Rutherford, B. R., & Roose, S. P. (2013). A model of placebo response in antidepressant clinical trials. American Journal of Psychiatry, 170(7), 723-733.

measure, and portions of patients do not respond to either drugs or psychotherapy. Researchers have invested their efforts into finding another therapy that may address these problems. A new technology that is being developed is called gene therapy. By modifying genes that contribute to psychiatric disorders, researchers are hoping to both alleviate symptoms already present as well as prevent psychiatry disorders that individuals are genetically predisposed to. Recently, there have been some exciting and promising results. For example, Dr. Lin Mei and his team at the Medical College of Georgia have successfully alleviated schizophrenia-like symptoms in adult mice by restoring the overexpressed Neuregulin-1 gene that caused the decrease of messenger chemicals glutamate and GABA (Mei, 2008). However, before homologous gene therapies can be clinically applied to human beings, we still have to address many technical and ethical issues.

The most profound technical problem about gene therapy for psychiatric disorders is the fact that neither disorders nor symptoms have a oneto-one relationship with gene expression abnormalities. The relationship between genes and psychiatric disorder symptoms are way more complex than that. Genetic abnormalities may be unique for every single patient and often more than one gene interact with each other and contribute to the symptoms. To further complicate the situation, these genes are all under the influence of environment. For now, researchers have not yet fully understood the genetic and environmental causes underlying any psychiatric disease. To advance genetic therapy, we need to have a deeper, clearer understanding of the pathology of psychiatric diseases. Even if all the technical issues have been addressed, how we address the ethical issues of human gene therapy is also vital. The techniques

that are used to conduct gene therapy are usually highly invasive, such as injecting genetically modified viruses into the target area in brains. It is not just a simple judgment of riskbenefit analysis, but rather an issue that appeals to the more holistic beneficence and nonmaleficence aspect of medicine’s ethic paradigm. It is a complicated call for the clinicians and it is even harder to explain it well to the patient for them to give informed consent. Furthermore, from the equality point of view, will the high-cost of genetic therapy make it only accessible to the wealthy and widen the gap that already exists? We have to admit that there are a lot of technical and ethical obstacles we must overcome before gene therapy can be performed as frequently as drug prescription. But with the encouraging progress that is being made continuously, that day will surely come.



References Adam, D. (2001). Gene therapy may be up to speed for cheats at 2008 olympics. Nature, 414(6864), 569-570. doi:10.1038/414569a [doi] Bob, P., & Svetlak, M. (2011). Dissociative states and neural complexity. Brain and Cognition, 75(2), 188-195. doi:10.1016/j.bandc.2010.11.014 [doi] Chiu, A. S., Gehringer, M. M., Welch, J. H., & Neilan, B. A. (2011). Does alpha-amino-beta-methylaminopropionic acid (BMAA) play a role in neurodegeneration? International Journal of Environmental Research and Public Health, 8(9), 3728-3746. doi:10.3390/ijerph8093728 [doi] Didigu, C. A., Wilen, C. B., Wang, J., Duong, J., Secreto, A. J., Danet-Desnoyers, G. A., . . . Doms, R. W. (2014). Simultaneous zinc-finger nuclease editing of the HIV coreceptors ccr5 and cxcr4 protects CD4+ T cells from HIV-1 infection. Blood, 123(1), 61-69. doi:10.1182/blood-2013-08-521229 [doi] Hacein-Bey-Abina, S., von Kalle, C., Schmidt, M., Le Deist, F., Wulffraat, N., McIntyre, E., . . . Fischer, A. (2003). A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. The New England Journal of Medicine, 348(3), 255-256. doi:10.1056/NEJM200301163480314 [doi] Lawrenson, T., Shorinola, O., Stacey, N., Li, C., Ostergaard, L., Patron, N., . . . Harwood, W. (2015). Induction of targeted, heritable mutations in barley and brassica oleracea using RNA-guided Cas9 nuclease. Genome Biology, 16, 258-015-0826-7. doi:10.1186/s13059-015-0826-7 [doi] Liang, P., Xu, Y., Zhang, X., Ding, C., Huang, R., Zhang, Z., . . . Huang, J. (2015). CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein & Cell, 6(5), 363-372. doi:10.1007/s13238-015-0153-5 [doi] Menashe, I., Larsen, E. C., & Banerjee-Basu, S. (2013). Prioritization of copy number variation loci associated with autism from AutDB-an integrative multi-study genetic database. PloS One, 8(6), e66707. doi:10.1371/journal.pone.0066707 [doi] Muramatsu, S. (2010). The current status of gene therapy for parkinson's disease. Annals of Neurosciences, 17(2), 92-95. doi:10.5214/ans.0972-7531.1017209 Reardon, S. (2016). First CRISPR clinical trial gets green light from US panel . Retrieved from -panel-1.20137 Russell, S. J., Peng, K., & Bell, J. C. (2012). ONCOLYTIC VIROTHERAPY. Nature Biotechnology, 30(7), 658-670. doi:10.1038/nbt.2287 Sanders, N. N., Van Rompaey, E., De Smedt, S. C., & Demeester, J. (2002). On the transport of lipoplexes through cystic fibrosis sputum. Pharmaceutical Research, 19(4), 451-456. Sharma, A., Easow Mathew, M., Sriganesh, V., & Reiss, U. M. (2016). Gene therapy for haemophilia. The Cochrane Database of Systematic Reviews, 12, CD010822. doi:10.1002/14651858.CD010822.pub3 [doi] W h a t i s g e n e t h e r a p y. R e t r i e v e d f r o m (]


In this age of exciting scientific research, it is not uncommon to be overwhelmed by the outpouring of results and methods from potentially life-altering research. For example, computational studies using linkage analysis and genome-wide associations are discovering genes and loci associated with autism (Menashe, Larsen, & Banerjee-Basu, 2013), researchers are linking certain environmental and pathogenic exposures to Alzheimer’s disease (Chiu, Gehringer, Welch, & Neilan, 2011), and scientists are striving to understand how consciousness is mediated by the firing patterns of our neurons (Bob & Svetlak, 2011). Gene therapy, in particular, has been widely acclaimed to be the solution to several intractable diseases such as Alzheimer’s disease, Neiman Pick disease, type C1, hemophilia, etc., yet most would be hard-pressed to describe the means by which gene therapy is effected, or the many uses, of gene therapy (Sharma, Easow Mathew, Sriganesh, & Reiss, 2016). The multifaceted nature of gene therapy ranges from its expected use in treating several genetic disorders to nontherapeutic gene doping by athletes crookedly striving to inch ahead of the competition (Adam, 2001). In general, gene therapy is the introduction of genetic material into someone’s cells for the purposes of

staving off or combatting disease. These cells could either be somatic cells or germline cells. To quickly recapitulate, somatic cells are non-dividing cells of the body (such as skin cells, neurons, cardiac myocytes) while germline cells are cells capable of dividing and giving rise to progeny (sperm or eggs). The gene, which usually replaces or inactivates a defective copy, is delivered via a vector, and the most common vectors are attenuated, non-pathogenic viruses, i.e. those that have had their pathogenic or diseasecausing genes removed. Due to the replicative nature of germ cells, any modifications done early in embryonic development are transmitted to all cells in the developing embryo, and as such, passed on to future progeny. Despite the ramifications of these effects, such as eliminating inherited diseases, critics often argue that it is akin to “playing God” and ethics proponents cite that there might be unforeseen, adverse effects. Somatic gene therapy, on the other hand, is often safer due to the therapeutic effects targeting only non-dividing cells and are not passed on to the future progeny. Most research in humans, to date, has been performed on somatic cells in disorders such as cystic fibrosis, muscular dystrophy, cancer, etc., and in the European Union, the highly controversial germline therapy has been prohibited (What is gene therapy.)



Virotherapy has been used to introduce genetic material into affected cells and this method is predicated upon the life cycle of viruses: infect living cell, hijack replicative machinery, assemble viral proteins, and disseminate. Several factors come into play such as the type, size, tropism, packaging capacity, or integrative ability of the virus to be used (Russell, Peng, & Bell, 2012). Typical viruses used for these purposes include lentiviruses, adenoviruses, adeno-associated virus, and so on. Several clinical trials have demonstrated the potential for Virotherapy. For example, several Parkinson’s patients improved following the infusion of an adeno-associated vector encoding Glutamic Acid Decarboxylase (GAD) in the subthalamic nucleus of the brain (Muramatsu, 2010). However, despite the successes, there have been a few lethal drawbacks in the history of gene Virotherapy. Two clinical trials for X-linked severe combined immunodeficiency went awry after 4 of the 20 patients with hematopoietic stem cell retro-virotherapy developed T-cell leukemia (HaceinBey-Abina et al., 2003). This insertional mutagenesis was due to the insertion of the gene in the viral vector integrating into a sensitive spot in the human genome, such as a tumor suppressor gene. Clinical trials investigating virotherapy are plagued with patients mounting immune responses, toxicity, and targeting issues. Despite these drawbacks, research into expanding or limiting the host cell range, increasing the efficacy and minimizing off-target effects and overexpression of the gene product show great promise on the use of viruses for therapeutic purposes.


One of the reasons for the rise of virotherapy was the inability of DNA to cross the cell membrane, due in large part to its size and charge. But with advances in biochemistry techniques, the efficiency of non-virotherapy gene transfer rivals virotherapy. Scientists can now inject DNA coated heavy metals, typically gold, into the cell with the use of a biolistic particle delivery system, or a gene gun. Now gaining momentum is the use of short strands of DNA to inactivate diseased genes. Short anti-sense oligonucleotides bind to the defective gene to prevent its transcription, while short-interfering RNA (siRNA) bind to and cleave specific sequences on the mRNA transcript thus preventing the translation of the gene (What is gene therapy.) Lipoplexes are lipids complexed with DNA to prevent the premature degradation of DNA. They have been used to transfect respiratory epithelial cells, and will be significant players in the battle against the genetic disease cystic fibrosis (Sanders, Van Rompaey, De Smedt, & Demeester, 2002). Finally, one cannot discuss gene therapy without including gene editing. The revolutionary brainchild of Jennifer Doudna, the UC Berkeley professor of chemistry and molecular and cell biology, CRISPR-Cas9, has upended the scientific community and has far-reaching effects into several industries ranging from agriculture to healthcare (Lawrenson et al., 2015). Simply put, the system consists of Cas9, a nuclease, and a guide RNA which directs the nuclease to the sequence to be cleaved. The versatility of CRISPR lies in the possibility of the guide RNA to target any 20 unique sequences in the genome. Using the cell’s DNA repair system, one can disrupt a gene and/or insert a functional gene. In June 2016, the US National Institute of Health approved the first use of CRISPR-Cas9 to

augment existing therapies relying upon T-cell. The T-cells of 18 patients with varying cancers will be edited to include a protein which enable T-cells to detect and target cancer cells, knockout genes for proteins that interfere with T-cell cancer detection and prevent the inactivation of T-cells by the cancer cells (Reardon, 2016). Despite the overwhelming potential of CRISPR-Cas9, there is a major caveat: off-target interactions. In 2015, a Chinese team concluded that CRISPR is not ready for clinical germline modification due to off-target effects and ‘untoward’ mutations after attempting to cure beta-thalassemia by altering the DNA of non-viable human embryos (Liang et al., 2015). Nevertheless, other gene editing tools existed prior to the conception of CRISPR-Cas9. Zinc-finger nucleases are artificial restriction enzymes formed by fusing a DNA binding domain to a nuclease domain. Like CRISPR-Cas9, it can be directed to specific sites in the genome. A 2014 study, which removed the genes for two proteins critical for HIV entry, CXCR4 and CCR5, had promising results on the use of gene editing to prevent HIV infection (Didigu et al., 2014). In conclusion, there are several types of gene therapy, each with its unique advantages and drawbacks. The popular use of viral vectors, mostly due to the natural propensity of viruses to infect host cells and deliver a genetic payload, has generated several promising results, and the continual refinement to increase gene transfer yield, coupled with the increasingly efficient non-viral, bioengineered vectors and the ease of use and versatility of gene editing techniques like CRISPR-Cas9 and zinc-finger nucleases have cast a bright light on the path forward to the once somber landscape that was the realm of genetic diseases and cancer. Image from








technology has recently become a boiling topic in scientific debates, while many researchers claim

that this game-changing technique is the future of science and treatment development, some tap into the ethical compilations that surround the use of gene editing. Most ethical concerns arise when humans are the target of CRISPR. Less focus is placed on model organisms, which might as well spark its own ethical debates regarding the release of genetically modified organisms into nature. However, given that research is conducted in a contained environment and numerous safety precautions are incorporated into research protocols, neuroscientists are less concerned about “CRISPRing” model organisms. The focus is on using CRISPR to unravel the most puzzling questions about brain mechanisms. In seeking answers to our questions about this novel technology, we had the pleasure to sit down with Dr. Tod Thiele, assistant professor in the department of biological sciences at University of Toronto Scarborough who studies the sensory motor integration in larval zebrafish, and get an expert opinion on the use of CRISPR technology in current neuroscience research. Dr. Thiele, Could you tell us a little about your current research? Sure! We are interested in how the brain uses sensory information to produce the movements necessary to perform behaviour, like hunting or escaping from predators, and we do this using the larval zebrafish which is an exciting system to work in. Why do you work with zebrafish in particular? We study seven-day-old fish whose brains have about 100,000 neurons compared to 86 billion neurons in the human brain. So, it is more simple but still very challenging! Given that we are studying a vertebrate brain and that the brain’s layout has remained similar throughout evolution, we are trying to make discoveries about brain circuits in the fish that will hopefully also be applicable to circuits in the mammalian brain. What do you look at specifically? We look at circuits in larval fish that are thought to be homologous to the striatum of the basal ganglia in humans. This is a major centre for decision making and behavioral control and is also affected in Parkinson’s disease. So, how do you use genetic tools to study this? Genetic tools are maybe the most important part of our research. We make transgenic fish which express various proteins that allow us to either record neuronal activity, manipulate activity, or kill specific neurons in cases where we want to do loss of function studies. To r e c o r d n e u r o n a l a c t i v i t y, w e e x p r e s s t h e calcium-sensitive protein GCaMP, a revolutionary protein, which gets brighter when a neuron is active. We a l s o u s e o p t o g e n e t i c t o o l s i n c l u d i n g channelrhodopsin, archaerhodopsin and halorhodopsin to increase or decrease the activity of neurons with light. Together, these make a powerful toolkit for mapping the circuits of the brain!

Now, let's be more specific and talk about CRISPR; how do you employ this technique in your research? The way we express these proteins in specific brain regions is to use regulatory DNA elements that direct protein expression in a subset of neurons. For our initial CRISPR project, we wanted to get genetic control over the neurons of the direct pathway within the striatum that express the type 1 dopamine receptor. In collaboration with the zebrafish core facility at SickKids, we used CRISPR genome editing to knock-in the transcriptional activator Gal4 into the endogenous type 1 dopamine receptor locus. Now we can use the Gal4/UAS system to express any protein we want in those cells and either ablate them, excite them, inhibit them or record from them. Could you explain the Gal4/UAS system that you mentioned? Gal4 is a transcription activator from the yeast. When Gal4 is translated, it binds to its target, the Upstream Activating Sequence (UAS), and drives the expression of whatever protein you put downstream of the UAS. In this way, all cells that express Gal4 will also express the protein you engineered after the UAS. The system was first developed to control gene expression in fruit flies but has also been used with great success in zebrafish.


If there wasn’t CRISPR, what would have been the alternative technique? Well, [CRISPR] is revolutionary and is what has made it possible! We couldn’t efficiently introduce genes into specific sites within the zebrafish genome before CRISPR technology came along. Even in the case of Gal4/UAS? No, Gal4/UAS system was always possible. The big revolution is that prior to the CRISPR technology, you would inject Gal4 DNA but would have no way to direct where it would end up in the genome. CRISPR technology directs the Gal4 DNA to integrate at a specific site. Thus, in theory we can recapitulate the expression pattern of any gene. Now let us look at the bigger picture, what is most fascinating about using these genetic tools in studying sensorimotor integration? The most fascinating thing is that we can make a manipulation of the genetic level and then study the circuits at the behavioral level. We can watch the activity of genetically defined populations of neurons during a variety of behaviours that fish produce like prey capture, predator avoidance or optomotor swimming and see how the behavioural piano in the brain is being played! Recently, CRISPR has become a very popular subject for scientific debates, could you tell us how easy and c h e a p i s i t t o e m p l o y t h i s t e c h n i q u e ?
 It is easy in the sense that you don’t need a lot of fancy machines and it uses fairly standard lab techniques. It just takes some expertise in how to design the guide RNAs that are used to specify the region of the genome to be edited. It is also an extremely inexpensive technology to use.

the type 1 dopamine receptor. Color codes for depth. Each neuron is ~ 5 microns in diameter.

Do you think there are potential harms that might be caused by the use of this method or after the o r g a n i s m i s g e n e t i c a l l y m o d i fi e d ?
 There is a lot of controversy in using CRISPR in humans, but in the zebrafish we are making changes to the genome that facilitate our biomedical research. We also ensure that these animals stay in the lab and are not introduced into the environment. How do you predict the use of this technique in n e u r o s c i e n t i fi c fi e l d s i n 1 0 y e a r s ?
 It could be used anywhere from curing childhood diseases to treating individuals suffering from schizophrenia. By editing the genes that contribute to

We watch the activity of genetically defined neurons during a variety of behaviours and see how the piano in the brain is being played!


Dorsal view of zebrafish’s brain showing neurons that express

such diseases it is hoped that one can reverse the disease state. It really has the promise to cure many devastating diseases. We could someday go to the doctor for a CRISPR injection to treat diabetes or depression… but, it is still early and lots needs to be worked out! A key thing is figuring out a way to direct the CRISPR machinery to the right tissue at the right time.

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Interneuron Issue 4.2  
Interneuron Issue 4.2  

February 2016: The Genetics of Neuroscience