Bowdoin Science Journal

Biology

Unpacking the Ethical Implications of Human Germline Editing

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On December 30th, 2019, biophysicist Dr. He Jiankui was sentenced to three years in prison for forging ethical review documents and misleading doctors into unknowingly implanting gene-edited embryos into two women in China (Normile 2019). One of these women had a set of twins and the other had a single child. These children are now the first genetically modified humans in history to be resistant to HIV. They can pass this modification to the next generation, and their whereabouts are still unknown (Greely 2019). Jiankui used CRISPR/Cas9, a gene editing tool, to modify their germlines and edit the CCR5 gene, a contributor to broad immune responses (his focus was its importance to HIV viruses).

Based on the National Natural Science Foundation of China, there has been a total of 3.7 billion yuan (roughly $576 million) government spending on embryonic stem cell research from 1997 to 2019 (Lou 2021). Germline gene editing research is allowed, but establishing a pregnancy with genetically modified embryos has been outlawed by multiple regulations (Xinqing 2014). Similarly to China, the National Institutes of Health within the US estimates that human embryonic stem cell research has received $1.48 billion in government funding since 2009. There are no current laws or regulations that ban germline gene editing conducted through private funding within the US, but it would have to be approved by the FDA for marketing and clinical studies. No proposals have been submitted (​​Genetic Literacy Project 2019). Dr. Jiankui has since been disgraced by the scientific community around the world for his actions. Some scientists believe that germline gene editing is not only an unethical practice but a potentially dangerous one that could lead to a new era of eugenics with irreversible harm (Genetic Literacy Project 2019). Little is known about the intricate details of Dr. Jiankui’s experiment, but what has been revealed poses several hard-hitting ethical questions. Should we manipulate the next generation of humans? What are the ethical dilemmas in the advancement of this technology? To create an informed opinion on the matter, it is helpful to first understand the intricacies of Dr. Jiankui’s experiment, and how exactly he made the first genetically modified children.

Dr. Jiankui conducted human germline genome editing, which refers to the technique of modifying not only the genetic information of a subject but also what can be passed down to the next generation (Normile 2019). Germ cells can create a new generation (ex. sperm and eggs). So germ cells, and cells that produce germ cells, are known as the germline. These are different from somatic cells, which are body cells that continuously divide throughout a person’s lifetime and play a minimal role in gene inheritance (Greely 2019). Certain alleles or alterations to CCR5 within the human genome can provide resistance to HIV. Thus, Jiankui used CRISPR to alter CCR5 in HIV-susceptible patients to make them resistant to the disease.

CRISPR, which stands for clustered regularly interspaced short palindromic repeats, is a technique originally found in bacteria as a defense system to render inserted viral DNA ineffective. It also allows for the modification of DNA (Rapini 2023). The enzyme Cas-9 can cut pieces of DNA and is guided to its location based on an attached strand of RNA (called the guide RNA strand). By cutting a targeted sequence of DNA, it can render protein-coding genes inactive or disfunctional. After Cas-9 has cut the target DNA, researchers can choose to place a modified DNA sequence in the vacant space (Fig 1).

Figure 1. The editing technique of CRISPR involves the guide RNA strand, guide RNA sequence, Cas-9 enzyme, and target DNA (Roach 2015).

While little is known about Dr. Jiankui’s specific technique in using the CRISPR/Cas-9 system, we know that he modified the gene CCR5 (Greely 2019). CCR5 is a gene on chromosome 3 that encodes for a protein called C-C chemokine receptor type 5 (also shortened to CCR5) (Normile 2019). CCR5, along with another receptor called CD4, is utilized by the HIV virus to bind to bacteria-detecting/destruction cells called macrophages (white blood cells) and infect them. The HIV virus protein envelope binds to the primary receptor CD4 on the macrophage with the gp120 protein, which are exterior “protective” protein around the HIV cell. If the CCR5 coreceptor is present as well, it can successfully enter the macrophage, release viral RNA and enzymes, and infect the macrophage. This infection alters the function of the macrophage, causing it to assemble and release viruses. Eventually, the gene that codes for the gp120 receptor is altered by a mutation and can now bind to a different co-receptor called CXCR4 which is found on the CD4 plus T-cells. (Prakash 2019). The same process of virus construction occurs within T-cells, but as the viruses leave the T-cell it ruptures the plasma membrane. This kills the T-cells, causing a weakened immune response and eventually resulting in the onset of AIDS.

Figure 2. The mechanism by which the HIV viral envelope infects the macrophage creates mutated viral cells with different gp120 receptors and destroys T-cells. (Prakash 2019) 

It is believed that Dr. Jiankui used the CRISPR/Cas-9 system to delete 32 base pairs of the CCR5 gene, therefore making it produce non-functional copies of the CCR5 protein. This was done in the hope that HIV would be unable to infect the white blood cells of the babies born from the embryos (Normile 2019). If Jiankui only modified somatic cells using this technique, the children may or may not have gained protection against HIV and could not pass it down to the next generation.

In 2015 (before He’s experiment was released) Jennifer Doudna, one of two researchers who published their findings on CRISPR, convened with the U.C Berkeley’s Institute for Genome Innovation, which was composed of Nobel laureates and esteemed professors of bioethics. They concluded that conducting germline editing would be irresponsible until matters such as balancing the potential risks and benefits have been concluded and that there is a broad societal consensus about the appropriate use of proposed applications of this technology (Cohan, 2018). Jennifer Doudna and Emmanuelle Charpentier created this technology to find ways to edit out faulty genes and cure diseases at the source—within our genetic code (Doudna 2019). However, after the release of He’s experiment, Doudna publicly stated her evolving concerns relating to this application of her discovery. During her national press tour in 2019, she shared a frightening dream she had in which a colleague of her’s asked about CRISPR but later turned out to be Hitler (Than 2019). This likely stems from the horrifying history of Nazis and the eugenics movement, which stemmed from the idea of improving the genetic quality of humans and sterilizing and or eliminating those with unfavorable traits. She also voiced that her opinion on germline genome editing has been evolving from an outright ban to warranting it in certain circumstances. However, her underlying opinion remains that it would have to be under transparent and safe circumstances and concern a medical need that was unmet by any technology. In addition to Doudna, countless other scientists have come out in disagreement with He Jianku’s work including researchers from Stanford University, Harvard Medical School, and the NIH (Cohen 2018). Specifically, a journalist from Science magazine states that representatives from eight countries who attended the International Summit on Human Genome Editing in Hong Kong came to a consensus that his actions were irresponsible, violated international norms, lacked transparency, and did not have sufficient medical justification (Cohen 2018).

Jennifer Doudna stated that her goal was to edit out faulty genes, but that definition leaves a lot to interpretation. How can we as a society come together to define the parameters of a faulty gene? From treating HIV to Down Syndrome, these conditions hold more than just health implications, but social ones as well. If the scientific community deems it accessible to prevent Down syndrome using human germline genome editing, are they stating that humans that currently possess this condition are “wrong” and have “faulty genes” in need of correction? Whoever holds the power to manipulate this technology essentially has the power to decide what the next generation of humans will be capable of and what genes are deemed “faulty” within our society. Jianku’s reckless use of technology was majorly deemed wrong for its lack of transparency, safety, and following set regulations upheld by currency bioethical standards. However, in situations like parents wanting their children to be healthy, it is completely understandable that human germline editing sounds like an intriguing way to protect their children. For these reasons, it is imperative to familiarize ourselves with the development of this technology, and its continued push for regulations across the world to maintain ethical standards. Further, it is also important to ask ourselves hard-hitting questions such as: should one person, scientist, or government have the power to determine what the next generation will possess, look like, or become? We as members of society must continue to stay informed in a world with the capacity to genetically modify human beings.

References:

  1. Bhanu Prakash. “How the HIV Infection Cycle Works.” Proceum Pvt. Ltd. January 2019. https://www.youtube.com/watch?v=GyofqO1TRjU
  2. Brianna Rapini, Sarina Peterson, “Genetic Engineering.” September 2023. https://www.youtube.com/watch?v=CDw4WPng2iE
  3. Dennis Normile. “Chinese scientist who produced genetically altered babies sentenced to 3 years in jail.” Science. December 2019.https://www.science.org/content/article/chinese- scientist-who-produced-genetically-altered-babies-sentenced-3-years-jail
  4. Deng Luo, Zihui Xu, Zhongjing Wang, Wenzhuo Ran. “China’s Stem Cell Research and Knowledge Levels of Medical Practitioners and Students.” Stem Cells International. 2021. https://doi.org/10.1155/2021/6667743.
  5. Doudna J. 2019. Faculty Research Page. Department of Molecular & Cell Biology. http://mcb.berkeley.edu/faculty/BMB/doudnaj.html.
  6. Dylan Roach, Tanya Lewis. “CRISPR, the gene-editing tech that’s making headlines, explained in one graphic.” Insider. December 2015. https://www.businessinsider.com /crispr-gene-editing-explained-2015-12
  7. Genetic Literacy Project. 2019 July 23. United States: Germline / Embryonic. Global Gene Editing Regulation Tracker. https://crispr-gene-editing-regs-tracker.geneticliteracy project.org/united-states-embryonic-germline-gene-editing/.
  8. Henry T Greely. “CRISPR’d babies: human germline genome editing in the ‘He Jiankui affair.’” Journal of Law and the Biosciences. Volume 6, Issue 1. October 2019, Pages 111–183, https://doi.org/10.1093/jlb/lsz010
  9. Indra Mani. “CRISPR-Cas9 for treating hereditary diseases.” Progress in molecular biology and translational science. Volume 181. February 2017. https://pubmed.ncbi.nlm.nih.gov/34127193/
  10. John Cohen. “After last week’s shock, scientists scramble to prevent more gene-edited babies.” Science. December 2018.https://www.science.org/content/article/after-last- weeks-shock-scientists-scramble-prevet-more-gene-edited-babies
    Join David Ignatius. “Walter Isaacson & Jennifer Doudna join Washington Post Live to discuss CRISPR.” Washington Post. March 2021. https://www.youtube.com/ watch?v=cKHuuALENZk
  11. Than K. 2019 Nov 12. AI and gene-editing pioneers to discuss ethics. Stanford News. [accessed 2023 Dec 3]. https://news.stanford.edu/2019/11/12/ai-gene-editing-pioneers- discuss-ethics/#:~:text=Doudna%20decried%20the%20act%20but.
  12. Yi Zheng et al. “Structure of CC Chemokine Receptor 5 with a Potent Chemokine Antagonist Reveals Mechanisms of Chemokine Recognition and Molecular Mimicry by HIV.” Immunity. Volume 46, Issue 6. June 2017. https://www.cell.com/immunity /pdf/S1074-7613(17)30218-2.pdf
  13. ‌Zhang Xinqing, Zhang Wenxia, Zhao Yandong. “The Chinese Ethical Review System and its Compliance Mechanisms.” TRUST. September 2014. https://trust-project.eu/ wp-content/uploads/2016/03/Chinese-Ethics-Review-System.pdf

Inverse Vaccines: A New Way to Treat Autoimmune Disorders

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Williams, 2023

Of the 8.1 billion people in the world, 1 in 10 have an autoimmune disorder. There are 8.1 billion people in the world, and 810 million of these people, or 1 in 10, have an autoimmune disorder (“1 in 10 people”, 2023). Autoimmune disorders are a category of conditions in which the body attacks itself. Although management systems for most of these types of conditions have been developed, autoimmune diseases still cannot be cured (“Autoimmune disorders”). In fact, even with management of their symptoms, up to 50% of patients with autoimmune disorders still experience impairment in their health-related quality of life (Pryce and Fontana, 2017). The development of so-called “inverse vaccines” may provide the much needed mechanism to help find a cure for this class of conditions by teaching the body not to attack itself. 

A traditional vaccine works because it helps the body learn to recognize parts of foreign pathogens and builds up the body’s immune response against these pathogens so that the response is stronger and happens more quickly after recognition. In people with autoimmune diseases, the body also forms an immune response against self molecules because it mistakenly identifies them as foreign antigens, or pathogenic molecules (usually proteins or sugars) that induce an immune response. The idea behind “inverse vaccines” is that instead of building up the immune system’s response to foreign antigens, they could suppress the response to self-antigens by helping to teach the body to recognize these misidentified molecules as self. 

A study led by Andrew Tremain at the University of Chicago’s Pritzker School of Medicine is one of the groups who are involved in this novel inverse vaccine research. The inverse vaccines they developed contain modified copies of the self-antigens that are targeted by the immune system which are attached to long chains of sugars called polysaccharides (Tremain et al., 2023). These polysaccharide chains guide the self-antigens to the liver, which plays an important role in the establishment of tolerance to these molecules. Once these modified self-antigens arrive at the liver, specialized immune cells pick them up and then inhibit the action of T cells against them through T cell uptake (Leslie, 2023). T cells are a type of immune cell that carry out part of the typical response against molecules identified as foreign or invaders through either cytotoxic or signaling based immune responses. The inhibition of the T cell response against misidentified self-antigens reduces or prevents the autoimmune response that causes the body to attack itself, thereby acting as a kind of “inverse vaccine”. 

Once Tremain et al. developed their inverse vaccine, they conducted testing to determine its efficacy and viability as a method for increasing tolerance to self-antigens in those with autoimmune disorders. First, they wanted to ascertain whether inverse vaccines could truly provide inhibition of an immune response. To do this, they injected an egg white protein into mice as an experimental foreign antigen to trigger a strong immune response. Then an inverse vaccine against the egg white protein was injected to suppress the response to the original dose of the protein. In their analysis, they found that the T cells that would’ve responded to the egg white protein were not present. These results suggested that the inverse vaccine blocked the typical immune response, demonstrating viability as a treatment method against stimulated immune responses (Tremain et al. 2023).

However, Tremain and his colleagues still had to demonstrate the efficacy of inverse vaccines in inhibiting an autoimmune response rather than one caused by a foreign antigen. To do this, they induced an autoimmune disease called experimental autoimmune encephalomyelitis (EAE) in mice. In EAE, the immune system attacks myelin, the substance responsible for forming insulation around nerve cell axons. EAE is a particularly informative experimental model in mice because it mimics multiple sclerosis (MS), a human autoimmune disorder. Once they induced EAE, Tremain et al. injected an inverse vaccine made up of a polysaccharide carrying part of a myelin protein. Physiological analysis after injection suggested that this treatment had stopped mice from developing EAE. Furthermore, injection of a different inverse vaccine targeting an alternate form of EAE showed prevention of symptom relapse. This means that Tremain et al. were able to demonstrate inhibition of autoimmune responses against two types of EAE in mice, representing two different types of MS, providing a heartening outlook on this research (Tremain et al., 2023). 

In summary, inverse vaccines had the ability to turn off immune responses to particular antigens in mice. These results are a promising sign for the ability of inverse vaccines to combat autoimmune diseases. Additionally, initial clinical trials testing the safety and efficacy of inverse vaccine strategy to increase tolerance to self-antigens in humans have had positive results so far for immune disorders such as multiple sclerosis and celiac disease where the misidentified self-antigens are known (Leslie, 2023). However research into tolerance-increasing strategies tends to stall both because we do not know which self-antigens are attacked in several autoimmune disorders, and because the mechanisms that produce tolerance after antigens are brought to the liver are not well understood (“Immune Tolerance”). This means that even if autoimmune vaccines prove to be a viable form of treatment for autoimmune disorders, they will only be able to treat diseases with known self-antigens until further research into the antigen and tolerance mechanisms is conducted. Nevertheless, inverse vaccine research is incredibly promising and has the potential to help hundreds of millions of people worldwide.

Works Cited

Pryce, CR., and Fontana, A. (2017). Depression in autoimmune diseases. Current Topics in Behavioral Neurosciences, 31. https://doi.org/10.1007/7854_2016_7 

1 in 10 people suffer from autoimmune diseases. (2023). Neuroscience News.  https://neurosciencenews.com/population-autoimmune-disease-23198/ 

Autoimmune disorders. (n.d.). Retrieved November 5, 2023, from  http://www.betterhealth.vic.gov.au/health/conditionsandtreatments/autoimmune-disorders

Tremain, A.C., Wallace, R.P., Lorentz, K.M. et al. Synthetically glycosylated antigens for the antigen-specific suppression of established immune responses. Nat. Biomed. Eng 7, 1142–1155 (2023). https://doi.org/10.1038/s41551-023-01086-2 

Leslie, M. (2023). ‘Inverse vaccine’ could help tame autoimmune diseases, Science. https://www.science.org/content/article/inverse-vaccine-could-help-tame-autoimmune-diseases 

Immune Tolerance in Autoimmune Disease, Immune Tolerance Network. (n.d). Retrieved November 5, 2023, from https://www.immunetolerance.org/researchers/clinical-trials/autoimmune-disease 

Understanding Multiple Sclerosis, Oregon Health and Science University. Retrieved November 5, 2023, from  https://www.ohsu.edu/brain-institute/understanding-multiple-sclerosis 

Williams, S. (2023). “Inverse vaccine” shows potential to treat multiple sclerosis and other autoimmune diseases. Pritzker School of Molecular Engineering,  The University of Chicago. https://pme.uchicago.edu/news/inverse-vaccine-shows-potential-treat-multiple-sclerosis-and-other-autoimmune-diseases

Plant Talk: Eavesdropping on Underground Plant Communication

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Have you ever looked at a tiny sapling, a winding vine, or a massive oak tree and felt like they have some sort of personality? With the rustle of some leaves or the snap of a twig it might seem like these plants are talking to each other. As it turns out, these fantasies aren’t too far from the truth. Vascular plants (which consist of most plants other than mosses and algae) can actually communicate. These plants can exchange messages through their root systems with the help of mycorrhizal fungi. These fungi exist in a mutualistic relationship with plants and, along with acting as a living walkie talkie, they provide many survival benefits to the plants they live with. 

To be clear, vascular plants aren’t chatting in some sort of plant language in the same way that we talk to each other. Instead, they communicate through the transfer of infochemicals. “Infochemical” is an umbrella term for substances released by one plant and detected by another (Chen 2018). Infochemicals can take the form of plant hormones or nutrients and are passed between plants through the soil. The problem with this system is that transport through the soil is incredibly inefficient. When infochemicals move from plant to plant, they can quickly be absorbed by organic material or degrade in the soil such that they do not reach the intended “listener” plant. This is where mycorrhizal fungi come into play.

Mycorrhizal fungi (MF) are distinguished from other fungi by the symbiotic relationship that they have with plant roots. MF attach to plant roots and perform beneficial services for the plant in return for the carbon necessary for MF’s survival. MF networks add large amounts of surface area to plant root systems, allowing for the more efficient uptake of nutrients to the plants such as nitrogen, phosphorus, and carbon. The MF relationship increases efficiency of water collection, enhances photosynthesis, and improves resistance to pathogens. (Barto 2012). 

When it comes to plant communication, MF act as “superhighways” for the infochemicals to travel from plant to plant. Instead of having to travel through the soil, infochemicals can be safely transported between plants through common mycorrhizal networks (CMNs). CMNs are made up of interconnected networks of fungal branches, which span the distance between plant roots (Chen 2018). These networks are not exclusive to one species of plant because MF are not host specific and therefore can associate with multiple species at the same time. This allows for messages, in the form of infochemicals, to be passed efficiently between plants of varying species. This method is exponentially more efficient than infochemical transport through the soil, allowing plants to communicate much easier.

You might be wondering, what do these plants have to talk about? It turns out, they have a whole lot to discuss. The world can be a dangerous place and plants use these CMN superhighways as an emergency warning system to let neighboring plants know about potential threats. A plant that experiences a disturbance, such as infection by a pathogen or herbivore attack, can send signals to surrounding plants to let them know of the potential danger. The plants receiving the message can then increase their defense to better prepare for the threat. This exact phenomenon has been observed in neighboring plants where one plant is infected with a pathogen, and then surrounding uninfected plants respond to infochemical signals by activating defense proteins (Chen 2018).

Beyond plant defense, there is still a lot to learn about how plants are communicating and what kinds of things they are “talking” about. There are still questions to be answered such as how plant relatedness impacts infochemical transfer or how far these networks can span underground. If we continue to eavesdrop on this “plant talk” then we can start to understand the interconnected nature of plant communities even better.

Works Cited

Barto, E. K., Weidenhamer, J. D., Cipollini, D., & Rillig, M. C. (2012). Fungal superhighways: do common mycorrhizal networks enhance below ground communication?. Trends in plant science, 17(11), 633–637. https://doi.org/10.1016/j.tplants.2012.06.007

Chen, M., Arato, M., Borghi, L., Nouri, E., & Reinhardt, D. (2018). Beneficial Services of Arbuscular Mycorrhizal Fungi – From Ecology to Application. Frontiers in plant science, 9, 1270. https://doi.org/10.3389/fpls.2018.01270

Bonazzi, D. (2021). The secret underground life of trees. Weizmann Compass. Retrieved December 3, 2023, from https://www.weizmann.ac.il/WeizmannCompass/sections/features/the-secret-underground-life-of-trees.

 

Ending the Biomedical Harvest: Synthetic Alternatives to Horseshoe Crab Blood for Bacterial Endotoxin Detection

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Did you know that horseshoe crabs have incredible immune systems? In fact, horseshoe crabs have the best immune systems out of all living invertebrates. Their secret? Blood. Horseshoe crab blood is very simple in composition, with only a single cell type in general circulation (the granular amebocyte) and only three proteins in the plasma of the blood (hemocyanin, C-reactive proteins, and a2-macroglobulin) (Armstrong et al., 2008). These proteins contribute to the horseshoe crab’s blood clotting system, protecting them from infection. Horseshoe crab blood has been found to be very sensitive to bacterial endotoxins found in illness-causing Gram-negative bacteria (Protecting Health). When horseshoe crab blood cells come into contact with bacterial endotoxin, they clot around it, preventing the bacterium from invading nearby cells (Natural History Museum 2020). 

With the rise of vaccine development, especially in the case of the Covid-19 pandemic, horseshoe crab blood plays an essential role in testing the safety of vaccines due to its endotoxin-detection properties. Additionally, large volumes of horseshoe crab blood can be collected easily, making it a convenient blood source (Armstrong et al., 2008). Despite all the beneficial applications of horseshoe crab blood, horseshoe crab bleeding leaves thousands of horseshoe crabs dead annually, causing their populations to be in decline (Maloney et al., 2018). A 2018 study has promoted a synthetic alternative to horseshoe crab blood, recombinant Factor C (rFC), and proven its efficacy in bacterial endotoxin detection. The use of rFC in vaccine development can eliminate the need for the use of actual horseshoe crab blood, sparing the horseshoe crab and promoting the conservation of this endangered species. 

Typically, horseshoe crab blood is collected by the direct puncture of the heart under sterile conditions that minimize contamination by bacterial endotoxins (Figure 1). A large horseshoe crab can produce between 200 – 400 mL of blood, and the blood clotting system can be studied microscopically. The limitation of contamination by bacterial endotoxins is extremely important in the blood collection process, because cell clotting will compromise the effectiveness of the blood for its intended use of developing vaccines. Only undamaged horseshoe crabs are selected for blood collection, and the animal is bled by the insertion of a needle into the heart through the outer hinge joint of the horseshoe crab (Figure 2). The animal is then squeezed gently so that as much blood as possible can be deposited into the collection tube (Armstrong et al., 2008).

Figure 1: Horseshoe crab bleeding on a larger scale, with precautions taken to ensure sanitary, endotoxin-free conditions for blood collection.

Figure 2: The three major components of the body of a horseshoe crab, including the prosoma (P), the opisthosoma (O), and the telson (T). The hinge (H) is where the prosoma meets the opisthosoma, and that is where the needle is inserted for blood collection. 

Following collection, horseshoe crab blood is ready for use in endotoxin detection (Armstrong et al., 2008). For example, in vaccine development, a Limulus amebocyte lysate (LAL) test detects the levels of clotting in horseshoe crab blood when it comes into contact with different vaccines. Horseshoe crab blood is very precise with detecting even small traces of endotoxin, making it an effective tool to identify small quantities of endotoxin present in potential vaccines (Protecting Health).  

Although horseshoe crab blood is effective in its ability to detect endotoxins, recombinant Factor C (rFC) can do the same job in a way that is more ecologically sustainable. Initially, rFC was discovered by scientists at the National University of Singapore, allowing them to visualize endotoxin detection using animal-free technology. Every year over 500,000 horseshoe crabs are captured and as much as ⅓ of their blood is drained, contributing to high mortality rates. On top of this, around 13% of the horseshoe crabs bled are later sold for bait, resulting in nearly 130,000 horseshoe crab victims to the biomedical industry. A 2018 study confirmed that the biomedical industry could reduce their use of horseshoe crab blood by nearly 90% if they were to employ rFC as a synthetic alternative for endotoxin detection processes. The 2018 study reviews multiple studies that show how rFC is just as effective as actual horseshoe crab blood in endotoxin detection, as rFC has been able to demonstrate the same high rate and sensitivity as horseshoe crab blood in detecting small amounts of endotoxin in a wide range of chemical structures. When endotoxin binds to a synthetic rFC molecule, it causes the rFC to fluoresce directly proportional to the concentration of endotoxin in a substance. rFC has even been able to demonstrate a higher rate of specificity for endotoxin detection (compared to horseshoe crab blood) in some studies (Maloney et al., 2018).

The most important next step of this research is to get synthetic rFC into the hands of the biomedical industry. Exposure to endotoxin can cause serious illness, making endotoxin detection for vaccines an essential part of the vaccine development process.  Even though there is ample evidence that rFC is equivalent to or better than horseshoe crab blood at detecting bacterial endotoxin, there are still limitations to the usage of rFC, as it is difficult for the biomedical industry to adopt new technologies quickly. Endotoxin detection testing is very highly regulated, so many pharmaceutical manufacturers may be hesitant to employ new detection technologies, as they may want to stick to traditional methods instead (Maloney et al., 2018). Despite these limitations, in order to progress towards horseshoe crab conservation, rFC should be produced and employed on a large scale so that the biomedical industry will no longer be solely reliant on the exploitation of horseshoe crabs for bacterial endotoxin detection.

Literature Cited

1. Armstrong, P., Conrad, M. Blood Collection from the American Horseshoe Crab, Limulus Polyphemus. J. Vis. Exp. (20), e958, doi:10.3791/958 (2008). 

2. “Horseshoe Crab Blood: The Miracle Vaccine Ingredient That’s Saved Millions of Lives.” Www.nhm.ac.uk, www.nhm.ac.uk/discover/horseshoe-crab-blood-miracle-vaccine-ingredient.html#:~:text=Horseshoe%20crab%20blood%20is%20bright.  

3. Maloney T, Phelan R, Simmons N. Saving the horseshoe crab: A synthetic alternative to horseshoe crab blood for endotoxin detection. PLoS Biol. 2018 Oct 12;16(10):e2006607. doi: 10.1371/journal.pbio.2006607. PMID: 30312293; PMCID: PMC6200278.  

4. “Protecting Health.” Www.horseshoecrab.org, www.horseshoecrab.org/med/health.html. Accessed 12 Nov. 2023.  



Pupil Mimicry Strengthens Infant-Parent Bonding

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Sometimes, it is a wonder how something so small can connect human beings on a deeper level, but that is what pupil mimicry does. Pupil mimicry describes the changes in pupil size that occur in both participants during eye contact, which can help with social bonding. It also reflects the different cognitive and emotional processes that occur during eye contact and socialization, such as showing social interest (Aktar et al., 2020). When pupil size synchronously dilates, meaning the pupil expands when eye contact is made, there is a promotion of trust and bonding between the two responders. The opposite is true for synchronous pupil constriction, which diminishes a positive social bond between the two responders.  

Pupil mimicry is an old, robust phenomenon (Prochazkova et al., 2018) that is modulated by oxytocin. This evolutionarily conserved neuropeptide acts as a hormone and neurotransmitter and facilitates social bonding (Aktar et al., 2020). Pupil mimicry has been observed in monkeys and chimpanzees, where it also increases trust and social familiarity (Kret et al., 2014). This effect occurs in infants as well, which suggests that pupil mimicry may help facilitate bonding between infants and their parents. But how can scientists measure that? 

It has been shown that young infants can differentiate between their own-race faces and other-race faces. Therefore, scientists hypothesized that infants would have quicker pupillary responses to pupils belonging to the same race as their parents when compared to other races. Researchers Aktar, Raijmakers, and Kret conducted a study with three aims to test this hypothesis: 

  1.  Do infants’ pupils react to dynamic videos of eyes with pupil sizes that change realistically? 
  2. Do parents and infants have the same speed in matching pupil size? 
  3. Do both the parents’ and infants’ pupils have differing rates of pupil mimicry between own-race faces and other-race faces

For the first aim, infants and parents watched black-and-white dynamic videos of same-race models (Dutch male and female) that had constricting, static, or dilating pupils while their pupillary reactions were tracked (Figure 1; Aktar et al., 2020). For the second and third aims, infants and parents watched black-and-white dynamic videos with two races: Dutch for the same-race category and Japanese for the other-race category (Aktar et al., 2020). The researchers compared the parents’ pupil mimicry speed to the infants’. 

Figure 1: Experimental set-up of infants and parents as they observe the stimuli (Aktar et al., 2020).

The researchers confirmed that both infants and parents were able to perform pupil mimicry. They also found that parents had quicker pupil response to dilated or constricted pupils than infants, possibly due to adults being more cognitively advanced than infants. Finally, they concluded that there was no significant difference in pupil mimicry response between own race and other races, but there were slight pupil mimicry delays. The researchers have several explanations for the slight delays. For instance, the infants’ pupils tend to stay dilated when they see a dilated pupil, regardless of race, since infants are still developing their pupil mimicry control. For adults, pupil mimicry tends to take about 2.5 milliseconds longer when given other-race stimuli. This may be from greater cognitive effort used to process other-race faces than own-race faces (Aktar et al., 2020). 

The key finding of the research is race does not affect the participants’ rate of pupil mimicry during emotionally neutral interactions (Aktar et al., 2020). So, though pupil mimicry helps strengthen parent-infant relationships, infants also have the skills to establish trust and awareness with strangers regardless of race. However, when infants are not in a neutral setting, meaning an environment where they feel unsafe and discontent, they are more likely to seek out their parents and less likely to make eye contact (Aktar et al., 2020). That is why, if the infants felt fussy or frightened, the researchers sat the parents right next to them to provide a feeling of safety (Figure 1). Eye contact conveys a great deal of information. Maybe the next time you make eye contact with someone, stare at them to see how their pupil responds to you!

References

Aktar, E., Raijmakers, M. E. J., & Kret, M. E. (2020). Pupil mimicry in infants and parents. Cognition and Emotion, 34(6), 1160–1170. https://doi.org/10.1080/02699931.2020.1732875

Kret, M. E., Tomonaga, M., & Matsuzawa, T. (2014). Chimpanzees and humans mimic pupil-size of conspecifics. PloS one, 9(8), e104886. https://doi.org/10.1371/journal.pone.0104886

Prochazkova, E., Prochazkova, L., Giffin, M. R., Scholte, H. S., De Dreu, C. K. W., & Kret, M. E. (2018, July 16). Pupil mimicry promotes trust through the theory-of-mind network – PNAS. Proceedings of the National Academy of Sciences. https://www.pnas.org/doi/10.1073/pnas.1803916115

CRISPR-izing the Mitochondrial Genome Is Here

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CRISPR-Cas9 has become a standard method for editing the nuclear genome, but researchers have long debated whether the mitochondrial genome could be edited too. At last, we have an answer: yes.

When you hear the word “genome,” what comes to mind? The collection of DNA present in the nucleus? While technically correct, our old friend, the “powerhouse of the cell,” would like to object: it too has a genome! In humans, the mitochondrial genome is a circularized strand of DNA that is around 16,500 base pairs long, and within a single cell, it can have hundreds to thousands of copies [1]. At first glance, mitochondrial DNA (mtDNA) doesn’t seem that impressive, especially compared to the 3.4 billion base pairs of the nuclear genome. However, it has significant relevance to human health: mtDNA mutations are associated with numerous diseases from the cardiomyopathy of Barth syndrome to the neurodegeneration of Leigh syndrome [2]. Thus, efforts have been made to “fix” these defects, particularly through CRISPR-Cas9 gene editing.

The CRISPR-Cas9 system consists of an enzyme (Cas9) and a guide RNA (sgRNA) that can locate a specific region of the genome and induce a double-stranded break in the DNA. Using DNA repair mechanisms, this can be exploited to delete or insert a new sequence. For instance, homology-directed repair (HDR) uses a DNA template, either natural or supplied by researchers, to rebuild the broken strand with that template’s sequence. This has been especially powerful for editing nuclear genomes, but it has been debated whether this method could be used for mtDNA as well [3].

This is where Bi et al. enter the scene with their recent study [4]. Their goal was simple: to demonstrate that the mitochondrial genome can, in fact, be “CRISPR-ized.” The first step was to design a CRISPR-Cas9 system capable of entering the mitochondrion. To accomplish this, they took an existing system for nuclear editing and inserted “localization signals” that tell the cell where the protein needs to go. One of these was the mitochondrial targeting signal (MTS) derived from a nuclear-encoded gene, COX8A, that allows the protein to travel to the mitochondrion. Next, an sgRNA was designed that targets the ND4 gene of the mitochondrial genome. Combining these, Bi et al. created a system that could be tested in human cells. Indeed, with the use of fluorescent markers and microscopy, they demonstrated that both the Cas9 protein and sgRNA could enter the mitochondrion (Figure 1).

Next, the researchers needed to determine whether this system could edit mtDNA once inside the organelle. In addressing this, they added the CRISPR-Cas9 system alongside a template strand (called an ssODN) that contained an inserted sequence, GAATTC, that they could screen for (Figure 1). Once the cells were treated, they confirmed the insertion through two methods: PCR and direct sequencing. The PCR method used a primer that could only bind to the inserted sequence, so if the mtDNA was amplified, that indicated a successful “knockin.” The sequencing method, “PCR-free 3rd generation sequencing,” provided additional confirmation without the risk of false-positives from PCR artifacts (Figure 1).

Finally, as a proof of concept, Bi et al. altered the sgRNA and template strand to insert a pathogenic mtDNA mutation (changing ACCTTGC to GCAAGGT on the ND1 gene). This is associated with mitochondrial myopathy (a disease that causes muscular problems), and if successfully inserted, it could provide researchers with a new cell that models this mutant. Indeed, PCR and sequencing confirmed its knockin, demonstrating the power of editing mtDNA.

The authors of this paper directly confirmed that mtDNA could be edited via CRISPR-Cas9 for the first time. This is in contrast to previous studies which have shown mitochondrial localization of the Cas9-sgRNA complex, but with no clear indication of knockin because of PCR-based artifacts or indirect screening [5]. However, the present study is not without its own limitations, particularly in the method’s feasibility at a clinical level. First and foremost, an individual cell has many more copies of mtDNA than nuclear DNA, so editing every strand is more difficult. Indeed, the “knockin efficiency,” or proportion of DNA with the GAATTC insertion, was only 0.03-0.05% (compared to the 20-30% seen in some nuclear genome editing studies) [6]. Furthermore, Bi et al. reported an increase of reactive oxygen species (ROS) formation upon the addition of CRISPR-Cas9, a class of reactive molecules that can degrade DNA and induce cell death. 

Work remains to be done, whether it is through improving the efficacy of the CRISPR-CAS9 system or confirming the method’s safety amidst ROS production. Nonetheless, Bi et al. give us a powerful starting point for treating mitochondrial dysfunction in ways never previously thought possible. In the coming years, CRISPR-izing mtDNA may even become a standard technique, just as we saw with nuclear DNA in the last decade.

References:

  1. Falkenberg M, Larsson NG, Gustafsson CM. (2007). DNA replication and transcription in mammalian mitochondria. Annu Rev Biochem, 76: 679-99. https://doi.org/10.1146/annurev.biochem.76.060305.152028. 
  2. Stenton SL, Prokisch H. (2020). Genetics of mitochondrial diseases: Identifying mutations to help diagnosis. EBioMedicine, 56: 102784. https://doi.org/10.1016/j.ebiom.2020.102784.
  3. Gammage PA, Moraes CT, Minczuk M. (2018). Mitochondrial Genome Engineering: The Revolution May Not Be CRISPR-Ized. Trends Genet, 34(2): 101-110. https://doi.org/10.1016/j.tig.2017.11.001. 
  4. Bi R et al. (2022). Direct evidence of CRISPR-Cas9-mediated mitochondrial genome editing. Innovation (Camb), 3(6): 100329. https://doi.org/10.1016/j.xinn.2022.100329. 
  5. Antón Z et al. (2020). Mitochondrial import, health and mtDNA copy number variability seen when using type II and type V CRISPR effectors. J Cell Sci, 133(18): jcs248468. https://doi.org/10.1242/jcs.248468. 
  6. Zhang JP et al. (2017). Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage. Genome Biol, 18(1): 35. https://doi.org/10.1186/s13059-017-1164-8. 

Retrieving Forgotten Memories With Optogenetics

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Alzheimer’s disease is a neurodegenerative disorder in which patients experience a progressive decline in memory (Roy et. al, 2016). The early stages involve struggles to remember mostly episodic memories, which are memories of personal experiences that are linked to activity in the hippocampus. To discover potential treatments, scientists have tried to better understand the biological mechanisms behind the disease. Optogenetic activation of engram cells and calculation of dendritic spine density are two methods to research the disease. Dendritic spines are extensions from dendrites, which are the structures that receive signals from other neurons and pass them to the cell body. Memory is associated with changes in dendritic spines or the formation of new ones, which makes scientists believe that they serve as sites for memory formation and storage (Roy et. al, 2016). Engram cells are biological traces of established memories that store the information and are reactivated when memories are retrieved (Ortega-de San Luis et al., 2022). Optogenetics is a technique in which genes for light sensitive proteins are injected into specific neurons in the brain so that they can be activated by laser lights that open ion channels (Figure 1). 

Figure 1. The process of optogenetics (Adapted from Buchen, 2010). 

A 2016 study affiliated with the Massachusetts Institute of Technology used optogenetics to activate engram cells in the hippocampus of mouse models of early Alzheimer’s disease (AD) (Roy et al., 2016). With direct activation of hippocampal cells, the mice were able to retrieve the memories they had forgotten, indicating that the issue is retrieving memories; the memories are still available in the brain, but there is a problem in recalling them. Amnesia in early AD mice is also correlated with a reduction of spine density of engram cells in the hippocampus. The study showed that restoring dendritic spine density with optogenetic activation allows for the retrieval of long-term memory. Spine density was restored as the optogenetic activation allowed sodium ions to flow into the neurons and consequently depolarized them (meaning the cell is less negatively charged). This depolarization triggers long term potentiation, which is the process of strengthening connections between neurons that underlies memory and learning. Thus, restoration of spine density could lead to an effective strategy for treating memory loss in patients with early Alzheimer’s disease. 

In the experiment, the scientists labeled engram cells in the mouse models of Early Alzheimer’s disease and tested memory recall with contextual fear conditioning and long-term memory testing. Contextual fear conditioning is when mice are trained to associate a particular context (a cage) with a shock, creating a memory of the association in the hippocampus. Long-term memory was tested by placing them back into the cage and measuring their freezing behavior, a commonly used measure of fear. They also calculated dendritic spine density. 

The researchers found that optogenetic activation of memory engrams restores fear memory in early AD mice and that reversal of engram-specific spine deficits rescues memory in early AD mice. The diagram below shows the process of fear conditioning (panel t): the mice were trained to associate a shock with a certain context, and then their long term memory was tested by placing them back into the cage and observing their “freeze response,” which demonstrates that they feel fear in the cage because of the memory of the shock in the cage. The graphs show that when the engrams were activated optogenetically, the mice froze more than they did when the engrams were not activated, indicating that the optogenetic activation of the engram cells restored the mice’s memory of the fearful context. 

Figure 2. Optogenetic activation of memory engrams restores fear memory in early AD mice (Adapted from Roy et al., 2016, Fig. 1). 

The results of this study provide directions and hope for future treatments for Alzheimer’s Disease. If the amnesia is due to retrieval impairments, memory could be restored by technologies involving brain stimulation, like the optogenetic activation did in the mouse models. However, optogenetics is currently a technique only possible in animals since it is invasive, so further research will have to be done to discover a technique employing the principle in a more plausible way for humans, perhaps by finding a way to restore spine density in engram cells. There are other limitations when it comes to future treatments for Alzheimer’s because of the fact that they studied only early AD mice and episodic memory. Even though they showed that amnesia in early AD mice impairs memory retrieval, long-term memory storage in advanced stages of AD may also be impaired and eventually lost as neuronal degeneration progresses. Also, humans with early AD often exhibit non-episodic memory deficits as well, which involve brain structures outside of the medial temporal lobe that the current study did not investigate. However, overall, the findings contribute to a better understanding of memory retrieval deficits in early cases of AD, which may also apply to other neurological diseases in which patients have difficulty with memory retrieval, such as Huntington’s Disease. 

 

Literature Cited

Buchen, L. (2010). Neuroscience: Illuminating the brain. Nature, 465(7294), Article 7294. https://doi.org/10.1038/465026a

Ortega-de San Luis, C., & Ryan, T. J. (2022b). Understanding the physical basis of memory: Molecular mechanisms of the engram. The Journal of Biological Chemistry, 298(5), 101866. https://doi.org/10.1016/j.jbc.2022.101866

Roy, D. S., Arons, A., Mitchell, T. I., Pignatelli, M., Ryan, T. J., & Tonegawa, S. (2016d). Memory retrieval by activating engram cells in mouse models of early Alzheimer’s disease. Nature, 531(7595), 508–512. https://doi.org/10.1038/nature17172

Yuhas, D. (n.d.). Forgotten Memories May Remain Intact in the Brain. Scientific American. Retrieved November 4, 2023, from https://www.scientificamerican.com/article/forgotten-memories-may-remain-intact-in-the-brain/

PedPRM Unveils Promising Treatment for Insomnia in Children with Autism Spectrum Disorder

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Getting enough sleep is widely considered crucial to our well-being. However, for some individuals with Autism, getting enough quality sleep is not as easy as it sounds. Autism Spectrum Disorder (ASD) is a developmental disorder that widely affects the U.S. population, as 1 in 36 children aged eight has been diagnosed with autism (CDC, 2023). The kind and severity of the symptoms that individuals with ASD may exhibit can vary along the ASD spectrum.  Sleeping, in particular, is a common challenge for those with ASD, as many suffer from problems related to the REM (Rapid Eye Movement) sleep phase, a phase critical for memory consolidation (Devnani & Hedge, 2015). This can quickly become a vicious cycle for some, as lack of sleep can increase the severity of other Autism-related symptoms but also affect both the individual and the quality of life of their family or caretakers. 

To investigate possible treatments, a recent study by Malow et al. focuses on the pharmacological approach of using melatonin to treat sleep disorders. Melatonin is a hormone produced by the body’s pineal gland to regulate circadian rhythm, allowing the body to relax according to the appropriate light-darkness cycles of the day. This study uses a small, long-release tablet (PedPRM) used to mimic endogenous melatonin secretion. It focused mainly on a younger population, from children to adolescents, who met two criteria. The first was that they had confirmed diagnoses of either Autism or Smith-Magenis syndrome and had also experienced sleep abnormalities. Smith-Magenis syndrome is also a developmental disorder that involves symptoms similar to those of ASD, affecting behavior, cognition, and sleep. They also had to have not previously seen improvements when using sleep hygiene treatments to be included in the study, such as establishing a strict bedtime routine and taking steps to provide a calm and comfortable sleep environment (CHOC, 2023).

With the study sample set, the study was conducted over 108 weeks and divided into four phases (Graph I). Throughout the four phases, participants’ caregivers would document sleep quality and total sleep time in a sleep and nap diary to record the efficacy of treatment. The study participants started the first stage with a 2-week period in which they were given placebos. If participants showed improvement while receiving a placebo, they would be removed from the study to reduce the possibility of external factors affecting the results (Scott et al., 2021). After clearing Phase 1, participants entered the second phase, which consisted of a double-blinded 13-week period in which they were randomly placed into either a placebo or treatment group (PedPRM). After this, Phase 3 comprised a longer 91-week open-label period in which both groups were combined. For the final phase of the study, participants were placed again in a 2-week single-blind placebo period to ensure that the drug had been completely removed from the participants with no adverse effects after stopping treatment.

Upon concluding the experimental period, the results to be considered for this study could be divided into three groups: participants’ sleep quality, caregivers’ well-being, and the participants’ growth development. With this data, the researchers found a significant

 decrease in sleep disturbance (Graph II-A) and an increase in caregiver satisfaction (II-B) and quality of life (II-C). These were most pronounced during the first half of the treatment. In the latter half of treatment, sleep disturbance continued to decrease but at a slower pace than the initial treatment phase. Fortunately, there were no reported deaths, and most adverse reactions included daytime fatigue and mood swings. However, the severity and extent of these were not detailed in the results of the study and offer the potential to be analyzed further.  

The study shows compelling initial evidence that PedPRM is an effective treatment for sleep disorders in individuals with ASD. However, as noted by the researchers, it also shows that constant active treatment is required as most sleep quality improvements are removed upon halting treatment. Since medications for children are generally more strictly controlled, PedPRM consistently demonstrates a possibility for effective pediatric treatment, even if for long-term medication. In particular, these findings are essential as it has been found that rapid-release melatonin is not helpful with maintaining sleep a couple of hours after administration, and it had long been considered a challenge to find small, swallowable prolonged-release tablets for children (Fliesler, 2022). As sleep interruption is something that mainly affects those with neurodevelopmental disorders, this is a significant step towards adequate treatment. However, it is essential to note that this pharmacological alternative should only be considered if behavioral interventions and sleep hygiene modifications have been attempted but have been found unsuccessful. 

 

 

Sources 

CDC. (2022, December 9). Centers for Disease Control and Prevention. https://www.cdc.gov/ncbddd/autism/facts.html 

CDC Newsroom. (2016, January 1). CDC. https://www.cdc.gov/media/releases/2023/p0323-autism.html 

CHOC – Children’s Hospital of Orange County. (2023, March 2). Autism and Sleep Hygiene – Children’s Hospital of Orange County. Children’s Hospital of Orange County. https://www.choc.org/programs-services/autism-neurodevelopmental/co-occurring-conditions-program/autism-and-sleep/ 

Devnani, P., & Hegde, A. U. (2015). Autism and sleep disorders. Journal of Pediatric Neurosciences, 10(4), 304. https://doi.org/10.4103/1817-1745.174438 

Fliesler, N. (2022, June 13). Melatonin for kids: Is it effective? Is it safe? – Boston Children’s Answers. Boston Children’s Answers. https://answers.childrenshospital.org/melatonin-for-children/#:~:text=There%20is%20some%20evidence%20to,the%20ability%20to%20swallow%20capsules. 

Furfaro, H. (2023, March 10). Sleep problems in autism explained. Spectrum | Autism Research News. https://www.spectrumnews.org/news/sleep-problems-autism-explained/ 

Lemoine, P., Garfinkel, D., Laudon, M., Nir, T., & Zisapel, N. (2011). Prolonged-release melatonin for insomnia – an open-label long-term study of efficacy, safety, and withdrawal. Therapeutics and Clinical Risk Management, 301. https://doi.org/10.2147/tcrm.s23036 

Malow, B. A., Findling, R. L., Schröder, C., Maras, A., Breddy, J., Nir, T., Zisapel, N., & Gringras, P. (2021b). Sleep, Growth, and puberty after 2 years of Prolonged-Release Melatonin in children with Autism Spectrum Disorder. Journal of the American Academy of Child and Adolescent Psychiatry, 60(2), 252-261.e3. https://doi.org/10.1016/j.jaac.2019.12.007 

Scott, A., Sharpe, L., Quinn, V. F., & Colagiuri, B. (2022). Association of single-blind placebo run-in periods with the placebo response in randomized clinical trials of antidepressants. JAMA Psychiatry, 79(1), 42. https://doi.org/10.1001/jamapsychiatry.2021.3204 

Signs & Symptoms | Autism Spectrum Disorder (ASD) | NCBDDD | CDC. (2023, January 11). Centers for Disease Control and Prevention. https://www.cdc.gov/ncbddd/autism/signs.html

mRNA: Unpacking the Research that led to the COVID-19 Vaccines and the 2023 Nobel Prize in Medicine

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        By 2023, 81.4% of the United States population have received at least one dose of the COVID-19 mRNA vaccine, and just about everyone has heard of these lifesaving shots (CDC, 2023). But how many of us actually know how they work? In general, vaccinations expose the body to a weakened strain of a virus that initiates an immune response so that the virus can be recognized, and quickly neutralized, in the future (Jain, 2021). This works because of the structure of our immune system, which is split up into innate immunity and adaptive immunity. Innate immunity is the immediate response that our bodies put up when exposed to a pathogen; it sends out macrophages to gobble up infected cells, creates an inflammatory response to kill off the infection, and uses a host of molecules and pathways to destroy the virus. Adaptive immunity involves the presence of B and T cells, which arise in a viral infection and create immunological memory through antibodies and memory cells so that a future infection of the same virus is immediately recognized and shut down. Vaccines work to stimulate this adaptive immunity, while minimizing innate immunity.

        mRNA vaccines use this same basic principle, but instead of injecting a weakened strain of the actual virus into someone, they instead inject a piece of mRNA, or messenger RNA, which codes for a viral protein that stimulates this immune response (Jain, 2021).

Figure 1: The methodology of mRNA vaccines. Adapted from Jain, et. al., 2021

mRNA vaccines are much easier to make than traditional vaccines because they don’t require growing and inactivating a virus, and they don’t carry the risk that live vaccines have of infecting the subject, but up until recently, they weren’t even remotely possible because in vitro mRNA would create a huge inflammatory response when entering the body (Karikó, 2005). However, Hungarian biochemist Katalin Karikó and her colleague Drew Weissman were undeterred, and their research to create mRNA without an inflammatory response paved the way for mRNA vaccines and won them the Nobel Prize in Medicine in 2023 (Karikó, 2005).

        Karikó and Weissman’s research focused on in vitro mRNA’s stimulation of Toll-like receptors (TLRs), which recognize pathogens and activate signaling pathways leading to an inflammatory response (Karikó, 2005). They discovered that mammalian mRNA, which doesn’t stimulate an immune response, contains many base pair modifications, whereas bacterial RNA, which is known to stimulate a response, has no modifications (Karikó, 2005). In vitro mRNA, grown in a lab rather than from mammalian cell cultures, also has no, or very few, modifications. This led the researchers to question whether base pair modifications were how cells distinguished between foreign and non-foreign RNA, and thus which pieces of RNA stimulate an immune response (Karikó, 2005). They discovered that in vitro RNA activates three main TLRs known for activating an inflammatory response, but that nucleoside modifications limit that activation. Most importantly, they discovered that the suppressed immune response is proportional to the number of nucleotide modifications, and that these modifications also increase the stability of in vitro mRNA in the body, another original roadblock in the usage of mRNA for vaccines. (Karikó, 2005).

Figure 2: The use of base modifications to suppress inflammatory response of RNA. Adapted from Nobelprize.org

        Combined with later research also by Karikó and Weissman discovering that modified base-pairs also create increased protein production and other research designing more stable lipid carriers to deposit the mRNA into human cells, mRNA became a promising new vaccine technology, eventually allowing the COVID-19 vaccines to be produced in record times, shortening the pandemic and granting all of us our lives back. But the road to Karikó’s research being recognized, and especially the road to the Nobel Prize, was not predetermined. Karikó’s research was long overlooked, in part because of her identity as a Hungarian immigrant. She was “demoted four times” at UPenn, being told that her research didn’t measure up to their standards, but she continued to persevere, making her both a scientific success story, and a personal one (Shrikant, 2023). The COVID-19 vaccines created by Pfizer and Moderna were the first mRNA vaccines to be released, but future research is focusing on creating a universal flu vaccine and possibly even an HIV vaccine, all using mRNA technologies, which could drastically improve the landscape of viral infections, both in the US and across the globe.  

 

Literature Cited

Karikó, K., Buckstein, M., Ni, H., & Weissman, D. (2005). Suppression of RNA Recognition by Toll-Like Receptors: The Impact of Nucleoside Modification and the Evolutionary Origin of RNA. Immunity, 23(2), 165–175. https://doi.org/10.1016/j.immuni.2005.06.008

 Karikó, K., Muramatsu, H., Welsh, F. A., Ludwig, J., Kato, H., Akira, S., & Weissman, D. (2008). Incorporation of Pseudouridine into mRNA yields Superior Nonimmunogenic Vector with Increased Translational Capacity and Biological Stability. Molecular Therapy, 16(11), 1833–1840. https://doi.org/10.1038/mt.2008.200

 Jain, S., Venkataraman, A., Wechsler, M. E., & Peppas, N. A. (2021). Messenger RNA-Based Vaccines: Past, Present, and Future Directions in the Context of the COVID-19 Pandemic. Advanced Drug Delivery Reviews, 179, 114000. https://doi.org/10.1016/j.addr.2021.114000

 The Nobel Prize in Physiology or Medicine 2023. (n.d.). NobelPrize.Org. Retrieved October 22, 2023, from https://www.nobelprize.org/prizes/medicine/2023/press-release/

 CDC. (2020, March 28). Covid Data Tracker. Centers for Disease Control and Prevention. https://covid.cdc.gov/covid-data-tracker

Shrikant, A. (2023, October 6). Nobel Prize Winner Katalin Karikó was “demoted 4 times” at her old job. How she persisted: “You have to focus on what’s next.” CNBC. https://www.cnbc.com/2023/10/06/nobel-prize-winner-katalin-karik-on-being-demoted-perseverance-.html 

 

The Possibilities of the CRISPR-Cas9 System

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          As humans continue to further explore the fields of science, they deepen their understanding of convoluted subjects through the use of advanced technologies. One of the notable technologies today is the CRISPR-Cas9 system, a highly precise DNA editing tool that allows for genome manipulation in humans, animals, and plants. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) works with a CRISPR-associated (Cas) nuclease, which was originally derived from a genome editing system used as immune defense by bacteria. To put it simply, if infected by a virus, the bacteria captures a small piece of the invading viruses’ DNA and inserts their own DNA to create a CRISPR segment (Tavakoli et al., 2021). With this segment, the bacteria can produce RNA segments that will attach to parts of the viruses’ DNA if it attacks again. The bacteria use the Cas9 enzyme to cut the viruses’ DNA apart and incapacitate it. 

          Scientists adapted the system so it could be inputted into the human genome and edit DNA. Researchers created small segments of RNA that have a “guide” sequence which binds to a target sequence in the DNA and attaches to the Cas9 enzyme (Tavakoli et al., 2021). The guide RNA (gRNA) identifies the intended DNA sequence and the Cas9 enzyme cuts the specific location, similar to how the process occurs in bacteria. The cell’s own DNA machinery is then used to add or delete pieces of the genetic material, known as gene knock-in and gene knockout, respectively. However, it can also alter the DNA by replacing segments with more customized sequences. CRISPR activation (CRISPRa) is used to up-regulate the expression of a gene while CRISPR interference (CRISPRi) down-regulates the expression of a cell (CRISPR Guide).

Figure 1. Gene Knockout
Figure 2. Activation of Target  Gene with CRISPRa

                                       Note. Adapted from CRISPR Guide, Addgene, 2022.

          There are many potential applications in cancer immunotherapy, treatment of genetic diseases, improving plant genetic variation, and more. For example, there have been advances towards mitigating the effects of blood disorders such as sickle cell disease (SCD). In those with SCD, the misshapen red blood cells can block blood vessels, thus slowing or stopping blood flow. Effects of this fatal disease are anemia, chronic pain, strokes, and organ damage. In this instance, CRISPR technology is not used to directly rectify the disease-inducing gene variant. Instead of restoring adult hemoglobin, it works to increase the levels of fetal hemoglobin, an oxygen carrier protein that only fetuses make in the womb (Henderson, 2022). Since it’s not affected by the sickle cell mutation, it can be a substitute for the defective adult hemoglobin in red blood cells (Henderson, 2022).

Figure 3. Hemoglobin Switching in Infants

Note. Adapted from CRISPR Clinical Trials: A 2022 Update, Henderson, 2022, Copyright 2022 by Innovative Genomics Institute.

         As seen in the diagram above, SCD symptoms begin during infancy when levels of fetal hemoglobin decrease. The blood stem cells are harvested from the blood and then the genomes are edited to activate the fetal hemoglobin gene. Next, chemotherapy eliminates the disease-inducing blood stem cells from the person’s body, and the genome-edited stem cells are put back into the bloodstream through an IV, creating a new blood stem cell population that produces fetal hemoglobin (Henderson, 2022). This technique is ex vivo genome editing, where the editing occurs outside the body to prevent the risk of lingering and unwanted CRISPR components in the body. Those treated for SCD in CRISPR clinical trials show normal to near-normal hemoglobin levels with at least 30% as fetal hemoglobin (Henderson, 2022).

          It raises the following questions: is it possible to select certain features in humans that are seen as favorable or desirable? Is it possible to have almost complete manipulation of physical features or to enhance the body’s biological processes to unlock superhuman abilities only seen in movies? These human experimentations would be best measured if implemented during the early stages of life so as to let the body integrate the changes and develop properly. Perhaps we could alter ourselves to increase muscle mass, heighten athletic ability, or augment intelligence. In regard to altering the human genome, there are certain limitations as to what can and can’t be done without violating certain ethical boundaries. 

          This can be seen in the case of He Jianku, a Chinese scientist who used CRISPR to edit two embryos, both of which are now living babies. Though there was little information that only came from He himself and the Chinese government, it has been said that sometime in late 2017, He injected the two embryos with a CRISPR construct that would delete a 32-base-pair in the CCR5 gene on chromosome 3, leading to a non-functional CCR5 protein (Greely, 2019). He sought to achieve humans who could not contract AIDS because they don’t have this functioning protein (Greely, 2019). Though this theory was later proved wrong and the CRISPR construct did not fulfill his intentions, he managed to induce never before seen changes that led to the production of non-functional proteins. His actions prompted the Chinese court to convict him on the basis of deliberately violating medical regulations (Greely, 2019). Could it be argued that pushing the boundaries are necessary for scientific growth and greater human evolution? How far is too far? As CRISPR and other gene editing tools continue to develop, we can keep imagining the unnerving yet exciting possibilities that are awaiting.

 

Literature Cited

CRISPR Guide. (n.d.). Addgene. 

Greely, H. T. (2019). CRISPR’d babies: human germline genome editing in the ‘He Jiankui affair’. Journal of Law and Biosciences, 6(1), 111-183.

Henderson, H. (2022). CRISPR Clinical Trials: A 2022 Update. Innovative Genomics Institute.

Tavakoli, K., Pour-Aboughadareh, A., Kianersi, F., Poczai, P., Etminan, A., & Shooshtari, L. (2021). Applications of CRISPR-Cas9 as an Advanced Genome Editing System in Life Sciences. BioTech, 10(3), 14. 

What are genome editing and CRISPR-Cas9? (2022). MedlinePlus.