Description

Drug addiction is a chronic condition characterized by compulsive drug seeking and use despite negative consequences. Extensive research shows that surges of dopamine during pleasurable activities affect the brain's reward circuits. G9a, an under-researched gene, regulates dopamine production and release, influencing how the brain responds to addictive substances. Elevated levels of G9a are observed in addiction, and our research aims to reduce these levels using RNAi technology, specifically Small-Interfering RNA. Through a three-component system involving plasmids altered for overexpression, siRNA, and a hypothetical lipid-nanoparticle therapeutic, we identified the optimal siRNA concentration for patients with varying G9a expression levels.

Addiction is a chronic condition characterized by the compulsive seeking of a certain substance or activity despite adverse consequences. Drug addiction impacted over 16% of Americans in the past year, with this percentage increasing annually. Addiction can start for various reasons, but two of the most common are the use of recreational drugs in social settings, and the misuse of prescription drugs (NIDA 2022). The mistaken belief that addiction can be simplified to a matter of personal choice has led to a common misunderstanding of the disease and delayed the development of effective treatments, leaving many patients unable to receive adequate help. In history, addiction has primarily been viewed as a lack of willpower or a moral weakness, and was only classified as a disease in the late 20th century. Research has now found that addiction is a multifaceted issue that changes the chemistry of the brain; thus, the path to recovery involves complex medical treatments instead of purely advising patients to discontinue their usage of certain drugs (Institute of Medicine).

The human brain is wired to remember pleasurable activities to increase the chances of its recurrence. This process works through signals sent by the neurotransmitter dopamine. When the brain undergoes a pleasurable activity, a surge of dopamine is released, influencing neural circuits involved in reward processing and forming memory associations. This encourages the brain to seek out similar dopamine-releasing experiences in the future. Drugs produce an intense feeling of euphoria, causing an unhealthy amount of dopamine production and release in the brain, and thereby creating a powerful relationship between the activity of taking drugs and the desire to do it again. These huge surges of dopamine have the potential to change the neural connectivity of the brain and permanently impact an individual’s health and well being (U.S. Department of Health 2017).

Los Angeles County is notorious for its rising rates of substance abuse. In fact, according to a July 2022 report from the County of Los Angeles Public Health, 7.6% of Los Angeles County residents aged 12 and above live with a substance use disorder, and 15.2% had used an illicit drug within the past month. Although the percentage itself does not seem like a significant amount, LA County hosts 10 million people, and thus the corresponding data would be approximately 760,000 and 1.5 million people respectively. This sparks hot debate within communities, districts, and schools.

Research has shown that G9a activity is responsible for regulating the genes that influence the production and release of dopamine; thus, the G9a enzyme impacts how the brain responds to addictive substances. In a 2018 study of male rats, scientists examined how reducing G9a expression in the nucleus accumbens, the brain region associated with reward responses, impacts addiction and anxiety-related behaviors. They found that reducing the levels of G9a in rats’ brains decreased their desire to consume cocaine, suggesting that G9a plays a crucial role in addictive behaviors. On top of that, reducing G9a also reduced the likelihood of the rats seeking cocaine again after a period of abstinence, reducing the chance for relapse. Given that a major challenge with current addiction treatment is the risk for relapse, this research suggests that G9a research is a promising way to help diminish that risk and enhance treatments. The decreased addictive behavior caused by G9a knockdown also led to reduced anxiety-like behaviors. Anxiety and addiction often occur concurrently in individuals, making it necessary to design treatments that can target both behaviors. Understanding the influence of G9a in both anxiety and addiction raises the possibility of developing treatments that can focus on both disorders (Anderson 2019).

RNAi is a natural process occurring in our cells millions of times a day to regulate our cell’s gene expression or translation by neutralizing the mRNA produced by our gene’s RNA Polymerase II. The molecule responsible for this neutralization is siRNA. Although siRNA is a molecule naturally produced as a part of our cell’s line of defense against irregular gene expression and viruses, many proteins do not have specific siRNA created by our cells to regulate them. G9a is one of these proteins, and we believe that using synthetic siRNA to lower excessive levels of G9a will not only be incredibly efficient, but also reliable and safe for users of all different addiction dispositions with a G9a linkage.

Recent hypotheses surrounding the topic of drug addiction and epigenetic factors point to the possibility of decreasing drug addiction by increasing levels of histone deacetylases and lowering levels of histone acetylation leading to an overall decrease in gene expression, which in turn could block the brain's reward response to drugs, making them less stimulating and addictive (Source). This epigenetic process (how gene expression can be influenced on/off without altering the DNA) mediated through siRNA would be extremely effective because of its targeting mechanism, specific region control, and natural properties. In the nucleus accumbens shell, G9a disrupts the CREB transcription factor causing irregular function of D1- type dopamine receptors, resulting in addiction symptoms. With current mainstream technology, we can inhibit these D1- type dopamine receptors through the usage of antagonist drugs. However, these antagonist drugs lack the targeting mechanism specific to siRNA meaning they not only will cause inhibition of receptors that should not be inhibited but they could also cause further disruption to the production of the CREB transcription factor. Secondly, without specific region control, these antagonist drugs are unable to differentiate specific minute subregions of the brain such as the nucleus accumbens shell where addiction receptors are most prominent. Lastly, because of its inaccuracy in targeting and regional impact, antagonist drugs are infamous for its side effects and withdrawal symptoms causing both an unpleasant treatment procedure for the patient and the physician who needs to constantly order more drugs to keep the patient off chemical substance dependency.

Being a natural cure to addiction, RNAi technology through the mediation of siRNA is able to specifically target and dismantle excess protein levels in specific hard to reach areas. Our research shows the optimal concentration to be administered to different patients with different levels of G9a Concentration found in their bloodstream. Through this therapeutic, addiction patients will experience a gradual, stable, natural form of recovery centering around protecting the patient from normal withdrawal symptoms and dreadful side effects of chemically developed antagonist drugs.

A patient with substance abuse addiction would have higher concentrations of the G9a protein in real cell tissue within the nucleus accumbens shell. To introduce this overexpression of G9a, we designed a three-component system to address all aspects of this problem more inclusively and comprehensively.

Our first is a safe alternative design that theoretically mimics addiction through the overexpression of our protein G9a. We set our sights on targeting the encoding gene of G9a, euchromatic histone lysine methyltransferase 2 (better known as EHMT2), in order to understand its effects. Our plan was to construct a plasmid through Gibson Assembly of three separate pieces which would be cultured in DH5α E-coli, validated through gel electrophoresis and plasmid sequencing. This plasmid construct would contain the CMV promoter which strongly drives protein production in many mammalian cells including ours, and a puromycin antibiotic resistance gene for an increase in the cells taking up the plasmid.

This details the planned plasmid construct in detail of the parts we were to use.

Simultaneously, we also did thorough research on RNAi technology, leading to a design in small interfering RNA (siRNA) which we believe has the potential to be implemented for patients as an effective method to bring the gene expression levels back down to a normally regulated amount. We thus transfected different concentrations of siRNA into HEK293-T cells with a similar plasmid to the one we constructed to best mimic the overexpression of G9a and fully understand the effect that this siRNA would have on this protein.

To turn this siRNA into a therapeutic to be applied for patient care, our team also planned a concept model on the creation of Lipid Nanoparticles which would utilize SORT molecules within their receptor membrane to identify and mediate through cells specific to the nucleus accumbens shell (Nash). Our LNP would shield the siRNA from degradation of the body and carry the siRNA to the Nash where it would be able to dissolve naturally and specifically silence the overexpressed G9a protein.

NIDA. "Most reported substance use among adolescents held steady in 2022." National Institute on Drug Abuse, 15 Dec. 2022, https://nida.nih.gov/news-events/news-releases/2022/12/most-reported-substance-use-among-adolescents-held-steady-in-2022
NIDA. "Treatment and Recovery." National Institute on Drug Abuse, 25 Sep. 2023, https://nida.nih.gov/publications/drugs-brains-behavior-science-addiction/treatment-recovery
NIDA. "Drugs and the Brain." National Institute on Drug Abuse, 1 Jul. 2011, https://nida.nih.gov/publications/drugs-brains-behavior-science-addiction/drugs-brain
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Anderson Sources specifically: Anderson, Ethan M et al. “Knockdown of the histone di-methyltransferase G9a in nucleus accumbens shell decreases cocaine self-administration, stress-induced reinstatement, and anxiety.” Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology vol. 44,8 (2019): 1370-1376. doi:10.1038/s41386-018-0305-4
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Anderson, Ethan M et al. “Overexpression of the Histone Dimethyltransferase G9a in Nucleus Accumbens Shell Increases Cocaine Self-Administration, Stress-Induced Reinstatement, and Anxiety.” The Journal of neuroscience : the official journal of the Society for Neuroscience vol. 38,4 (2018): 803-813. doi:10.1523/JNEUROSCI.1657-17.2017
Anderson, Ethan M et al. “The histone methyltransferase G9a mediates stress-regulated alcohol drinking.” Addiction biology vol. 27,1 (2022): e13060. doi:10.1111/adb.13060