Team:Hong Kong HKUST/hp/article/genet

Overview

The Hong Kong University of Science and Technology (HKUST) iGEM 2013 team has built a fatty acid inducible system that enables inducible glyoxylate shunt to serve as an artificial futile cycle in human liver cells. Our vision is to introduce genes necessary for the cycle into the human body, with the aim of ultimately increasing energy expenditure in response to high circulating fatty acid levels. The idea is this would help obesity patients increase expenditure of calories and alleviate health complications such as cardiovascular disease, diabetes, and cancer. Because introducing genes into the human body is currently not a common therapeutic method, we thought it would be necessary to research on the details of how our project could be actually applied. Even though the application of our project may not occur in the near future, we thought it necessary to evaluate our project’s application as a potential biotechnology product. We have investigated a possible method that is under serious research in the science community: gene therapy. In this article, we will introduce the approach in more detail, examine the biosafety and bioethical issues, and lastly examine whether our project could be applied using this method.

Gene Therapy

We designed to introduce an inducible system that allows tunable fatty acid uptake by sensing fatty acid concentrations. Fatty acids uptake was to be quantified to compare the activities of wild type cells and cells expressing inducible glyoxylate shunt.

To facilitate expression of inducible glyoxylate shunt in human hepatoma cell line (HepG2 cell), cell viability at different sodium palmitate concentration was measured. While a high fatty acid level is known to lead apoptosis, the cell viability test ensured maintenance of a stable cell line for transfection.

We used MTT assay to test cell viabilities in different fatty acid concentrations. The objective was to determine a range of optimal concentrations of fatty acids to be introduced into HepG2 cell and achieve more than 60% viability after 24 hours incubation and/or more than 50% in 48 hours.

Introduction

Gene therapy is a method that uses gene to treat or prevent disease. In general, inserting a gene can replace a mutated gene, inactivate a mutated gene, or introduce a new gene into the body to assist fighting a disease. Currently, scientists are trying to make gene therapy more safe and effective. In the future, effective gene therapy might replace the use of drugs or surgery (Handbook help me, 2013).

History

In 1966, Edward Tatum first published a paper suggesting the use of viruses in somatic-cell genetics and genetic therapy. The first clinical trial was conducted by Rogers et al. to cure urea cycle disorder by introducing the gene for arginase using Shope papilloma virus. The result of the trial was negative. They later found out that was so because Shape papilloma virus genome actually does not encode an arginase. Even with series of negative result for clinical trials, gene therapy received great attention from many scientists until the death of Jesse Gelsinger. In 1999, Jesse Gelsinger, an omithine transcarbamylase (OTC) deficiency patient, took part in a gene therapy clinical trial at the University of Pennsylvania in Philadelphia. High dose of adenovirus was administered and Gelsinger’s immune system responded instantly. He died because of multirogran failure. This case was very important in the study of gene therapy because Gelsinger became the first patient who showed that viral vector for gene therapy can cause death to the patients (Wirth, Parker & Ylä-Herttuala, 2013).

Gene therapy slowly got its attention back and currently more than 1800 approved gene therapy clinical trials all around the world have been conducted or are still ongoing. Caner is the most common disease treated by gene therapy. Over 60% of clinical gene therapy trials worldwide are for cancer treatment. Gene therapy is being clinically used in China. In 2003, China became the first country to approve gene therapy. Adenoviral vectors, retroviral vectors and naked plasmid have been used most for gene transfer vectors in clinical trials (Wirth, Parker & Ylä-Herttuala, 2013).

Methods

In general, there are two categories of gene therapy: germ line gene therapy and somatic gene therapy. The difference is whether the genetic materials inserted will be passed to next generation. In somatic gene therapy, genetic material is not passed down while the genetic material is passed down in germ line gene therapy (Wirth, Parker & Ylä-Herttuala, 2013). Both of these methods require a carrier called a vector that can transport the new gene into the desired cell (Handbook help me, 2013). Adenoviral vectors, retroviral vectors and naked plasmid have been used most for gene transfer vectors in clinical trials (Wirth, Parker & Ylä-Herttuala, 2013). When virus vectors are used viruses are modified so that they won’t infect the subjects. Different types of virus vectors can produce different outcomes. For example, if retroviruses are used a vector, the genetic material gets integrated into the subject’s chromosomes. If adenoviruses are used, DNA is introduced into the nucleus of the cell, but the DNA is not integrated into the chromosome (Handbook help me, 2013).

Non-viral vectors can also be used for gene therapy. Free plasmid can delivered into the cells using physical methods such as: electroporation, gene gun, ultrasound and hydrodynamic pressure. Non-viral methods were at disadvantage because of their low efficiency, but with the development of more efficient vector technology, non-viral vectors are becoming more popular; they can reduce problems that occur while using viral vectors such as endogenous virus recombination, oncogenic effects and unexpected immune response (Huang & Niidome, 2002).

Biosafety & Bioethics

As much as gene therapy looks promising, there are many biosafety issues that it raises. The indisputable concerns are whether transferring genes is safe and whether the transferred gene have effect on the germ line and ultimately to offspring. Technical issues also exist. For example, the use of retroviruses raised criticism from the start. Because of integration transgene that can cause mutation, the use of retroviruses can impose threat to patients. The main argument against gene therapy is that in the worst scenario, unexpected change in the genetic makeup of the gene therapy patient maybe passed down onto offspring. To refute this argument, some scientists say that other therapies such as mutagenic drugs and radiation therapy can cause genetic alteration that can be passed down onto the offspring. They argue that these therapies are being used without extensive biosafety concerns (Wirth, Parker & Ylä-Herttuala, 2013).

Bioethical issues are also a great concern surrounding gene therapy. The first concern is that there is no clear boundary on what is a “good” or “bad” use of gene therapy. It is also very hard to determine what traits are normal and which traits are should consider as disorders. Gene therapy might also be used not only to cure a disease, but also to enhance basic human traits such as height and intelligence causing social disparity between the poor and the rich (Handbook help me, 2013).

Our Project & Gene Therapy

With the background information mentioned above, we have investigated the application of gene therapy for our project. It seems like gene therapy is a feasible method to deliver desired genes into the destined human cells. Currently, gene therapy, however, is only tested and used for incurable diseases such as cancer and rare genetic diseases (Handbook help me, 2013). The complication that we want to alleviate, obesity, at this current stage of scientific research is not considered as an incurable disease. In fact, many people disagree to even consider obesity as a disease. The severity of obesity, nonetheless, is slowly being recognized by the science community and the American Medical Association in June 2013 finally decided to classify obesity as a disease (Dailey, 2013).

Application of gene therapy for our project is indeed an invasive method to treat obesity. Presently, many people will be reluctant to use this method unless obesity and its complications are life threatening (Yu, 2013). On the contrary, if we consider other method that is currently used to cure diseases such as medicine and surgery, these methods are also invasive. The difference is that these invasive techniques have gained enough trust from public through serious for clinical trials and tests. If gene therapy becomes safer and publicly accepted and if the severity of obesity constantly increases, the use of our project through gene therapy does not seem very improbable in the future.

Conclusion

Gene therapy is a promising method to cure diseases. Although currently, the public has doubts about this invasive method, scientists are eagerly trying to make this therapeutic method more safe and efficient. In the future, when gene therapy become more accepted by the public, we speculate that this method can be even be used for obesity considering the fact that more people are starting to recognize the severity of obesity.

References

Dailey, K. (2013, June 25). Obesity is a disease in the us. should it be?. BBC. Retrieved from http://www.bbc.co.uk/news/23011804

Handbook help me understand genetics gene therapy. In (2013). Genetics home reference.

Huang, L., & Niidome, T. (2002). Gene therapy progress and prospects: nonviral vectors. Gene Therapy,9(24), 1647-1652. Retrieved from http://www.nature.com/gt/journal/v9/n24/abs/3301923a.html

Wirth, T., Parker, N., & Ylä-Herttuala, W. (2013). History of gene therapy. Elsevier-Gene , 525(2), 162-169. Retrieved from http://ac.els-cdn.com/S0378111913004344/1-s2.0-S0378111913004344-main.pdf?_tid=42ad73a2-108c-11e3-a4a5-00000aab0f6c&acdnat=1377768215_ab7a2ccc7dc6f1f218759a92fccfc8ba

Yu, C. H. (2013, July 16). Interview by J.H. Lee ]. Hkust igem 2013 team informative interview with professor yu.