Team:TAS Taipei/Practices
Human Practice Summary
Introduction
A project without direction and impact is not something that will be relevant in the eyes of the public. The goal of the policy and practice portion of our project is to ensure that we are headed in the right direction in terms of our project’s global impact and relevance. We have gathered data from researchers, medical doctors, veterinarians, cataract patients and public opinions to find out the best way to develop our project so that it can benefit as many people as possible.
Our Policy and Practice is separated into three categories, Research, Outreach and Impact. In the Research category, we collect and analyze all of the data from researchers, doctors, vets, investors, and the general public. This information shapes the direction of every part of our project, including the construct design, nanoparticle prototype synthesis and delivery, biological modeling and the development of a marketing plan for selling our final product. In the Outreach category, we educated the public and spread awareness of our cataracts project, synthetic biology and science in general. We have also collaborated and communicated with several iGEM teams to help solve problems together. Through educating and raising awareness, we hope not only to get more people involved in finding a solution to cataracts, but also to get more people involved and interested in synthetic biology research. The final category, Impact, includes working with organizations and infrastructure that is already in place to actually help people who suffer from Cataracts. We have raised funds, through bake sales and other events, to donate to the Himalayan Cataracts Project, a non-profit organization based in Nepal. With the help of our business and economics teacher, we have also created a marketing plan and started discussions with several biotech investors about the possibility of getting our project to market.
Research
Professional Help
In order to improve how we develop our prototype we needed to get in contact with experts in the fields of eye surgery and cataracts research. Our first step was to contact eye doctors to discuss any problems associated with current cataracts treatment. Finally, while developing our project we realized that cataracts is a major issue in pets and other animals as well as people. As a result, we contacted local veterinarians to discuss what pet owners do when their pets contract cataracts. As our project developed, we needed more specific information regarding our genes of interest, cataracts development, and our delivery mechanism. We contacted scientists doing research similar to our own to get their opinion on our projects progress.
Contact with Eye Doctors
Eye doctors are on the front lines, delivering cataracts surgery, some privately and others for charity in organizations such as the Himalayan Cataracts Projects. We contacted local Taiwanese Eye Doctors to ask them about cataracts surgery. Here is a list of the doctors we contacted along with the information they provided:
Dr. Wei-Chi Wu is a retina specialist and an associate professor at Chang Gung memorial hospital in Taiwan. According to Dr. Wu current cataracts surgery methods are efficient and effective, but are not without their issues. Besides the issue of price, there can also be several different post-surgery complications such as infection, hemorrhaging, or secondary glaucoma. In regards to our project, he said one of the biggest issues we would face is non-invasive delivery. Currently, injections and incisions are the only methods for delivery because all current potential methods of noninvasive delivery either lack efficiency or induce with side effects.
Dr. Tsu Chieh Cheng is a optomologist at Chang cheng eye hospital in Taiwan. Dr. Cheng points out that aside from possibly causing complications such as astigmatism, myopia, and hyperopia, cataracts surgeries have risks, for wound infections, dislodgement of lens, and massive bleeding during surgeries aren’t uncommon. He remarked that our project sounded very promising, but the effect the drug has on other parts of the eyes must be checked. Furthermore, he suggested us to use rabbits or dogs as animal models, because they can be observed more easily.
Contact with Veterinarians
Cataracts trouble millions of people’s lives as a result of aging and diseases; however, many pet owners come across the problem of their pets suffering from cataracts as well. To alleviate this problem, the benefits of our solution for preventing and treating cataracts can be applied to domestic animals as well.
As we continue to develop our project with consideration for the application on other animals, we realize that the slight dissimilarities among the lenses of different animals must be accounted for. Therefore, we consulted with veterinarians at clinics around Taipei. We visited two veterinarian clinics, and asked various others about relevant questions regarding the viability of our project solution. From the interviews we gained valuable insight on the eyedrops that pharmaceutical companies have developed to mitigate the cataracts in the lenses. One of the drugs developed is called Ocluvet(R).
Science Researchers
Researchers’ firsthand knowledge, regardless of how many papers one reads on PubMed, provides a better understanding on the topic of research. Thus, in order to gain a better view of our project, we have contacted professional researchers ,via email, who have in depth knowledge on 25 hydroxycholesterol, which is the main candidate for the cause of formation of cataracts.
Our questions were primarily focused on how 25HC works on the molecular level. We asked questions such as: what are the current researches conducted that uses 25HC? To what extent is 25 HC responsible for reversing protein aggregation? And what are effective methods for 25HC storage? Both professors gave thorough responses on these questions, which we then used to support our choice of methods. One response that was particularly helpful from Dr. Cyster was his suggestions on effective ways to store 25HC, in which he provided us with a recommended storage temperature. On the other hand Dr. Gestwicki provided us with two published papers that addressed our concerns about 25HC, and this helped elucidate our understanding of 25HC.
Public Opinion
Bioethics Panel
We hosted a bioethics panel, where we invited teachers from various fields to engage in a conversation about bioethics and thoughts on our project in particular. The reason for including teachers from different fields is to allow our project and the topic of bioethics to be discussed from different aspects. Since we did this at the start of our project, the opinions shared and advice given during the panel shaped the direction of our project.
Survey
During TAS’s Spring Fair we conducted a survey, from which we derived a general sense of how willing people are going to buy products that are produced by GMOs, and our eyedrops in specific.
Interviews with Cataracts Patients
We did interviews with two cataracts patients about their past experiences in different cataracts treatment, and extracted this information along with the responses from survey, and incorporated into some of the ideas in our marketing plan. Something in common that the two people we interviewed had was that they both suffered from inconveniences due to the lens options, which would either make one nearsighted or farsighted. Furthermore, complications due to the surgery varied among individual cases; one suffered from dryness and irritation of the eye, while the other had no complications at all. Nevertheless, both of them stated that the surgery process was efficient and effective; however, if given the choice to apply effective eye drops instead of surgery, they’d be willing to use it despite some may fear the idea of having nanoparticles in one's eyes.
Education and Outreach
Science is often intimidating to many, but with little explanation and some experience, it isn’t scary at all. Thus, aside from the goal to raise awareness of cataracts, the promotion of science and synthetic biology in particular, are also topics we aim elucidate. Here are a list of activities and programs that serve our purpose.
Prevention: GSR-HIS
Science is often intimidating to many, but with little explanation and some experience, it isn’t scary at all. Thus, aside from the goal to raise awareness of cataracts, the promotion of science and synthetic biology in particular, are also topics we aim elucidate. Here are a list of activities and programs that serve our purpose.
We designed some easy experiments to let the Kindergarten students try playing with. We did this for several times, and the kids loved it. The experiments were usually very simple yet demonstrates important scientific concepts, for instance, we teached them how to use the microscope, why light reflects through prisms, how static electricity works etc.
Treatment: CH25H-HIS
We want to extract alpha crystallin B protein, one of the main proteins in the lens, to see if we can create the same aggregation as cataracts lenses.The construct here contains similar components, except the open reading is replaced by CRYAB and 10x Histidine tag. We wanted to express CRYAB, one of the two main crystallin proteins in the lens, to test if h2o2 can aggregate crystallin proteins. CRYAB cDNA was ordered from Origene. We designed primers that were synthesized by Tri-I biotech to move CRYAB into iGEM backbone. The full construct is shown in figure #.
Impact
The current method of treatment for cataracts is surgery, which is not only invasive and costly, but also possibly unavailable in areas with limited medical facilities. Taking biosafety, cost, and delivery effectiveness into account, we designed and built a prototype with two distinct steps. First, proteins must be purified and separated from bacteria, and then packaged in nanoparticles that aid delivery through the cornea and into the lens.
Step 1: His-Tag Protein Purification
Our survey results show that people are reluctant to put anything bacteria-related into their bodies, so we aimed to separate our protein products from the bacteria that produced them. Once isolated, pure proteins can be directly used to avoid introducing foreign bacteria into our bodies. As shown in Figure A, both constructs for prevention and treatment include a downstream 10x histidine tag. The encoded GSR-HIS or CH25H-HIS proteins can then be isolated using a commercial kit (Capturem His-tagged Purification Miniprep Kit from Clontech).
Step 2: Packaging in Nanoparticles
The cornea is the outermost layer of the eye which protects the eye by preventing entry of foreign materials, but this function also largely prevents drugs from reaching the lens (Gaudana et al., 2010). This problem has challenged researchers and driven them to search for a way to deliver drugs through the cornea and into the lens. Currently, the most promising ocular drug delivery method is using chitosan nanoparticles as drug carriers (Cholkar et al., 2013).
We selected chitosan as the optimal material to make nanoparticles for several reasons. First, chitosan nanoparticles can embedded in and adhere to the cornea, this minimizes drainage loss. Also, these nanoparticles can penetrate the cornea and deliver the drug directly to the target area via (Campos et al., 2005). Its low toxicity to somatic cells makes it safe, and its biodegradability allows the drug to be released continuously in the eye (Enriquez de Salamanca et al., 2006).
We dissolved chitosan into 1% by volume glacial acetic acid aqueous solution. We adjusted the pH of the chitosan solution to 5.5 by adding 1M NaOH in order to account for the stability of the desired proteins when they are added. We dissolved sodium tripolyphosphate (TPP) in distilled water. Chitosan and TPP were dissolved in equal volumes and their mass ratio was 3:1. We stirred the chitosan solution at 600 rpm while adding 3 ml of TPP solution dropwise. To collect the nanoparticles, we centrifuged the suspension at 17,000 xg for 40 minutes at 4C, and nanoparticles were collected as pellets. The specific protocol can be found in our lab notebook.
To ensure our nanoparticles were correct in size and configuration, we imaged the nanoparticles using scanning electron microscopy and atomic force microscopy.
Step 2i: Protein Encapsulation
Next, we wanted to load our protein into the nanoparticles. In order to determine how successfully our nanoparticles encapsulated proteins, we measured the change in protein concentration in the supernatant before and after nanoparticle formation. By performing a Bradford Assay with the nanoparticle supernatant (Bio-Rad), we found the encapsulation efficiency to be 50%.
More BSA remained in the supernatant after nanoparticle formation when Chitosan was added to TPP. It is inferred that more proteins are encapsulated in the nanoparticles for those made from TPP added to Chitosan.
We synthesized nanoparticles containing fluorescent proteins to prove that our nanoparticles can be used to encapsulate our protein drugs. To do so, we lysed bacteria expressing green fluorescent protein (GFP), red fluorescent protein (RFP), and green pigment (from pGRN, Bba_K274003). We then extracted and encapsulated the proteins in nanoparticles using the procedure described above.
Step 2ii: Protein Release
As nanoparticles degrade, they release the proteins inside. We measured and mathematically modeled the release rate to determine the optimal frequency of drug administration.
To measure release rate, we suspended nanoparticles containing bovine serum albumin (BSA), our standard protein, in phosphate buffered saline (PBS). PBS models the conditions of our cornea on which the nanoparticles that we deliver will be embedded to degrade and release the proteins. We measured protein concentration of the solution outside the nanoparticles over time. We performed trials at three different temperatures, -20C, 4C, and 37C to reflect long term storage, short term storage, and drug application.
Procedure for this experiment is illustrated in our lab notebook.
Step 3: Application - Eye drop and Contact Lens
We engineered nanoparticles to deliver glutathione reductase (GSR) and cholesterol 25-hydroxylase (CH25H) to the lens through a safe, cost effective, and non-invasive method. We purified the proteins from bacteria using histidine tag purification method to minimize toxicity and loaded them into nanoparticles to maximize drug delivery. We designed two drug delivery prototypes to apply our nanoparticles: eye drops and contact lenses.
Citations
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