Section 1: Context and Professional Development

Context & background

My name is Cham Ting Chien, a Pharmaceutical Healthcare final year student in DIT. In the past 3 years, I have developed broad understandings in pharmaceutical as well as laboratory skills. For example, chemistry, cell culture, characterization of samples with pharmaceutical instrument (HPLC, FTIR, NMR). Drugs can be delivered in various pathway, but the three field that I am interested and putting extra effort in are gene therapy, CRISPR and stem cells.

1.1 Gene therapy

Figure 1. Gene transfection

My first experience in biotechnology was the transformation of antibiotic resistant plasmid into E. coli, even though the experiment was good enough for a lab report write up, the transformation efficiency was not ideal, not to mention the high cell death due to heat shock. Later then, I came across various theories and techniques on gene delivery that shows promising therapeutic effects.

Since I have immense interest in biotechnology and stem cells therapy, I did my work placement in Charles Institute of Dermatology in UCD led by Wenxin Wang. Wang group is a research group who focuses on polymer synthesis, hydrogel and gene therapy to treat Epidermolysis bullosa. It is a genetic defect affecting the keratin genes, causing patients to have fragile skins, where the rate of skin regeneration is very slow. After the 4-month placement, I am still working with Dezhong Zhou whenever I have time in the first semester, till now for my FYP.

Figure 2. Wang group, Charles Institute of Dermatology, UCD. (wenxinwang.ie, 2016)

My placement project was to synthesis a novel cationic-polymer for gene delivery. There are barriers that makes gene delivery difficult;

1. Gene should not be degraded once entered the cell

2. DNA has to be delivered into the nucleus, passing through an extra membrane barrier

3. DNA/RNA promotes or knock down gene expression temporarily - continuous administration is required

4. Off target modification might occur where selectivity is crucial

*Please refer to section 2 for projects done.

1.2 Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)

Prokaryotes do not have RNAi, CRISPR is first discovered in Streptococcus pyogenes which provides adaptive immune system by degrading foreign nucleic acids obtained via horizontal gene transfer (HGT).​ CRISPR locus is rich of short palindrome repeats, each repeats are separated by spaces, where each spacer is unique, and a set of CRISPR-associated gene (cas gene) located at the 3' end. The activation of CRISPR-Cas system consists of three crucial stages: spacer acquisition, CRISPR-Cas expression and DNA interference. Any genetically modified germ cells has the probability of 50% to be pass down to the next generation; however, the CRISPR locus is 100% inherited to offspring, which allows elimination of terrible family inherited disease.

Figure 3. Overview of CRISPR-Cas adaptive immune systems. (Jiang and Doudna, 2015)

Let's make a big hypothesis that human beings are genetically engineered with CRISPR locus in one of the chromosome, any introduced gene in any form (transformation, transduction, transfection, conjugation) will be incorporated within repeats as spacer, and it will be expressed as the transcription factor binds on the promoter; or degrade foreign genes that code the same as spacer.

1.3 Career goals

To date, stem cells are one of the most potential therapy that scientist all around the world invest for research, however, even if it succeeds in treating a disease, who is able to afford such expensive treatment? I know it sounds crazy but what if a cell line of stem cells is cultivated, that is genetically modified to carry general characteristics that suits EVERYBODY in the world? It can be produced in bulk, which means cheaper and affordable for everyone!

That sounds a little too far, it is very interesting that a designed drug is capable of targeting its specific site of action. My target in this semester is to learn how to design a new drug, and control its parameter and characteristics to have the optimum pharmacokinetic and pharmocodynamic; the exact dose that achieve desired therapeutic and least toxic effect.

Professional development

2.1 disulphide-cationic polymer

During my work placement, I made a polymer that delivers DNA into cells. The polymer has several properties that were thought to enhance gene transfection.

1. Overall polymer is positively charged, therefore capable of forming a stable complex with DNA/RNA; as well as the capability to approach the negatively charged cellular surface.

2. Guanidine group from arginine enhance formation of endosome.

3. Amidazole ring has pKa of 6, protonated and prevents lysozyme degradation before it is released into cytosol; achieved by proton sponge effect.

4. HeLa cells were used. It is known that tumor cells have relatively higher GSH in the cytoplasm, high enough to reduce disulphide bonds into thiol groups, disintegrating the polymer and gene is released.

Figure 4. Structure of polymer.

2.2 improved version disulphide cationic polymer

From what we failed in the 2.1, we are planning to change the method on polymerisation, as well as adding new features to the nanoparticle. Gene is coiled at the core of nanoparticle, while the initiator is altered to allow bond formation between initiator (red dashed line) that forms a protection to the core.

Figure 5. Formation of nanoparticle with 5 polymers.

2.3 self-assembly dendrisomes

The aim of the project is to prove the ability of polymers to self-assembly into vesicle like hollow nanoparticle, as shown in figure 6.

Figure 6. A golf ball that has structure of hollow sphere. (thorouglyreviewed.com, 2016)

Since the polymer was not designed to be positive, it is not restricted to deliver negatively charged nucleic acids, but also drugs, protein, hormone etc. It exist as a hyperbranch in a homogeneous DMSO solution, when added dropwise into drug solution, it self-assembly into vesicles that encapsulate the solution content (coacervation). In my project, it shows that the concentration and molecular weight do affect its structure of vesicle.

My future

Before I started this course, I've always wanted to work as a quality control technician. However, upon the experience I gained in UCD, I was inspired and excited on taking the challenge of research. Besides that, end-product quality analysis will be outdated soon, being substituted by Process Analytical Technology (Lean, Six Sigma etc).

To be honest, I am not sure what I would like to do after graduation. I was given a chance to talk to a Process Engineer for MSAT cell culture from BioMarin International in GradCareer Fair. Since I am interested in many fields, he told me that the MSAT would suit me because it requires research on how to scale up the production; and through the process, PAT should be taken into consideration in order to achieve Quality by Design (QbD).

This module provides insight on how to design a drug that would suite the body, techniques in protecting the API from biodegradable enzymes/harsh pH and methods of synthesizing protection (coating/capsules/emulsion etc). It is very important to apply the knowledge I've learn from the course, especially Medicinal Chemistry, Drug discovery and synthesis, Drug delivery, Organic Chemistry and Pharmaceutical Formulation; that even if I do not work as a researcher in future, I believe that what I learn now would allow better understandings in the other pharmaceutical area.

1. Wenxin Wang.ie, 2016

2. Jiang, F., Doudna J. (2015) The Structural Biology of CRISPR-Cas Systems. Science Direct. 30, 100-111.

3. throughlyreviewed.ie (2016) A golf ball that has structure of hollow sphere.