-Notebook-

Week 1: Inspiration from other teams

• Eg. Tsinghua 2023, McGill 2023
• Combined other teams’ ideas with our previous ideas
• Protein level (antibody) → Genetic level (promoters and reporter genes)
• Tsinghua: AND gate system
• using multiple promoters to increase specificity
• Eg. PSA promoter + PSMA promoter, PSA promoter + AR promoter
• Tsinghua: PD-L1 nanobody
• With the aid of immune system kills cancer cells
• McGill: induced pyroptosis of cancer cells
• PLKLFC 2023: PSMA has a high correlation with PCa
• + AND gate is difficult to design
• Decided to use PSMA promoter only
• PSMA promoters should only be activated inside cells
• Does not cause harm to nearby cells

Week 2: Delivery of plasmid to prostate

• Aim: to make it as convenient and painless as possible
• Inject plasmids into the bloodstream by intravenous injection → how to make it travel to the prostate? 1
• Plasmids degrade in the blood 3
• Need “carriers” to transport them
• Idea: polymers (small molecules of plastics) to prevent degradation 5
• Potential polymer 1: Hyaluronic acid (found in the fluids in the eyes and joints) 8
• Binds to CD44 (overexpressed at tumour) and releases plasmid inside 9
• Problem: unspecific to PCa 10,11
• ✓ Use promoters inside the plasmid to confirm the location
• 👎🏻 Decrease in [plasmid] before reaching prostate
• Potential polymer 2: Chitosan - sugar from the outer skeleton of shellfish
• Potential polymer 3: PAMAM (Poly(amidoamine) dendrimers) - plastics
• Utilised in previous research regarding gene therapy for prostate cancer (Tai et al., 2020)
• Problem: cytotoxic to cells 17
• Solution: Attach polyethylene glycol (PEG) to reduce cytotoxicity (Guo, X., Wang, 2017) 20

Week 3: Guides of polymers - Aptamers

• How to target prostate with the polymer? - Aptamers1
• Sections of DNA/RNA/peptides that target specific molecules 3,4
• High specificity 5,6
• Produced in a batch → reduces production costs
• Reversible denaturation (unlike antibodies)
• Less immunogenic than antibodies
• Can be attached to nanoparticles/polymers
• Potential aptamer 1: EpDT3 16
• Binds to EpCAM (epithelial cell adhesion molecule, overexpressed in some tumour cells) 17
• 👍🏻EpCAM is highly expressed in metastasized PCa cells → detect later stages of cancer 19
• 👎🏻Still non-specific to PCa
• Dual aptamer design 23
• A10-3.2: PSMA +ve (but only 80% of PCa patients have PSMA)
• DUP-1: PSMA -ve
• Used in a lot of research previously
• We can add a constitutive promoter inside our plasmid to express the reporter genes.

• PD-L1 nanobody
• Normal cancer cells: have PD-L1 to “block” attacks from T-cells
• PD-L1 nanobody: binds to PD-L1 so that cancer cells have no way to block attacks
• Problem:
• requires immune cells to kill
• At later stages, immune system may be compromised
• Cannot kill cancer cells effectively
• Bax gene(2./2.1)
• BCL2 associated X, apoptosis regulator
• If Bax protein is transcribed, it promotes apoptosis (type of programmed cell death) in cancer cells
• Bax has been chosen

Week 1: Formulation of initial plan

1. Construction of plasmids
2. Purchase of A and B separately

1. A plasmid containing our gene that is non-mammalian; and
2. Another plasmid that is mammalian but lacks any promoter genes (pENTR1A)

• A and B will be digested, then ligated together using BamHI and HindIII enzymes
• Colony PCR → confirm size of plasmids are correct 1.e

1. Testing of PSMA promoter
• Evaluate the activity of PSMA promoter in different cell lines
• GFP (commonly used and known to work) as reporter gene

• Plasmid A: pENTR1A-PSMA-GFP

• Transfected to cancer and non-cancerous cell lines
1. Testing of Gluc reporter gene
• Measure luminescence given out by reaction w/ Gluc as substrate

• Plasmid B: pENTR1A-PSMA-Gluc

• Transfected to cancer and non-cancerous cell lines
• Testing of polymer delivery
• Make sure polymers can correctly transport plasmids to cancer cells

• Polymer plasmid conjugate X:

• PAMAM polymer

• A10-3.2 & DUP-1 aptamers

• Plasmid C: PB-Gluc

1. Testing of killing function
• Confirm that Bax gene can kill cancer cells

• Polymer plasmid conjugate Y:

• PAMAM polymer

• A10-3.2 & DUP-1 aptamers

• Plasmid D: PB-Gluc-Bax

Week 2: Figuring out protocols used

1. Plasmid construction
   1. Plasmids in powder form: transformed into DH5a
   2. Colonies are picked → plasmids extracted
   3. Restriction digestion: linearise genes (BamHI and HindIII)
   4. Gel electrophoresis: visualise the results of digestion
   5. Gel purification: obtain the digested plasmids
   6. Ligation: stick two genes together
   7. Colony PCR: confirm that ligated genes have the correct size
2. Polymers
   1. PEG is attached to PAMAM to form PAMAM-PEG conjugate
   2. Aptamers are attached to PAMAM-PEG
   3. BODIPY are introduced to some of the PAMAM-PEG
      1. Microscopes are used to view BODIPY fluorescence
      2. Confirm that aptamers can bring plasmids to cells
   4. Plasmids C & D are introduced to other PAMAM-PEG
   5. Polymer-plasmid conjugates are mixed with cells
      1. MTT Assay are performed to quantify the death of cancer cells (required absorbance measurement)
      2. Luminescence of Gluc are detected

Week 3: Required equipment and apparatus

• Cell lines ∵ environment for prostate cancers are hard to simulate with chemicals (purchased)
• Plate readers: measure luminescence (Gluc), fluorescence (GFP), absorbance (MTT assay) (equipped but only absorbance can be measured)
• Live cells monitoring microscopes: monitor the situation of cell lines (equipped)
• PCR Thermal Cycler: perform colony PCR (equipped but is aged)

Week 4: Evaluation of initial plan

• ❓Construction of plasmids: colony PCR can be tried but cannot confirm that PCR machine will work
• Contingency plan: extract the ligated plasmids, digested using only 1 enzyme, then gel run to visualize the size
• ❓Testing GFP and Gluc light: required to borrow at other labs but possible
• ✔️Testing Bax killing function: feasible
• 🔜Polymers: the technical difficulty is too high