What is Reverse Transcriptase PCR (RT-PCR)?
A way of amplifying specific RNA by converting it to cDNA and then running a PCR
This uses reverse transcriptase to form the cDNA, with help from a olgio-dT primer
What is the most common culprit for human cancers?
How does a combination of Thymidine Kinase and Ganciclovir function as a suicide gene?
Thymidine kinase activates ganciclovir via phosphorylation (due to it having a high efficiancy in doing the first phosphorylation compared to other kinases), before cellular kinases phophorylate it again
Once it has been phosphorlyated 3 times, it can act as a guanosine mimic and be incorporated into DNA, causing DNA to be damaged and the cell to die
What are the 2 different types of non-viral nucleic acid delivery?
Physical Techniques --> Injection, gene gun, ultrasound, electroporation
Chemical Techniques --> Encapsulation and Complexation which leads to nanoscale particulates
What is a common co-treatment given with cancer vaccines?
Checkpoint inhibitors such as drugs to block the PDL-1
How can Microarray Technologies be used?
These are microchips that contain many probes that will bind with a complementary strand from the patient
So we can see if the patient has a specific strand of DNA if it flashes up (as the patients strands are labelled)
What are Cell Penetrating Peptides (CPPs)?
Short sequences of HIV-TAT protein that are arginine (R) rich
These have the potential to deliver liposomes, nanoparticles and other biological entities across membranes (due to its positive charge) --> Espeically oligonucleotides
They have low cytotoxicity and can be conjugated to carriers
How can Oligonucleotides be used as a nucleic acid-based therapy?
Decoy Oligo --> Can prevent transcription by binding to the promoter region of the gene
Antisense Oligo --> Can prevent translation
siRNA --> The 21-25bp siRNA can bind to mRNA, causing it to cleave
- We must know the gene sequence to do this
miRNA --> A 22 nucleotide ssDNA that causes mRNA destabilisation
- Low levels can cause cancer as they are useful in regulation
How does Oncorine function?
An oncolytic virus
Can't replicate in healthy cells due to the presence of p53, however in tumour cells there is no p53, so it can replicate and function!
Usually in adenoviruses there is E1B, which inhibits p53, but by remvoving this we can make a tumour selective drug
What are the pros and cons of vascular and stromal ablation?
Pros --> Very selective, the mAb can couple with various toxins, they are relatively stable
Cons --> High doses needed, can have off-target effects
Why are breast ducts susceptible to cancer?
As they are hyper-mutatable (divide quickly)
What is good about Mesenchymal Stem Cell Therapy?
And what are the 4 ways in which it can be used?
They are easy to isolate (from adipose and bone marrow) and anti-inflammatory, hypoimmunogenic and can home to the site of injury
By increasing the immune response it can make the situation worse (as inflammation can go through the roof), so we add on cancer drugs to these as they can get close to the tumour
Vehicle for cancer drugs
When are orphan cancer drugs used most?
In rare cancers where the production of a normal cancer drug isnt financially viable
Also allows the pharma to get safety/efficacy data quickly, which saves them money --> Useful if it turns out the drug can be used in another condition eventually
What are the most common cancers in children?
Cancer of the bone
This is because they are growing rapidly, so DNA errors are more likely to occur
Treatments are also difficult to find due to the lack of clinical trials, and many ethical dilemas
What are the benefits of High-Throughput screening?
Hundreds of samples per hour
Multiple analytes can be quantifies at a time
Sensitive and accurate
What are the advantages and disadvantages of CRISPR/CAS9?
Advantages --> Small, inexpensive, specific and versatile
Disadvantages --> Delivery, off-target effects and potential abuse
How does the Affymetrix GeneChip work?
Restriction enzymes digest the DNA into fragments
We then ligate adapters to the fragments, allowing PCR to occur
We then label the fragments, and put them through a microchip which checks the fragments against many different probes
What's the difference between Sanger Sequencing and Illumina Sequencing Technology?
Sanger --> ddNTPs are labelled with a fluorophore and act as chain terminators
Illumina --> Once the ddNTPs are added, we can record the colour and then wash it off, allowing the chain to carry on growing until another terminator binds
How does Electrochemiluminescence work?
A sample with the protein of interest is added to a biotin labelled antibody and a ruthenium labelled antibody
Both of these antibodies will bind to the protein of interest before the biotin binds strongly to a magnetic bead
The complex is then passed through a flow cell, in which the magnetic bead is attracted to the large magnet at the bottom
Ruthenium will give off photons, so the more photons that are given off and detected by the photomultiplier, the more of the target protein that is present
What are the advantages and disadvantages of non-viral vectors?
Advantages --> Easy to prepare and modify, large capacity, less immunogenic
Disadvantages --> Poor efficiency and transient (short acting)
What is Allelle-Specific PCR (ASPCR)?
This can detect point mutations without replying on changes in restriction sites
The primer at the 3' end that is inserted will only bind to the complementary strand, and not any mutated version. So if the point mutation occurs, it wont bind and so either will the DNA polymerase....so PCR amplification won't occur
What is the difference between Direct and Cell-Based gene delivery?
Direct --> The therapeutic gene is packaged into something like an retrovirus and injected into the patient
Cell-Based --> Embryonic Stem Cells (ESCs)/Adult Stem Cells are genetically modified and differentiated before being grown. These are then injected into the patient
- This can also be used as a way of increasing the number of retroviruses for direct delivery
What is TK Cell Therapy?
Haemopoietic Stem Cells (HSCs) normally find their niche in the bone marrow and repopulate the cells that are needed
T cells from a donor can often be differentiated into TK cells which can become alloreactive and cause graft-host disease, which creates a massive immune response
- This is treated with the use of suicide genes such as ganciclovir to kill of the alloreactive TK cells
What is Capillary Gel Electrophesis (CGE)?
Where the sample moves through capillaries and are detected by lasers
The strands are still seperated by charge and molecular weight
What are the 3 main reasons for why humans are prone to cancer?
Bad genetics --> Cell defects and epigenetics (eg, smoking)
Viral DNA --> 8% of our genome is viral, and so these elements can be transcribed under certain conditions
Explain the following genetic therapies
Killing of Specific Cells
Gene Augmentation --> Insertion of a functional gene to the cell
- Could mean that the new cell could code for a therapeutic mAb!
Gene Inhibiton --> Introducing a blocking gene into the cell which prevents the faulty gene from functioning
Killing of Specific Cells --> Adding in a suicide gene that produces toxins and kill the cell.....or adding in a marker gene that causes proteins to be expressed on the cell surface, attracting the immune response
What are the postives and negatives of using mAbs to treat cancer?
Advantages --> Can bind to cell surface receptors and engage lymphocytes to kill the cell, can be easy to make, can make lots of money from them!
Disadvantages --> More complicated mAbs are very hard to make reproducibly and in the quantity needed, multi-dosing also often needed...which is bad from a compliance/cost point of view
Why can dirty cancer drugs actually be quite useful?
In respect to BRAF
As if the dirty drug can inhibit several parts of the same pathway, if one target mutates...we can still target the others!
This is especially true in the BRAF pathway as we struggle to target RAS specifically
- So we target the PIK3 (parallel) pathway also
What is a Dendrimer?
A non-viral vector that is built up via nitrogen chemistry
The given surface groups can be used for targetting, or to avoid/stealth the immune system
They can also be designed to release under specific conditions
What is major problem with kinase inhbitors? Like those that are used to treated EGFR mutations?
They arent that specific, and kinases are everywhere in the body! So off-site actions are inevitable