Molecular Diagnostics and Biological Therapies Flashcards
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?
K-Ras
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
Anti-angiogeneis
Cytokine delivery
miRNAs
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
Low volume
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
Hyper-mutable genome
Explain the following genetic therapies
Gene Augmentation
Gene Inhibition
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
How does the self-test PSA kit work?
Free anti-PSA antibodies (Ab1) contain dye, and will bind to any PSA that is present in the sample (urine)
If PSA has binded to these, it will then bind to the tethered anti-PSA antibodies in the result area….producing a colour from the dye
Ab1 will also bind to the tethered anti-Ab1 antibodies in the top result area, which shows that the test is functioning (acts as a control)
What type of genome does a retrovirus have?
And how can they cause cancer?
ssRNA genome
The viral DNA can promote proto-oncogenes, or split apart a tumour suppressor gene (TSG)….both of which will promote tumour formation
How do the PCR reaction occur?
The region to be amplified is heated to 94 degrees for 2 mins, splitting the dsDNA to ss
Pre-made primers of 18-22 nucleotides long, anneal once cooled to 30-55 degrees for 30 seconds
dNTPs are added with Taq/Pfu Polymerase (heat stable) to synthesise the new strands (at 65-75 degrees for 2-5mins)
What is the difference with Sandwich ELISA?
Used for low concetration samples of the target protein (dirty samples)
A capture antibody binds to the plate, which catches the target protein
The rest is identical to ELISA
Why is killing the Cancer Stem Cell (CSC) in a tumour cell vital?
Explain the 2 theories
As it increases the chance of the tumour going into to regression
Theory 1 –> If you only give standard chemo you will kill off all cancer cells except the CSC…which will cause the cancer to relapse as the stem cells will just make a new tumour
Theory 2 –> If not killed then more CSCs may form, making treatment much more difficult…and cause the tumour to become metastatic
Why do we inhibit PDL-1 and/or CTLA-1?
As normally they will bind with tumour cells….and reduce the immune response
So by inhibiting them we increase the immune response
What is ELISA?
Enzyme-Linked Immunosorbant Assay (ELISA) is used both qualitatively and quantitatively to detect a specific antigen or antibody in a sample
A mixture of proteins are adsorbed onto the plate, and then blocking molecules added to prevent false positives
A detection antibody then binds to the target protein, with all other proteins being washed off
A second antibody (with an enzyme attached) binds to the first detection antibody
The enzyme then converts a substrate into a flurogenic compound like Alkaline Phosphotase (AP) or Horseradish Perioxidase (HRP) that can be detected
What binds to what activation region out of YESCARTA and KYMRIAH?
YESCARTA –> CD28
KYMRIAH –> 4-1BB
How does Real-Time Sequencing microarrays work?
Each base enters a nanopore and fluoresces (but only in that very specific pore) allowing us to see what base is being added
This base is then cleaved and another added, which flashes
This allows us in real time to see what base is being added
How many base pairs are used in the Cancerseek blood test?
61bp
As above this the results start to plateau
For a cancer vaccine to function, where does it need to get to?
And how does it do this?
The lymph nodes
Does this by the peptides being broken down by either proteasomes or Cathepsins to make them small enough to be taken up my MHCs
What is a Lipoplex?
A mixture of cationic liposomes and DNA/RNA
Due to its high charge it can be toxic
Also has a relatively short duration of action
How are T-Cells activated?
And what is the role of APCs in this?
Tc cells are activated by APCs (such as dendritic cells) which prime them for specific sites, like tumour cells
- This is done via the MHC of the APC and the TCR/CD3 of the T cell
The future is looking towards manufacturing artificial APCs to stimulate the immine system by being able to activate the T-cells
How can FISH be used to detect if a chromosomal inversion/fusion (like EML4-ALK) has occured?
By the FISH ‘Break Apart’ Assay
By adding the fluorescent probe to specific parts, we can track the changes in the positions of the colours
So for inversions of the EML4 region of the chromosome, the signals will move further apart, whilst if there is also a deletion (as well as the inversion), only the red signal will appear
How does Flow Cytometry work?
A method for detecting specific molecules on and within cells
A laser passes through the sample, with it being dimmed for longer with larger samples, which is detected by the forward scattering cell (FSC)
The side scatter cell (SSC) detects the amount of laser that bounces off the samples granular parts –> Which tells us how granular the cell is
In modern flow cytometers you can quantify multiple antigens at once
What is FlSH?
And what can it be used to detect?
Fluorescence In Situ Hybridization
Use of a probe that fluoresces or has a label that will bind with a specific piece of ssDNA
Chromosomal translocations/inversions
Gene Deletions
Gene Amplifications
What is an RFLP?
And what role does this have in terms of EGFR and how PCR can be used to spot them?
Restriction Fragment Length Polymophisms (RFLPs) are where the restriction site is either deleted or a new one created
In EGFR mutations (G719X or L859R) the Apa I and Msc I restriction sites are lost, which increases the bp size of the product
So if Apa I is lost, a product of 237bp will be produced as the DNA isn’t split…which PCR would find!
How does GaINAc-siRNA enter the cell?
The GaINAc has an affinity for cells ASGPR receptors (in a clathrin-coated pit), allowing the siRNA to get into the cell
Explain Slab Gel Electrophesis?
And what is a mutliplex reaction?
DNA is put into a gel and the DNA moves towards the cathode, with larger bits of DNA not moving as far/fast through the gel
Ethidium Bromide is normally used to stain the nucleic acid in agarose ge, allowing it to flouresce under UV light
We can also use a fluorescent marker or a conjugate enzyme
A mutliplex reaction is where more than 1 set of primers is used on each gel, so we get a multitude of products
What are dentritic cell cancer vaccines?
Extraction of the patients dendritic cells, of which are loaded with several antigens before being matured
This means that when re-injected back into the body they can help recruit more different T-cells to kill different cancers
What are the 2 major challenges to genetic material delivery?
Formation –> We need to complex DNA with the vector, keeping the DNA stable and able to pass into the cell….which is very hard!
Degredation –> Lots of things are present inside of the cell that can degrade both the vector and genetic material
What is a DNA Polyplex?
A cationic polyer that forms a Toroid, which protects the nucleic acid from the immune system
What are the 4 general stages of cancer grading?
Early Stage
Localised –> First movement into the lymph
Regional Spread –> Moved into distant lymph nodes
Distant Spread –> Also known as metastatic cancer
Most specific cancers will have their own specific grading system like the Dukes scale for colorectal
What is Real-Time PCR (q-PCR)?
And how does it work?
A type of PCR that allows us to quantify how much of the desired DNA we actually have in real time
Works by having a flourescent reporter on the forward strand that is quenched. It is removed by the DNA polymerase when the DNA is being synthesised, which causes it to fluoresce….which can be quantified
Natrually as the number of PCR cycles increases, and the concentration of the DNA strand that we want, as does the fluorescence (so we can see if overexpression of a gene has occured as more of the dsDNA will be formed in less cycles!)
What is Immunohistochemistry (IHC)?
And how can it be used as part of CISH?
IHC –> Requires surface antigens on which the antibody binds, and then secondary antibodies can bind to amplify the signal
- This complex can then be visuallised
Chromogenic In Situ Hybridisation (CISH) –> A combination of FISH and IHC
A probe from FISH is used which is bound to by an antibody and then a secondary antibody with a conjugated enzyme. The enzmyatic rection causes a coloured precipitate to be created (eg, when gold is made = GOLD-FISH)
How is it possible to get both lobular and ductal cancer from a specific breast cell line?
Different drivers will cause the cell to become cancerous in a specific way (whether that be lobular or ductal) despite the original cell being the same
How can CRISPR/CAS9 be used to control gene expression?
We can bind specific activators or inhibitors to dCas9, which guides it to specific genes where transcription takes place
Depending on what is bound, the gene will either be transcribed or not
These activators or inhibitors will cause a mutation in the nuclease or not, which is what causes the gene expression to occur or not
How many cycles of PCR does it take to create 2 brand new dsDNA?
How do we calculate how many strands of dsDNA that we have full stop?
3 cycles
The number of dsDNA that are present is 2^n
So after 5 cycles you have 32 (2^5 = 35)
How does tumour escape function?
The tumour cells release cytokines, causing changes in the antigens of the tumour cells
This means that tumour cells appear that cant be targetted as they have different targets!
- These have low levels of MHCs and tumour cell recognition
What are CAR T cells?
Genetically engineered T cells that express a chimeric antigen receptor (CAR)
This means that we dont need MHC presentation from APC, as we already have the antigen presented already
Name 6 different ways that mAbs can be used to treat cancer
Tumour specific antibodies
Inhibit angiognesis by using Anti-VEGF antibodies
Checkpoint blockaids via PDL1/CTLA4 to recruit the immune system
Radioimmunology (attaching radiotheraputics to the mAb)
Effector cells can be brought closer to the tumour to aid killing them (bispecific antibodies)
Attatching drugs to the mAb to increase selcetivity of the drug
What is a teratoma?
A tumour that is made up several different tissues
What are the 4 different types of ways that small molecular kinase inhibitors can bind to their target?
Active conformation
Inactive conformation
Allosteric adjacent
Allosteric remote
How can the CRISPR/CAP9 mechanism be used for gene editing?
This is used to create gRNAs to a specific part of DNA to cleave it –> This is permament
It can also be used in viral/non-viral delivery and in the modification of T-cells
We can use it to form a plasmid with all the genes we cant and then insert it into the nucleus
We can deliver the gRNA to the site needed to allow translation into the complex we want
What is Hybridization?
And why can probes be used using the principle?
The joining of 2 complementory ssDNA strands
Occurs only at tempretures below Tm (which is greater for G+C due to more H-bonds being present)
We can use probes to bind to the completementory strands to find out what strands are present
What are bispecific antibodies?
Antibodies that can target 2 different oncogenes/tumours
