Molecular Diagnostics and Biological Therapies

Question 1

What is Reverse Transcriptase PCR (RT-PCR)?

Answer 1

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

Question 2

What is the most common culprit for human cancers?

Answer 2

K-Ras

Question 3

How does a combination of Thymidine Kinase and Ganciclovir function as a suicide gene?

Answer 3

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

Question 4

What are the 2 different types of non-viral nucleic acid delivery?

Answer 4

Physical Techniques –> Injection, gene gun, ultrasound, electroporation

Chemical Techniques –> Encapsulation and Complexation which leads to nanoscale particulates

Question 5

What is a common co-treatment given with cancer vaccines?

Answer 5

Checkpoint inhibitors such as drugs to block the PDL-1

Question 6

How can Microarray Technologies be used?

Answer 6

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)

Question 7

What are Cell Penetrating Peptides (CPPs)?

Answer 7

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

Question 8

How can Oligonucleotides be used as a nucleic acid-based therapy?

Answer 8

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

Question 9

How does Oncorine function?

Answer 9

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

Question 10

What are the pros and cons of vascular and stromal ablation?

Answer 10

Pros –> Very selective, the mAb can couple with various toxins, they are relatively stable

Cons –> High doses needed, can have off-target effects

Question 11

Why are breast ducts susceptible to cancer?

Answer 11

As they are hyper-mutatable (divide quickly)

Question 12

What is good about Mesenchymal Stem Cell Therapy?

And what are the 4 ways in which it can be used?

Answer 12

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

Question 13

When are orphan cancer drugs used most?

Answer 13

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

Question 14

What are the most common cancers in children?

Answer 14

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

Question 15

What are the benefits of High-Throughput screening?

Answer 15

Hundreds of samples per hour

Multiple analytes can be quantifies at a time

Low volume

Sensitive and accurate

Question 16

What are the advantages and disadvantages of CRISPR/CAS9?

Answer 16

Advantages –> Small, inexpensive, specific and versatile

Disadvantages –> Delivery, off-target effects and potential abuse

Question 17

How does the Affymetrix GeneChip work?

Answer 17

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

Question 18

What’s the difference between Sanger Sequencing and Illumina Sequencing Technology?

Answer 18

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

Question 19

How does Electrochemiluminescence work?

Answer 19

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

Question 20

What are the advantages and disadvantages of non-viral vectors?

Answer 20

Advantages –> Easy to prepare and modify, large capacity, less immunogenic

Disadvantages –> Poor efficiency and transient (short acting)

Question 21

What is Allelle-Specific PCR (ASPCR)?

Answer 21

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

Question 22

What is the difference between Direct and Cell-Based gene delivery?

Answer 22

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

Question 23

What is TK Cell Therapy?

Answer 23

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

Question 24

What is Capillary Gel Electrophesis (CGE)?

Answer 24

Where the sample moves through capillaries and are detected by lasers

The strands are still seperated by charge and molecular weight

Question 25

What are the **3 main reasons for why humans are prone to cancer**?

Answer 25

**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**

Question 26

Explain the following genetic therapies **Gene Augmentation** **Gene Inhibition** **Killing of Specific Cells**

Answer 26

**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

Question 27

What are the **postives and negatives of using mAbs** to treat cancer?

Answer 27

**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

Question 28

Why can **dirty cancer drugs actually be quite useful**? In respect to BRAF

Answer 28

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

Question 29

What is a **Dendrimer**?

Answer 29

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

Question 30

What is **major problem with kinase inhbitors**? Like those that are used to treated EGFR mutations?

Answer 30

They arent that specific, and kinases are everywhere in the body! So off-site actions are inevitable

Question 31

How does the **self-test PSA kit** work?

Answer 31

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)

Question 32

What genome does **adeno-associated viruses** have? And what drug is on the market for treatment of lipoprotein lipase deficiency (LPLD)

Answer 32

**ssDNA** **Glybera** --\> A small IM injection to the leg

Question 33

What type of genome does a **retrovirus** have? And how can they cause cancer?

Answer 33

**ssRNA** genome The viral DNA can promote proto-oncogenes, or split apart a tumour suppressor gene (TSG)....both of which will promote tumour formation

Question 34

How do the **PCR** reaction occur?

Answer 34

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)

Question 35

What is a **range of variation**?

Answer 35

The differences between different peoples genes Eg, some people will have a chromosome that means a certain mutation is inevitable - This usually occurs due to a single nucleotide polymorphism (SNP)

Question 36

What is the difference with **Sandwich ELISA**?

Answer 36

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

Question 37

Why is killing the **Cancer Stem Cell (CSC)** in a tumour cell vital? Explain the 2 theories

Answer 37

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**

Question 38

Why do we inhibit **PDL-1 and/or CTLA-1**?

Answer 38

As normally they will bind with tumour cells....and reduce the immune response So by inhibiting them we increase the immune response

Question 39

What is **ELISA**?

Answer 39

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

Question 40

What binds to what activation region out of **YESCARTA** and **KYMRIAH**?

Answer 40

**YESCARTA** --\> CD28 **KYMRIAH** --\> 4-1BB

Question 41

How does **Real-Time Sequencing microarrays** work?

Answer 41

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

Question 42

How many **base pairs** are used in the **Cancerseek blood test**?

Answer 42

**61bp** As above this the results start to plateau

Question 43

For a **cancer vaccine to function, where does it need to get to**? And how does it do this?

Answer 43

**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

Question 44

What is a **Lipoplex**?

Answer 44

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

Question 45

How are **T-Cells activated**? And what is the role of **APCs** in this?

Answer 45

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

Question 46

How can **FISH** be used to detect if a **chromosomal inversion/fusion** (like EML4-ALK) has occured?

Answer 46

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

Question 47

How does **Flow Cytometry** work?

Answer 47

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

Question 48

What is **FlSH**? And what can it be used to detect?

Answer 48

**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

Question 49

What is an **RFLP**? And what role does this have in terms of EGFR and how PCR can be used to spot them?

Answer 49

**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!

Question 50

How does **GaINAc-siRNA** enter the cell?

Answer 50

The GaINAc has an affinity for cells ASGPR receptors (in a clathrin-coated pit), allowing the siRNA to get into the cell

Question 51

Explain **Slab Gel Electrophesis**? And what is a **mutliplex reaction**?

Answer 51

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

Question 52

What are **dentritic cell cancer vaccines**?

Answer 52

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

Question 53

What are the **2 major challenges to genetic material delivery**?

Answer 53

**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

Question 54

What's the difference between **Electrochemiluminescence and Luminex Technology**?

Answer 54

**Luminex** has several beads, each being a different colour and so we can look at several different target proteins, and see what colour is given off

Question 55

What is a **DNA Polyplex**?

Answer 55

A cationic polyer that forms a Toroid, which protects the nucleic acid from the immune system

Question 56

What are the **4 general stages of cancer grading**?

Answer 56

**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

Question 57

What is **Real-Time PCR (q-PCR)?** And how does it work?

Answer 57

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!)**

Question 58

What is **Immunohistochemistry (IHC)**? And how can it be used as part of **CISH**?

Answer 58

**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)

Question 59

What type of genome does an **adenovirus** have? And name a drug that relies on an adenovirus

Answer 59

**dsDNA** **Gendicine** --\> A p53 injection

Question 60

How is it possible to get **both lobular and ductal cancer from a specific breast cell line**?

Answer 60

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

Question 61

How can **CRISPR/CAS9 be used to control gene expression**?

Answer 61

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

Question 62

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?

Answer 62

3 cycles The number of dsDNA that are present is 2^n So after 5 cycles you have 32 (2^5 = 35)

Question 63

How does **tumour escape function**?

Answer 63

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

Question 64

What are **CAR T cells**?

Answer 64

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

Question 65

What makes **BRCA1/2** more likely to cause cancer?

Answer 65

They are **autosomal dominant**, so a mutation in only one copy will increase the risk of cancer greatly

Question 66

Name **6 different ways that mAbs can be used** to treat cancer

Answer 66

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

Question 67

What is a **teratoma**?

Answer 67

A tumour that is made up **several different tissues**

Question 68

What is a "**Common Theme/Thread**"?

Answer 68

Where there is a common link between cancers in different parts of the body Eg, 40% of cancers are caused by EXON 2 driver of K-RAS - So if we can treat the EXON 2 driver, we can treat 40% of cancers It is for this reason that we need to study the cause of the cancer and not just its location, as we may be able to treat both with one drug if they are cancerous for the same reason

Question 69

How is **Gioblastoma multiforme (GMB)** treated?

Answer 69

Due to VEGF being upregulated (a pro-angiogenic growth factor), a gene for soluble a VEGF receptor is inserted, which mops up VEGF and so reduces angiogensis Can also use siRNA to form nanoplexes which can work against VEGF

Question 70

What are the **3 types of mutation that PCR can detect**?

Answer 70

**Point mutations (SNPs)** **Deletions** **Insertions**

Question 71

What are the **4 different types of ways that small molecular kinase inhibitors can bind to their target**?

Answer 71

**Active conformation** **Inactive conformation** **Allosteric adjacent** **Allosteric remote**

Question 72

How can the **CRISPR/CAP9 mechanism** be used for **gene editing**?

Answer 72

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

Question 73

What is **Hybridization**? And why can **probes** be used using the principle?

Answer 73

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

Question 74

What are **bispecific antibodies**?

Answer 74

Antibodies that can target 2 different oncogenes/tumours