Therapeutic macromolecules : Intro To Gene Therapy

Question 1

What is gene therapy?

Answer 1

It is a treatment method based on the delivery of nucleic acids to cells (transfection) in order alter protein expression in order to treat a medical condition

Question 2

Why is gene therapy used to alter gene expression?

Answer 2

Lots of indications and diseases are due to incorrect gene expression and thus incorrect production of protien where either too much or too little protein is produced or the wrong protein is coded etc.
By altering gene expression to produce the right protein we can treat the disease.

Question 3

How does gene therapy aim to treat medical conditions ? ( 4 ways)

Answer 3

➕introduce a new gene
✍️ edit a mutated gene
👯 replace a mutated gene
🤐 silence a gene that is not working well

Question 4

What are introns?

Answer 4

Introns are non - coding sections of the DNA that are transcribed into pre - mRNA but are removed to produce mature mRNA by splicing.

Question 5

What are exons?

Answer 5

Exons are coding mRNA that are transcribed into pre - mRNA and are retained in the mature mRNA after the introns are removed.

Exons contain information about how to make protien and the folding of the protein etc which is necessary in producing the correct and functional protein.

Question 6

What is splicing and when does it take place?

Answer 6

• removal if non coding introns from pre - mRNA to produce mature mRNA which only has coding exons present.

Question 7

What is a gene? 🧬

Answer 7

A small section of DNA - ie small section of nucleic acids

Question 8

What are the 3 main goals for gene therapy?

Answer 8

• addition of a new gene
• repacking a faulty or problematic gene with a new one
• silencing a problematic gene

Question 9

What are 3 examples of gene therapy agents?

Answer 9

• DNA therapy
• RNA therapy
• Oligonucleotides

Question 10

How is DNA therapy delivered?

Answer 10

In a plasmid - its plasmid DNA therapy.

Question 11

What are the aims of DNA plasmid therapy?

Answer 11

✅ replace a defective gene
✅ introduce a new gene

Question 12

Is DNA plasmid therapy used to silence genes that are faulty or problematic etc?

Answer 12

❌ NO

Question 13

Describe the structure of DNA plasmid gene therapy?

Answer 13

• Circular double stranded DNA (dsDNA)

Question 14

What is the site of action for DNA plasmid therapy?

Answer 14

• the nucleus

Question 15

Does DNA plasmid therapy require transcription and translation?

Answer 15

YES BOTH ARE REQUIRED

Question 16

Briefly describe the mechanism of action of DNA plasmid gene therapy?

Answer 16

1. The plasmid will be packaged into a delivery system
2. The delivery system with the DNA double strand will enter the cell via endocytosis
3. The endosome matures - some will be degraded by lysosomal degradation
4. Those that do not degrade will release the DDS via endosomal escape
5. Some may be degraded by cystolic degradation, the rest will be translocate and transfection into the nucleus.
6. In the nucleus the gene will be transcribed , then translated outside the nucleus buy the RNA to express the correct functional genes and subsequent proteins : )

Question 17

DNA gene therapy can exist in the nucleus isolated from chromosomes - what does this mean?

Answer 17

It’s not necessarily integrated into the patients chromosome and genome, however it is passed onto the daughter cells in cell division as the DNA replicates

Question 18

Give 3 examples of DNA therapeutics and what they are used to treat.

Answer 18

Hemgenix 🩸 - haemophilia B
Zolgensma 🩻 - ppl with spinal muscular atrophy
Luxturna 👁️ - vision loss in px with inherited retinal dystrophy caused by biallic RPE 65 mutations

Question 19

What is the main site of action for RNA therapy?

Answer 19

The cytoplasm - there is no requirement for it to get into the nucleus usually.

Question 20

Is RNA therapy integrated into the genome?

Answer 20

NO

Question 21

What types of RNA therapy can we use?

Answer 21

• mRNA therapy -> allows for protein production
• microRNA - targets multiple mRNAs
• small activating RNA ( saRNA)
• suppressor tRNA
• RNA aptamers

Question 22

What is the main aim in terms of gene therapy using RNA ?

Answer 22

• 🤐 to silence mutated genes and thus STOP gene and protein expression.

Question 23

Give two examples if RNA silencing therapies.

Answer 23

1. Small - interfering RNA ( siRNA)
2. Short - hairpin RNA (shRNA)

Question 24

Give 4 examples of SiRNA therapies used in practice?

Answer 24

Patisiran -stage 1 or 2 polyneuropathy in px with hATTR
Givosiran
Inclisiran
Vutrisiran - stage 1 or 2 polyneuropathy in px with hATTR

Question 25

What is the difference in siRNA and shRNA

Answer 25

ShRNA is like a prodrug of siRNA which ultimately works in the cytoplasm to prevent the expression of a protein

Question 26

What kinds of diseases do we use silencing gene therapy for?

Answer 26

Rare diseases where there is a small population of ppl that have the mutation that can be targeted.

Question 27

Mechanism of action for RNA interference by si and shRNA?

Answer 27

ShRNA is the prodrug - present in a plasmid and will need to get into the nucleus (only to replicate it so that we dont need multiple administrations).

shRNA processed in the nucleus and then enters cytoplasm where the “loop” on it’s structure is removed by cleaving DICER, releasing the active “siRNA” form!

SiRNA works in the cytoplasm, and will form a complex with RISC, which separates its strands into antisense and sense. The sense strand will then , whist still in ca complex with RISC bind to the target mRNA labelling it for degradation and cleavage and thus preventing the production of a protein.

Question 28

Site of action for siRNA and shRNA therapies?

Answer 28

ShRNA - nucleus
SiRNA - cytoplasm

Question 29

Are RNA therapies used to replace faulty genes or to incorporate new genes into the DNA?

Answer 29

❌ NO - they are used to SILENCE genes in order to prevent protein expression :)

Question 30

What are Oligonucleotides?

Answer 30

They are short double stranded DNA/RNA strands that target RNA

Question 31

What is the mechanism of action of Oligonucleotides in gene therapy?

Answer 31

They either :
1. Silencing - They trigger the degradation of mRNA that would produce the protein of interest - thus prevent the production of a protein.

2. Splicing - where they remove the introns so that the order of exons can be altered and so will change the type of protein being produced.

Question 32

What is the target for oligonucleotide therapy?

Answer 32

RNA
They act on it to prevent protein expression by either splicing or silencing it.

Question 33

What kinds of genetic diseases are oligonucleotide therapies used to treat?

Answer 33

Single gene diseases - ie very rare

Question 34

Give 3 examples of oligonucleotide therapies used in clinic and what they are used to treat?

Answer 34

1. Tegesdi —> stage 1 or 2 polyneuropathy in px with hATTR in adult patients
2. Waylivra —> px with genetically confirmed FCS who are at high risk for pancreatitis.
3. Spinraza —> 5q SPinal Musclular Atropy.

Question 35

What are the two ways that gene therapy can be administered?

Answer 35

• in vivo
• ex vivo

Question 36

What is ex vivo gene delivery?

Answer 36

This is when the gene therapy is not delivered directly into the patient.
Stem cells are removed from the patient.

The gene therapy (+/- packaging in a vector) is then inserted into the stem cell and grown in a Petri dish.

Once the cells express the target gene they are re injected back into the patient
It is an autologous process.

Question 37

What is a major drawback of ex - vivo gene delivery?

Answer 37

❌ it will take a long time to do 🕰️

Question 38

What is in vivo gene delivery?

Answer 38

This is where we package the gene therapy into a vector 📦, and then directly inject it into the patient via parenteral administration, where it will circulate in the blood until it finds its target.

Question 39

What is a major drawback of in - vivo gene delivery?

Answer 39

• ❌ poor stability in blood circulation.
• ❌ risks associated with delivery to target cells - need to make sure that its accurate and precise

Question 40

What are the main properties we would want the vector that we use in gene therapy to have?

Answer 40

• ✅ high transfection rate - ie low dose can transfection a lot of cells as possible
• ✅ non - immunogenicity or pathogenic
• ✅ allow for sustained expression of the gene, or silencing if using interfering RNA
• ✅ selectively / actively target desired cells and tissues
• ✅ easy to produce and load with genetic material (ie if too small for our large gene - not efficient)

Question 41

Describe the main barriers to delivery of gene therapy? (2)

Answer 41

1. Enzymatic degradation
2. Cellular uptake

Question 42

How does enzymatic degradation pose as a barrier to gene therapy?

Answer 42

❌ RNA and DNAases will cleave respective genetic material making them non functional as they are unable to differentiate them from genetic material that needs to be degraded and medication.
❌ can occur intra or extracellularly

Question 43

How does cellular uptake pose as a barrier for delivery of gene therapy?

Answer 43

❌ Gene therapy involves the use of large hydrophilic molecules (that have-ve charge) —> may not be able to cross cell membranes adequately to enter cells to have effect.
- and prior to entering the cell, they may have even been susceptible to degradation by enzymes extracellularly !!!!😭

Question 44

Are all sites for gene therapy delivery equal in susceptibility to enzymatic delivery?

Answer 44

🙅‍♀️ NO
Oral delivery has a lot of proteolytic activity compared to pulmonary where its very little.

Question 45

How can we overcome the barriers to gene delivery>

Answer 45

To improve cellular uptake:
- ✅ we can increase hydrophobicity ie by adding an alkyl chain. This will improve the interaction with the cell membrane.
- ✅ add permeation enhancers to the formulation such as surfactants, cationic polymers, calcium chelations, cell penetrating peptides.

** To avoid enzymatic degradation: **
- ✅ encapsulate the biolgical drug in nanomedicine however this also has its own challenges ⚠️

Question 46

How do permeation enhancers work to improve cellular uptake of gene therapy? What is the drawback?

Answer 46

They will increase the space between the phospholipids in the plasma membrane of targeted cells allowing for >er uptake of larger sized molecules.

❌ this method isnt really widely used as it can also allow for other molecules such as bacteria etc to enter the cell….

Question 47

What are some challenges to gene therapy that may arise in regards to the disease?

Answer 47

1. We need to be able to identify the genetic targets clearly (ie to prevent off target effects etc).
   - we need to know whether the mutation is germ line (ie inherited) or somatic ie environmentally accumulated).
2. We need to understand the pathophysiology of the genetic mutations as it will affect our decision in regards to replacing or silencing the mutated gene.

Question 48

What are some challenges to gene therapy that may arise in regards to the drug/ gene therapy ?

Answer 48

1. We need to ensure that the method used for delivery must be safe and efficient - ie we need to ensure that the gene therapy gets to the right cell, right receptor at the right dose to have the right effect. (Need to get to the right site of action, ie nucleus or cytoplasm).
2. The effect must be specific - ensure that we have low risk of off target effects or DDIs.
3. We want a sustained effect not a transient one - to avoid multiple and frequent injections and admin over the year.
4. Consider the risk of introducing somatic mutations into the genome… essp with DNA therapy.
5. Ethical considerations in regards to testing - ie difficult to test for some rare diseases bc not a lot of population have such mutations causing the disease etc ====> this may affect the regulatory body decision to authorise the treatment ie if there is a lack of evidence.
6. Cost and regulatory considerations

Question 49

What ethical considerations do we need to make in regards to gene therapy?

Answer 49

• performance enhancing gene therapy - not ethical in field of sports competition (ie using it to increase expression of EPO and growth factor (IGF -1) ——> if formulated as an injection it would be difficult to now tell whether levels of EPO are intrinsic or due to doping.
• cost and accessiblity of treatment —> very expensive and so they need to deffo be accurate and must work. Also if they are very expensive, this means that it’s not really that available to populations that have a lower GDP - can prohibit use.
• there are also regulatory hurdles that need to be overcome in order to approve gene therapy use .

Question 50

What is the difference between gene therapy and genetic engineering?

Answer 50

Gene therapy is medical treatment of a genetic disease whilst genetic engineering is enhancement of genes for a desired outcome (ie GM crops are bigger than regular).