Non - Viral Gene Delivery

Question 1

What are the main formulation challenges we face when delivering gene therapy to patients?
- ie what must we consider?

Answer 1

• the formulation should not trigger an immune response
• needs to be specific - ie get to the target site at the right dose at the right time (ie nucleus or cytoplasm)
• avoid off target effects
• needs to be stable - ie liable to enzymatic degradation and poor cellular uptake
• needs to be able to cross the cell membrane

Question 2

True or false : non viral gene delivery nanoparticles can be organic or inorganic.

Answer 2

True

Question 3

What charge do all gene therapy nanoparticle carriers need to have on their surface and why?

Answer 3

+ve charge - ie they need to be cationic.

All gene therapy has an intrinsic negative charge (ie both RNA and DNA) and loading of the gene therapy into the DDS is usually via electrostatic attraction and so a +ve charge is needed.

Question 4

Give an example of a type of nano carrier that has an intrinsic cationic charge?

Answer 4

Peptide nanoparticles- their amine groups have a +ve charge

Question 5

Give an example of a type of nano carrier that has been modified to have a cationic charge?

Answer 5

Inorganic nanoparticles

Question 6

A major advantage of gene therapy is that it is really potent - what does this mean?

Answer 6

This means it is very effective at low doses - can give smaller doses
- better bc lack of wastage and also cheaper

Question 7

What are the 4 types of nanoparticles we can use in non - viral gene delivery?

Answer 7

🧈 lipids - ie DOTAP, DOPE
🥩 peptides - Poly(L -lys)
⛓️ inorganic nanoparticles (ie augNP, AgNP,CNTs mesoporous silica).
🫧 Polymers (ie polyplexes) —> PEI, dendrimers, chitosan

Question 8

What are organic nano-complexes?

Answer 8

These are the DDS which have been complexed with gene therapy.

Question 9

What are the 3 types of organic nanocomplexes that we can use to deliver gene therapy and what they originate from?

Answer 9

Polymer + gene therapy = polyplex

Dendrimer + gene therapy = dendriplex

Lipid/liposome + gene therapy = lipoplex

Question 10

What is often needed in order to use inorganic NPs as a delivery system for gene therapy?

Answer 10

We would need to modify the surface so that it carries a positive charge so that we can load the drug onto the surface.

Question 11

What is the mechanism in which nanocomplexes enter cells?

Answer 11

Endocytosis

Question 12

Describe some extracellularbarriers that may creep up when administering a nanocomplexex containing gene therapy to a patient, systemically?

Answer 12

1. Liable to enzymatic degradation
2. In health tissue - tight junctions may prevent endocytosis
3. Positive charge (+)ve, on the surface of the nano complex may mean that it interacts with other endothelial cells that are NOT the target, or some other plasma proteins.

Question 13

Describe some intracellularbarriers that may creep up when administering a nanocomplexex containing gene therapy to a patient, systemically?

Answer 13

We know that the NC will enter via endocytosis and thus will be encapsulated within an endosome inside the cell.
1. The endosome forms a barrier where the NC needs to escape it prior to maturation of the endosome where its liable to lysosomal degradation

Question 14

What are polyplexes?

Answer 14

Gene therapy + polymer

Question 15

Give 3 examples of polyplexes.

Answer 15

PEI ( Polyethylene imine)
Dendrimers
Chitosan

Question 16

Which is the most commonly used polymer used to carry gene therapy?

Answer 16

Poly ethylene imine (PEI).

Question 17

Once the DDS and gene therapy are inside the cell via endocytosis, what must occur so that they can have action in the cytoplasm or nucleus?

Answer 17

Endosomal escape

Question 18

Is endocytosis size dependant?

Answer 18

Yes - larger molecules are more difficult to enter cells (however can use permeation enhancers , ie surfactant).

Question 19

What is meant when we say there are different types of endocytosis mechanisms?

Answer 19

There are different ways the nanoplexes can enter the cells via endocytosis.
- some size dependant
- receptor mediated
- receptor independent

Question 20

How does the pH of an endosome change as it matures (both for those that have or dont have any gene therapy within)?

Answer 20

It decreases - ie it becomes more acidic inside.

• facilitates lysosomal degradation in mature endosome (lysosome) which is a very acidic environment.

Question 21

What is a polyamine?

Answer 21

A type of polyplex where the repeating unit of the polymer DDS we are using has an amino function.

Question 22

Are polyamides basic or acidic.

Answer 22

They are basic and will accept H+

Question 23

Why is the structure (ie primary, secondary, tertiary) of the amine present in the polyamine we use very important in terms buffering capacity for endosomal escape.

Answer 23

Buffering capacity = the ability to resist changes in pH
We know that the pH of the endosome becomes increasingly acidic as it matures.
We thus need a polymer that can withstand these changes to pH, ie accepting the H+ so that the gene isn’t degraded.

Primary and secondary amines have more space to accept H+ compared to tertiary amines which have 3 R - groups and thus very low ability to accept a H+ and thus has a low buffering capacity.

Question 24

What are the 3 proposed ideas for endosomal escape?

Answer 24

🧽 Proton sponge effect (ie osmotic gradient)
⚛️ ionisation
🫧 dissociation of the polyplexes

Question 25

What is endosomal escape?

Answer 25

This is basically how the nanoplex (and thus gene therapy within) will be released from the endosome into the cytoplasm where it will either go to nucleus (ie DNA therapy) or remain (siRNA).

It often involves some level of disruption fo the endosomal membrane.

Question 26

Describe the endosomal escape mechanism : proton sponge effect? 🧽

Answer 26

We know that the endosome becomes increasingly acidic as it matures into a lysosome.

In an endosome with a polyplexes, the polymer choice will affect buffering capacity. If the polymer chosen for the polyplexes has primary or secondary amines, these will accept the H+ that are increasing in concentration in the endosome. The endosome will try bring in more H+ into the endosome in futile attempt however effect is offset by the buffering capacity of the polymer amine groups.

As a result the inside of the endosome increases [H+] to a greater extent to cytoplasm - creating a osmotic gradient. H2O from cytoplasm will move into endosome down the concentration gradient via osmosis, causing the endosome to swell up and rupture, releasing the polyplexes.

Question 27

What is the charge of the phosphilpids that make up the endosome?

Answer 27

They are anionic - remember, the resting membrane potential is usually negative.

• hence cationic nanoplexes can complex with both the gene therapy AND cell membranes to form endosomes !:)

Question 28

Describe how polyplexes will use ionisation as a mechanism for endosomal escape?

Answer 28

As we know the charge of the endosomal bilayer is pretty anionic (-ve) as they come form the cell membrane.

We also know that the charge of our polyplex should be positive.

They interact causing disruption to the membrane structure by forming pores from which the polyplex can escape.

• local escape compared to rupture

Question 29

Describe how polyplexes will use dissociation of the polyplex as a mechanism for endosomal escape?

Answer 29

Here the gene therapy is released from the polyplex within the endosome.
This makes the inside of the endosome more ++++ (because they are no longer being neutralised by the gene therapy).

There are thus greater interactions of the positively charged (cationic) polymers with the anionic endosome membrane —-> disrupts membrane by creating pores from which gene therapy is released into the cytoplasm

— ⚠️ risk of cystolic degradation

Question 30

Describe two lipid based nanoparticles that we could use to form lipoplexes (ie lipid based gene therapy DDS)?

Answer 30

A liposome that is made up of helper lipid, and cationic (+ve) phospholipid with the RNA inside.

Lipid nanoparticle : made out of ionisable cationic lipid (ie it becomes cationic in low pH), helper lipid, RNA, cholesterol, PEG, and RNA.

Question 31

Ionisable cationic lipid and helper lipid become cationic and positively charged at which pH?

Answer 31

Low pH - acidic conditions

Question 32

Describe the mechanism of endosomal escape of gene lipoplexes from endsomes?

Answer 32

The lipoplex has a +ve surface charge (ie via use of ionisable cationic lipid , or a cationic lipid).
Once in the liposome, as the pH decreases and becomes more acidic, the conformation fo the phospholipids in both the lipoplex and the endosome change.
They interact with each other (electrostatic) forming hexagonal phase of neutral phospholipid pauses, as the the gene therapy is displaced as they interact and is released into the cytoplasm in the cell.

Question 33

Give an example of a drug that is formulated in a lipoplex?

Answer 33

Onpattro - patisiran, a siRNA gene therapy.

Question 34

What can lipoplexes be used to deliver into cells?

Answer 34

Gene editing tools such as crispr/cas 9
MRNA vaccines

Question 35

We mentioned that we need to make the surface of the lipoplex (and our nanoplexes in general) cationic to allow for interaction and loading of our gene therapy via electrostatic interaction. What is the implication of administering a +vly charged lipoplex systemically and how can we over come this?

Answer 35

• immune system detects large, hydrophobic and CHARGED molecules - thus lipoplex may be liable to clearance by MPS or opsoninisation.
• we can overcome this by using PEG which masks charge, and hydrophobicity.

Question 36

What considerations do we need to make when using PEG?

Answer 36

They avoid the immune system by stealth however they do interact with immune cells making allergy a risk of use.

• they may cause IgE mediated degraulaiton of basophils - can lead to anaphylaxis
• can lead to complement activation related pseudo allergy

We thus need to consider the type of PEG we use to reduce allergic side effects.

Question 37

What should we co - prescribe patients who are taking pegylated surface modified lipoplexes?

Answer 37

• ✅ anti - histamines (suppress inflammation)
• ✅ corticosteroids (suppress inflammation and immune system)

Question 38

Which are used more, polyplexes or lipoplexes in practice?

Answer 38

Lipoplexes - commonly as a research tool.

Question 39

What is the N : P ratio?

Answer 39

N = amine function of the carrier (+ve charge).
P = phosphate on the gene therapy agent ( -ve charge)

A greater N:P ration indicates the presence of more carrier and a reduced N:P indicates less encapsulation of the DNA (bc its naked and would have a low ratio).

Question 40

What should we assess in regards to a physical mixture of the carrier and the gene therapy agent?

Answer 40

• the stability in the presence of anionic macromolecules
• efficacy of loading into the carrier

Question 41

What do we use to assess gene expression? (Ie mRNA expression)

Answer 41

PCR

Question 42

What do we use to assess protein expression and HOW ?

Answer 42

Western blot
It does so by detecting protein of interest using antibodies and electrophoresis ?

Question 43

What is the rationale for using a “scrambled sequence (SCR)”?

Answer 43

It has the same composition (ie size and length) as the therapeutic gene molecule hwever it doesnt target the gene we want to treat or affect - the sequence is scrambled.

✅ It is used to check for non - specific effects (ie the genotoxicity of the carrier).
- it ensures that the effect seen is deffo due to the drug and not the carrier or something else.

Question 44

Is PCR qualitative or quantitative?

Answer 44

Both
- RT- PCR is qualitative (reverse transcriptase)
- qRT - PCR - quantitative

Question 45

What is the function of housekeeping genes?

Answer 45

They are the control variables - we use their expression as a reference for the levels of protein expression

Question 46

What is ELISA and what is it used for?

Answer 46

Enzyme linked immunosorbent assay.
- it is a quantitative method that is used to measure the presence and levels of antibodies, antigens, glycoproteins proteins in biolgical samples

Question 47

How can we make ELISA quantitative?

Answer 47

Calibration curve.

Question 48

How does ELISA work?

Answer 48

It relies on the use of an antibodie that is linked to a dye or fluorescence - this will then specifically bind to and recognise the protein of interest and will produce a signal which is what is measured.