Gene therapy

Question 1

How long as gene therapy been around for?

Answer 1

~ 30 years

Question 2

How have the views on gene therapy changed?

Answer 2

Gene therapy has suffered fromm overhype in the early days, premature advancement to poorly controlled clinical trials and safety concerns with viruses that stopped the hype

Question 3

Why was there initial hype over gene therapy?

Answer 3

In the early 90s scientists said it was going to cure many conditions
Lancet reported on a tumour that ws injected with virus that reduced tumour size
VEGF was used to reover a patient from peripheral vascular disease (since then VEGF has not been successful)

Question 4

What major event caused the public to lose faith in gene therapy?

Answer 4

Death of Jesse Gelsinger in 1999
Jesse had OTC deficiency, meaning he could not metabolise ammonia
The wildtype corrective gene was placed into Jesse via adenoviral vector
He died after 4 days due to a cytokine storm induced by the virus- the cytokines caused multiorgan failure

Question 5

Why did the death of Jesse Gelsinger happen?

Answer 5

Cytokine storm
It happened due to poor knowledge of virus:host actions and patient variability
There was failure to disclose that monkeys had died in the trial

Question 6

Why is gene therapy not feasible for cystic fibrosis?

Answer 6

The virus is unable to pass into cells due to the thick mucus

Question 7

What is the criteria for successful gene therapy?

Answer 7

• There is currently unmet clinical needs e.g heart failure which are no effective pharmacological strategy
• Need a detailed understanding of the pathogenesis and genes involved
• Need an effective and safe delivery system
• And little/no off-target effects
• Need targeted strategy to treat the condition for the right length of time in the appropriate cells and organs.

Question 8

What are the different methods that can be used for gene therapy?

Answer 8

Lentivirus
Adeno-associated virus AAV
Human Adenovirus HAdV
Lipid nanoparticles
Future- CRISPR/Cas9

Question 9

When are lentiviruses used for gene therapy?

Answer 9

Used when long-term expression needed, such as ex vivo stem cell applications

Question 10

When is AVV suitable as a vector for gene therapy?

Answer 10

Gives long term delivery, however genome of the virus is small which provides a problem for large gene transfer

Question 11

What is AAV currently used for in gene therapy?

Answer 11

Some current uses include ocular- used a needle to inject the virus at the back of the eye to treat genetic eye disease
There has been substantial advances in muscle and liver gene therapy but heart and brain are attractive oppurtunities

Question 12

What is AAV?

Answer 12

Adeno-associated virus, it is not associated with any known human disease
Induces a T cell response that can be managed clinically. Should be safe

Question 13

What is HAdV?

Answer 13

Human Adenovirus
A large family of human and non-human viruses

Question 14

When is HAdV mainly used?

Answer 14

Used in COVID-19 vaccines and HIV and Ebola vaccines
It is most effective in CV and cancer
Not useful for providing a gene that is missing as cannot give long term delivery
It is associated with self-limiting infections (however there is exceptions, this type of virus killed Jesse Gelsinger)

Question 15

What are the pros and cons of lipid nanoparticles for delivering genes?

Answer 15

They are not very efficient but since they are not a virus they can be delivered multiple times

Question 16

What are lipid nanoparticles?

Answer 16

Lipid nanoparticles are spherical vesicles made of ionizable lipids, which are small enough to be taken up into cells by endocytosis
They are a novel pharmaceutical drug delivery system, and a novel pharmaceutical formulation.
LNPs as a drug delivery vehicle were first approved in 2018 for the siRNA drug Onpattro

Question 17

What is the future technique in gene therapy?

Answer 17

CRISPR/Cas9 in vivo gene editing
2021 paper in the New England Journal of Medicine
- Gene editing for Transthyretin amyloidosis, a life-threatening disease characterized by progressive accumulation of misfolded transthyretin (TTR) protein in tissues, predominantly the nerves and heart.
- Can edit the gene and cut out parts using the CRISPR Cas9 system that cuts parts of the DNA to correct the gene
- First study found little adverse effects and a reduction of TTR protein so fairly successful in 6 patients.

Question 18

What was the first successful gene therapy case?

Answer 18

In 1990 doctors partially reversed a case of severe combined immune deficiency, or SCID, also known as “bubble boy syndrome,” in a young American girl, Ashanthi DeSilva.

Question 19

What is bubble boy syndrome?

Answer 19

X linked disease
X-linked severe combined immunodeficiency (SCID)
Immune system deficiency that means infections cannot be fought
SCID results from a mutation in the interleukin 2 receptor gamma (IL2RG) gene
There is a mutation in the gamma-c. This causes no immune signalling, absent T and NK lymphocytes and abnormal B lympocytes
- 1 in 50,000-100,000 births so rare
- Causes severe diarrhoea, pneuomonia, fungal infection, failure to thrive and usually death within the first year of life.

Question 20

How is Bubble boy syndrome treated?

Answer 20

It is fully curable by a fully matched bone marrow transplant (usually from a sibling)
High mortality rate of ~50% if the donor is not fully matched
Gene therapy given to treat this

Question 21

What is CRISPR/Cas9 in simple terms?

Answer 21

CRISPR is a fundamentally new way to change genes. The basic technology consists of an enzyme that cuts DNA and a segment of guide RNA that tells the enzyme where to snip. The package may include other components, such as a new piece of DNA code to plug into the edited area. The cell’s natural repair mechanism completes the edit
Helps in gene therapy to stop the genes from randomly integrating near oncogenes and causing cancer

Question 22

When was the phase 1 trial for gene therapy in SCID and what were the outcomes?

Answer 22

Started in 2000 in Paris
Cells were taken from 2 patients, infected with a MMLV (murine leukemia virus) and the cells returned to the patients.
- This method seemed to work well, overall in many patients there was an increase in lymphocytes.
- However NK cells and B cell defects were only partially restored.

Question 23

What is the downside to gene therapy treatment in SCID gene?

Answer 23

The virus integrates randomly in the genome and can insert itself in the wrong place- e.g. into an oncogene that gets activated
This led to leukaemia in one of the patients being tested with gene therapy in Novemeber 2002.
There was a 20% leukaemia rate out of 20 patients treated in Paris and London with 1 death.
The long term survival rate for gene therapy was 86%

Question 24

What is an example of gene therapy used to treat CNS?

Answer 24

MLD=
Neuronal white matter degeneration with rapid and irreversible loss of motor and cognitive function
Orchard theraputics removed hematopoietic stem cells from patients and using a lentivirus replaced back with the corrected gene
The survival rates were much higher in those treated with the ARSA cell gene therapy, and there was a rapid improvement in cognitive function
There was less brain degeneration seen in patients treated with gene therapy as wel

Question 25

What is MLD?

Answer 25

Metachromatic leukodystrophy
- It causes neuronal white matter degeneration with rapid and irreversible loss of motor and cognitive function
- In its most severe form, children pass away around 5 years after symptom onset
- The mutated ARSA gene means there is a build up in toxins
- 1 in 100,000 live births

Question 26

What is the name of the gene therapy used to treat MLD?

Answer 26

Libmeldy is now a EU approved medicine for treatment
There was debate in the FDA as there was already exisiting treatments however, after input from patients who emphasised that their quality of life was better, it got approved.

Question 27

What is haemophilia?

Answer 27

Blood condition in which essential clotting factors are (partly) missing
Main problem is internal bleeding into joints, muscles and soft tissues.
There are two types of haemophilia, the most common being haemophilia A, in which Factor VIII is lacking. In haemophilia B, Factor IX is lacking.

Question 28

What is the current treatment of haemophilia?

Answer 28

Usually replacement therapy of a missing factor. In severe cases it is via prophylaxis and in more mild cases it is via injections

Question 29

What were the results of preclinical trials testing gene therapy in haemophillia?

Answer 29

Early studies used AAV expressing factor IX
- Treatment in haemophilia B mice, there was a long term increase of factor 9.
- After it as confirmed it worked in mice, it was then tested in dogs. A liver-directed infusion with AVV virus and the corrected FIX gene gave correction for over 8 years
- Clotting factor did not reach normal levels however do not need to completely correct it to see results.

Question 30

What was the set up and results of of the phase 1 and 2 trials testing gene therapy in haemophiliacs?

Answer 30

Dose escalation trial where the gene was administered via hepatic artery infusion of the AVV.
- No adverse effects reported.
- However, this trial and the field became stalled as it was found that factor 9 decreased overtime.
- Concluded that more efficient vectors were needed

Question 31

What is a more efficient AAV vector?

Answer 31

New family of AA-viruses called AAV8.
- The AAV8 family have a low pre-existing immunity in humans.
- There is also distinct biology- rapid uncoating and degradation of the viral capsid.
- Trials with AAV8 in haemophilia commenced in 2010.

Question 32

Where are we at now with gene therapy in haemophilia?

Answer 32

• Near-to-complete correction of both haemophilia A and B have now been achieved by hepatic in vivo gene transfer.
• However, mild liver toxicities have been observed in some patients receiving high vector doses.
• There is several gene therapies that have now been licensed for haemophilia, including biomartin’s Roxaparvovec AAV gene therapy.

Question 33

What does the FDA expect to be the number of gene therapies approved every year?

Answer 33

From 2025 onwards there will be upto 20 new market approvals per year for gene therapy

Question 34

How is angiogenesis being targeted for gene therapy?

Answer 34

Initially some results had been promising- pivotal study by Yla-Herttuala and Alitalo found that gene therapy could induce greater angiogenesis.
- No single positive result from a phase two trial as of yet.
- Blood vessel formation is a complex process that will be hard to treat with a singular gene therapy.

Question 35

What trial looked at gene therapy for heart failure?

Answer 35

CUPID clinical trial
- In heart failure there is not efficient calcium pumping due to failing myocytes.
- A gene, SERCA2a is downregulated in HF and the CUPID trial looked at whether delivery of this gene could help HF.
- The early results were favourable however the phase IIb trial failed.
- This was likely due to a delivery issue to the diseased heart. Delivered the gene via a catheter to the coronary artery but most of the virus was probably washed away. need to deliver to the heart muscle itself.
- There is now several studies ongoing with new AAV vectors that have better cardiac uptake.

Question 36

What is a clinical problem involving coronary artery bypass grafting?

Answer 36

As of 2019, there was 15,000 operations in the UK
In the short term it is an effective procedure to restore blood flow to the heart but in the long term 30-50% of saphenous vein grafts fail
The reason so many grafts fail is that the vein is hard and in alot of the cases remodelling to an artery fails and it leads to atherosclerosis (get injured in the high blood pressure)

Question 37

What gene is being investiagted to stop atheroscelrosis in coronary artery bypass graft?

Answer 37

TIMP- tissue inhibitor of metalloproteinase-3
TIMP lowers MMP activity which alters the cell matrix and is seen in atheroscelorsis
Overexpression of TIMP to re-balance MMP activity has been tested.

Question 38

Where is the TIMP gene therapy treatment at now?

Answer 38

• This is adenovirus-associated
• There is a clinical trial testing this in Glasgow starting this year.
• Giving TIMP-3 virus vs placebo.
• This treatment has been developed from 2013 and is predicted to go on until 2026.

Question 39

What are RNA based therapies?

Answer 39

RNA based therapies can be used to introduce an exogenous mRNA into the cel or inhibit the translation of endogenous mRNA
Can replace a defective protein (gene replacement therapy) or introduce transcripts not naturally present in the cell (antigens for vaccination, chimeric antigen receptors for cell therapy).

Question 40

What are the different RNA therapies?

Answer 40

Antisense oligonucleotides (ASO) bind to mRNA sequences and influence processing or stability of RNA molecules
miRNA can mimic endogenous miRNA, non coding RNA sequences that regulate gene expression. May also compete with endogenous miRNAs to bind to target mRNAs leading to decreased protein expression
siRNA- degrades mRNA after transcription, preventing translation

Question 41

What are examples of RNA therapies?

Answer 41

ASO- mipomersen
siRNA- incliseran

Question 42

What is mipomersen

Answer 42

Antisense oligonucleotide, binds to ApoB-100 and inhibits translation into protein
Decreases lipoproteins (LDL)
It was discontinued from clincical use in 2013 by the European medicines agency over severe liver toxicity

Question 43

What is used instead of mipomersen?

Answer 43

A phase II trial studying volanesorsen
Volanesorsen is an antisense oligonucleotide which inhibits the formation of the apolipoprotein apoC–III, thereby lowering serum triglycerides

Question 44

What is incliseran?

Answer 44

A siRNA therapy targeting PCSK9
Tested in ORION trials.
Inclisiran has met all primary and secondary endpoints across three Phase III trials, had a favorable safety profile, and matched the LDL-lowering efficiency of antibody-based PCSK9 inhibitors alirocumab when given in a twice annual injection

Question 45

How may RNA therapies be used in revasculisation?

Answer 45

• AZD8601 is a VEGF-A modified RNA under development as a novel modality for local production of human VEGF-A protein and is developed for the treatment of diabetic patients with ulcers.
• AZD8601 EPICCURE Phase II trial demonstrated safety and tolerability in patients with heart failure
• Created by moderna, is just isolated RNA not conjugated to anything. Epicardial injections directly into the heart to produced vascular endothelial growth factor A

Question 46

How do miRNAs help in cardiac function?

Answer 46

miRNAs are key for function- miRNA was removed by removing the dicer enzyme Chen et al 2008, these KO mice developed hypertrophy
When miRNA were knocked out of mice, VEGF levels were lower and angiogenesis rates were lower

Question 47

What miRNA may be involved in revascularisation of the heart?

Answer 47

miR-92 inhibition caused revasculisation of the heart
In mouse models of limb ischemia and myocardial infarction, systemic administration of anti-miR designed to inhibit miR-92a led to enhanced blood vessel growth and functional recovery of damaged tissue.
Phase I trial currently in recruitment phase

Question 48

What are the challenges with miRNA therapies?

Answer 48

One miRNA effects multiple targets, so regulating one could impact on a wide variety of molecular pathways
RNA are very unstable and need to be destroyed within hours if not modified
A lack of effective organ-specific (e.g. cardiac-specific) delivery methods is another problem for both miRNA mimics and inhibitors.
Also poor bioavilability, rapid degradation, poor cellular uptake, immunogenicity, off-target effects need to be considered

Question 49

What are micro bubbles?

Answer 49

Can put miRNA into a gas bubble then blow it through veins using ultrasound. Burst it using ultrasound
Was a nice idea but did not work efficiently

Question 50

What is an example of a RNA therapy that worked too well?

Answer 50

Giving miR-199a to pigs caused a decrease in infarct size and fibrotic area
The proliferation was uncontrolled however and the heart became hypertrophic

Question 51

When was the therapeutic effect of synthetic mRNA first demonstrated?

Answer 51

1992- mRNA encoding vasopressin injected in the hypothalamus of rats with central diabetes insipidus transiently reversed the disease

Question 52

What is the favourable delivery method for RNA based drugs?

Answer 52

Lipid nanoparticals have started to replace viral vectors as the chosen delivery method for RNA-based drugs due to their favorable safety profile, low immunogenicity, and ease of manufacture.

Question 53

What is the future of lipid nanoparticles?

Answer 53

Leukosomes
Leukosomes are assembled by integrating membrane proteins from leukocytes into the lipid nanoparticles.
The leukosomes target activated endothelium and exhibit superior accumulation in atherosclerotic vessels as well as tumor vessels in mouse models

In ApoE -/- mice , leukosomes localized to the diseased aorta to a substantially greater degree than standard Lipid nanoparticles

Question 54

What do we know about lncRNAs?

Answer 54

Long non-coding RNAs
- Predictions of the number of lncRNAs encoded by the human genome are in the tens of thousands, yet only hundreds have been functionally validated and characterized, possibly representing <1% of all lncRNAs.
- A number of large-scale projects center around annotating lncRNAs including FANTOM, NONCODE, GENCODE

Question 55

How effective is gene therapy at promoting angiogenesis after an MI?

Answer 55

• VEGF and FGF: have been used in gene therapy trials in patients with ischaemic heart disease
• However, no agent has been identified as a front runner for sustained clinical translation (treatment works in animals but not humans). Reasons for this include:
  
  • Delivery method may not be ideal
  • Small sample sizes/ underpowered
  • Growth factor isoforms
  • Short half life of the proteins
    However, very few adverse effects seen in the trials

Question 56

What are the pros and cons of mRNA in cardiac therapy?

Answer 56

PROS
- Low toxicity as does not act on the nucleus
- Direct injection as do not lipid-based carrier
- Spontaneous immediate reaction
CONS
- mRNA is not stable and degraded rapidly- need a stable cap
- Toxicity
- Triggers innate immune system

Question 57

How many gene trails are currently occuring right now?

Answer 57

896 trials, although only 4 treatments are approved by the US Food and Drug Adminstration

Question 58

How many of the population are infected by adenovirus?

Answer 58

As many as 60% of some populations are seropositive for recombinant AdV-2

Question 59

What has been done to improve mRNA delivery efficiency?

Answer 59

Vehicles such as lipid nanoparticles have been developed
In vitro experiments have indicated that encapsulating mRNA into a vehicle increased the amount of intact mRNA by more than 10,000 times compared to naked mRNA after incubating in serum
Delivering VEGF mRNA via lipofectamine improved angiogenesis and cardiac repair in vivo

Question 60

What method of gene delivery appears to be the most cardiotrophic?

Answer 60

In viral vehicles, the recombinant AAV serotype 9 (rAAV9) appears to be the most cardiotropic. Notably, over 20-times more vector copies were still found in the liver compared to the heart following systemic delivery of rAAV9- this is a problem as may cause toxicity e.g. mipomersen when given chronically

Question 61

What is the most efficient way to deliver cardiac gene therapy?

Answer 61

Direct intramyocardial injection
The percutaneous approach is less invasive and feasible in a stable patient so is used more often

Question 62

What was the first trial to use gene therapy to treat CVD and what were the outcomes?

Answer 62

The CUPID trial was the first clinical attempt to use AAV gene therapy to treat HF. CUPID was a Phase II, blinded, randomized, placebo-controlled, multicenter study enrolling 39 patients
The results from this showed reduction in GF hospitalisation
CUPID2 was then ran- phase IIb, treatment failed to improve clinical outcomes.

Question 63

What is a advantage of using AAV over adenovirus?

Answer 63

Adeno-associated viruses are not a highly immunogenic (or its immunogenicity can be easily controlled by pharma- cological treatment at the time of vector administration). However, the use of these vectors is only indicated when permanent expression of the transgene is desirable.