Gene Therapy

Question 1

What is ATMP and what does it entail ??

Answer 1

Based on cell and gene therapy

Specifically tailored- specific patient/specific gene

Question 2

Gene therapy-

Answer 2

Aim is to modify genetic material of living cells for therapeutic purposes

Question 3

How does gene therapy modify genetic material ??

Answer 3

• Viral vectors
  Lenitiviral vectors /Adenovirus
• Non- viral vectors- plasmids/nano particles
  -Genetically modify virus to make it safe
  Remove harmful components – use it as a carrier
  Make them replication incompetent when in the cell

Question 4

Lentiviral vectors

Answer 4

Type of retrovirus

1. Primate based lentivirus
2. non – primate based lentivirus

Question 5

Neurons

Answer 5

Non- dividing

Question 6

Glia

Answer 6

Dividing

• Integrate genes into chromosome of target cells
• Long term expression – issue with random integration in the genome
• No toxicity
• Large cloning capacity is large

Question 7

AAV- how is it used ? What are the issues?

Answer 7

most promising for brain and spinal cord application

• Efficient
• -effective
• Long term expression
• No toxicity
• But low cloning capacity – issue’s with size of gene – cant package into virus

Question 8

What is important about AAV ?

Answer 8

Can cross the Blood brain barrier

Question 9

Making the gene in the lab

Answer 9

Vector -Transfer genome
Gag-pol for packaging
Envelope for virus 
Transfect cells to produce the virus- 3 plasmid co-transfection 
Collect concentrated viral titre

Question 10

Gene therapy- Hype or hope ??

Answer 10

• Successful therapy for SCID -X1 disease
• 2 children cured and off treatment
• Issue- host randomly into host genome – caused leukaemia > one death
  Problem with the vector

Question 11

Issue with targeting brain and spinal cord –

Answer 11

Only 8% of drugs tested enter the market
-Often low efficiency
Inefficiency of therapy deliver to the cns because of factors like the BBB

Question 12

Gene transfer to Nervous system –

Answer 12

Virus produce – reporter gene
In vitro transduction efficiency
Test it in Hippocampal, DRG and organotypic slice with GFP
Test efficiency in brain – inject tiny amount into mouse > shows expression – lac z in striatum efficiency of virus to target striatum and SN – implicated in Parkinsons

Question 13

What does AAV Stand for ??

Answer 13

Adeno-associated vectors

Question 14

Gene Therapy

Answer 14

Gene therapy uses virus carriers which have been modified to remove all harmful properties to transfer therapeutic genes into patients.

Question 15

CNS Vector Targeting

Answer 15

Use specific promoter

Question 16

Therapeutic Strategies for PD

Answer 16

Target dopaminergic neurons
1st time patient presents with Parkinson’s – already lost 80% of neurons gone
Neuroprotection – protective GDNF
Restorative > Aim to replace dopamine – L-dopa – severe side effects

Question 17

Neuroprotection

Answer 17

• Lentiviral factors – GDNF
• How can we replace dopamine ?
• L dOPa problematic as disease progresses
• Aim to make cells that don’t express dopamine – add 3 main enz in dopamine synthesis in order to increase dopamine > made it to trial

Question 18

Spinal muscular atrophy

Answer 18

Childhood form of MND
Depletion of SMN protein
50% of children die before age of 2

Question 19

Gene therapy for SMA

Answer 19

Increase the levels of SMN in cells using gene therapy- gene transferred using viral vectors
SMA – cerebral MN die and patients can no longer breath

Question 20

Systemic delivery of SCAAV9 expressing SMN

Answer 20

prolongs survival in SMA model
- Tried in SMA patients
- Avexis Trial – video on website
- Children survived over 2 years – walk run/ breathe
Haemophilia B – trial inject AAV8 – persistent FIX expression