Small molecules treating CF - 2

Question 1

PULSE CHASE EXPERIMENT - outline main procedure and what did the results show?

Answer 1

HEK cells (overexpressing CFTR) exposed to radioactive compounds, so CFTR is produced with radioactive marker. 
Run along gel and see ratio of mature:immature bandss 
- More mature bands present when treated with VX-809 !! But this doesn't prove that this is a functional protein --> also need patch clamp for this!!!

Question 2

What did results from patch clamp (with VX-770 and VX-809 treatment?) - what does patch clamp measure?

Answer 2

Patch clamp is a measure of open probability of CFTR channel –> Po is almost the same as WT level when VX-809 added and HIGHER than WT with VX-890 + VX-770

Increased short circuit current through CFTR seen when cAMP added – > can tell this is due to CFTR because this decays away when a CFTR inhibitor added

Question 3

Clinical studies of the drugs – conditions of trial?

Answer 3

Randomised, double-blind placebo conditions
18-19 patients homozygous for dF508 mutation given varying doses of drug

19 patients took a placebo

Question 4

Clinical study - main results?

Answer 4

No improvement in lung function was seen despite strong in vitro evidence
8mM decrease in sweat [Cl-] with just VX-809
Both drugs in combination gave a 55mM reduction in sweat [Cl-]

Question 5

PHASE 3 CLINICAL TRIALS - how many patients, what was their genotype ? Results after 2 weeks?

Answer 5

1108 patients homozygous for dF508 given varying doses of both drugs

• highest dose of all 3 drugs saw maximum reduction in sweat [Cl-] of 13mM
• slight positive impact of lung function after 2 weeks on highest dose
• fewer developments of cough or infection and hospitalisations

Question 6

Overall impacts of VX-809 and VX-770?

Answer 6

VX-809 can traffick to membrane but this alone doesn;t improve symptoms
VX-770 POTENTIATES the effects of VX-809 and enhances its effects to alleviate symptoms

Question 7

What is the problem with VX-809 and VX-770?

Answer 7

VERY VERY EXPENISVE - effects might not outweigh the costs saved by treating patients with the drugs – can cost up to £189,000 per year per patient!!!

Question 8

What is the problem with gene therapy and this in treating CF?

Answer 8

Airway cells are very high turnover - constantly replacing -new cells don’t keep the WT CFTR induced by gene therapy as not part of patient genome!!

Question 9

Gene therapy by injection of liposome complexes - problem?

Answer 9

Issue of immune response –> may come with immune side effects!

Question 10

New method of gene therapy? What does it involve and what stage of trials ?

Answer 10

Viral techniques – at phase 2B clinical trials

Question 11

Conditions of viral gene therapy trials??

RESULTS??

Answer 11

140 patients –> 60 given placebo of 0.9% nebulised saline solution
70 patients given nebulised liposome with CFTR coding sequence (deeply breathed in)

Patients have stablised airway function (no worsening but only very small improvement seen)
Slight improvements in - lung function - lung volume - Cl- secretion and decreased gas trapping seen

Question 12

Nebulised liposome CFTR (inhaled) – what is the problem?

Answer 12

-Placebo control not the best but using scrambled WT CFTR would be unethical as this could cause the patients’ symptoms to worsen
-Genetic differences could cause differing responses to gene therapy
MORE WORK NEEDED