Small molecules and CF

Question 1

Give 3 examples of current treatments, their effects and why they are not the future of CF treatment.

Answer 1

Nebulised antibiotics - tobramycin, fights infection, careful when using antibiotics as they don’t want CF patient to develop antibiotic resistence.

Mycolytics - break down mucous

Nebulised hypertonic saline - concentrated saline attracts water and hydrates the ASL

None of these treat the cause - only the symptoms

Question 2

What two small molecule drugs have been developed and what are their targets

Answer 2

VTX-770 (Ivakaftor) is classed as a potenitiator and is used in G551D patients

VTX809 (Lumakaftor or Orkambi) Classes as a corrector and targets the deltaF508 mutant.

Question 3

Describe the G551D mutant

Answer 3

Glycine to Aspartate

type III mutant (regulation mutant)

Gating defect - doesn’t open in response to normal regulation

Severe symptoms

Question 4

How is the G551D mutation screened for

Answer 4

Use a cell-based fluorescence Vm assay
cells expressing the mutant protein are loaded with a fluorescent dye that emits a certain amount of light at a certain Vm. Opening a Cl channel should drive the Vm down so it was hypothesised that changes in fluorescence due to the addition of a compound reflects a change in Vm due to Cl opening. Tested 225,000 compounds and ones that induced a Vm change were further investigated to see if it was due to an increase in G551D activation. Got VTX-770

Question 5

Describe the delta F508 muation

Answer 5

Phenylalanine deletion

type II mutation (trafficking)

Up to 90% allele frequency

Severe symptoms

It’s sent for degredation. If the mutant reaches the membrane it can function

Question 6

How is the delta F508 mutant screened for

Answer 6

Cell based immunoblot assay that measures the ratio of mature (glycosylated) Vs immature (non-glycosylated) CFTR. Again, investigated if compounds were capable of trafficking it to the membrane (tested 164,000) VTX-809, Lumakaftor, Orkambi gave the strongest response.

Question 7

What was the first experiment done to show the efficacy of VTX-770 (Ivakaftor)

Answer 7

In Fisher rat thymoid cells
Measured the SCC of the cells when unstimulated, and when stimulated through the addition of forskolin. In G551D mutants there is very little response to forskolin compared to WT. When forskoin is added alongside VTX-770 there is a larger increase to around 20uA. This is not the same as WT (45uA) but as we only need around 15 -20% of CFTR function for normal function in airway cells, this result was promising

Question 8

What was the second experiment done to investigate the efficacy of VTX-770 (Ivakaftor)

Answer 8

Again in Fisher rats thymoid cells, looked at single channel currents of CFTR in the presence of PKA and ATP in G551D mutants which did not yield many downward deflections compared to WT. Addition of VTX-770 increased the number of deflections (increased Po)

Question 9

What was the third experiment done on primary human bronchial epithelium from a G551D and delta F508 heterozygous CF patient.

Answer 9

Measured the SCC from the patients epithelia and compared to control
Firstly they blocked ENaC, then added forskolin - which only mildly increased the SCC. Then added increasing concentrations of VTX-770 - saw increasing SCC

Then added CFTRinh-172 (CFTR inhibitor) which reduced the current showing the increase was due to increased CFTR function.

In WT - FSK addition increases SCC by 56uA/cm - in CF patient it was only a 2.9uA increase. Addition of VTX-770 made it 27uA which is 48% of WT.

Question 10

What was the fourth and final experiment done with VTX-770

Answer 10

Look at beat frequency of cilia and the ASL height over time.

The ASL height was set too high - then added VIP (a CFTR activator) and compared G551D/delta F508 hetero mutant and WT cells. In the mutant the beat frequency and ASL height goes very low compared to WT. Addition of VTX-770 with VIP brings the volume of the ASL toward WT and increases the beat frequency.

Question 11

What observations were made in the VTX-770 clinical trial

Answer 11

Increase in FEV1 in just two weeks (48 week trial)

Proptortion of patients that were event free after 48 weeks increased compared to placebo (67% compared to 41%)

Sweat Cl concentration was measured and after two weeks the treated patients were down to around 50mM. Not WT (15-20mM) but lower than the 60mM threshold used to diagnose CF.

Electrode stuck up the nose to measure the nasal Vte in response to CFTR stim by isoprotenerol a B receptor antagonist that activates cAMP via PKA. Reduction in Vte shows Cl secretion. Placebo showed little shift compared to VTX-770 (only at higher concentrations - 75 and 150mg)

Question 12

What pulse chase experiment was performed to investigate VTX-809 in HEK cells

Answer 12

HEK cells overexpressed either WT or F508 CFTR

The cells were incubated with radioactive methionine/cysteine, both of which are taken up by the cell to make proteins. Next the radioactive compounds are removed from the solution and the cells are left for different amounts of time.

For CFTR, it matures to its glycosylated form, as we know the MW for both these forms of CFTR, calculate the amount of radiation at each form of CFTR over time.

Question 13

How long does it take for the mature form of CFTR to show in WT cells

Answer 13

30 mins

Question 14

How long does it take for all immature CFTR to be glycosylated in WT cells

Answer 14

120 mins

Question 15

What is seen in the pulse chase for F508 mutants

Answer 15

At time 0 there is lots of immature CFTR, which disappears over time. Unlike WT however, it doesn’t form much of the mature band as the immature CFTR is sent for degredation.

Question 16

What is the result of incubating the F508 mutants with VTX-809 in the pulse chase experiment

Answer 16

See the same amount of immature and over time there is more mature CFTR appearing.

i.e. exposing cells to VTX-809 drives some of the F508 mutant channels into the mature form, once at the membrane F508 mutants have a reduced Po but do still function.

Question 17

What was the result of a VTX-809 clinical trial

Answer 17

Trial was stopped early due to the lacking effect of thre drug at any concentration. No way near the same effect as VTX-770 in G551D mutants. Tried a combination therapy of VTX-770/809

Question 18

What was the full clinical study findings after initial findings supporting combination therapy with VTX-770/809

Answer 18

% change in FEV1 was slightly increased in the two test groups of around 4%. Both groups showed similar results regardless of VTX-809 concentration. Most patients showed little improvement in lung function, some not at all and some by >10%. Even with such a mixed response, concluded from a forest plot that the treatment was having a positive impact on all populations, even if its very limited. Placebo patients also suffered from a pulmonary event sooner and more often then treateed groups.

Question 19

What conclusion can be made from the combination therapy of VTX-770/809

Answer 19

VTX-809 trafficks F508 mutant CFTR to the membrane and its functional. However, in F508 patients, this is not sufficient to induce an improvement in symptoms.
If administered with VTX-770 then this improves as it enhances the function of the mutant CFTR.

Downside is cost

Question 20

What gene therapy approaches have been made for CF treatment

Answer 20

Liposome complexes containing the DNA for CFTR to introduce it into the airway

Question 21

What was the problem with the control used for gene therapy

Answer 21

0.9% nebulised saline - best option would be to use scrabled CFTR gene sequence but this is not an ethical thing to do.

Question 22

What was the result of the gene therapeutic treatment for CF

Answer 22

FEV1 change was maintained in treated patients whereas placebo lost function. There were also less patients that lost lung function in the treated group

Forest plot concluded that overall impact was positive.

Tested the bronchial DNA score to see if CFTR DNA was entering the cells - It was

This suggested that the therapy stabilised lung function, worked heterogenously (which could be due to liposome formulation) and requires more investigation.