L6 - Alternative Treatments in CF

Question 1

Why did gene therapy fade after its initial investigation in the early 1990s?

Answer 1

Faced challenges

• Efficiency of transfection
• How long transfection lasted – upper airway cells replaced all the time

Question 2

What did early gene therapy investigations use?

Answer 2

Adenovirus

Question 3

What has recent gene therapy investigations used?

Answer 3

Liposome complexes

Question 4

What did the first trial of non-viral CFTR gene therapy involve?

Answer 4

Used liposome complexes 
140 patients 
- 62 placebo - 0.9% saline nebulised 
- 78 test - 78 pGM169/GL67A nebulised 
  -- WT material encoding CFTR 
28 day intervals and 9 doses

Question 5

In the first trial of non-viral CFTR gene therapy what results did they see for airway function?

Answer 5

Placebo – steady decline in lung function
Gene therapy – able to stabilise lung function
- Didn’t improve function but stopped decline
- Still more improvements in group that had therapy

Question 6

In the first trial of non-viral CFTR gene therapy why was there such a range in how people responded?

Answer 6

Could be due to

• How much WT CFTR was actually being produced
• Difference in genetic background of patients make them more likely to respond to the gene therapy

Question 7

How do gas trapping and the results of gene therapy link?

Answer 7

Gas trapping secondary to obstruction of the small airways
(lower respiratory tract) is likely to occur early in infants with CF
Anything that decreases likelihood of gas trapping is a strong indicator that we have an improvement with gene therapy

Question 8

In the first trial of non-viral CFTR gene therapy what results did they see for change in Cl response?

Answer 8

Measured nasal transepithelial potential of patients then injected Cl free Isoproterenol solution
Gene therapy –> more Cl secretion in nasal epithelium –> improvement in CFTR function

Question 9

Overall what 3 things did the first trial of non-viral CFTR gene therapy show?

Answer 9

Stabilisation of lung function
Heterogeneous population - some responded well, others less well
- Effectiveness of gene therapy treatment
- Differences in the patients themselves
0.9% saline control not the best

Question 10

What would be a better gene therapy control than 0.9% saline

Answer 10

Ethical reasons prevent using scrambled message for CFTR in vivo
In vitro have liposome with scrambled CFTR = doesn’t make any CFTR

Question 11

What happens to ENaC in CFTR patients?

Answer 11

ENaC function enhanced in upper airway

• CFTR activity inhibits ENaC
• Mutations in CFTR lead to a reduction in this inhibition
• Enhances Na absorption from ASL –> ASL even lower

Question 12

Why would you target ENaC for CFTR treatment when it is CFTR that is mutated?

Answer 12

If you block ENaC –> block excessive Na absorption –> increase ASL height

Because mutation in CFTR means less inhibition of ENaC

Question 13

What happened when they tested amiloride on CF patients?

Answer 13

Absence of amiloride 
- Lung function down 
Amiloride 
- No improvement in lung function 
- Using Amiloride to target ENaC was put to the side

Question 14

What is SPLUNC1?

Answer 14

Secreted protein short palate lung and nasal epithelial clone 1

Question 15

What does trypsin do to ENaC?

Answer 15

Under physiological conditions ENaC undergoes proteolytic cleavage by trypsin

Question 16

What are the two ways to up regulate ENaC?

Answer 16

Increase number of ENaC channels in membrane
Trypsin cleaves ENaC to increase its open probability
- Increases ENaC currents –> enhances Na absorption

Question 17

How do SPLUNC1 and ENaC interact?

Answer 17

SPLUNC1 binds to ENaC and prevents cleavage

Suppressed Na uptake across apical membrane

Question 18

Results - ENaC and trypsin

Answer 18

Large increase in ENaC current

Question 19

Results - ENaC and SPLUNC1

Answer 19

Prevents endogenous proteases from cleaving protein
Decrease in current
Less activation of ENaC by proteolytic cleavage

Question 20

Results - ENaC, trypsin and SPLUNC1

Answer 20

Still a decrease in current

Less decrease than in absence of trypsin

Question 21

After the success of SPLUNC1 in decreasing ENaC currents how did they exploit this to develop a new treatment for CF?

Answer 21

Derived a peptide from SPLUNC1 – S18

• Mimics SPLUNC1 binding
• ENaC inhibitory domain of SPLUNC1

Question 22

How did they test if S18 and SPLUNC1 had the same properties and effect on ENaC?

Answer 22

Relative fluorescence experiment 
- If SPUNC1 doesn’t bind fluorescence does not occur  
Tested how much SPLUNC1 binds in 
- Absence of S18
- Presence of S18 
- Investigated pH dependence of it

Question 23

What where the results of the fluorescence experiment with SPLUC1 and S18?

Answer 23

pH 6
- Fluorescence low – binding of SPLUNC1 to ENaC low
- S18 - no difference
- Acidification inhibits binding of SPLUNC1
pH 7.5
- Fluorescence high - binding of SPLUNC1 to ENaC high
- S18 – fluorescence lowered
- S18 binds to ENaC at the same site as SPLUNC1 – competitive binding
- Has the same role as SPUNC1 –> inhibits ENaC cleavage

Question 24

Overall how do S18 and SPLUNC1 interact?

Answer 24

S18 binds to HBE cells and blocks pH-dependent binding of SPLUNC1

Question 25

What were the results of an experiment looking at how S18 affected the ASL height?

Answer 25

Height of ASL increases 
- Because ENaC is being inhibited 
Transepithelial potential 
- ENaC contributes -4mV 
- With S18 – ENaC contributed 1mV

Question 26

What were the results of an experiment looking at how S18 affected amiloride ISC?

Answer 26

With S18 reduced –> inhibits proteolytic cleavage –> open probability low –> low Na reabsorption –> reduced currents –> transepithelial potential reduced

Question 27

What were the results of an experiment looking at how S18 affected the ASL height in alveolar cells?

Answer 27

Large increase in ASL height

- Bad –> height could be too big

Question 28

How does Na and Cl handling differ in the upper airways and alveolar?

Answer 28

Upper airways

• Cl out
• Na in

Alveolar

• CL in
• Na in

CHECK

Question 29

What role does ENaC play in the collecting ducts?

Answer 29

Na absorption by principal cells in collecting ducts
Determines total Na content of body –> determines extracellular fluid volume –> role in determining BP
Affects plasma K

Question 30

What are the systemic effects of S18?

Answer 30

No impact on urine flow rate
No impact on Na or K excretion
Fewer systemic effects –> not inhibiting ENaC in the kidney
Much better than amiloride

Question 31

What are the systemic effects of amiloride?

Answer 31

Increase in urine flow rate and Na excretion –> blocks ENaC in kidney –> block water and Na reabsorption
Decrease in excretion of K –> Na absorption drives K secretion
- Increased risk of cardiac arrhythmias –> impacts nerve excitability

Question 32

How did S18 work in a CF mouse model (ENaC beta subunit over expression)?

Answer 32

Treatment with S18
- Increase in height in WT and CF model
S18 – alleviated the impact of overexpressing ENaC
However not a perfect CF mouse model so also tested on sheep

Question 33

How did S18 work in a CF sheep model?

Answer 33

If you block CFTR
- Tracheal mucous velocity decreases
Add amiloride
- Transient recovery of mucous clearance – not maintained
Add S18
- Recovery of mucous clearance almost back to baseline – maintained

Question 34

What method did they use to look at S18 in a sheep CF model?

Answer 34

Roentgenographic method to look at tracheal mucous velocity

- Pharmacological inhibition to block CFTR in upper airway

Question 35

Consider whether S18 would be an ideal molecule to use in CF patients?

Answer 35

S18 inhibited ENaC in HBE and alveolar cells
HBE cells – this would be a positive thing – could reverse the low PCL height seen in CF patients
Alveolar cell – normal function of CFTR – CL absorption, activation ENaC – fluid uptake from apical surface
CF patients often have alveolar oedema
If S18 inhibits ENaC this might make the oedema worse
- Maybe in the alveoli ENaC is already inhibited due to CFTR mutation
- The patient might actually need an ENaC activator in alveoli

Question 36

How can you get a drug that only targets the upper airway?

Answer 36

The smaller the particle size the further down the airway it goes – get a drug that just targets upper airway

Question 37

Overall what is the effect of S18?

Answer 37

S18 is a SPLUNC1 derived peptide that has antagonistic effects on ENaC
S18 has less systemic effects compared to amiloride
S18 has positive effects on lung function in CF models