L6 - Alternative Treatments in CF Flashcards
Why did gene therapy fade after its initial investigation in the early 1990s?
Faced challenges
- Efficiency of transfection
- How long transfection lasted – upper airway cells replaced all the time
What did early gene therapy investigations use?
Adenovirus
What has recent gene therapy investigations used?
Liposome complexes
What did the first trial of non-viral CFTR gene therapy involve?
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
In the first trial of non-viral CFTR gene therapy what results did they see for airway function?
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
In the first trial of non-viral CFTR gene therapy why was there such a range in how people responded?
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
How do gas trapping and the results of gene therapy link?
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
In the first trial of non-viral CFTR gene therapy what results did they see for change in Cl response?
Measured nasal transepithelial potential of patients then injected Cl free Isoproterenol solution
Gene therapy –> more Cl secretion in nasal epithelium –> improvement in CFTR function
Overall what 3 things did the first trial of non-viral CFTR gene therapy show?
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
What would be a better gene therapy control than 0.9% saline
Ethical reasons prevent using scrambled message for CFTR in vivo
In vitro have liposome with scrambled CFTR = doesn’t make any CFTR
What happens to ENaC in CFTR patients?
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
Why would you target ENaC for CFTR treatment when it is CFTR that is mutated?
If you block ENaC –> block excessive Na absorption –> increase ASL height
Because mutation in CFTR means less inhibition of ENaC
What happened when they tested amiloride on CF patients?
Absence of amiloride - Lung function down Amiloride - No improvement in lung function - Using Amiloride to target ENaC was put to the side
What is SPLUNC1?
Secreted protein short palate lung and nasal epithelial clone 1
What does trypsin do to ENaC?
Under physiological conditions ENaC undergoes proteolytic cleavage by trypsin
What are the two ways to up regulate ENaC?
Increase number of ENaC channels in membrane
Trypsin cleaves ENaC to increase its open probability
- Increases ENaC currents –> enhances Na absorption
How do SPLUNC1 and ENaC interact?
SPLUNC1 binds to ENaC and prevents cleavage
Suppressed Na uptake across apical membrane
Results - ENaC and trypsin
Large increase in ENaC current
Results - ENaC and SPLUNC1
Prevents endogenous proteases from cleaving protein
Decrease in current
Less activation of ENaC by proteolytic cleavage
Results - ENaC, trypsin and SPLUNC1
Still a decrease in current
Less decrease than in absence of trypsin
After the success of SPLUNC1 in decreasing ENaC currents how did they exploit this to develop a new treatment for CF?
Derived a peptide from SPLUNC1 – S18
- Mimics SPLUNC1 binding
- ENaC inhibitory domain of SPLUNC1
How did they test if S18 and SPLUNC1 had the same properties and effect on ENaC?
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
What where the results of the fluorescence experiment with SPLUC1 and S18?
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
Overall how do S18 and SPLUNC1 interact?
S18 binds to HBE cells and blocks pH-dependent binding of SPLUNC1
What were the results of an experiment looking at how S18 affected the ASL height?
Height of ASL increases - Because ENaC is being inhibited Transepithelial potential - ENaC contributes -4mV - With S18 – ENaC contributed 1mV
What were the results of an experiment looking at how S18 affected amiloride ISC?
With S18 reduced –> inhibits proteolytic cleavage –> open probability low –> low Na reabsorption –> reduced currents –> transepithelial potential reduced
What were the results of an experiment looking at how S18 affected the ASL height in alveolar cells?
Large increase in ASL height
- Bad –> height could be too big
How does Na and Cl handling differ in the upper airways and alveolar?
Upper airways
- Cl out
- Na in
Alveolar
- CL in
- Na in
CHECK
What role does ENaC play in the collecting ducts?
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
What are the systemic effects of S18?
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
What are the systemic effects of amiloride?
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
How did S18 work in a CF mouse model (ENaC beta subunit over expression)?
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
How did S18 work in a CF sheep model?
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
What method did they use to look at S18 in a sheep CF model?
Roentgenographic method to look at tracheal mucous velocity
- Pharmacological inhibition to block CFTR in upper airway
Consider whether S18 would be an ideal molecule to use in CF patients?
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
How can you get a drug that only targets the upper airway?
The smaller the particle size the further down the airway it goes – get a drug that just targets upper airway
Overall what is the effect of S18?
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
