lecture 8

Question 1

what are the 3 approaches for treating the basic anion permeability defect in CF?

Answer 1

1. genetic therapy
2. CFTR modulator therapy
3. alternate channel therapy

Question 2

how muchCFTR function needs to be corrected?

Answer 2

-autosomal recessive- 50% function should be helpful
5% = lung disease
1% = pancreatic disease

Question 3

how is a new therapy assessed in vivo?

Answer 3

endpoints to measure:
1. lung function (FEV1.0)
2. sweat chloride (sweat test)
parameters: hospitalisations, BMI & weight gain, quality of life

Question 4

what 2 parts are sweat glands composed of?

Answer 4

1. secretory coil
2. reabsorptive duct epithelium

Question 5

what is the secretory coil?

Answer 5

-embedded in skin
-primary secretion
-NaCl rich secretion from acinar (CC) cells
-Cl- secretion from TMEM16A (only some CFTR dependency)
-activated by beta-adrenergic stimulation

Question 6

what is the reabsorptive duct epithelium?

Answer 6

-transcellular NaCl absorption = hypotonic fluid
-epithelium has low water permeability so water doesn’t move
-cells express both ENaC and CFTR and involved in NaCl absorption

Question 7

how does ion and fluid transport in normal sweat glands occur?

Answer 7

-ENaC and CFTR present
-electrochemical gradient for Cl- moves down gradient driven by Na+ reabsorption
-leads to salt absorption but no water following

Question 8

how does ion and fluid transport in CF sweat glands occur?

Answer 8

-don’t have ability to transport chloride
-loss of CFTR = prevents NaCl absorption = isotonic solution (120mM)

Question 9

what is genetic therapy?

Answer 9

therapeutic approaches that:
-deliver copies of healthy gene using gene addition (DNA or mRNA)
-fix the chromosomal DNA using gene editing (CRISPR-CAS)

Question 10

how was gene therapy first tested?

Answer 10

-put gene/code for CFTR into virus and get virus to infect the cells and reduce CFTR

Question 11

what are the main problems with gene therapy?

Answer 11

physical and/or immune barriers
-body rejects the cells as recognises as being infected

Question 12

what is the new approach to gene therapy?

Answer 12

-use liposome (bag of lipid) make it charged and mix with the cDNA for CFTR
-come together electrostaticly
-some gets into cell acne eventually makes CFTR
-inefficient - isn’t targeted

Question 13

how can you improve the efficacy of gene therapy?

Answer 13

gene transfer agents:
-lentivirus - long term, stable correction as gene integrated into DNA
-modify virus to make them less immunogenic
-nanoparticles

Question 14

what are the problems?

Answer 14

-need to target correct cells in lungs
-mucus is important barrier to gene therapy still
-potential for disruption of other genes

Question 15

what are some other genetic and cell based approaches?

Answer 15

-mRNA vaccines

Question 16

what is antisense oligonucleotide (ASO) mediated therapy?

Answer 16

-used to overcome splicing and frameshift mutations

Question 17

how is CFTR modulator therapy used?

Answer 17

-uses chemicals (drugs) to correct mutant CFTR
-can be used alone or in combination with gene therapy or alternate channel therapy
-required effect depends on mutation
-designer (personalised) therapy may be required

Question 18

what aspect of mutant CFTR activity can be altered?

Answer 18

-chloride movement produces electrical current
-channels open due to chloride movement and charge
-CFTR is part of ABC transporter family - has NBD - needs to be phosphorylated by protein kinase A and ATP to be bound

Question 19

how is total Cl- current calculated?

Answer 19

N x Po x i
N = number of channels in cell
Po = probability of how long open
I = chloride current

Question 20

what can cause increases in single channel activity?

Answer 20

increases number of CFTR channels at cell surface eg. class ll
enhancing CFTR channel gating (Po) eg. class III
increase ion flux eg. class IV

Question 21

what is the CFTR drug discovery process?

Answer 21

-high throughput screening -> screen chemicals that improve
-medicinal chemistry
-biology activity in cell cultures
-animal studies
-human studies