lecture 8 Flashcards
what are the 3 approaches for treating the basic anion permeability defect in CF?
- genetic therapy
- CFTR modulator therapy
- alternate channel therapy
how muchCFTR function needs to be corrected?
-autosomal recessive- 50% function should be helpful
5% = lung disease
1% = pancreatic disease
how is a new therapy assessed in vivo?
endpoints to measure:
1. lung function (FEV1.0)
2. sweat chloride (sweat test)
parameters: hospitalisations, BMI & weight gain, quality of life
what 2 parts are sweat glands composed of?
- secretory coil
- reabsorptive duct epithelium
what is the secretory coil?
-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
what is the reabsorptive duct epithelium?
-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
how does ion and fluid transport in normal sweat glands occur?
-ENaC and CFTR present
-electrochemical gradient for Cl- moves down gradient driven by Na+ reabsorption
-leads to salt absorption but no water following
how does ion and fluid transport in CF sweat glands occur?
-don’t have ability to transport chloride
-loss of CFTR = prevents NaCl absorption = isotonic solution (120mM)
what is genetic therapy?
therapeutic approaches that:
-deliver copies of healthy gene using gene addition (DNA or mRNA)
-fix the chromosomal DNA using gene editing (CRISPR-CAS)
how was gene therapy first tested?
-put gene/code for CFTR into virus and get virus to infect the cells and reduce CFTR
what are the main problems with gene therapy?
physical and/or immune barriers
-body rejects the cells as recognises as being infected
what is the new approach to gene therapy?
-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
how can you improve the efficacy of gene therapy?
gene transfer agents:
-lentivirus - long term, stable correction as gene integrated into DNA
-modify virus to make them less immunogenic
-nanoparticles
what are the problems?
-need to target correct cells in lungs
-mucus is important barrier to gene therapy still
-potential for disruption of other genes
what are some other genetic and cell based approaches?
-mRNA vaccines
what is antisense oligonucleotide (ASO) mediated therapy?
-used to overcome splicing and frameshift mutations
how is CFTR modulator therapy used?
-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
what aspect of mutant CFTR activity can be altered?
-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
how is total Cl- current calculated?
N x Po x i
N = number of channels in cell
Po = probability of how long open
I = chloride current
what can cause increases in single channel activity?
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
what is the CFTR drug discovery process?
-high throughput screening -> screen chemicals that improve
-medicinal chemistry
-biology activity in cell cultures
-animal studies
-human studies
