Novel therapies for genetic disorders Flashcards
What is a nonsense mutation?
Premature stop
What cellular response is induced by truncated proteins?
Degradation via ERAD
What cellular responses are induced by misfolded proteins?
- UPR: an effort to force correct folding and reduce translation
- ubiquination and proteolytic degradation
What cellular responses are induced by nonsense mutations?
- nonsense mediated decay of mRNA
2. if translation does occur: ERAD
What are the requirements of a protein used in gene product replacement?
- Stable in blood and intracellularly
- Targeted to correct cell type and organelle
How can proteins be modified to improve their properties for gene product replacement?
- Modification of glycosylation pattern
- Fusion proteins
What enzyme is used to treat Gaucher’s and what modifications are made to increase its uptake?
- Glucocerebrosidase = deficient enzyme
- Needs to be taken in by Kupffer cells (liver macrophages)
- The native carb side chain of glucocerebrosidase is cleaved to expose terminal mannose residues, which are recognized by the macrophage lectin receptor
What is the genetic cause of achondroplasia?
- Normal: when bound by FGF, FGFR3 receptor inhibits bone growth
- Achondroplasia: constitutive activation of the receptor and thus constitutive inhibition of growth
What pathway inhibits the activity of FGF/FGFR3?
- NPR-B receptor binds CNP => signaling cascade => inhibition of inhibition of bone growth
Why was native CNP not a viable treatment for achondroplasia and how was it modified?
- Very short half life
- Added 2 N-ter residues and greatly increased 1/2 life
What is the therapeutic benefit of creating a fusion protein?
- May improve uptake
- May increase stability
What is hypophosphatasia?
- Alkaline phosphatase deficiency (PPi => Pi + Pi)
- Causes poor mineralization of bones
How is hypophosphatasia treated?
Gene product replacement = fusion of alkaline phosphatase + Ig molecule + deca-asp tag to promote uptake
Pharmacological chaperones
- Chemicals which bind to active site to help enzymes fold normally, even if primary sequence is changed
- Normally folded protein will be transported to lysozyme
- In lysozyme, chaperone will be released and enzyme can act on native substrate
How can enzyme inhibition be used to treat enzymatic deficiency?
- Inhibit an earlier step to reduce accumulation of a product
What is “read-through”?
- When a chemical prevents termination of translation when a stop codon is encountered
- A different AA is substituted for the stop but translation is continued, potentially preserving some protein function
How can alternative splicing be used to treat an enzyme deficiency?
Spinal muscular atrophy:
- a pseudogene is very similar to the mutated gene in spinal muscular atrophy but lacks functionality due to an exon spliced out
- hnRNP modifies splicing to make pseudogene more active and able to replace the defective gene
How can exon skipping be used to treat disease caused by mutant protein?
Duchenne’s muscular dystrophy:
- Antisense oligo masks splice site on pre-mRNA of gene with the problematic mutation
- Exon will be excluded, resulting in a less problematic protein
What nucleic acid products are used for exon skipping?
Antisense oligos:
- 20-30 nt
- modified bases prevent nuclease digestion
Morpholinos:
- methylenemorpholine rings with phophorodiamidate linkages
What are other potential novel therapies for genetic disorders?
- inhibitory mRNA to silence mutant alleles (Huntington’s)
- drug/protein/mRNA delivery in exosomes
What is the blood-brain barrier?
- Highly selective permeability barrier that separates blood from brain fluid
- Formed by capillary endothelial cells joined by tight junctions
- Permits passage of: water, some gases, passive diffusion of lipid soluble molecules, selective transport of glucose, AAs, hormones
What is a method that could be used to transport drugs across the BBB?
- Link drugs to molecules that are normally transported across the barrier with specific receptors
- ex: LRP1 transported in via an endosome
