7 and 8. Molecular Therapies Flashcards
What type of defects can occur aberrant gene expression?
1) Defect in controlling region
2) Error in coding region
3) Known unknowns
- Trans-acting factors
- Modifying genes
- epigenetics
What therapy/intervention targets what stage of processing the code?
Transcription (DNA) = Gene therapy + Cell transplant
Transcription (pre-mRNA) = Conventional pharmaceuticals
Splicing (mature RNA) = Molecular therapy
Protein (translation) = Readthrough antibiotics + conventional pharmaceuticals
What are the two modes of treatment for inherited disorders?
Direct (molecular) treatments to address cause
- Mutation dependent
Indirect treatments (address impact of disease)
- reduce inflammation etc
Do all diseases have a genetic component?
Basically yes
Is cystic fibrosis a monogenic inherited disease?
Yes
Does environment play a bigger role in monogenic or multiple gene diseases?
Polygenic diseases
What are some ways in which gene therapy works?
Replace defective genes
Deliver genes that cause destruction of cancer cells
Provide genes that impede tissue growth
What type of gene transfer therapy is available?
Somatic cell gene transfer
When did gene therapy begin?
1980s
How many people die in the US due to adverse drug reactions (ADR)?
100 000 deaths annually
- Stroke
- Heart disease
- Cancer
- ADR
How many people effected by rare diseases in Australia?
400 000
What is the difference between gene therapy and molecular therapies?
Gene therapy
- Gene replacement
- Gene repair
Molecular therapies
- Modify gene output
- Stop codon read-through
- Use another gene
What are the first steps taken in any therapy?
1) Correct diagnosis
2) Identifying cause and mechanism
3) Rational design to replace/compensate/by - pass the problem
4) Can only be done if mutation is properly understood
What happens in a point mutation?
Single base is substituted, can result in any number of things happening to protein (truncated)
What occurs in a frameshift mutation?
A insertion of deletion of a number of nucleotides NOT divisible by three (e.g. 1) resulting in a shift in reading frame
What is an in-frame deletion?
Deletion of multiples of 3 codons
What are the potential molecular / genetic therapeutic approaches?
1) Replace missing or mutated genes
2) Repair faulty genes
3) Modify gene expression
How is gene expression modified?
1) Molecules that alter transcription
2) Alteration of pre mRNA splicing
3) Down regulate/destroy specific mRNAs
4) Enhance mRNA stability
5) Prevent transcription
Alter splicing
Down regulate
Enhance
Prevent
What is an antisense therapy?
Antisense transcripts occur naturally - contribute to cellular regulation
Synthetic antisense oligonucleotides (AOs) are used to Alter mRNA splicing
Degrade transcripts
Block translation
How do synthetic anti-sense oligonucleotides in antisense therapies work?
Alter mRNA splicing
Degrade transcripts
Block translation
How is mRNA splicing altered using anti-sense oligonucleotides?
Exons are skipped via splicing
Only really useful is shortened product is still functional
This is being used in Duchenne muscular dystrophy
How are antisense sequences used to block translation?
Antisense translational blockade
Antisense sequences, target translation start sites
Examples are Neugene, Phosphorodiamidate morpholino oligomer (PMO)
Used to study zebrafish
What diseases could be treated with translational blockade?
Bird flu
SARS
Ebola
Hep C
How can transcript degradation be induced by AOs?
RNaseH - DNA antisense oligo annealed to RNA - induces RNaseH degradation of target RNA
RNA silencing
What does siRNA mean?
Small interfering or Silencing RNA
How does gene silencing work via siRNA?
~21 - 24 nucleotide double stranded RNA, induced degradation of mRNA:Dicer-RISK complex
What are two case studies associated with molecular therapies?
Duchenne muscular dystrophy
Spinal muscular atrophy
What are some issues associated with treating DMD?
Multi-system disorder
Multiple isoforms
Effects all muscle mass
2.4 megabase gene
Many mutations
60% frameshifting mutationsWhat are the two types of muscular dystrophy and what is the severity of each?
DMD = fatal X-linked muscle wasting (1/3500 boys)
- Frame shifting/nonsense mutation in dystrophin
gene
- Ultimate caridac and respiratory failure
- Truncated protein
BMD = milder allelic disorder
- In fram deletion, dystrophin levels are > 3% normal
- Large deletions may be associated with
mild asymptomatic disease
What is the dystrophin associated complex?
Multiprotein complex including dystrophin
How much dystrophin do carriers of DMD begin with?
50% of muscle fibers
Some dystrophin is better than none
What does MD prevent?
Reconstruction of muscle due to lack of dystrophin protein
What systems do MD effect?
CNS
Endocrine
What dystrophin isoforms are there?
Long - Skeletal, cardiac, smooth muscle, brain
Smaller - CNS, retina, kidney
How could DMD be repaired molecularly?
Improve muscle repair
Put patch over gene lesion
- AVI 4658
How is AVI 4658 treating DMD? (Eteplirsen)
nucelic acid analogue targeting dystrophin exon 51
Now in phase 2b
What is the main test to diagnose DMD?
6 minute walk test
What is eteplirsen a product of?
Sarepta therapeutics
What does SMA mean?
Spinal muscular atrophy
What causes SMA?
Loss of SMN1 on chromosome 5
What is SMN?
Survival motor neuron
Loss of gene is incompatible with life
What is type 1 SMA caused by?
SMN1 deleted
What is SMA type 2 caused by?
Multiple SNM2 copies
What is the current treatment for SMA?
Antisense oligomers - alter pre-mRNA processing to generate BMD like mRNA and protein
What does BMD stand for?
Becker muscular dystrophy
