20.03.23 Genetic treatments: mutation specific therapy Flashcards
What are antisense oligonucleotides (ASOs)
-Small single stranded DNA or RNA molecules (~20bp)
What stops ASOs from being degraded by nucleases
- Chemical modifications
- Most common are 2-O-methyl RNA phosphorothioate
Mechanisms of ASOs
- Blockage of cryptic splicing
- Alternative splicing
- Exon inclusion
- Exon skipping
- Alter transcript translation
How are ASOs used to block cryptic splicing
-Beta-thalassemia is caused by mutations in beta-globin gene which activate cryptic splice sites.
2-O-methyl RNA ASOs block access of the spliceosome to these sites enabling normal beta-globin splicing and normal haemoglobin expression.
-Only transient
-ASO needed for each type of mutation (generate, validate, trial). Increases costs and time for production.
How are ASOs used to skip exons
- DMD deletions and duplications can disrupt ORF and cause loss of protein expression. Leads to severe duchenne phenotype. Dels/dups that don’t disrupt ORF cause milder Becker phenotype.
- ASOs used to mask specific exons to restore ORF and partial dystrophin function.
- Transient. Low efficacy in heart tissue (many DMD patients die due to heart complications)
- Due to mutation hotspots, skipping of a small number of exons is applicable to a large number of patients.
- E.g. exon 51 skipping is used for 14% of DMD cases. Drisapersen
- New class of ASOs tricyclo-DNA (tcDNA) have improved tissue uptake.
How are ASOs used to cause alternate splicing
- Exon skipping of exon 27 in APOB leads to a reduction in LDL particle secretion and an increase in LDL affinity to LDL receptor
- LMNA missense mutation shifts alternative splicing, increasing production of truncated lamin A known as progerin. Accumulation of progerin causes Hutchinson-Gilford progeria (HGPS). Patients have accelerated aging and a shortened lifespan. ASO masks splice site restoring normal splicing.
How are ASOs used to enhance splicing
- SMA is caused by deletions of SMN1.
- SMN2 exon 7 is often skipped due to a C>T transition.
- Spinraza is an ASO that blocks the ISS (intronic splicing silencers) to increase exon 7 inclusion in SMN2 transcripts, increasing SMN protein production
How are ASOs used to downregulate transcript translation
- Suppress translation of HTT mRNA containing a pathological CAG repeat expansion in Huntington’s disease.
- However, also targeted all CAG containing genes, not specifically HTT.
- To improve specificity, targeted for HTT-dependent SNPs
- e.g. IONIS-HTTRx
What are the challenges of using ASOs
- Delivery to target tissue
- To achieve sustained effect. Re-administration required
- Hard to completely inhibit process. Large quantities of mRNA compared to ASO
What is RNA interference (RNA i)
A cellular mechanism of post-transcriptional gene silencing
What are siRNAs
small double stranded RNAs (20-25nt)
What enzyme catalyses the production of siRNAs
Dicer
How do siRNAs silence genes
-siRNAs are incorporated into the RNA induced silencing complex (RISC) which binds to mRNA and cleaves it.
Benefits of siRNA as a potential therapy
- High sequence specificity
- Currently no RNAi-based therapeutics available
Example of an isoform specific RNAi
- VEGF has 5 alternatively spliced isoforms. VEGF165 is strongly implicated in tumour angiogenesis.
- Silencing VEGF165 isoform can be done using RNAi that targets sequences at exons 5-7.
- Benefits- other VEGF isoforms remain functional
Example of an allele-specific RNAi
- Designing siRNAs that can discriminate between wildtype and mutant alleles.
- Could be used for dominant gain-of-function disorders, where mutated allele is targeted.
- E.g. COL1A1 in Osteogenesis imperfecta. Targets common SNPs rather than designing for each rare mutation.
Challenges of RNAi
- Delivery. Virally expressed, lipid-based delivery agents, nanoparticle technology.
- Non-specific off-target effects.
- siRNAs likely to elicit immunological responses against vector components
Describe CRISPR-Cas9 process
- CRISPR (clustered regularly interspaced short palendromic repeats) are sequences play a role in the defense system of prokaryotes.
- Cas9 (CRISPR-associated protein 9) an enzyme that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence
- Cost effective
- Trials for beta thalassemia (CTX100)
Issues with CRISPR-Cas9 / gene editing
- Ethics of editing a genome
- Delivery to target cells
- Off target mutagenesis. Use of guide RNAs should reduce this.
Examples of small molecules as therapeutics
- Zinc finger: create artificial restriction enzymes by fusing a zinc finger to DNA-binding domain. Difficult to design and target sites are limited.
- TALENS (Transcription activator-like effector nucleases). Artificial restriction enzymes made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain. More specific but larger, so delivery is more challenging.
Drugs that allow read-through of premature termination codons (PTCs)
- 10% of human diseases are caused by nonsense mutations
- Aminoglycoside antibiotics allow ribosomes to read through PTCs in mRNA, leading to production of full length protein.
- Efficiency varies due to neighbouring sequence, drug, nature of PTC.
- Ataluren induces ribosomes to read through PTCs but not normal stop codons. Targets mRNAs, so a problem if patient has low mRNA levels. Used for DMD, CF in phase III trials.
Molecules that promote correct folding/transport/alter activation
- Ivacaftor= improves transport of chloride through ion channels in patients that carry Gly551Asp.
- Lumacaftor enhances correct folding of Phe508del so it can reach cell membrane
