L11: ASO Therapy Flashcards
What are Antisense Oligonucleotides (ASO)?
Antisense: complementary to messenger RNA strand
Oligo: small (<25 nucleotides)
Antisense oligonucleotides (ASOs) are short, synthetic, single-stranded oligodeoxynucleotides that can alter RNA and reduce, restore, or modify protein expression through several distinct mechanisms.
Antisense oligonucleotides (ASOs) were first discovered to influence RNA processing and modulate protein expression over two decades ago. What has hampered translating these agents into the clinic?
Progress translating these agents into the clinic has been hampered by inadequate target engagement, insufficient biological activity, and off-target toxic effects.
Describe first generation ASOs
First-generation ASOs were short, synthetic, single-stranded oligodeoxynucleotides, typically 8–50 nucleotides in length, which bound by complementary base pairing to a target mRNA and led to endonuclease-mediated transcript knockdown and, consequently, to reduction of the levels of a deleterious protein
Why were early expectations of these agents remained largely unfulfilled?
First generation oligonucleotides mostly failed to meet therapeutic end points in clinical trials, mainly owing to their fast turnover and inability to achieve sufficient intracellular concentrations to suppress target genes
What did second and third generation ASOs change to improve these issues?
Since the early 1990s, a range of second-generation and third-generation ASOs with modified backbones that confer enhanced pharmacological properties have been developed.
What other benefits did second and third generation ASOs bring to the table? (3)
These improved ASOs can function via alternative mechanisms — for example, they can:
Alter pre-mRNA splicing by sterically blocking splicing factors
Block mRNA translation by preventing ribosome recruitment.
Furthermore, antisense molecules can be designed to bind non-coding RNAs and toxic RNAs associated with disease pathogenesis, which greatly expands the numbers and types of selectable targets.
What about these new ASOs made them ideal candidates for therapy development for neurological conditions?
Several features of this new class of drugs, including high specificity, ability to address targets otherwise inaccessible with traditional therapies, and reduced toxicity owing to limited systemic exposure, make these molecules ideal candidates for therapy development for human neurological conditions.
What is one of the most substantial obstacles for ASO therapies in the CNS?
The fact that oligonucleotides do not readily cross the blood-brain-barrier (BBB), and therefore require invasive delivery methods such as intrathecal (an injection into the spinal canal, or into the subarachnoid space so that it reaches the cerebrospinal fluid (CSF)) or intraventricular routes, remains one the most substantial obstacles for the clinical application of oligonucleotides in CNS disorders.
How is the method of delivery problem currently being worked on?
A large amount of work is currently being carried out to develop chemical modifications and vehicles that will improve ASO delivery and target engagement
What happened over the past few years to spark excitement in the field of ASO therapy?
In the past few years, two antisense agents have gained approval by the FDA for Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA), representing a landmark for the field and fuelling unprecedented excitement for the potential of this strategy in the treatment of human diseases.
What specifically caused the issues associated with the first generation of ASOs?
The first in vivo applications of ASOs showed limited clinical potential because of the high susceptibility of ASOs with an unmodified phosphoribose backbone to rapid degradation by endonucleases and exonucleases
Describe the chemical modifications which have now been applied to ASOs, resulting in improved pharmacological characteristics
In the past few decades a number of chemical modifications to the phosphodiester backbone have been made to improve antisense oligonucleotide (ASO) pharmacokinetic properties, tolerability profile, and target binding affinity.
Backbone modifications:
Phosphorothioate DNA, phosphorodiamidate morpholino (PMO), and peptide nucleic acid designs all confer resistance to nucleases and enhanced uptake in cells, resulting in increased potency of the ASO.
Tricyclo-DNAs (tcDNA) are conformationally constrained DNA analogues with increased potency and enhanced uptake in tissues after systemic administration.
2’-Sugar sing modifications:
Ribose substitutions, including 2′-O-methyl (2′-OMe), 2′-O-methoxyethyl (2′-MOE), and locked nucleic acid, are frequently used in combination to further increase stability, enhance target binding, and generally confer less toxicity than unmodified designs.
What effects have been seen in these second generation ASOs?
increased hybridization affinity to their target RNA, increased resistance to nuclease degradation, and reduced immunostimulatory activity compared with their unmodified counterparts.
What kind of molecules can ASOs target?
mRNA
MiRNA
lncRNA
What can the downstream effects of this targetting be?
Modified gene expression in terms of the proteins resulting
What are the benefits of ASOs? (3)
Design Precision: High specificity/stability
High target affinity = minimize off-target effects
Personalised treatment
1 injection = 6 months treatment
What are the three decay functions?
mRNA decay
Inhibition of nonsense-mediated decay
Activation of no-go decay
What are the three masking functions?
Splice modulators
AntimIR
Release of sequestered molecule
Modulate gene expression
What is the aim of gene therapy?
To treat/prevent diseases through modification of the expression of specific genes
How are ASOs relevant to gene therapy?
ASO gene therapy can be used to modulate gene expression/ modify diseases and have undergone success in clinical trials
Describe how ASOs can modulate gene expression through mRNA degradation?
Once bound to the RNA through Watson–Crick base pairing, ASOs can form an RNA–DNA hybrid that becomes a substrate for RNase H, resulting in target mRNA degradation
What effect does RNase H have and is it necessary?
The RNase H family consists of ubiquitously expressed enzymes that hydrolyse the RNA strand of an RNA–DNA duplex. RNase H1 is the necessary mediator and the rate-limiting step for ASO activity
In terms of the ASO, what is required for RNase H activity?
A minimum of five consecutive 2′-deoxy residues is sufficient for RNase H activation in vitro
How can ASOs suppress translation?
ASOs targeting the AUG start site can sterically block the binding of RNA binding protein complexes, such as ribosomal subunits, suppressing translation of target mRNA
How can ASOs combat diseases caused by a toxic RNA gain-of-function mechanism?
ASOs designed to bind complementarily with high-affinity to untranslated regions can prevent binding and sequestration of RNA-binding proteins by steric hindrance
How can ASOs affect splicing?
ASO binding to splice sites or to exonic or intronic inclusion signals results in skipping or inclusion of the targeted exon
If gene has a mutation so that there is aberrant splicing resulting in intron retention and no protein production, ASOs can bind to the site of the gene mutation of the RNA and induce correct splicing and restored protein production.
How can ASOs increase the amount of protein translated from a gene?
Translation of the upstream open reading frames (uORFs) typically inhibits the expression of the primary ORF. ASOs binding to the uORFs are able to increase the amounts of protein translated from a downstream ORF.
Binding to certain mRNA regions can also increase the stability of that transcript.
So give three ways it can carry out disease modification
- Correct aberrant splicing
- Induce Exon Skipping
- Regulate disease-associated
genes
Give 6 types of mRNA splicing types
Constitutive splicing: normal
Exon skipping/ inclusion
Alternative 5’ splice sites
Alternative 3’ splice sites
Intron retention
Mutually exclusive exons
Describe the historical development of ASOs
- Sense & antisense RNA can
hybridize, inhibiting synthesis of
gene products - ASO approach proposed
- First ASO therapy proposed for DMD
ASO promotes exon skipping - 1st ASO drug approved: Fomivirsen: Treats cytomegalovirus (CMV) retinitis
2014: CRISPR/Cas9 system
can target ssRNA
2018: ASO drug approved:
Eteplirsen: Duchenne muscular dystrophy
(DMD)
Nusinersen: Spinal muscular atrophy
(SMA)
- Golodirse: DMD
ASO drug - Vitolarsen: DMD ASO drug
2022? Casimersen: DMD ASO
drug
Aside from blocking RNA binding proteins, what two other functions of ASOs are considered steric hindence?
Blocking translation from ribosomes
miRNAs can have regulatory role on gene expression. ASOs can sequester miRNAs to reverse effects
What is Duchenne Muscular Dystrophy (DMD)?
Genetic disorder of progressive muscle degeneration due to dystrophin protein alterations, which normally anchors proteins from the cytoskeleton to those in the myofibre membrane
What is the prevalence of DMD?
Prevalence : 1 in 3500 males
When is the onset of DMD?
Childhood onset
What are the consequences of this muscle degeneration?
Descreased heart function and cardiomyopathy leads to heart failure.
Weak diaphragm can lead to respiratory failure.
Loss of muscle mass, inflammation and fibrosis means they require a wheelchair.
Describe the inheritance pattern of DMD
X-linked recessive inheritance discovery of DMD - gene on X chromosome encodes dystrophin protein. If a mother is heteroxygous, can have a daughter who is heterozygous or a son who is homozygous (or either without). Only homozygous is affected.
What is the mechanism of action of DMD?
A translational reading frame shift can lead to a premature stop codon which prohibits dystrophin production. The most common DMD-causative mutations lead to the loss of the DMD open reading frame (ORF).
How sucessful has DMD gene therapy been?
Sucessful! There have been two successful therapys for DMD to skip exon 53 (2019,2020) and one to skip exon 24 (2022)
How do ASOs correct out-of frame mRNAs?
Through exon splicing, results in the expression of a functional — albeit partially shortened — protein, by skipping DMD-causative mutations. Most of the crucial functional domains of dystrophin, which lie at the N-terminal and C-terminal of the protein, are typically unaffected by internal exon skipping
What is spinal muscular atrophy?
Disorder affecting motor neurons (nerve cells in the spinal cord) - responsible for muscle movement. A mutation in the SMN1 gene leads to a deficiency in the SMN protein which leads to splicing defects in motor neurons. Loss of motor neurons in the anterior horns of the spinal cord prevents signalling between the brain and skeletal muscles; this leads to progressive muscle weakness and atrophy
How prevalent is SMA?
1 in 8000
How does ASO aid SMA?
Spinal muscular atrophy: ASO corrects exons skipping in
SMN2, increasing SMN protein production. It is injected locally into the spinal canal and binds to exon seven, preventing its skipping leading to improved motor skills.
Give 6 administration routes for ASOs
(A) intravitreal,
(B) intranasal,
(C) subcutaneous,
(D) intravenous (most common),
(E) intraventricular
(F) intrathecal injections.
Name four strategies to enhance delivery & efficiency in clinical trials
Antisense oligonucleotides can be delivered into the cells more efficiently using
(A) viral vectors,
(B) conjugated peptides, antibodies, and other ligands (e.g., aptamers and acetylgalactosamine (GaINAc)),
(C) nanoparticles
(D) extracellular vesicles.
Clinical trials are trying to figure out if they can add delivery partners such as viruses to increase the efficacy
Which DMD patients are not treatable with current ASOs?
DMD patients with deletions not treatable with current ASOs (targeting exons 51, 53, 45) = no treatment options.
What is a big challenge with ASOs and their development in clinical trials?
ASO Toxicity:
Binding to off-target mRNAs => reduce proteins/alter structure to impair function
Chemical modifications => ASO-protein interactions lead to immune reaction => fever…
Serious : nephrotoxicity toxic effects on kidneys
Therefore give 6 ethical considerations for ASOs
Off-Target Effects
Access & Affordability Long-Term Effects & Follow-up
Equity in Research
Collaboration & Transparency
Patient Privacy
Why did ARID1B catch attention of the lab?
Seemed to be involved in a range of neurological and psychiatric disorders
Describe a rare neurodevelopmental disorder associated with ARID1B
ARID1B-RD: Coffin Siris is a rare neurodevelopmental disorder. It is associated with coarse facial features, microcephaly, growth deficiency, intellectual disability, hypoplastic/ absent fifth finger/ toenails, epilepsy.
Comment on the inheritence of ARID1B-RD: Coffin Siris
Spontaneous mutations occurs during formation of egg or sperm cell during embryonic development. This causes haploinsufficiency, reduced transcript and Intellectual disability, Autism, Coffin Siris Syndrome etc
What is the role of ARID1B?
ARID1B gene (Chr 6: 434,754 bp) protein is involved in chromatin
remodelling, therefore regulating gene expression (Tightly packed DNA = gene expression).
What are the aims of FAR?
Foundation for ARID1B Research
- Develop ARID1B-related disorders(AD) treatments
- Take candidate therapies to clinical trial
What does the ARID-1B research so far consist of?
- ARID1B common cause of ID
- De novo ARID1B mutations cause CSS
ARID1B haploinsufficient mouse models:
ASD/ID behavioural phenotypes
How can we increase ARID1B gene expression to help CSS patients?
Oligonucleotide therapy (ONT)
How have ASOs been used in alzheimers disease?
ONT in action: Alzheimer’s disease (ASO corrects deregulated alt splicing).
