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
