Read with caution!

This post was written during early stages of trying to understand a complex scientific problem, and we didn't get everything right. The original author no longer endorses the content of this post. It is being left online for historical reasons, but read at your own risk.

Antisense therapy (Wikipedia)

Antisense therapy is a form of treatment for genetic disorders or infections. When the genetic sequence of a particular gene is known to be causative of a particular disease, it is possible to synthesize a strand of nucleic acid (DNA, RNA or a chemical analogue) that will bind to the messenger RNA (mRNA) produced by that gene and inactivate it, effectively turning that gene “off”. This is because mRNA has to be single stranded for it to be translated. Alternatively, the strand might be targeted to bind a splicing site on pre-mRNA and modify the exon content of an mRNA.[1]

This synthesized nucleic acid is termed an “anti-sense” oligonucleotide because its base sequence is complementary to the gene’s messenger RNA (mRNA), which is called the “sense” sequence (so that a sense segment of mRNA ” 5′-AAGGUC-3′ ” would be blocked by the anti-sense mRNA segment ” 3′-UUCCAG-5′ “).

Antisense drugs are being researched to treat cancers (including lung cancer, colorectal carcinoma, pancreatic carcinoma, malignant glioma and malignant melanoma), diabetes, Amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy and diseases such as asthma and arthritis with an inflammatory component.


miRNA blog:


“Single treatment of RNAi against prion protein rescues early neuronal disfunction and prolongs survival in mice with prion disease,” White et al., Proc Natl Acad Sci (2008)

Prion diseases are fatal neurodegenerative conditions for which there is no effective treatment. Prion propagation involves the conversion of cellular prion protein, PrP(C), to its conformational isomer, PrP(Sc), which accumulates in disease. Here, we show effective therapeutic knockdown of PrP(C) expression using RNAi in mice with established prion disease. A single administration of lentivirus expressing a shRNA targeting PrP into each hippocampus of mice with established prion disease significantly prolonged survival time. Treated animals lived 19% and 24% longer than mice given an “empty” lentivirus, or not treated, respectively. Lentivirally mediated RNAi of PrP also prevented the onset of behavioral deficits associated with early prion disease, reduced spongiform degeneration, and protected against neuronal loss. In contrast, mice receiving empty virus or no treatment developed early cognitive impairment and showed severe spongiosis and neuronal loss. The focal use of RNAi therapeutically in prion disease further supports strategies depleting PrP(C), which we previously established to be a valid target for prion-based treatments. This approach can now be used to define the temporal, quantitative, and regional requirements for PrP knockdown for effective treatment of prion disease and to explore mechanisms involved in predegenerative neuronal dysfunction and its rescue.


“Therapy for prion diseases: insights from the use of RNA interference,”  White et al., Prion (2009)

Insights into the molecular basis and the temporal evolution of neurotoxicity in prion disease are increasing, and recent work in mice leads to new avenues for targeting treatment of these disorders. Using lentivirally mediated RNA interference (RNAi) against native prion protein (PrP), White et al. report the first therapeutic intervention that results in neuronal rescue, prevents symptoms and increases survival in mice with established prion disease.(1) Both the target and the timing of treatment here are crucial to the effectiveness of this strategy: the formation of the neurotoxic prion agent is prevented at a point when diseased neurons can still be saved from death. But the data also give new insights into the timing of treatment in the context of the pattern of spread of prion infection throughout the brain, with implications for developing the most effective treatments.

The conversion reaction itself is critical to neurotoxicity in prion diseases: neither loss of PrPC function,35 nor deposition of PrPSc is sufficient to cause pathology. However, the precise identity of the neurotoxic prion species and the exact mechanism of neurotoxicity are unknown.

A significant advantage of the RNAi therapeutic approach in prion disease is its applicability to all known strains of prion disease. Within any species the primary sequence of PrPC, and PrPSc, is the same for all strains: thus RNAi should be an effective treatment for all variants. This is in contrast to many previous candidate treatments for prion disease, which have suffered from inconsistent results dependant upon the prion strain involved. This should also apply  to familial prion diseases that arise from a coding mutation in the gene encoding PrPCPRNPIt is likely that allele-specific silencing strategies to reduce expression of the mutant would be effective here. Genetic testing can identify these patients during the preclinical phase so successful treatment of this category may be possible through preventative silencing of the mutant PRNP allele expression prior to development of pathology.

Lowering the amount of PrPC available for conversion may enhance the ability of other agents to inhibit the disease process when administered in combination. For example, by combining RNAi against the prion protein with a drug to increase endogenous clearance of PrPSc it may be possible to delay disease progression indefinitely.
The major obstacle to the use of therapeutic RNAi in neurodegenerative disease remains the problem of delivery to the brain. The blood-brain-barrier (BBB) restricts passive entry of molecules from the peripheral circulation meaning active transport across this barrier, transient disruption of the BBB’s impermeability, or direct injection into the brain are currently required for delivery to the CNS. New technologies recently developed offer hope by targeting receptors in the BBB to mediate activate transport of interfering RNA42,71 or utilising viruses capable of transversing the BBB unaided.72
Another caveat to the use of RNAi in vivo is the need to avoid both off-target effects and interference with processing of endogenous miRNAs due to over-loading of the RNAi pathway. Off-target effects can include silencing of unintended genes with limited sequence complementarity to the siRNA guide strand, cytotoxicity or activation of interferon responses. Careful design, in vitro screening and selection of the most potent sequences for RNAi reduces off-target effects and cytotoxicity in vivo,73 and minimizes competition for endogenous miRNA machinery.74 In prion disease, since only partial reduction of PrPC expression is likely to be required for a therapeutic benefit, low doses of RNAi should be sufficient, minimising the potential for unintended side effects.
One drawback of this approach from a public health perspective is that it aims to eliminate neurotoxicity rather than abolish prion replication altogether. While PrPSc continues to be produced, infectivity remains, so the problem of potential transmissibility persists.
Also, whilst the ablation of PrPC expression in adult mice is well tolerated, the consequences of reducing PrPC in humans remain unknown. Initial attempts should proceed with caution and the use of transient silencing through infusion of siRNA duplexes or expression of shRNAs from inducible viral vectors should be considered so that treatment can be halted if unforeseen adverse effects develop. In the end, the balance of possible adverse effects of PrP loss against the benefits of improved survival and protection against neuronal loss in key brain areas, will determine future therapies for prion and other neurodegenerative disorders.



“Promising therapy for ALS delivers antisense drug directly to nervous system,” Kain, UCSD News Release, 2006

The study conducted in the laboratory of Don Cleveland, Ph.D., UCSD Professor of Medicine, Neurosciences and Cellular and Molecular Medicine and member of the Ludwig Institute for Cancer Research, shows that therapeutic molecules known as antisense oligonucleotides can be delivered to the brain and spinal cord through the cerebrospinal fluid (CSF) at doses shown to slow the progression of ALS in rats. The study will be published July 27 in advance of publication in the August issue of Journal of Clinical Investigation.

With colleagues Timothy Miller, M.D., Ph.D., UCSD Department of Neurosciences, and Richard A. Smith, M.D., of the Center for Neurologic Study, Cleveland found that when effective doses of the antisense therapy were delivered, far less of a protein that causes a hereditable form of amyotrophic lateral sclerosis was produced.

“Limiting mutant damage to microglia robustly slowed the disease’s course, even when all motor neurons were expressing high levels of a SOD1 mutant,” said Cleveland. “Our research suggests that what starts ALS and what keeps it going are two separate phases; it also suggests that with the right therapy, ALS could become a manageable, chronic disease.”

Within a year, Cleveland hopes the first clinical trial will be initiated in humans. In order to deliver the antisense drug directly to the nervous system, surgeons will insert a small pump into a patient using a fairly routine surgery that has already been approved for management of pain. A small catheter is then implanted into the area surrounding the spinal cord, in order to pump antisense oligonucleotide drugs directly into the nervous system.

The investigators noted that if the antisense approach works for ALS – by delivering therapeutic agents for neurodegenerative diseases across the highly impermeable blood-brain barrier – it would likely also work in other neurodegenerative conditions, including Alzheimer’s, Parkinson’s and Huntington’s diseases.

“We know we’re on target with the pathogenic mechanism,” said Cleveland. The remaining question is whether the genetic-based therapy will be tolerated. “If tolerated, this sets the stage for broader treatment of neurodegenerative disease, especially Huntington’s disease, where there is currently no treatment, but key genes involved in promoting disease are known.”


ALS Clinical Trials – now enrolling


“Antisense Oligonucleotide therapy for neurodegenerative diseases,” Smith et al., J Clin Invest (2006)

Neurotoxicity from accumulation of misfolded/mutant proteins is thought to drive pathogenesis in neurodegenerative diseases. Since decreasing levels of proteins responsible for such accumulations is likely to ameliorate disease, a therapeutic strategy has been developed to downregulate almost any gene in the CNS. Modified antisense oligonucleotides, continuously infused intraventricularly, have been demonstrated to distribute widely throughout the CNS of rodents and primates, including the regions affected in the major neurodegenerative diseases. Using this route of administration, we found that antisense oligonucleotides to superoxide dismutase 1 (SOD1), one of the most abundant brain proteins, reduced both SOD1 protein and mRNA levels throughout the brain and spinal cord. Treatment initiated near onset significantly slowed disease progression in a model of amyotrophic lateral sclerosis (ALS) caused by a mutation in SOD1. This suggests that direct delivery of antisense oligonucleotides could be an effective, dosage-regulatable means of treating neurodegenerative diseases, including ALS, where appropriate target proteins are known.


Q&A with K 1.11.12

Q:  I have a question about how antisense therapy works (or aspires to work.)

Say I have two copies of chromosome 20 hanging out in each of my cell nuclei, and the two copies have two point differences.  The healthy one from Dad has an aspartic acid (D) at position 178 and a valine (V) at position 129.  The unhealthy one from Mom has an asparagine (N) at position 178 and a methionine (M) at position 129.

My understanding is that antisense therapy aims to block the expression of the faulty gene by intercepting the mRNA it produces pre-translation.  My question is: are antisense agents clever enough to bind to only mRNA from the subtly different Mom chromosome, thus blocking production only of the misfolding-inclined PrP(c)?  Or would they also bind to mRNA from the healthy Dad chromosome, essentially suppressing production of all PrP(c)?  Basically, are those two tiny differences enough to distinguish the chromosomes from each other for the purposes of the therapy?


A:  I’d have to look into how exactly antisense therapy is being set up these days, but assuming it is used analogously to natural microRNAs (miRNAs) in human cells, here are some thoughts. Most miRNAs naturally target sequences in the 3′UTR (untranslated region) of mRNAs. (as background, the 5′UTR is the first part of the message transcribed, before the translation start codon, the open reading frame (ORF) is the region of mRNA that directly encodes the protein, running from the start codon to the stop codon, and the 3′UTR is a region after the stop codon, but before the poly(A) tail that is added to human RNAs. The 3′ UTR (and the 5′ UTR, for that matter) often contain regulatory information, and the vast majority of characterized human miRNA binding sites are in the 3′ UTRs of mRNAs. This position may be important mechanistically for miRNAs triggering deadenylation of the mRNA (which impairs translation of the mRNA, and also destabilizes it (makes it prone to degradation)). Alternatively, the position in the 3′ UTR of miRNA binding sites rather than in ORFs may be because there is less constraint on these sequences (i.e. they don’t have to encode codons which code for protein). SO I am guessing that antisense also targets the 3′UTR and therefore would target both variants of mRNA.

Now it is a good question that we can look more into whether or not you CAN make effective antisense RNAs that target the ORF. (Maybe one issue would be that translating ribosomes are pretty powerful, and could knock the miRNA complex off…). But the other thing to know about human miRNAs is that they almost all have one or two mismatches to the sequence they are targeting within their ~21 nucleotide (nt) sequence. Because mismatches are well-tolerated by miRNA machinery, it would not be trivial to make a miRNA that would be great at distinguishing the single point mutation in the two mRNAs. The exception to the rule of mismatches being tolerated, however, is in the 6-nt “seed” sequence that I believe is located between nt 2 and nt 7 of the miRNA: this is the most important region for targeting miRNAs and the region where mismatches would be the least tolerated. SO, if there is precedence for targeting antisense to the ORF of mRNAs, the best bet would be to try putting this single point mutation into the “seed sequence” or the miRNA.

(The other assumption I am making is that antisense therapy in humans is taking advantage of the miRNA pathway of RNAi, rather than the siRNA pathway – I think this is a good assumption because I don’t think human cells have the appropriate machinery for siRNA. But siRNAs have complete complementarity to their targets (no mismatches) and typically function by cleaving the mRNA at the siRNA site, rather than by translationally repressing / destabilizing the mRNA).