Date Published: October 5, 2017
Publisher: BioMed Central
Author(s): Rhia Ghosh, Sarah J. Tabrizi.
Gene suppression approaches have emerged over the last 20 years as a novel therapeutic approach for the treatment of neurodegenerative diseases. These include RNA interference and anti-sense oligonucleotides, both of which act at the post-transcriptional level, and genome-editing techniques, which aim to repair the responsible mutant gene. All serve to inhibit the expression of disease-causing proteins, leading to the potential prevention or even reversal of the disease phenotype. In this review we summarise the main developments in gene suppression strategies, using examples from Huntington’s disease and other inherited causes of neurodegeneration, and explore how these might illuminate a path to tackle other proteinopathy-associated dementias in the future.
Gene suppression approaches refer to targeted molecular genetic therapies that serve to lower the expression of specific genes. The term “gene silencing” has also been used to describe these methods; however, this term should be considered a misnomer as complete gene inactivation does not occur, and might not be desirable. Such techniques have made enormous progress over the last 20 years, and show great promise for the treatment of inherited neurodegenerative diseases arising from a known genetic mutation.
Post-transcriptional gene suppression refers to approaches that trigger the cleavage, enhanced degradation or translational suppression of the target mRNA. They include RNAi, ASOs and catalytic nucleic acids (ribozymes and DNA enzymes). Ultimately all of these mechanisms serve to modulate translation efficiency, thus lowering the amount of protein expressed  (see Fig. 1).Fig. 1Mechanisms of post-transcriptional gene suppression. a Ribozymes act in the cytoplasm where they hybridise complementary mature mRNA sequences and induce catalytic cleavage. b ASOs bind to complementary mRNA targets, leading to RNAse H1-induced mRNA cleavage. They are able to target both pre-mRNA in the nucleus and mature mRNA in the cytoplasm. c RNAi occurs in the cytoplasm and leads to the degradation of mature mRNA via a complex and highly regulated process. ASO: anti-sense oligonucleotide, AS-siRNA: antisense short interfering RNA, RISC: RNA-induced silencing complex, RNAi: RNA interference (Reproduced from Godinho et al.  with permission from Elsevier)
Gene suppression can also be achieved at the transcriptional level, using effectors that bind to specific DNA sequences. One such approach uses ZFPs which contain a zinc finger domain that can be manipulated synthetically to bind a DNA sequence of interest, fused to a functional protein domain. Zinc finger nucleases (ZFNs) cleave DNA at a particular target site, and zinc finger transcriptional repressors (ZFTRs) are transcription factor DNA-recognition motifs fused to a transcriptional repressor domain. Delivery of such agents would be through the use of viral vectors.
ASOs can be developed to target mRNA from both the mutant allele and the wild-type allele equally or to favour the mutant allele over the wild-type allele. The former, non-allele-specific, approach has the advantages of addressing the entire HD population and maintaining flexibility for ASO design across the entire gene, increasing the likelihood of identifying a specific, potent and well-tolerated ASO. The potential disadvantage for non-allele-specific ASOs lies in reduction of the wild-type allele. An allele-specific approach can be achieved by targeting either the CAG expansion or a sequence containing a specific single nucleotide polymorphism (SNP) found only on the mutant allele. Allele-specific approaches have the advantage of preferential reduction of the mutant protein. Potential disadvantages include blunted potency and tolerability due to severe limitations on sequence space, undesirable reduction of other proteins containing CAG repeats not linked to disease (with CAG-targeting ASOs) and addressing only a subset of the disease population (with SNP-targeting ASOs).
The first human clinical trial of intrathecal delivery of ASOs for the treatment of a neurodegenerative condition was carried out in patients with amyotrophic lateral sclerosis (ALS) . Inherited mutations in SOD1 account for 13% of familial ALS and 2% of all ALS, and cause disease through a toxic gain-of-function of SOD1 protein. An ASO targeting SOD1 mRNA was shown to reduce both wild-type and mutant SOD1 (i.e. non-allele selective) in transgenic mice and in human cell models of ALS [95, 96]. A phase 1 trial carried out by Ionis Pharmaceuticals has since demonstrated the safety and tolerability of intrathecal infusion of this drug in ALS patients and has proven that the drug is distributed to spinal cord tissues following injection and is detectable in the CNS 3 months following injection . Having demonstrated the feasibility of this approach, Ionis Pharmaceuticals, in conjunction with Biogen, is currently undertaking a phase 1/2a clinical study of a more potent ASO (ISIS-SOD1Rx) in ALS associated with a SOD1 mutation .
Recently DeVos et al.  have shown that treatment with tau-reducing ASOs prevents neuronal loss in a mouse model of tauopathy. Tau is a microtubule-associated protein in neurons, which becomes hyper-phosphorylated, misfolds and forms insoluble neurofibrillary tangles (NFTs) in AD. The close temporal and spatial relationship between tau pathology and neurodegeneration is thought to underlie the widespread neuronal loss and progressive dementia seen in this condition. Indeed mutations in the MAPT gene, which encodes tau protein, are causative for another neurodegenerative disease—frontotemporal lobar degeneration (FTLD). This study demonstrated improved survival and histopathology in a mouse model of tauopathy, and also showed tau protein reduction in CNS tissues and CSF in non-human primates following intrathecal bolus administration of ASOs targeting MAPT mRNA. This opens up the exciting potential for gene suppression treatments of tau as a therapeutic avenue for AD and other tauopathies such as progressive supranuclear palsy (PSP), and clinical trials of tau-lowering therapies are imminent.
Gene suppression technologies have made enormous progress in the last 20 years. Clinical trials of ASOs are already underway and trials of RNAi and genome editing using ZFNs are not far behind. The relative advantages of ASOs and RNAi are summarised in Table 1. Lessons learnt from work carried out in the field of HD, as well as ALS and SMA, can be applied to other neurodegenerative diseases in which the causative gene mutation or disease protein is known.Table 1Relative advantages of RNAi and ASOs as a strategy to achieve gene suppressionAdvantages of different approaches to post-transcriptional gene suppressionRNA interferenceAnti-sense oligonucleotides• siRNAs do not cross the BBB and if introduced into CSF cannot achieve widespread distribution in the CNS parenchyma. However, siRNA effector sequences can be constitutively delivered through miRNA expression systems expressed from a viral vector.• siRNAs can lead to very high levels of translational suppression and lowering of target protein, if this is desired.• siRNAs have prolonged effects in terms of gene suppression and so there is potentially minimal or no need for repetitive administration. However, this is a type of gene therapy, which is irreversible, and there are no antidotes.• Through peripheral administration in neonates or very early infancy, global or larger CNS areas can be targeted.• ASOs do not cross an intact BBB. However, ASOs are soluble in artificial CSF and can be delivered directly into the CSF space. Once introduced into the CSF, modified ASOs achieve widespread distribution in the CNS parenchyma and enter neuronal and glial cells.• ASOs have predictable, dose-dependent pharmacokinetics, enabling modulation of dose to achieve the desired level of pharmacological activity; with intrathecal bolus dosing every few months in symptomatic patients and potentially less frequently in pre-symptomatic patients.• ASO pharmacological effects are reversible and gene suppression reverses when treatment is stopped.• In addition to exonic regions, ASOs are able to target intronic regions as they bind to pre-mRNA rather than only mature transcripts. Thus they have more mRNA “real estate” from which to find the ideal ASO drug candidate, and can be used to treat a wider range of diseases.• Avoids saturation of RNAi pathways which can lead to liver toxicity.• No need for viral vector delivery, and therefore avoids the generation of an immune response.ASO anti-sense oligonucleotide, BBB blood–brain barrier, CSF cerebrospinal fluid, CNS central nervous system, miRNA micro-RNA, RNAi RNA interference, siRNA small interfering RNA