With CRISPR/Cas in the picture how does antisense oligonucleotide-mediated splice modulation fit in?
The rapid development and adaptation of CRISPR/Cas9 nucleases which can be engineered to target and edit nearly any DNA sequence over the past few years has been phenomenal. The use of CRISPR/Cas9 nucleases has had a huge impact on biomedical research and raised hopes that in the future any disease-causing mutation could be specifically corrected and consequently the disease would be treated permanently.
Studies in mice and monkeys have shown its feasibility in animal models and first-in-man studies may begin as early as 2017.1 However, serious questions remain about off-target effects such as seen in a study using CRISPR/Cas9 to edit the β-globin gene in human zygotes.2 Another often unstated problem is the issue of delivery as a guide RNA, CRISPR/Cas9 nuclease as well as a specific repair template all need to be supplied to cause sequence-specific gene editing.
Thus, first-in-man studies will likely concentrate on CRISPR/Cas9′ ability to induce one or more double strand breaks (DSBs) in the desired DNA sequence resulting in the excision or unspecific modification of a DNA sequence instead of relying on precise homology directed repair (HDR) to correct a disease-causing mutation. These first treatments will be limited to relatively easily accessible target tissues such as the eye, blood and bone marrow, tissues that have been the targets of gene addition strategies for the last 20 years.
Where then does antisense oligonucleotide-mediated splice modulation fit in?
One of the main differences between gene editing and splice modulation is that gene editing makes permanent changes in the genome while splice modulation temporarily alters the ratios of existing or newly induced splice isoforms, thereby requiring repeat administration for longer-term effects. This is not necessarily a disadvantage as short-term modulation of gene expression may be sufficient during treatment of certain diseases, such as cancers or for immunomodulation. Delivery of antisense oligonucleotides, while still an area of ongoing improvement, is simpler than delivering the components of the CRISPR/Cas9 system and can be achieved without resorting to viral vectors and their attendant problems. A key limitation of the CRISPR/Cas technology is that specific HDR-mediated gene editing is only possible in the S and G2 phases of the cell cycle and terminally differentiated cells like neurons are therefore not targetable in this way. However, non-homologous end joining (NHEJ) based applications of CRISPR/Cas, such as deletions of genes or exons are still possible.
Thus, there is unquestionably a future for splice modulation therapies in tissues made up by terminally differentiated cells where it would be impossible to gene edit precursor cells as is the case in the central nervous system. Also, targets requiring exon inclusion or changes in the delicate balance between different endogenous splice isoforms may be ideal for a splice modulation approach.3
Currently, the most advanced splice modulation therapies are eteplirsen and drisapersen for the treatment of Duchenne muscular dystrophy (DMD; for a fun explanation on how these work please go here4). Drisapersen has recently been denied marketing approval by the FDA in America, it is still being considered by the EMA, while eteplirsen is awaiting a decision from FDA and further trials skipping other exons in the DMD gene are underway.
However, perhaps the most promising current splice modulation therapy is nusinersen for the treatment of spinal muscular atrophy (SMA). SMA is caused by loss of function or deletion of the survival of motor neuron 1 (SMN1) gene, leading to death of motor neurons and muscle wasting. Severity of the disease is dependent on the number of SMN2 genes in the genome of affected people, but ranges from extreme muscle weakness (Type I – death in infancy due to respiratory failure) to intermediate (Type II – children can sit but not walk independently or Type III – children may walk for a few years) to mild adult-onset muscle weakness (Type IV).
Nusinersen is an antisense oligonucleotide that increases inclusion of exon 7 in the SMN2 transcript, a paralogue of SMN1 that normally cannot compensate for loss of SMN1 due to exclusion of exon 7 from 80-90% of its transcripts. In February, Chiriboga et al.5 reported the results of a phase I study in 28 children aged 2-14 years with Type II and III SMA. The aim was to establish safety and tolerability as well as to investigate the pharmacokinetics in cerebrospinal fluid and blood in patient cohorts treated with ascending single-dose levels of 1, 3, 6 and 9 mg. Other efficacy endpoints studied were the Hammersmith Functional Motor Scale Expanded (HFMSE) and Pediatric Quality of Life Inventory.
Results were encouraging. In the 6 and 9 mg groups, SMN protein levels more than doubled, although this was not quite statistically significant (p = 0.06 in the 9 mg group). The 9 mg group showed improved HFMSE scores from baseline at day 85 (mean increase of 3.1 points or 17.6% increase; p = 0.016) and at 9-14 months (mean increase of 5.8 points or 32.8% increase; p = 0.008; n = 8). No other statistically significant improvements were found and the small number of participants allows for the possibility of false positives. The treatment was well tolerated and there were no serious drug related adverse events. Phase II and III clinical trials with nusinersen are ongoing and data from an open label phase II study in infants with type I SMA are extremely encouraging with significant increases in median event free age and motor function scores compared to natural history controls.6
The requirement for repeated intrathecal injection is somewhat of a concern with nusinersen as systematically administered antisense oligonucleotides do not cross the blood brain barrier in sufficient concentration. In animal models nusinersen’s half-life in the CNS was 4-6 months and encouragingly this long half-life was confirmed in this human study, supporting infrequent administration of the drug. In a companion paper, Hache et al.7 evaluated the safety and side effect profile of the intrathecal injection procedure in more detail. Although 32% of the procedures were associated with treatment related side effects (mainly headache, back-pain and post–lumbar puncture syndrome), none of these were serious and all resolved with conservative treatment.
Nusinersen demonstrates the feasibility of antisense-oligonucleotide treatment for splice modulation in the central nervous system with direct delivery into the cerebrospinal fluid. Another very exciting target in the CNS recently reported by Hinrich et al.8 is the apolipoprotein E receptor 2 (ApoER2). The paper demonstrates that inclusion of exon 18 in ApoER2 mRNA is reduced in the brain of human Alzheimer’s disease patients and that increasing inclusion of the equivalent exon in a mouse model using an antisense oligonucleotide rescues cognitive defects caused by amyloid-β.
Antisense oligonucleotide-mediated splice modulation therapeutics clearly have a future alongside CRISPR/Cas9 and it is going to be very interesting to see how these two approaches are positioned in therapeutic oligonucleotide drug development going forward.
- MIT Technology Review. (2016). CRISPR Gene Editing to Be Tested on People by 2017, Says Editas. [online] Available at: http://www.technologyreview.com/s/543181/crispr-gene-editing-to-be-tested-on-people-by-2017-says-editas/ [Accessed 1 Apr. 2016].
- CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes.
Liang P, Xu Y, Zhang X, Ding C, Huang R, Zhang Z, Lv J, Xie X, Chen Y, Li Y, Sun Y, Bai Y, Songyang Z, Ma W, Zhou C, Huang J.
Protein Cell. 2015 May;6(5):363-72.
- Antisense-mediated exon skipping: taking advantage of a trick from Mother Nature to treat rare genetic diseases.
Veltrop M, Aartsma-Rus A.
Exp Cell Res. 2014 Jul 1;325(1):50-5.
- Dmd.nl. (2016). Department of Human Genetics : redirection page. [online] Available at: http://www.dmd.nl/gt/dance [Accessed 1 Apr. 2016].
- Results from a phase 1 study of nusinersen (ISIS-SMNRx) in children with spinal muscular atrophy.
Chiriboga CA, Swoboda KJ, Darras BT, Iannaccone ST, Montes J, De Vivo DC, Norris DA, Bennett CF, Bishop KM.
Neurology. 2016 Mar 8;86(10):890-7.
- Videonewswire.com. (2016). Isis Pharmaceuticals’ Webcast to Discuss Data From ISIS-SMN Rx Phase 2 Study in Infants With SMA. Registration Page. [online] Available at: http://www.videonewswire.com/event.asp?id=102440 [Accessed 1 Apr. 2016].
- Intrathecal Injections in Children With Spinal Muscular Atrophy: Nusinersen Clinical Trial Experience.
Haché M, Swoboda KJ, Sethna N, Farrow-Gillespie A, Khandji A, Xia S, Bishop KM.
J Child Neurol. 2016 Jan 27.
- Therapeutic correction of ApoER2 splicing in Alzheimer’s disease mice using antisense oligonucleotides.
Hinrich AJ, Jodelka FM, Chang JL, Brutman D, Bruno AM, Briggs CA, James BD, Stutzmann GE, Bennett DA, Miller SA, Rigo F, Marr RA, Hastings ML.
EMBO Mol Med. 2016 Feb 22.