Development of antisense oligonucleotides with therapeutic potential for treating amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease characterised by degenerative changes to both upper and lower motor neurons. Current treatment options for ALS are based on symptom management and respiratory support, with the only approved medications prolonging survival for just a few months. Over 60 clinical trials for drugs to treat ALS have ended in disappointment in the last decades, the majority of which were for small molecule-based treatments. Antisense oligonucleotides (AONs) are short nucleic acid analogues that can be designed to directly and specifically target the pathological hallmarks of disease at the RNA level. AONs can work via several mechanisms including strategies that rely on the endogenous RNase H enzymes to degrade target transcripts or strategies that involve the steric block of splicing factors or cellular machinery. Developments in AON based medicine in recent years has led to several new AON therapies receiving approvals to treat various conditions. The work outlined in this thesis has involved the development and assessment of AONs with therapeutic potential for treating ALS. AONs with the phosphorodiamidate morpholino oligomer (PMO) chemistry were preferred in this work due to their strong safety profile and clinical approvals for Duchenne muscular dystrophy. AONs that rely on RNase H require chemical modifications such as the phosphorothioate backbone (PS) modification to increase their stability and cellular uptake. PMOs are nucleic acid analogous that are inherently stable but are not compatible with RNase H activity, instead requiring a steric blocking strategy. ALS is a heterogenous disease, however a common feature is the cytoplasmic aggregation of proteins. The transactive response DNA-binding protein 43 (TDP-43) encoded by the TARDBP gene was identified in 2006 as a primary protein component of intracellular inclusions in most ALS cases, occurring in more than 90% of patients. As TDP-43 is autoregulated, its cytoplasmic mislocalisation in ALS leads to its upregulation. In this work, AONs with the PMO chemistry that utilise a splice-switching strategy were developed that can reduce the expression of TDP-43. It has recently been discovered that mislocalisation of TDP-43 leads to the downregulation of Stathmin-2, an important neuronal protein involved in axonal development and repair. AONs that can prevent the down-regulation of Stathmin-2 in response to TDP-43 nuclear depletion in human cells were also successfully developed. These AONs alone or in combination could have therapeutic potential to treat the majority of sporadic ALS patients. iii A subset of ALS patients, including some with early-onset ALS, have pathogenic variants in the FUS gene which leads to FUS protein mislocalisation and aggregation. There is also mounting evidence that FUS may play a role in the pathogenesis of sporadic ALS. Another focus of this study was the development of AONs that can modify expression of FUS. Two strategies were explored, including a splice switching strategy, to reduce total FUS expression. A steric blocking PMO AON was developed that can reduce FUS expression in human cells. As FUS-ALS is usually inherited in an autosomal dominant manner, a second strategy aimed to selectively knock down expression of only one FUS allele. This therapeutic strategy would allow expression of the normal allele in FUS-ALS patients, reducing potential complications due to insufficiency of the protein. Several AON design parameters and chemistries were explored with AONs using the newer thiophosphoramidate morpholino oligomer chemistry showing promise in achieving this aim. The work presented in this thesis has provided evidence that splice-switching AON based therapeutics that can reduce expression of TDP-43 while preventing the down-regulation of Stathmin-2 is feasible. Like-wise both splice-switching and RNase H utilising allele selective, FUS targeted AONs have been developed that have therapeutic potential in treating FUS-ALS. With further development, there is hope that this work may contribute to the availability of disease altering antisense based therapeutics for ALS patients