Selective DNAzyme-mediated cleavage of AChR mutant transcripts by targeting the mutation site or through mismatches in the binding arm

Many dominantly inherited disorders are caused by missense amino acid substitutions resulting from a single nucleotide exchange in the encoding gene. For these disorders, where proteins expressed from the mutant alleles are often pathogenic and present throughout life, gene silencing, through intervention at the mRNA level, holds promise as a therapeutic approach. We have used mutations that underlie the slow channel congenital myasthenic syndrome (SCCMS) as a model system to study allele-specific gene silencing of RNA transcripts by DNAzymes. We tested the ability of DNAzymes to give allele-specific cleavage for i) mutations that create cleavage sites, and ii) mutations located close to a DNAzyme cleavage site that create a potential mismatch in the binding arms. For both we demonstrate selective cleavage of mutant transcripts under simulated physiological conditions. For DNAzymes with binding arm mismatches the degree of selectivity for mutant over wild type may be enhanced by optimising the mismatch position as well as the binding arm length. The optimal sites for mismatches are 1.1 and 1.2 in arm I, and 16.2 in arm II. Asymmetric binding arm DNAzymes with a shorter arm I are more discriminative. Our results show it should be possible to apply DNAzyme-mediated cleavage of mutant alleles even when the mutant does not itself create a putative cleavage site. This therapeutic approach may be well suited to dominantly inherited disorders such as SCCMS, where loss of some wild type transcripts is unlikely to have pathogenic consequences

Selective cleavage of AChR cRNAs harbouring mutations underlying the slow channel myasthenic syndrome by hammerhead ribozymes

Slow channel congenital myasthenic syndrome (SCCMS) is a dominant disorder caused by missense mutations in muscle acetylcholine receptors (AChR). Expression from mutant alleles causes prolonged AChR ion-channel activations. This ‘gain of function’ results in excitotoxic damage due to excess entry of calcium ions that manifests as an endplate myopathy. The biology of SCCMS provides a model system to investigate the potential of catalytic nucleic acids for therapy in dominantly inherited disorders involving single missense mutations. Hammerhead ribozymes can catalytically cleave RNA transcripts in a sequence-specific manner. We designed hammerhead ribozymes to target transcripts from four SCCMS mutations, αT254I, αS226F, αS269I and εL221F. Ribozymes were incubated with cRNA transcripts encoding wild type and mutant AChR subunits. The ribozymes efficiently cleaved the mutant allele cRNA transcripts but left the wild type cRNA intact. Cleavage efficiency was optimised for αS226F. We were able to demonstrate robust catalytic activity under simulated physiological conditions and at high Ca2+ concentrations, which is likely to be accumulated at the endplate region of the SCCMS patient muscles. These results demonstrate the potential for gene therapy applications of ribozymes to specifically down-regulate expression of mutant alleles in dominantly inherited disorders

Design of RNAi Hairpins for Mutation-Specific Silencing of Ataxin-7 and Correction of a SCA7 Phenotype

Spinocerebellar ataxia type 7 is a polyglutamine disorder caused by an expanded CAG repeat mutation that results in neurodegeneration. Since no treatment exists for this chronic disease, novel therapies such post-transcriptional RNA interference-based gene silencing are under investigation, in particular those that might enable constitutive and tissue-specific silencing, such as expressed hairpins. Given that this method of silencing can be abolished by the presence of nucleotide mismatches against the target RNA, we sought to identify expressed RNA hairpins selective for silencing the mutant ataxin-7 transcript using a linked SNP. By targeting both short and full-length tagged ataxin-7 sequences, we show that mutation-specific selectivity can be obtained with single nucleotide mismatches to the wild-type RNA target incorporated 3′ to the centre of the active strand of short hairpin RNAs. The activity of the most effective short hairpin RNA incorporating the nucleotide mismatch at position 16 was further studied in a heterozygous ataxin-7 disease model, demonstrating significantly reduced levels of toxic mutant ataxin-7 protein with decreased mutant protein aggregation and retention of normal wild-type protein in a non-aggregated diffuse cellular distribution. Allele-specific mutant ataxin7 silencing was also obtained with the use of primary microRNA mimics, the most highly effective construct also harbouring the single nucleotide mismatch at position 16, corroborating our earlier findings. Our data provide understanding of RNA interference guide strand anatomy optimised for the allele-specific silencing of a polyglutamine mutation linked SNP and give a basis for the use of allele-specific RNA interference as a viable therapeutic approach for spinocerebellar ataxia 7

Allele-Specific Knockdown of ALS-Associated Mutant TDP-43 in Neural Stem Cells Derived from Induced Pluripotent Stem Cells

TDP-43 is found in cytoplasmic inclusions in 95% of amyotrophic lateral sclerosis (ALS) and 60% of frontotemporal lobar degeneration (FTLD). Approximately 4% of familial ALS is caused by mutations in TDP-43. The majority of these mutations are found in the glycine-rich domain, including the variant M337V, which is one of the most common mutations in TDP-43. In order to investigate the use of allele-specific RNA interference (RNAi) as a potential therapeutic tool, we designed and screened a set of siRNAs that specifically target TDP-43(M337V) mutation. Two siRNA specifically silenced the M337V mutation in HEK293T cells transfected with GFP-TDP-43(wt) or GFP-TDP-43(M337V) or TDP-43 C-terminal fragments counterparts. C-terminal TDP-43 transfected cells show an increase of cytosolic inclusions, which are decreased after allele-specific siRNA in M337V cells. We then investigated the effects of one of these allele-specific siRNAs in induced pluripotent stem cells (iPSCs) derived from an ALS patient carrying the M337V mutation. These lines showed a two-fold increase in cytosolic TDP-43 compared to the control. Following transfection with the allele-specific siRNA, cytosolic TDP-43 was reduced by 30% compared to cells transfected with a scrambled siRNA. We conclude that RNA interference can be used to selectively target the TDP-43(M337V) allele in mammalian and patient cells, thus demonstrating the potential for using RNA interference as a therapeutic tool for ALS

Gene therapy for congenital myasthenic syndromes

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Allele-specific siRNAs targeting TDP-43^M337V mutant allele.

<p><b>A.</b> Schematic representation of TDP-43 protein containing two RNA-recognition motifs (RRM1 and RRM2), a bipartite nuclear localization signal (NLS), a nuclear export signal (NES) and a glycine-rich domain in the carboxy-terminal. The M337V mutation localization is indicated. Five allele-specific siRNAs were designed to contain mismatches at positions 9 (M9), 3 (M3), or 17 (M17); double mismatches at positions 8 and 9 (M8-9) or multiple mismatches at positions 5, 7, 10 and 16 (M5U). <b>B.</b> Representative western blot image showing the effects of allele-specific siRNA on cells transfected with GFP-TDP-43^wt and GFP-TDP-43^M337V. The allele-specific siM9 reduces the levels of GFP-TDP-43^M337V specifically whereas GFP-TDP-43^wt levels remain unchanged. FLAG-tagged protein was used as a control for transfection efficiency. <b>C.</b> Densitometry analysis of relative GFP-TDP-43 normalised to GAPDH. Mean from three independent experiments. Error bars represent standard error of the mean (SEM). (One way ANOVA, * P<0.05; *** P<0.001).</p

Allele-specific siRNA silences the mutant allele specifically and reduces cytoplasmic inclusions in HEK293 cells.

<p><b>A.</b> HA-TDP-43^wt stably expressing HEK293 cells were co-transfected with C-terminal GFP-TDP-43^wt or GFP-TDP-43^M337V and siRNAs for 48 hours. Cells were fixed and stained with HA antibody (red) and DAPI (blue). GFP-TDP-43 cytosolic and nuclear inclusions of different sizes were seen. C-terminal GFP-TDP-43^wt and GFP-TDP43^M337V inclusions co-localized with full length HA-TDP-43^wt (arrows), however some inclusions did not recruit full length HA-TDP-43^wt (arrowhead). Scale bars = 20 µm. <b>B.</b> Percentage of cells with GFP aggregates. siM9 reduced the number of cells with aggregates in GFP-TDP-43^M337V – expressing cells. More than 70 000 cells were counted from three independent experiments. Error bars represent SEM (Student's T-test,*** P<0.001).</p

Allele-specific siM9 decreases total TDP-43 transcripts and protein levels in neuralised cells.

<p><b>A.</b> Representative Western blot image showing M337V knockdown in M337V lines. <b>B.</b> Densitometry analysis of relative TDP-43 protein normalised to GAPDH. <b>C.</b> qPCR display unchanged levels of total TDP-43 in the control lines transfected with siM9, whereas M337V lines showed a reduction. Error bars represent SEM (One way ANOVA, * P<0.05, ** P<0.01, *** P<0.001).</p

Allele-specific knockdown of M337V allele on neural stem cells.

<p><b>A.</b> NSCs were transfected with allele-specific siM9 and stained for TDP-43. Images were acquired using identical parameters and analysed using Metamorph software. Representative confocal immunolabeling images showing allele-specific M337V knockdown in M337V lines. The allele-specific siM9 reduces endogenous TDP-43^M337V expression in all compartments (cytosolic (<b>B</b>), nuclear (<b>C</b>) and total (<b>D</b>) TDP-43) (n = 3 independent experiments. ** P<0.01 and *** P<0.001).</p