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1.  RNA Toxicity from the ALS/FTD C9ORF72 Expansion Is Mitigated by Antisense Intervention 
Neuron  2013;80(2):415-428.
A hexanucleotide GGGGCC repeat expansion in the noncoding region of the C9ORF72 gene is the most common genetic abnormality in familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The function of the C9ORF72 protein is unknown, as is the mechanism by which the repeat expansion could cause disease. Induced pluripotent stem cell (iPSC)-differentiated neurons from C9ORF72 ALS patients revealed disease-specific (1) intranuclear GGGGCCexp RNA foci, (2) dysregulated gene expression, (3) sequestration of GGGGCCexp RNA binding protein ADARB2, and (4) susceptibility to excitotoxicity. These pathological and pathogenic characteristics were confirmed in ALS brain and were mitigated with antisense oligonucleotide (ASO) therapeutics to the C9ORF72 transcript or repeat expansion despite the presence of repeat-associated non-ATG translation (RAN) products. These data indicate a toxic RNA gain-of-function mechanism as a cause of C9ORF72 ALS and provide candidate antisense therapeutics and candidate human pharmacodynamic markers for therapy.
PMCID: PMC4098943  PMID: 24139042
2.  Nonsense-mediated decay as a terminating mechanism for antisense oligonucleotides 
Nucleic Acids Research  2014;42(9):5871-5879.
Antisense oligonucleotides (ASOs) are synthetic oligonucleotides that alter expression of disease-associated transcripts via Watson–Crick hybridization. ASOs that function through RNase H or the RNA-induced silencing complex (RISC) result in enzymatic degradation of target RNA. ASOs designed to sterically block access of proteins to the RNA modulate mRNA metabolism but do not typically cause degradation. Here, we rationally design steric blocking ASOs to promote mRNA reduction and characterize the terminating mechanism. Transfection of ASOs complementary to constitutive exons in STAT3 and Sod1 results in greater than 70% reduction of mRNA and protein. The ASOs promote aberrant exon skipping and generation of premature termination codon (PTC)-containing mRNAs. We inhibit the nonsense-mediated mRNA decay (NMD) pathway and show that the PTC-containing mRNAs are recognized by the UPF1 ATPase, cleaved by the SMG6 endonuclease and degraded by the XRN1 cytoplasmic exonuclease. NMD surveillance, however, does not entirely explain the mechanism of decreased STAT3 expression. In addition to exon skipping, ASO treatment causes intron retention and reduction of chromatin-associated STAT3 mRNA. The application of steric blocking ASOs to promote RNA degradation allows one to explore more nucleotide modifications than tolerated by RNase H or RISC-dependent ASOs, with the goal of improving ASO drug properties.
PMCID: PMC4027159  PMID: 24589581
3.  Antisense Reduction of Tau in Adult Mice Protects against Seizures 
The Journal of Neuroscience  2013;33(31):12887-12897.
Tau, a microtubule-associated protein, is implicated in the pathogenesis of Alzheimer's Disease (AD) in regard to both neurofibrillary tangle formation and neuronal network hyperexcitability. The genetic ablation of tau substantially reduces hyperexcitability in AD mouse lines, induced seizure models, and genetic in vivo models of epilepsy. These data demonstrate that tau is an important regulator of network excitability. However, developmental compensation in the genetic tau knock-out line may account for the protective effect against seizures. To test the efficacy of a tau reducing therapy for disorders with a detrimental hyperexcitability profile in adult animals, we identified antisense oligonucleotides that selectively decrease endogenous tau expression throughout the entire mouse CNS—brain and spinal cord tissue, interstitial fluid, and CSF—while having no effect on baseline motor or cognitive behavior. In two chemically induced seizure models, mice with reduced tau protein had less severe seizures than control mice. Total tau protein levels and seizure severity were highly correlated, such that those mice with the most severe seizures also had the highest levels of tau. Our results demonstrate that endogenous tau is integral for regulating neuronal hyperexcitability in adult animals and suggest that an antisense oligonucleotide reduction of tau could benefit those with epilepsy and perhaps other disorders associated with tau-mediated neuronal hyperexcitability.
PMCID: PMC3728694  PMID: 23904623
4.  Pathological impact of SMN2 mis-splicing in adult SMA mice 
EMBO Molecular Medicine  2013;5(10):1586-1601.
Loss-of-function mutations in SMN1 cause spinal muscular atrophy (SMA), a leading genetic cause of infant mortality. The related SMN2 gene expresses suboptimal levels of functional SMN protein, due to a splicing defect. Many SMA patients reach adulthood, and there is also adult-onset (type IV) SMA. There is currently no animal model for adult-onset SMA, and the tissue-specific pathogenesis of post-developmental SMN deficiency remains elusive. Here, we use an antisense oligonucleotide (ASO) to exacerbate SMN2 mis-splicing. Intracerebroventricular ASO injection in adult SMN2-transgenic mice phenocopies key aspects of adult-onset SMA, including delayed-onset motor dysfunction and relevant histopathological features. SMN2 mis-splicing increases during late-stage disease, likely accelerating disease progression. Systemic ASO injection in adult mice causes peripheral SMN2 mis-splicing and affects prognosis, eliciting marked liver and heart pathologies, with decreased IGF1 levels. ASO dose–response and time-course studies suggest that only moderate SMN levels are required in the adult central nervous system, and treatment with a splicing-correcting ASO shows a broad therapeutic time window. We describe distinctive pathological features of adult-onset and early-onset SMA.
PMCID: PMC3799581  PMID: 24014320
adult-onset SMA; pathology; SMN2; spinal muscular atrophy; splicing
5.  Rescue of hearing and vestibular function in a mouse model of human deafness 
Nature medicine  2013;19(3):345-350.
Hearing impairment is the most common sensory disorder, with congenital hearing impairment present in ~1 in 1000 newborns1, and yet there is no cellular cure for deafness. Hereditary deafness is often mediated by the developmental failure or degeneration of cochlear hair cells2. Until now, it was not known whether such congenital failures could be mitigated by therapeutic intervention3-5. Here we show that hearing and vestibular function can be rescued in a mouse model of human hereditary deafness. An antisense oligonucleotide (ASO) was used to correct defective pre–mRNA splicing of transcripts from the mutated USH1C.216G>A gene, which causes human Usher syndrome (Usher), the leading genetic cause of combined deafness and blindness6,7. Treatment of neonatal mice with a single systemic dose of ASO partially corrects USH1C.216G>A splicing, increases protein expression, improves stereocilia organization in the cochlea, and rescues cochlear hair cells, vestibular function and hearing in mice. Our results demonstrate the therapeutic potential of ASOs in the treatment of deafness and provide evidence that congenital deafness can be effectively overcome by treatment early in development to correct gene expression.
PMCID: PMC3657744  PMID: 23380860
6.  Antisense-based therapy for the treatment of spinal muscular atrophy 
The Journal of Cell Biology  2012;199(1):21-25.
One of the greatest thrills a biomedical researcher may experience is seeing the product of many years of dedicated effort finally make its way to the patient. As a team, we have worked for the past eight years to discover a drug that could treat a devastating childhood neuromuscular disease, spinal muscular atrophy (SMA). Here, we describe the journey that has led to a promising drug based on the biology underlying the disease.
PMCID: PMC3461520  PMID: 23027901
7.  Manipulation of PK-M mutually exclusive alternative splicing by antisense oligonucleotides 
Open Biology  2012;2(10):120133.
Alternative splicing of the pyruvate kinase M gene involves a choice between mutually exclusive exons 9 and 10. Use of exon 10 to generate the M2 isoform is crucial for aerobic glycolysis (the Warburg effect) and tumour growth. We previously demonstrated that splicing enhancer elements that activate exon 10 are mainly found in exon 10 itself, and deleting or mutating these elements increases the inclusion of exon 9 in cancer cells. To systematically search for new enhancer elements in exon 10 and develop an effective pharmacological method to force a switch from PK-M2 to PK-M1, we carried out an antisense oligonucleotide (ASO) screen. We found potent ASOs that target a novel enhancer in exon 10 and strongly switch the splicing of endogenous PK-M transcripts to include exon 9. We further show that the ASO-mediated switch in alternative splicing leads to apoptosis in glioblastoma cell lines, and this is caused by the downregulation of PK-M2, and not by the upregulation of PK-M1. These data highlight the potential of ASO-mediated inhibition of PK-M2 splicing as therapy for cancer.
PMCID: PMC3498831  PMID: 23155487
alternative splicing; antisense oligonucleotides; cancer
9.  Antisense Oligonucleotides Delivered to the Mouse CNS Ameliorate Symptoms of Severe Spinal Muscular Atrophy 
Science translational medicine  2011;3(72):72ra18.
Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by mutations in the SMN1 gene that result in a deficiency of SMN protein. One approach to treat SMA is to use antisense oligonucleotides (ASOs) to redirect the splicing of a paralogous gene, SMN2, to boost production of functional SMN. Injection of a 2′-O-2-methoxyethyl–modified ASO (ASO-10-27) into the cerebral lateral ventricles of mice with a severe form of SMA resulted in splice-mediated increases in SMN protein and in the number of motor neurons in the spinal cord, which led to improvements in muscle physiology, motor function and survival. Intrathecal infusion of ASO-10-27 into cynomolgus monkeys delivered putative therapeutic levels of the oligonucleotide to all regions of the spinal cord. These data demonstrate that central nervous system–directed ASO therapy is efficacious and that intrathecal infusion may represent a practical route for delivering this therapeutic in the clinic.
PMCID: PMC3140425  PMID: 21368223
10.  Functional Coupling of Last-Intron Splicing and 3′-End Processing to Transcription In Vitro: the Poly(A) Signal Couples to Splicing before Committing to Cleavage▿ †  
Molecular and Cellular Biology  2007;28(2):849-862.
We have developed an in vitro transcription system, using HeLa nuclear extract, that supports not only efficient splicing of a multiexon transcript but also efficient cleavage and polyadenylation. In this system, both last-intron splicing and cleavage/polyadenylation are functionally coupled to transcription via the tether of nascent RNA that extends from the terminal exon to the transcribing polymerase downstream. Communication between the 3′ splice site and the poly(A) site across the terminal exon is established within minutes of their transcription, and multiple steps leading up to 3′-end processing of this exon can be distinguished. First, the 3′ splice site establishes connections to enhance 3′-end processing, while the nascent 3′-end processing apparatus makes reciprocal functional connections to enhance splicing. Then, commitment to poly(A) site cleavage itself occurs and the connections of the 3′-end processing apparatus to the transcribing polymerase are strengthened. Finally, the chemical steps in the processing of the terminal exon take place, beginning with poly(A) site cleavage, continuing with polyadenylation of the 3′ end, and then finishing with splicing of the last intron.
PMCID: PMC2223410  PMID: 17967872

Results 1-10 (10)