BACKGROUND: Amyotrophic Lateral Sclerosis (ALS) is a fatal and progressive neurodegenerative disorder with identified genetic causes representing a significant minority of all cases. A GGGGCC hexanucleotide repeat expansion (HRE) mutation within the C9ORF72 gene has recently been identified as the most frequent known cause of ALS. The expansion leads to partial heterochromatinization of the locus, yet mutant RNAs and dipeptide repeat proteins (DPRs) are still produced in sufficient quantities to confer neurotoxicity. The levels of these toxic HRE products positively correlate with cellular toxicity and phenotypic severity across multiple disease models. Moreover, the degree of epigenetic repression inversely correlates with some facets of clinical presentation in C9-ALS patients. Recently, bacterial artificial chromosomes (BAC) have been used to generate transgenic mice that harbor the HRE mutation, complementing other relevant model systems such as patient-derived induced pluripotent stem cells (iPSCs). While epigenetic features of the HRE have been investigated in various model systems and post-mortem tissues, epigenetic dysregulation at the expanded locus in C9-BAC mice remains unexplored. METHODS AND RESULTS: Here, we sought to determine whether clinically relevant epigenetic perturbations caused by the HRE are mirrored in a C9-BAC mouse model. We used complementary DNA methylation assessment and immunoprecipitation methods to demonstrate that epigenetic aberrations caused by the HRE, such as DNA and histone methylation, are recapitulated in the C9-BAC mice. Strikingly, we found that cytosine hypermethylation within the promoter region of the human transgene occurred in a subset of C9-BAC mice similar to what is observed in patient populations. Moreover, we show that partial heterochromatinization of the C9 HRE occurs during the first weeks of the mouse lifespan, indicating age-dependent epigenetic repression. Using iPSC neurons, we found that preventing R-loop formation did not impede heterochromatinization of the HRE. CONCLUSIONS: Taken together, these observations provide further insight into mechanism and developmental time-course of epigenetic perturbations conferred by the C9ORF72 HRE. Finally, we suggest that epigenetic repression of the C9ORF72 HRE and nearby gene promoter could impede or delay motor neuron degeneration in C9-BAC mouse models of ALS/FTD.

OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of motor neurons, resulting in progressive muscle weakness, paralysis, and death within 5 years of diagnosis. About 10% of cases are inherited, of which 20% are due to mutations in the superoxide dismutase 1 (SOD1) gene. Riluzole, the only US Food and Drug Administration-approved ALS drug, prolongs survival by only a few months. Experiments in transgenic ALS mouse models have shown decreasing levels of mutant SOD1 protein as a potential therapeutic approach. We sought to develop an efficient adeno-associated virus (AAV)-mediated RNAi gene therapy for ALS. METHODS: A single-stranded AAV9 vector encoding an artificial microRNA against human SOD1 was injected into the cerebral lateral ventricles of neonatal SOD1(G93A) mice, and impact on disease progression and survival was assessed. RESULTS: This therapy extended median survival by 50% and delayed hindlimb paralysis, with animals remaining ambulatory until the humane endpoint, which was due to rapid body weight loss. AAV9-treated SOD1(G93A) mice showed reduction of mutant human SOD1 mRNA levels in upper and lower motor neurons and significant improvements in multiple parameters including the numbers of spinal motor neurons, diameter of ventral root axons, and extent of neuroinflammation in the SOD1(G93A) spinal cord. Mice also showed previously unexplored changes in pulmonary function, with AAV9-treated SOD1(G93A) mice displaying a phenotype reminiscent of patient pathophysiology. INTERPRETATION: These studies clearly demonstrate that an AAV9-delivered SOD1-specific artificial microRNA is an effective and translatable therapeutic approach for ALS.

Neurologic diseases tend to target various areas of the central nervous system (CNS) and can therefore result in paralysis, dementia, and death. Neurodegenerative diseases distinguish themselves from other diseases by affecting nerve cells, which unlike many other cells in our body cannot regenerate when severely injured. The discovery of RNA interference (RNAi) has enabled scientist to design new therapeutic approaches based on specific gene silencing rather than the canonical gene therapy through gene augmentation. Two types of molecules can be used for viral vector-mediated gene silencing: short hairpin RNAs (shRNAs) and artificial microRNAs (miRNAs) that have the ability to enter the RNAi pathway. Although both shRNAs and miRNAs can be used to silence genes, they enter the RNAi pathway at different points. Unlike shRNAs, miRNAs require an additional cleavage step inside the nucleus before being exported to the cytoplasm. These molecules can then be incorporated into the RNA-induced silencing complex (RISC) which utilizes sequence complementarity to recognize target mRNAs and activate either translational repression, in the case of partial complementarity, or induce mRNA cleavage in the case of complete complementarity. Elevated amounts of shRNAs, which are commonly driven by strong polymerase III promoters, can cause saturation of the endogenous RNAi machinery due to competition between endogenous and artificial molecules. Switching to a DNA polymerase II promoter is an alternative to reduce shRNA production, thereby reducing toxicity. Even though the molecules are designed to target specific mRNAs there may be off-target effects due to nonspecific binding that must be accounted for during the design process. In this chapter we discuss the design and in vitro screening of shRNAs and artificial miRNAs.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease; survival in ALS is typically 3-5 years. No treatment extends patient survival by more than three months. Approximately 20% of familial ALS and 1-3% of sporadic ALS patients carry a mutation in the gene encoding superoxide dismutase 1 (SOD1). In a transgenic ALS mouse model expressing the mutant SOD1(G93A) protein, silencing the SOD1 gene prolongs survival. One study reports a therapeutic effect of silencing the SOD1 gene in systemically treated adult ALS mice; this was achieved with a short hairpin RNA, a silencing molecule that has raised multiple safety concerns, and recombinant adeno-associated virus (rAAV) 9. We report here a silencing method based on an artificial microRNA termed miR-SOD1 systemically delivered using adeno-associated virus rAAVrh10, a serotype with a demonstrated safety profile in CNS clinical trials. Silencing of SOD1 in adult SOD1(G93A) transgenic mice with this construct profoundly delayed both disease onset and death in the SOD1(G93A) mice, and significantly preserved muscle strength and motor and respiratory functions. We also document that intrathecal delivery of the same rAAVrh10-miR-SOD1 in nonhuman primates significantly and safely silences SOD1 in lower motor neurons. This study supports the view that rAAVrh10-miR-SOD1 merits further development for the treatment of SOD1-linked ALS in humans.

A non-coding hexanucleotide repeat expansion in the C9ORF72 gene is the most common mutation associated with familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). To investigate the pathological role of C9ORF72 in these diseases, we generated a line of mice carrying a bacterial artificial chromosome containing exons 1 to 6 of the human C9ORF72 gene with approximately 500 repeats of the GGGGCC motif. The mice showed no overt behavioral phenotype but recapitulated distinctive histopathological features of C9ORF72 ALS/FTD, including sense and antisense intranuclear RNA foci and poly(glycine-proline) dipeptide repeat proteins. Finally, using an artificial microRNA that targets human C9ORF72 in cultures of primary cortical neurons from the C9BAC mice, we have attenuated expression of the C9BAC transgene and the poly(GP) dipeptides. The C9ORF72 BAC transgenic mice will be a valuable tool in the study of ALS/FTD pathobiology and therapy.