Summary 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). Patients harboring this expansion develop several unique histopathological hallmarks, including intranuclear foci composed of either sense or antisense RNA transcripts from the expanded repeats and dipeptide repeat proteins generated by non-canonical translation of the expanded RNA transcripts. To further 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 that are the hallmark of C9ORF72 ALS/FTD, including sense and antisense intranuclear RNA foci and poly(glycine-proline) dipeptides repeat proteins. Finally, using a synthetic 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.

The ability of Mycobacterium tuberculosis to infect, proliferate, and survive during long periods in the human lungs largely depends on the rigorous control of gene expression. Transcriptome-wide analyses are key to understanding gene regulation on a global scale. Here, we combine 5′-end-directed libraries with RNAseq expression libraries to gain insight into the transcriptome organization and post-transcriptional mRNA cleavage landscape in mycobacteria during log phase growth and under hypoxia, a physiologically relevant stress condition. Using the model organism Mycobacterium smegmatis , we identified 6,090 transcription start sites (TSSs) with high confidence during log phase growth, of which 67% were categorized as primary TSSs for annotated genes, and the remaining were classified as internal, antisense, or orphan, according to their genomic context. Interestingly, over 25% of the RNA transcripts lack a leader sequence, and of the coding sequences that do have leaders, 53% lack a strong consensus Shine-Dalgarno site. This indicates that like M. tuberculosis , M. smegmatis can initiate translation through multiple mechanisms. Our approach also allowed us to identify over 3,000 RNA cleavage sites, which occur at a novel sequence motif. To our knowledge, this represents the first report of a transcriptome-wide RNA cleavage site map in mycobacteria. The cleavage sites show a positional bias toward mRNA regulatory regions, highlighting the importance of post-transcriptional regulation in gene expression. We show that in low oxygen, a condition associated with the host environment during infection, mycobacteria change their transcriptomic profiles and endonucleolytic RNA cleavage is markedly reduced, suggesting a mechanistic explanation for previous reports of increased mRNA half-lives in response to stress. In addition, a number of TSSs were triggered in hypoxia, 56 of which contain the binding motif for the sigma factor SigF in their promoter regions. This suggests that SigF makes direct contributions to transcriptomic remodeling in hypoxia-challenged mycobacteria. Taken together, our data provide a foundation for further study of both transcriptional and posttranscriptional regulation in mycobacteria.

SignificanceChronic obstructive pulmonary disease affects 10% of the worldwide population, and the leading genetic cause is a genetic disease, α-1 antitrypsin (AAT) deficiency. Humans have only one gene that codes for the AAT protein, but mice have up to six, which made it impossible for decades to create a mouse model of the disease. Here we succeeded in creating this mouse model using CRISPR technology to target all of the mouse genes at once. Importantly, this mouse model spontaneously develops lung disease and recapitulates many aspects of the human disease. We anticipate that this model will be highly relevant not only to the preclinical development of therapeutics for AAT deficiency, but also to emphysema and smoking research.

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease caused by degeneration of motor neurons leading to rapidly progressive paralysis. About 10% of cases are caused by gain-of-function mutations that are transmitted as dominant traits. A potential therapy for these cases is to suppress the expression of the mutant gene. Here, we investigated silencing of SOD1, a gene commonly mutated in familial ALS, using an adeno-associated virus (AAV) encoding an artificial microRNA (miRNA) that targeted SOD1. In a superoxide dismutase 1 (SOD1)–mediated mouse model of ALS, we have previously demonstrated that SOD1 silencing delayed disease onset, increased survival time, and reduced muscle loss and motor and respiratory impairments. Here, we describe the preclinical characterization of this approach in cynomolgus macaques (Macaca fascicularis) using an AAV serotype for delivery that has been shown to be safe in clinical trials. We optimized AAV delivery to the spinal cord by preimplantation of a catheter and placement of the subject with head down at 30° during intrathecal infusion. We compared different promoters for the expression of artificial miRNAs directed against mutant SOD1. Results demonstrated efficient delivery and effective silencing of the SOD1 gene in motor neurons. These results support the notion that gene therapy with an artificial miRNA targeting SOD1 is safe and merits further development for the treatment of mutant SOD1-linked ALS.

Huntington’s disease is a devastating, incurable neurodegenerative disease affecting up to 12 per 100,000 patients worldwide. The disease is caused by a mutation in the Huntingtin (Htt) gene. There is interest in reducing mutant Huntingtin by targeting it at the mRNA level, but the maximum tolerable dose and long-term effects of such a treatment are unknown. Using a self-complementary AAV9 vector, we delivered a mir-155-based artificial miRNA under the control of the chicken β-actin or human U6 promoter. In mouse brain, the artificial miRNA reduced the human huntingtin mRNA by 50%. The U6, but not the CβA promoter, produced the artificial miRNA at supraphysiologic levels. Embedding the antisense strand in a U6-mir-30 scaffold reduced expression of the antisense strand but increased the sense strand. In mice treated with scAAV9-U6-mir-155-HTT or scAAV9-CβA-mir-155-HTT, activated microglia were present around the injection site 1 month post-injection. Six months post-injection, mice treated with scAAV9-CβA-mir-155-HTT were indistinguishable from controls. Those that received scAAV9-U6-mir-155-HTT showed behavioral abnormalities and striatal damage. In conclusion, miRNA backbone and promoter can be used together to modulate expression levels and strand selection of artificial miRNAs, and in brain, the CβA promoter can provide an effective and safe dose of a human huntingtin miRNA.

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The mechanisms and regulation of RNA degradation in mycobacteria have been subject to increased interest following the identification of interplay between RNA metabolism and drug resistance. Mycobacteria encode multiple ribonucleases that are predicted to participate in mRNA degradation and/or processing of stable RNAs. RNase E is an endoribonuclease hypothesized to play a major role in mRNA degradation due to its essentiality in mycobacteria and its role in mRNA degradation in gram-negative bacteria. Here, we defined the impact of RNase E on mRNA degradation rates transcriptome-wide in the non-pathogenic model Mycolicibacterium smegmatis. RNase E played a rate-limiting role in the degradation of at least 89% of protein-coding genes, with leadered transcripts generally being more affected by RNase E repression than leaderless transcripts. There was an apparent global slowing of transcription in response to knockdown of RNase E, suggesting that M. smegmatis regulates transcription in responses to changes in mRNA degradation. This compensation was incomplete, as the abundance of most transcripts increased upon RNase E knockdown. We assessed the sequence preferences for cleavage by RNase E transcriptome-wide in both M. smegmatis and M. tuberculosis, and found a consistent bias for cleavage in C-rich regions. Purified RNase E had a clear preference for cleavage immediately upstream of cytidines, distinct from the sequence preferences of RNase E in gram-negatives. We furthermore report a high-resolution map of mRNA cleavage sites in M. tuberculosis, which occur primarily within the RNase E-preferred sequence context, confirming RNase E as a broad contributor to M. tuberculosis transcriptome structure.