Comparison of scAAV9-CßA and scAAV9-U6 driven artificial miRNAs targeting human huntingtin

ABSTRACT: 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 ß-actin or human U6 promoter. In mouse brain, the artificial miRNA reduced the human huntingtin mRNA by 50%. The U6, but not the CßA 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-CßA-mir-155-HTT, activated microglia were present around the injection site one-month post-injection. Six-months post-injection, mice treated with scAAV9-CßA-mir-155-HTT were indistinguishable from controls. Those that received scAAV9-U6-mir-155-HTT showed behavioural 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 CßA promoter can provide an effective and safe dose of a human huntingtin. We also performed RNAseq on total RNA. In the mice injected with AAV9-U6-anti-HTT-6433, 44 transcripts were significantly downregulated while 30 were significantly upregulated when compared to the naïve control. In the AAV-CßA-anti-HTT-6433 group, 12 transcripts were downregulated and 4 were upregulated. Overall, transcripts containing a seed sequence target are downregulated in the scAAV-U6-anti-HTT-6433 group but not the scAAV9-CßA-anti-HTT-6433 group.

ORGANISM(S): Mus musculus

PROVIDER: GSE97353 | GEO | 2017/04/24

SECONDARY ACCESSION(S): PRJNA381487

REPOSITORIES: GEO