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Phosphorodiamidate morpholino oligomers suppress mutant huntingtin expression and attenuate neurotoxicity.

ABSTRACT: Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin (HTT) gene. Disease pathogenesis derives, at least in part, from the long polyglutamine tract encoded by mutant HTT. Therefore, considerable effort has been dedicated to the development of therapeutic strategies that significantly reduce the expression of the mutant HTT protein. Antisense oligonucleotides (ASOs) targeted to the CAG repeat region of HTT transcripts have been of particular interest due to their potential capacity to discriminate between normal and mutant HTT transcripts. Here, we focus on phosphorodiamidate morpholino oligomers (PMOs), ASOs that are especially stable, highly soluble and non-toxic. We designed three PMOs to selectively target expanded CAG repeat tracts (CTG22, CTG25 and CTG28), and two PMOs to selectively target sequences flanking the HTT CAG repeat (HTTex1a and HTTex1b). In HD patient-derived fibroblasts with expanded alleles containing 44, 77 or 109 CAG repeats, HTTex1a and HTTex1b were effective in suppressing the expression of mutant and non-mutant transcripts. CTGn PMOs also suppressed HTT expression, with the extent of suppression and the specificity for mutant transcripts dependent on the length of the targeted CAG repeat and on the CTG repeat length and concentration of the PMO. PMO CTG25 reduced HTT-induced cytotoxicity in vitro and suppressed mutant HTT expression in vivo in the N171-82Q transgenic mouse model. Finally, CTG28 reduced mutant HTT expression and improved the phenotype of Hdh(Q7/Q150) knock-in HD mice. These data demonstrate the potential of PMOs as an approach to suppressing the expression of mutant HTT.

SUBMITTER: Sun X

PROVIDER: S-EPMC4222366 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Phosphorodiamidate morpholino oligomers suppress mutant huntingtin expression and attenuate neurotoxicity.

Sun Xin X Marque Leonard O LO Cordner Zachary Z Pruitt Jennifer L JL Bhat Manik M Li Pan P PP Kannan Geetha G Ladenheim Ellen E EE Moran Timothy H TH Margolis Russell L RL Rudnicki Dobrila D DD

Human molecular genetics 20140704 23

Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin (HTT) gene. Disease pathogenesis derives, at least in part, from the long polyglutamine tract encoded by mutant HTT. Therefore, considerable effort has been dedicated to the development of therapeutic strategies that significantly reduce the expression of the mutant HTT protein. Antisense oligonucleotides (ASOs) targeted to the CAG repeat region of HTT transcripts have been ...[more]

PMID: 25035419

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Project description:In late 2015, the first example of a transferrable polymyxin resistance mechanism in Gram-negative pathogens, MCR-1, was reported. Since that report, MCR-1 has been described to occur in many Gram-negative pathogens, and the mechanism of MCR-1-mediated resistance was rapidly determined: an ethanolamine is attached to lipid A phosphate groups, rendering the membrane more electropositive and repelling positively charged polymyxins. Acquisition of MCR-1 is clinically significant because polymyxins are frequently last-line antibiotics used to treat extensively resistant organisms, so acquisition of this mechanism might lead to pan-resistant strains. Therefore, the ability to inhibit MCR-1 and restore polymyxin sensitivity would be an important scientific advancement. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) are antisense molecules that were designed to target mRNA, preventing translation. Peptide conjugation enhances cellular entry, but they are positively charged, so we tested our lead antibacterial PPMOs by targeting an essential Escherichia coli gene, acpP, and demonstrated that they were still effective in mcr-1-positive E. coli strains. We then designed and synthesized two PPMOs targeted to mcr-1 mRNA. Five clinical mcr-1-positive E. coli strains were resensitized to polymyxins by MCR-1 inhibition, reducing MICs 2- to 16-fold. Finally, therapeutic dosing of BALB/c mice with MCR-1 PPMO combined with colistin in a sepsis model reduced morbidity and bacterial burden in the spleen at 24 h and offered a survival advantage out to 5 days. This is the first example of a way to modulate colistin resistance with an antisense approach and may be a viable strategy to combat this globally emerging antibiotic resistance threat.IMPORTANCE Polymyxin use has been increasing as a last line of defense against Gram-negative pathogens with high-level resistance mechanisms, such as carbapenemases. The recently described MCR-1 is a plasmid-mediated mechanism of resistance to polymyxins. MCR-1 is currently found in Gram-negative organisms already possessing high-level resistance mechanisms, leaving clinicians few or no antibacterial options for infections caused by these strains. This study utilizes antisense molecules that target mRNA, preventing protein translation. Herein we describe antisense molecules that can be directly antibacterial because they target genes essential to bacterial growth or blockade of MCR-1, restoring polymyxin sensitivity. We also demonstrate that MCR-1 antisense molecules restore the efficacies of polymyxins in mouse models of E. coli septicemia. Considering all things together, we demonstrate that antisense molecules may be effective therapeutics either alone when they target an essential gene or combined with antibiotics when they target specific resistance mechanisms, such as those seen with MCR-1.

Dataset Information

Phosphorodiamidate morpholino oligomers suppress mutant huntingtin expression and attenuate neurotoxicity.

Publications

Phosphorodiamidate morpholino oligomers suppress mutant huntingtin expression and attenuate neurotoxicity.

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