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Systemic Injection of Peptide-PMOs into Humanized DMD Mice and Detection by RT-PCR and ELISA
Dyanna Melo1, Rika Maruyama1, Toshifumi Yokota2,3
1Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada.
Abstract:
Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder due to the lack of dystrophin production. The disease is characterized by muscle wasting, with the most common causes of death being respiratory failure or heart failure. Recently, exon skipping via phosphorodiamidate morpholino oligomers (PMOs) are used as an FDA approved treatment for DMD. Peptide-conjugated PMOs (PPMOs) are used to increase exon skipping efficacy in the heart and are a promising therapy for DMD. Researchers have previously relied on high-performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC/MS) methods for detecting PPMO uptake, but an enzyme-linked immunosorbent assay (ELISA) has been shown to have greater sensitivity. Here, we present methodologies to determine the uptake efficiency of a PPMO into the heart and efficacy of exon 51 skipping by a PPMO injected retro-orbitally into a humanized DMD mouse model via ELISA and RT-PCR, respectively.
Insights
Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) show promise for Duchenne muscular dystrophy (DMD). This study presents new methods to measure PPMO uptake and exon skipping efficacy in a DMD mouse model.
Area of Science:
- Biochemistry
- Genetics
- Pharmacology
Background:
- Duchenne muscular dystrophy (DMD) is a severe X-linked disorder caused by dystrophin deficiency, leading to muscle degeneration.
- Current treatments include exon skipping with phosphorodiamidate morpholino oligomers (PMOs), with peptide-conjugated PMOs (PPMOs) enhancing cardiac efficacy.
- Accurate measurement of PPMO uptake and exon skipping is crucial for therapeutic development.
Purpose of the Study:
- To establish sensitive methodologies for quantifying PPMO uptake in the heart.
- To assess the efficacy of exon 51 skipping induced by PPMOs in a humanized DMD mouse model.
- To compare the sensitivity of ELISA with traditional methods for PPMO detection.
Main Methods:
- Development and application of an enzyme-linked immunosorbent assay (ELISA) to measure PPMO cardiac uptake.
- Utilizing reverse transcription polymerase chain reaction (RT-PCR) to quantify exon 51 skipping efficiency.
- Employing a retro-orbital injection route in a humanized DMD mouse model.
Main Results:
- ELISA demonstrated high sensitivity for detecting PPMO uptake in cardiac tissue.
- RT-PCR confirmed successful and measurable exon 51 skipping.
- The developed methodologies provide a robust system for evaluating PPMO-based therapies.
Conclusions:
- Sensitive ELISA and RT-PCR methods enable precise evaluation of PPMO therapeutic strategies for DMD.
- These techniques are vital for advancing PPMO development and optimizing treatment for DMD patients.
- The study highlights the potential of PPMOs as a targeted therapy for cardiac complications in DMD.
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