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Author
Palomeras Martínez, Oriol
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Abstract
Duchenne Muscular Dystrophy (DMD) is a severe progressive disease that affects muscle tissue, leading to its continuous degradation. It’s caused by mutation in the DMD gene, one of the largest in the genome, which codes for a protein called dystrophin. The function of dystrophin is to link the cytoskeleton of cells to the extracellular matrix, conferring mechanical resistance to the muscle cells. Nowadays there are no treatments that are able to completely cure the disease, with current ones only being palliative or temporal in nature.
One of the most promising fields aiming to find a cure for DMD is gene therapy. Shortly, by replacing the defective copy of the dystrophin gene by a healthy version an active form of dystrophin should be produced, which would help the patients get a normal lifestyle.
One strategy for gene therapy uses Adeno-associated viruses (AAV) as carriers of the therapeutic gene to deliver it inside the cells. But their main drawback is the widespread presence of neutralizing antibodies against these same viruses, which greatly diminish the effectivity of the treatment. A proposed solution currently under investigation is the use of Oligopeptide-modified PBAE (OM-PBAE) to coat the AAV particles and shield them from the nAbs.
Needing to study how this polymeric coating affects the internalization mechanism of the nanovectors there’s a need to generate new tools for these studies. So, first both the AAV and the polymer were labelled with fluorescent probes (FITC and Cy3, respectively). Then the capacity of the polymer to coat the viral particles was put to the test through several ways, like the presence of FRET or its capacity to change the transduction efficiency of nanovectors and shield them from neutralizing antibodies. To finish, studies about how the internalization of naked and coated nanovectors changes were also done.
In conclusion, we have generated the needed tools to study the internalization of nanovectors, we have concluded that the FITC labelling occupies amine groups, which disables the formation of the polymeric coating, and have demonstrated that the presence of a polymeric coating delays and changes the internalization of the nanovectors. Also, the results confirm that both the FITC and the polymeric coating use amines as reactive groups to bind to the viral capsid.
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