Peptide-Functionalized Dendrimer Nanocarriers for Targeted Microdystrophin Gene Delivery.

José Manuel Condor Capcha,Jessica Hersh,Avinash Kaur,Mohit Singh,Sapna K. Deo,Mauricio Noguera,Joaquín J. Jiménez,Camila Iansen Irion,Guerline Lambert,Emre Dikici,Lina A. Shehadeh,Sylvia Daunert

doi:10.3390/pharmaceutics13122159

José Manuel Condor Capcha, Jessica Hersh + Show 10 more

Open Access

https://doi.org/10.3390/pharmaceutics13122159

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Abstract

Gene therapy is a good alternative for determined congenital disorders; however, there are numerous limitations for gene delivery in vivo including targeted cellular uptake, intracellular trafficking, and transport through the nuclear membrane. Here, a modified G5 polyamidoamine (G5 PAMAM) dendrimer–DNA complex was developed, which will allow cell-specific targeting to skeletal muscle cells and transport the DNA through the intracellular machinery and the nuclear membrane. The G5 PAMAM nanocarrier was modified with a skeletal muscle-targeting peptide (SMTP), a DLC8-binding peptide (DBP) for intracellular transport, and a nuclear localization signaling peptide (NLS) for nuclear uptake, and polyplexed with plasmid DNA containing the GFP-tagged microdystrophin (µDys) gene. The delivery of µDys has been considered as a therapeutic modality for patients suffering from a debilitating Duchenne muscular dystrophy (DMD) disorder. The nanocarrier–peptide–DNA polyplexes were prepared with different charge ratios and characterized for stability, size, surface charge, and cytotoxicity. Using the optimized nanocarrier polyplexes, the transfection efficiency in vitro was determined by demonstrating the expression of the GFP and the µDys protein using fluorescence and Western blotting studies, respectively. Protein expression in vivo was determined by injecting an optimal nanocarrier polyplex formulation to Duchenne model mice, mdx4Cv. Ultimately, these nanocarrier polyplexes will allow targeted delivery of the microdystrophin gene to skeletal muscle cells and result in improved muscle function in Duchenne muscular dystrophy patients.

Highlights

Many diseases are caused by single-gene mutations [1,2]
The charge ratios are defined as the ratio between the four components based on their surface charges and are written as G5 PAMAM–skeletal muscle-targeting peptide (SMTP):DLC8-binding peptide (DBP):nuclear localization signaling peptide (NLS):pμDys
The polyplexes with a charge ratio of 5:0.5:0.5:1 (G5-PAMAM–SMTP/DBP/NLS/pμDys) and greater were able to fully bind pμDys. This is indicated by the fact that the polyplex, when run on an agarose gel, did not migrate much further from the loading well when compared to free unbound pμDys, which is able to migrate on the agarose gel. This shows that the plasmid encoding the μDys gene was successfully loaded onto the PAMAM dendrimer nanocarrier (Figure 2a)

Summary

Introduction

Many diseases are caused by single-gene mutations [1,2]. Duchenne muscular dystrophy (DMD) is a devastating congenital disorder caused by an X-linked recessive mutation in the DMD gene, inducing an absence or decreased expression of dystrophin in skeletal muscle and cardiac muscle. The dystrophin protein links the cytoskeleton of muscle fibers to the extracellular matrix and serves as an important support protein in skeletal muscle cells [3]. The first signs of muscle weakness are detected in individuals, prominently males, when they are still very young. These patients, have a poor quality of life and an average life expectancy of 19 years [4]

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