Peptides vs. Polymers: Searching for the Most Efficient Delivery System for Mitochondrial Gene Therapy

Abstract

Together with the nucleus, the mitochondrion has its own genome. Mutations in mitochondrial DNA are responsible for a variety of disorders, including neurodegenerative diseases and cancer. Current therapeutic approaches are not effective. In this sense, mitochondrial gene therapy emerges as a valuable and promising therapeutic tool. To accomplish this goal, the design/development of a mitochondrial-specific gene delivery system is imperative. In this work, we explored the ability of novel polymer- and peptide-based systems for mitochondrial targeting, gene delivery, and protein expression, performing a comparison between them to reveal the most adequate system for mitochondrial gene therapy. Therefore, we synthesized a novel mitochondria-targeting polymer (polyethylenimine–dequalinium) to load and complex a mitochondrial-gene-based plasmid. The polymeric complexes exhibited physicochemical properties and cytotoxic profiles dependent on the nitrogen-to-phosphate-group ratio (N/P). A fluorescence confocal microscopy study revealed the mitochondrial targeting specificity of polymeric complexes. Moreover, transfection mediated by polymer and peptide delivery systems led to gene expression in mitochondria. Additionally, the mitochondrial protein was produced. A comparative study between polymeric and peptide/plasmid DNA complexes showed the great capacity of peptides to complex pDNA at lower N/P ratios, forming smaller particles bearing a positive charge, with repercussions on their capacity for cellular transfection, mitochondria targeting and, ultimately, gene delivery and protein expression. This report is a significant contribution to the implementation of mitochondrial gene therapy, instigating further research on the development of peptide-based delivery systems towards clinical translation.

Emerging Landscape of Cell-Penetrating Peptide-Mediated Organelle Restoration and Replacement

“…Both invasive and noninvasive strategies have been used to facilitate the delivery of therapeutic agent to intracellular organelles. Invasive techniques such as electroporation, biolistic transfections, sonoporation, and microfluidic device-based electroporation may cause damage to the cell membranes, − while noninvasive strategies such as nanoparticles, liposomes and quantum dots suffer from poor bioavailability, low efficiency and poor specificity when administered for macromolecules delivery. − Notably, CPP, as a noninvasive drug delivery system, meets stringent requirements to ensure efficient and targeted drug delivery to intracellular organelles with high biocompatibility and low side effects. , …”

Delivery Systems for Mitochondrial Gene Therapy: A Review

“…Many of the dysfunctions that occur in mitochondria come from mutations in their genome, thus gene therapy emerges as a very promising approach to the treatment of mitochondrial diseases. Mitochondrial gene therapy arises from the need to find treatments for mitochondrial dysfunctions, as the solutions available on the market only serve to alleviate the symptoms and do not provide an effective cure [ 87 ]. The main advantage of this approach, to conventional treatments, is that it focuses the problem on its origin replacing the mutated mitochondrial gene and restoring mitochondria function.…”

“…Moreover, these delivery systems showed greater mitochondria targeting ability depending on the PEI molecular weight and the PEI/pDNA ratio used. The internalization of these PEI–DQA/TAT/pND1 polymeric systems in the mitochondria was demonstrated through confocal microscopy followed by mRNA evaluation (RT-PCR), and quantification of ND1 protein expression (through a specific kit) within the mitochondrial cell extracts [ 87 ].…”

“…The expression and quantification of ND1 and GFP proteins were demonstrated by these researchers using an Elisa kit, proving that the developed systems promote gene and protein expression, a relevant contribution to mitochondrial gene therapy [ 124 ]. The same group also compared PEI-DQA/TAT systems with carriers based on peptides modified with a mitochondrial-targeting sequence (MTS), also designed/developed to create new strategies for mitochondrial gene delivery and expression [ 87 , 104 ]. The peptides used were cell-penetrating peptides (CPP) conjugated to MTS that encapsulated the mitochondrial gene ND1 plasmid DNA (MTS-CPP-pND1) [ 104 ].…”

“…This fact contributes to the physicochemical properties exhibited by MTS-CPP-pND1 such as small size and high zeta potential, which resulted in higher cellular uptake, mitochondria targeting, and accumulation when compared to the PEI-DQA/TAT systems. Furthermore, cells transfected with the peptide-based systems demonstrated higher ND1 gene expression, and, consequently, the respective protein was produced to a higher extent compared to those observed with the PEI-DQA/TAT/pND1 systems [ 87 ]. These examples of mitochondrial ND1 gene delivery systems are depicted in Figure 5 .…”