Mitochondrial Gene Therapy: Development of a mitochondrial targeted peptide/plasmid DNA vector

Publications

Publication . Faria, Rúben Miguel Ribeiro ; Costa, Diana Rita Barata; Sousa, Ângela Maria Almeida de; Boisguérin, Prisca

Mitochondria are cellular organelles measuring approximately 1 micron that can be found in large numbers in eukaryotic cells. These small organelles play a crucial role in cellular activity, being essential in intracellular signaling processes, apoptosis mechanisms, and energy production, among others. Mitochondria generate 90% of all energy consumed in cells, through the oxidative phosphorylation system that produces energy in the form of adenosine triphosphate (ATP) molecules. Mitochondria, similar to the nucleus, have their own genome, called mitochondrial DNA (mtDNA). Human mtDNA is composed of double-stranded circular DNA molecules, with each strand having its own composition and encoding different ribonucleic acids (RNA). The guanine-rich strand encodes 14 tRNAs, 2 rRNAs, and 12 polypeptides, while the lighter strand has information to transcribe only 8 tRNAs and one polypeptide. In total, mtDNA consists of just 37 genes that encode 13 mRNA, giving rise to 13 proteins that are part of the electron transport system and the ATPase complex. The oxidative phosphorylation system is composed of 5 complexes (NADH-ubiquinone reductase complex (complex I); succinate dehydrogenase complex (complex II); ubiquinol-cytochrome c oxidoreductase (complex III); cytochrome-C oxidase complex (complex IV) and ATP synthase (complex V)), which form the respiratory chain. mtDNA is much more susceptible to mutations when compared to the nuclear genome. Changes in mtDNA compromise the normal functioning of cells, mainly affecting neuronal and muscle tissues. The higher frequency of mutations in mtDNA can be explained by the fact that it does not have telomeres or introns in its constitution. Mitochondrial dysfunctions lead to the emergence of multisystem diseases, which can affect the normal functioning of the immune response, motor and brain function, and metabolic regulation and lead to aging. The vast majority of pathologies originating from mitochondria are inherited from maternal mtDNA. However, environmental factors such as stress and the consequent presence of reactive oxygen species, also contribute to the emergence of mutations in mtDNA. The most common mitochondrial diseases are Leber's hereditary optic neuropathy (LHON), mitochondrial encephalomyopathy, Pearson's syndrome, Parkinson's, Huntington's disease, Alzheimer's, and some types of cancer (breast, kidney, and colorectal). Complex I of the mitochondrial respiratory chain is the main entry point for electrons into the electron transport chain. Due to this fact, this complex is very important in the normal functioning of mitochondria. It is in complex I that the transfer of electrons from Nicotinamide Adenine Dinucleotide + Hydrogen (NADH) to ubiquinone occurs, the transport of protons across the inner mitochondrial membrane and is the main source of reactive oxygen species (ROS). Mutations in mitochondrial genes responsible for structural and assembly proteins of this complex lead to increased ROS production and loss of functions. One of these genes is the mitochondrial gene ND1 (NADH dehydrogenase 1). The mt-ND1 protein plays a crucial role in the structure of complex I. Mutations in mt-ND1 are associated with the emergence of LHON; Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS); progressive cardiomyopathy, and some types of cancer. Data from 2020 revealed that 1 in every 250 people have mutations in mtDNA and that 1 in every 5,000 have serious pathologies associated with mitochondrial dysfunction. However, currently, the medications available on the market only serve to mitigate the symptoms. No drug approved by the FDA or in development has been able to cure or slow the progression of mitochondrial diseases. Although there are approaches such as the use of antioxidant agents and other drugs to alleviate symptoms, the ineffectiveness of current medications highlights the urgent need for more effective treatments. Mitochondrial gene therapy is a promising approach that can focus its action directly on the cause of mitochondrial diseases and develop therapies tailored to the type of mutation. Gene therapy consists of the application of recombinant DNA techniques in which functional genes are used to replace defective genes and restore their normal functioning. As most mitochondrial diseases originate from mutations in mtDNA, mitochondrial gene therapy appears as a very promising strategy for treating this type of disease. Mitochondrial gene therapy makes it possible to attack the problem at its source and restore normal function to the affected mitochondrial gene. However, this type of therapy needs delivery systems that are effective in protecting and delivering genetic material to target cells/organelles. The greatest difficulty in applying gene therapy has been the development of nanocarriers that can effectively deliver genetic material. For mitochondrial gene therapy, the difficulty has been even greater, as the systems need to cross more barriers and be able to deliver only to that organelle. Thus, the main objective of this thesis is to develop delivery systems that have an affinity for mitochondria and can effectively deliver mitochondrial genes for the treatment of mitochondria-associated pathologies. The work carried out consisted of the development of delivery systems based on peptides (cell-penetrating peptides (CPP)) and polymers (polyethylenimine (PEI)), to deliver the mitochondrially encoded NADH dehydrogenase 1 protein (ND1) gene. To achieve this, these delivery systems were functionalized with ligands that allow specific targeting of mitochondria. The ligands used were triphenylphosphonium (TPP) and dequalinium chloride (DQA) to functionalize PEI and for CPP a mitochondrial targeting sequence (MTS) was used. The first step for the PEI-TPP/pND1 polymeric systems was to evaluate, through an experimental design, the optimal conditions for the formulation of nanoparticles. These systems were then characterized in terms of size, surface charge, and morphology. These delivery systems demonstrated the ability to internalize into cells and, through confocal microscopy, their preferential accumulation in mitochondria was demonstrated. Furthermore, these systems have demonstrated the ability to deliver the ND1 gene to mitochondria and lead to its transcription. The PEI-DQA/pND1 polymeric systems developed also demonstrated excellent physicochemical properties, showing the ability to transfect and internalize into cells. These nanocarriers delivered the ND1 gene directly into the mitochondria, leading to transcription of the gene of interest and production of the ND1 protein. However, the peptide-based systems (MTS-CPP) exhibited superior performance in terms of cellular internalization and targeting to mitochondria. Its greater ability to complex pND1 led to the formulation of nanoparticles with smaller sizes and consequently greater delivery of the gene of interest and protein expression. Showing better in vitro results, the MTS-CPP systems were tested in in vivo models (zebrafish embryos (ZF)). The peptide systems demonstrate the ability to internalize and distribute throughout the ZF organism, without causing any toxicity in these in vivo models. In short, the work carried out during this doctoral thesis sought to find solutions to the lack of effective delivery systems in mitochondrial gene therapy, to make this therapy viable for the treatment of mitochondrial diseases. The results obtained during the thesis demonstrate that the delivery systems developed are very promising for the development of mitochondrial gene therapy protocols. This work contributed to progress and innovation in an area of research that is still little explored, such as mitochondrial gene therapy. In the case of peptide-based systems, these systems have the potential to be considered in future investigations, to evaluate their translation to the clinic. The nanocarriers developed during this thesis were optimized for the delivery of the mitochondrial ND1 gene, however, these systems can be easily adapted for the delivery of any mitochondrial genes that are involved in pathologies associated with mtDNA mutations.

2025-04-14Doctoral thesis

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