Mitochondrial Genetic Disorders : Challenges & Opportunities

Due to the close proximity of the mitochondrial genome to the respiratory chain complex producing ATP, it is believed to be more susceptible to get damaged by the reactive oxygen species (ROS) than nuclear DNA. In addition to this, the poorly developed mitochondrial DNA (mtDNA) repair machinery and the lack of protective histones, unlike in nuclear genome are also believed to be the major factors for high mutation rate [6-8].


Introduction
Mitochondria perform some of the most vital functions in the life cycle of a cell; it regulates thermogenesis, calcium homeostasis [1], it's the site for the ATP production and also plays a key role in apoptosis [2], which is a mechanism of programmed cell death.The proteins required for the successful functioning of the mammalian mitochondrial machinery are synthesized in two separate genomes.While, the majority of the mitochondrial proteins are synthesized in the nuclear genome, expressed in the cytoplasm and then gets transferred to the mitochondria by a complex protein import machinery [3,4]; few, yet extremely important proteins get synthesized and expressed exclusively in the mitochondrial matrix.So, it is quite imperative that any defect in the mitochondrial genome would result in the production of the faulty proteins inside the mitochondrial matrix and thus, would have a direct impact on the overall performance of the mitochondria.

The Mitochondrial Genome
The mitochondrial genome is a closed, circular double stranded DNA (dsDNA) molecule, with a size of about 16.5kb [5].It is devoid of introns and the only non-coding portion it has, expression of biochemical and clinical defects are observed.The maternal inheritance and stochastic segregation of mtDNA along with an incomplete knowledge of the mitochondrial genome transcription machinery further add to the complexity of mtDNA diseases [15,16].

Biological barriers
One important facet of targeted-delivery is the specific delivery of cargo to its intended location, followed by its action.In this case, we take one step forward and discuss the idea to develop a safe non-viral, nanocarrier system, which not only delivers the bio-molecules to the target cells but to the specific organelle of the cell, in this case, a mitochondrion.However, to achieve the same, there are several cellular & sub-cellular barriers that a delivery system has to overcome [17].The extracellular barriers include the stability of the nanocarrier in blood serum, ability to escape the reticuloendothelial system (RES), and binding to target cell types.Intracellularly, the nanocarrier must be able to escape the endo-lysosomal barrier, traffic its way inside the highly viscous cytosolic matrix, and binds to the target organelle mitochondrion.The final step in this process is overcoming the mitochondrial membrane barrier, and the release of bioactive molecules inside the mitochondrial matrix.In case of the mtDNA specific genes, they further needs to be taken up the mitochondrial genome for the successful production of a functional protein.

DNA delivery to mitochondria
To deliver DNA specifically to mitochondria, several nanocarrier-mediated targeting approaches have been developed & tested on cultured cells.The first approach is the use of delivery vehicles having mitochondrial affinity, such as DQAsomes, which are liposome-like vesicles [18].They have been successful in delivering DNA and short DNA-conjugated sequences to the mitochondria based on its DNA condensation and mitochondriotropic properties [19,20].Other technique was the utilization of mitochondriotropic cationic amphiphiles, like triphenyl phosphonium ion (TPP), which utilizes the high membrane potential of the mitochondrial membrane and has been shown to successfully deliver PNA oligomer to the mitochondria after conjugation to TPP [21].In a less toxic alternative, mitochondriotropic liposome's modified by amphiphilic cations displayed cargo delivery to mitochondria and a promise for gene delivery applications [22].The liposome's prepared with isolated mitochondrial fraction were also shown to deliver DNA to the mitochondria of mammalian cells [23].Based on the ability of certain lipids to fuse with the mitochondrial membranes, another class of nanocarrier, MITO-Porters and more recently dual function MITO-Porters, having both endosome and mitochondria fusogenic envelope showed the delivery of GFP to human cells [24,25].In all the above stated cases, though the system ensures intracellular delivery and subsequently promoted enhanced colocalization of the delivered DNA with mitochondria; whether the DNA actually got inside the organelle needs to be established.
In addition to this, the mitochondrial protein import pathway has also been exploited to direct DNA into the mitochondria [26].In one of the notable studies, the complex of mitochondrial targeting peptide conjugated to peptide nucleic acid (PNA), and then annealed to DNA oligonucleotide were shown to be capable of uptake by isolated mammalian mitochondria and the mitochondria of cultured mammalian cells, when permeabilized to allow cytosolic uptake [27].In protofection-based strategy, a complex of DNA loaded with mitochondrial transcription factor-A (TFAM), combined with mitochondrial targeting sequence (MTS) and protein transduction domain (TD) was successful in partially restoring mitochondrial functions for Parkinson's disease cybrid model cells [28].
More recently, a new set of nanoparticle delivery systems, engineered to specifically target mitochondria have been reported in recent studies.This includes biodegradable polymeric nanoparticle platforms, such as PLGA-PEG nanoparticles, surface modified with TPP moiety [29,30]; mitochondrial targeting gold peptides [31]; TPP conjugated poly (amidoamine) dendrimers [32]; multifunctional enveloped mesoporous silica nanoparticles (MSNs) modified with TPP, peptides and coated with pH-sensitive PEG-PLL (DMA) polymer [33]; rhodamine-based plasmid DNA nanoparticles [34] and functional DOX-nanoparticles prepared with folate-terminated polyrotaxanes (FPRs) and dequalinium (DQA) [35].These nanocarrier formulations represent some novel techniques delivering various small molecules, peptides & DNA to mitochondria and along with previously mentioned delivery systems offer promise and potential to routinely target mitochondria as a sub-cellular target.

Conclusion
Based on all the above-discussed factors, it is imperative that to further our understanding regarding the mitochondrial genome and for its plausible therapeutic application to treat its genetic disorders in the future, first and foremost, there is a need to develop robust mitochondria-targeted nanocarrier devices, which could deliver the therapeutic genes and drugs specifically & routinely to the mitochondria.Second, there is a need to develop sophisticated bioassays, which could actually establish the delivery of DNA inside the mitochondria.Lastly, there is also an urgent need to develop a mitochondria-specific functional reporter system for easy expression and identification inside mitochondria.