Mitochondriotropic Cationic Vesicles A Strategy Towards Mitochondrial Gene Therapy

Abstract

The number of diseases found to be associated with defects of the mitochondrial genome has grown significantly over the last decade. Despite major advances in understanding mtDNA defects at the genetic and biochemical level, there is no satisfactory treatment for the vast majority of patients available. This is largely due to the fact that almost all mitochondrial DNA defects involve the final common pathway of oxidative metabolism making it impossible to bypass the defect by giving alternative metabolic carriers of energy. These seemingly objective limitations of conventional biochemical treatment for patients with detects of mtDNA warrant the exploration of gene therapeutic approaches. However, mitochondrial gene therapy still appears only theoretical and speculative. Any possibility for gene replacement is dependent on the use of a yet unavailable mitochondria-specific transfection vector. Based upon an analysis of the self-assembly behavior of dequalinium, a cationic single-chain bolaamphiphile which is known to selectively accumulate in mitochondria, we have developed a whole new strategy for mitochondria-specific DNA delivery. We have succeeded in preparing vesicles made of dequalinium, which we termed DQAsomes (U.S. Patent 6,090,619). We have shown that DQAsomes efficiently bind and protect DNA and we could demonstrate that DQAsome/DNA complexes selectively release DNA at cardiolipin-rich liposomes mimicking both, the inner and the outer mitochondrial membrane. Based on the intrinsic property of dequalinium to preferentially accumulate in mitochondria in response to the electrochemical gradient at the mitochondrial membrane and based on the selective DNA release at mitochondria-like membranes we propose DQAsomes as the first mitochondria-specific vector to deliver DNA to mitochondria in living cells.

From Serendipity to Mitochondria-Targeted Nanocarriers

“…Electron microscopic images of DQAsome-DNA complexes, i.e. DQAplexes (27), strikingly resembled the "spaghetti-and-meatball" shapes observed earlier with lipoplexes (44). It was further demonstrated that DQAplexes containing DNA at a molar ratio of DQA/DNA 6:1 completely protect the DNA against DNAse activity (45), and as shown earlier for lipoplexes (46) the addition of negatively charged lipids also releases intact DNA from the DQAsome-DNA complex (45).…”

“…Therefore, the selfassociation properties of DQA were studied in more detail. Monte Carlo computer simulations applied to a system of DQA-like single-chain bola-form amphiphiles, in a coarsegrained model, revealed the formation of vesicles, micelles, cylinders, disks, and planar aggregates as well as the possible co-existence of horseshoe and stretched conformation, all depending on lipid concentration and temperature used in that model (27,28).…”

Bolaamphiphile-based nanocomplex delivery of phosphorothioate gapmer antisense oligonucleotides as a treatment for <em>Clostridium difficile</em>

“…The results are in perfect agreement with previously published data. 53 Oligreen dye-exclusion assays were performed The effects of 12-cyclohexyl-DQA gapmer nanocomplexes and equivalent empty bolasome treatments on the growth of log-phase C. difficile cultures were replicated and further measured using quantitative CFU counts in triplicate. CFU results are expressed as average CFU/mL growth normalized to initial inoculum CFU counts ( Figure 9).…”

Transduction of Human Recombinant Proteins into Mitochondria as a Protein Therapeutic Approach for Mitochondrial Disorders

“…Vitamin E, conjugated to TPP (a lipophilic triphenylphosphonium cation) was also delivered selectively to mitochondria (65). "Mitochondriotropics," a term proposed by Weissig and Torchilin to describe the ability of molecules to diffuse into the intramitochondrial sites, has been an interesting concept (66,67). According to this, the properties of each one of the proposed therapeutics for mitochondrial disorders can influence its "trafficking" towards specific mitochondrial sites and in a state to "deliver" its intended therapeutic action.…”