A Muratovska | PubMed Health

PubMed2001

An antigenomic strategy for treating heteroplasmic mtDNA disorders.

In mammals, mitochondrial DNA (mtDNA) is the only autonomously replicating source of DNA outside the nucleus. Housed in the mitochondrial matrix, this molecule encodes thirteen polypeptides, all of which are believed to be essential components of the mitochondrial respiratory chain. Defects of the mitochondrial genome can cause severe neurological and multi-systemic disorders. As the genetic defect causes a dysfunction in the terminal stage of oxidative metabolism, there is little potential for pharmacological intervention. Thus, there is currently no effective therapy for these chronic progressive disorders. In the disease state, pathogenic mtDNA molecules often cohabit the same cell and tissue with wild type mtDNA, a situation termed heteroplasmy. Manifestation of biochemical and clinical defects occur only when a threshold level of heteroplasmy has been passed. The mitochondrial genome must be continually turned over. Consequently, if a pathogenic mtDNA molecule were to be targeted to prevent it from replicating, the wild type copy would be given a propagative advantage. Over time, therefore, the biochemical and, potentially, the clinical deficiency could be reversed. This manuscript summarises our attempts to identify such an antigenomic molecule, to localise this molecule to mitochondria and to assess its function in whole cells. Finally, we discuss the importance of identifying and designing new antigenomic molecules which may prove effective in treating patients with disorders of the mitochondrial genome.

DNA Replication↗

PubMed2001

Targeting large molecules to mitochondria.

Mitochondrial function is central to a range of cell processes and mitochondrial dysfunction contributes to a number of human diseases. Consequently there is growing interest in delivering large molecules such as nucleic acids, proteins, enzyme mimetics, drugs and probes to mitochondria within cells. The reasons for doing this are to understand how mitochondria function in the cell and to develop therapies for diseases involving mitochondrial damage. Here we review the methods that have been used to target large molecules to mitochondria and discuss some approaches under development.

Animals↗

PubMed2001

Targeting peptide nucleic acid (PNA) oligomers to mitochondria within cells by conjugation to lipophilic cations: implications for mitochondrial DNA replication, expression and disease.

The selective manipulation of mitochondrial DNA (mtDNA) replication and expression within mammalian cells has proven difficult. One promising approach is to use peptide nucleic acid (PNA) oligomers, nucleic acid analogues that bind selectively to complementary DNA or RNA sequences inhibiting replication and translation. However, the potential of PNAs is restricted by the difficulties of delivering them to mitochondria within cells. To overcome this problem we conjugated a PNA 11mer to a lipophilic phosphonium cation. Such cations are taken up by mitochondria through the lipid bilayer driven by the membrane potential across the inner membrane. As anticipated, phosphonium-PNA (ph-PNA) conjugates of 3.4-4 kDa were imported into both isolated mitochondria and mitochondria within human cells in culture. This was confirmed by using an ion-selective electrode to measure uptake of the ph-PNA conjugates; by cell fractionation in conjunction with immunoblotting; by confocal microscopy; by immunogold-electron microscopy; and by crosslinking ph-PNA conjugates to mitochondrial matrix proteins. In all cases dissipating the mitochondrial membrane potential with an uncoupler prevented ph-PNA uptake. The ph-PNA conjugate selectively inhibited the in vitro replication of DNA containing the A8344G point mutation that causes the human mtDNA disease 'myoclonic epilepsy and ragged red fibres' (MERRF) but not the wild-type sequence that differs at a single nucleotide position. Therefore these modified PNA oligomers retain their selective binding to DNA and the lipophilic cation delivers them to mitochondria within cells. When MERRF cells were incubated with the ph-PNA conjugate the ratio of MERRF to wild-type mtDNA was unaffected, even though the ph-PNA content of the mitochondria was sufficient to inhibit MERRF mtDNA replication in a cell-free system. This unexpected finding suggests that nucleic acid derivatives cannot bind their complementary sequences during mtDNA replication. In summary, we have developed a new strategy for targeting PNA oligomers to mitochondria and used it to determine the effects of PNA on mutated mtDNA replication in cells. This work presents new approaches for the manipulation of mtDNA replication and expression, and will assist in the development of therapies for mtDNA diseases.

Binding Sites↗