Importation of mitochodrial protein by an enhanced allotopic approach

an allotopic approach and mitochondrial technology, applied in the field of cell biology, molecular genetics, medicine, can solve the problems of requiring optimization, affecting the efficiency of allotopic expression as a therapeutic approach, and affecting the pathology of mitochondrial cells, so as to achieve efficient and stable importation of proteins, enhance allotopic approach, and enhance efficiency and stability

Inactive Publication Date: 2015-03-26
INST NAT DE LA SANTE & DE LA RECHERCHE MEDICALE (INSERM)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a better way to import proteins into the mitochondrion, a critical organelle in cells. We use a method called allotopic expression, which involves delivering a specific piece of RNA to the surface of the mitochondrion, where it is translated and brought back into the cell to perform its function. This method is more efficient and stable than previous techniques, and can be used to treat genetic mutations that cause cellular dysfunction. By controlling the sorting of the RNA to the mitochondrion, we can guide the delivery of the protein to the correct location and help it function properly in the cell. Overall, this invention allows for more effective treatment of mitochondrial diseases.

Problems solved by technology

Therefore, mitochondrial pathologies are considered among the most common genetically determined diseases, and are a major health issue since they remain inaccessible to both curative and palliative therapies.
Hence, up today important limitations are found to the allotopic expression as a therapeutic approach and require optimization to overcome the significant hurdles before it can be applied in genetic therapy.

Method used

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  • Importation of mitochodrial protein by an enhanced allotopic approach
  • Importation of mitochodrial protein by an enhanced allotopic approach
  • Importation of mitochodrial protein by an enhanced allotopic approach

Examples

Experimental program
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Effect test

example 1

Allotopic Expression of the ATP6 Mitochondrial Gene is Significantly Improved by the Localization of its mRNAs to the Surface of Mitochondria Leading to an Efficient Import of the Precursor

[0307]Abstract:

[0308]It is clear that impairment of mitochondrial energy metabolism is the key pathogenic factor in a growing number of neurodegenerative disorders. With the discovery of mtDNA mutations, the replacement of defective genes became an important goal for mitochondrial geneticists worldwide. Unfortunately, before the present invention, it was still not possible to introduce foreign genes into the mitochondria of mammalian cells.

[0309]To circumvent this problem, allotopic expression in the nucleus of genes encoded by mitochondrial DNA (mtDNA), became an attractive idea. However, for most mitochondrial genes tested, there were important limitations related to the high hydrophobicity of the corresponding proteins, which impedes their mitochondrial translocation.

[0310]We herein elucidate t...

example 2

Correct Mitochondrial Localization of the Recoded Mitochondrial ND1 and ND4 Genes in Fibroblasts from LHON Patients

[0366]The three most common pathogenic mutations from LHON affect complex I ND1, ND4 and ND6 genes with the double effect of lowering ATP synthesis and increasing oxidative stress chronically.

[0367]Since we have demonstrated that reengineered mitochondrial Atp6 proteins were successfully translocated inside the mitochondria in HeLa cells (see example 1 above), we decided to synthesize recoded mitochondrial genes ND1 and ND4. To ensure the efficient import of the allotopically expressed proteins we appended to them signals which will direct the corresponding mRNAs to the mitochondrial surface. We have chosen to use the MTS of COX10 gene alone or in combination with its entire 3′UTR. FIGS. 5A and 5B illustrate the constructs obtained and the full-length sequences inserted in the pCMV-Tag 4A vector.

[0368]Material and Methods:

[0369]Cell Culture and Transfection:

[0370]Fibrob...

example 3

Rescue of Mitochondrial Deficiency Causing Human Diseases (Transfection of Fibroblasts from a NARP Patient)

[0384]We also determined whether the reengineered ATP6 protein would be able to rescue mitochondrial deficiency in cells having a mutated ATP6 gene.

[0385]We obtained fibroblasts from a patient presenting NARP disease caused by the T8993G mutation in the ATP6 gene.

[0386]Fibroblasts were cultured on media containing sodium pyruvate and relatively high amounts of FBS, more particularly:[0387]on a medium containing glucose (D-MEM with L-glutamine, 4500 mg / L D-glucose, 110 mg / L sodium pyruvate 2.5 mM, FBS 15%, uridine 28 microM), or[0388]on a medium, which does not contain glucose, but contain galactose (liquid D-MEM (1×), with L-Glutamine without Glucose, sodium pyruvate 2.5 mM, galactose 10 mM, FBS 15%, uridine 28 microM).

[0389]Stably transfected cells expressing the nuclear version of ATP6 associated with either SOD2 MTS alone, or in combination with SOD2 3′UTR, were obtained. Re...

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Abstract

An expression vector containing appropriate mitochondrion-targeting sequences (MTS) and appropriate 3′UTR sequences provides efficient and stable delivery of a mRNA encoding a protein (CDS) to the mitochondrion of a mammalian cell. The MTS and 3′UTR sequences guide the CDS mRNA from the nuclear compartment of the cell to mitochondrion-bound polysomes, where the CDS is translated. This provides an efficient translocation of a mature functional protein into the mitochondria. A method of targeting mRNA expressed in the nuclear compartment of a mammalian cell to the mitochondrion is also provided. The vector and methods can be used to treat defects in mitochondrial function.

Description

FIELD OF THE INVENTION[0001]The present invention relates to the field of cell biology, molecular genetics, and medicine. It more particularly relates to the importation of proteins into the mitochondrion of animal and human cells.BACKGROUND OF THE INVENTION[0002]Mitochondria occupy a central position in the overall metabolism of eukaryotic cells; hence the oxidative phosphorylation (OXPHOS), the Krebs's cycle, the urea cycle, the heme biosynthesis and the fatty acid oxidation take place within the organelle. Recently, another major role for mitochondria in determining the cellular life span was established, as they are recognized to be a major early mediator in the apoptotic cascade. Mitochondria are also a major producer of reactive oxygen species (ROS) causing oxidative stress and therefore inducers of cell death.[0003]Primary defects in mitochondrial function are implicated in over 120 diseases and the list continues to grow, they encompass an extraordinary assemblage of clinica...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/02C07K14/47C12N9/10
CPCC12N9/0089C07K14/47C12N9/1085A61K48/005A61K48/0058C07K2319/07C12N9/0053C12N2810/80C12Y109/03001C12N15/85A61P43/00A61K48/0066C12N15/8509C12N2800/22
Inventor CORRAL-DEBRINSKI, MARISOLSAHEL, JOSE-ALAINKALTIMBACHER, VALERIEBONNET, CRYSTEL
Owner INST NAT DE LA SANTE & DE LA RECHERCHE MEDICALE (INSERM)
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