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US-20260125707-A1 - METHOD FOR INTRODUCING NUCLEIC ACID INTO MITOCHONDRION

US20260125707A1US 20260125707 A1US20260125707 A1US 20260125707A1US-20260125707-A1

Abstract

The purposes of the present invention are to provide a method of easily introducing a gene to mitochondria from the outside of a cell, and to provide a method of introducing a relatively large nucleic acid to mitochondria. A carrier peptide for introducing a nucleic acid to mitochondria is provided, which comprises a mitochondrial-targeting sequence and a polycationic sequence, wherein said mitochondrial-targeting sequence consists of an N-terminal region comprising a transmembrane domain in a mitoNEET protein.

Inventors

  • Keiji Numata
  • Naoto Yoshinaga

Assignees

  • RIKEN

Dates

Publication Date
20260507
Application Date
20231004
Priority Date
20221004

Claims (15)

  1. 1 . A carrier peptide for introducing a nucleic acid to mitochondria, comprising a mitochondrial-targeting sequence and a polycationic sequence, wherein said mitochondrial-targeting sequence consists of an N-terminal region comprising the transmembrane domain in a mitoNEET protein.
  2. 2 . The carrier peptide of claim 1 , wherein said mitochondrial-targeting sequence consists of the amino acid sequence MSLTSSSSVRVEWIAAVTIAAGTAAIGYLAYK (SEQ ID NO: 2).
  3. 3 . The carrier peptide of claim 1 , wherein said polycationic sequence comprises at least three cationic amino acid residues.
  4. 4 . The carrier peptide of claim 3 , wherein said cationic amino acid residues are lysine (K) residues, arginine (R) residues, histidine (H) residues, and/or derivatives of any of them.
  5. 5 . The carrier peptide of claim 1 , wherein said polycationic sequence comprises: five or more consecutive lysine (K) residues, arginine (R) residues, histidine (H) residues, or derivatives of any of them; seven or more consecutive lysine (K) residues, arginine (R) residues, histidine (H) residues, and/or derivatives of any of them; or a sequence formed by repeating the amino acid sequence KH or RH three to twenty times.
  6. 6 . A nucleic acid encoding the carrier peptide of claim 1 .
  7. 7 . A carrier peptide/nucleic acid complex for introducing a nucleic acid to mitochondria, comprising the carrier peptide of claim 1 and the nucleic acid.
  8. 8 . The carrier peptide/nucleic acid complex of claim 7 , wherein the number of amino groups and guanidino groups derived from said carrier peptide/the number of phosphate groups derived from said nucleic acid (N/P ratio) is 0.2 or more and 100 or less.
  9. 9 . The carrier peptide/nucleic acid complex of claim 7 , wherein said nucleic acid encodes a mitochondrial protein.
  10. 10 . The carrier peptide/nucleic acid complex of claim 7 , wherein said nucleic acid is DNA or RNA.
  11. 11 . The carrier peptide/nucleic acid complex of claim 10 , wherein said DNA is 1 kbp to 20 kbp.
  12. 12 . The carrier peptide/nucleic acid complex of claim 10 , wherein said DNA is a mitochondrial DNA.
  13. 13 . A pharmaceutical composition for treating a mitochondrial disease, comprising the carrier peptide/nucleic acid complex of claim 7 .
  14. 14 . A method of producing a carrier peptide/nucleic acid complex, comprising: a complex formation step of mixing the carrier peptide of claim 1 with a nucleic acid to form a carrier peptide/nucleic acid complex comprising said carrier peptide and said nucleic acid.
  15. 15 . A method of introducing a nucleic acid to mitochondria of an isolated target cell, comprising: a contact step of contacting the carrier peptide/nucleic acid complex of claim 7 with an isolated target cell, and a transfer step of culturing said target cell after said contact step to transfer said carrier peptide/nucleic acid complex to mitochondria.

Description

TECHNICAL FIELD The present invention relates to a carrier peptide for introducing a nucleic acid to mitochondria, a carrier peptide/nucleic acid complex, a pharmaceutical composition for treating a mitochondrial disease, a method for producing a carrier peptide/nucleic acid complex, and a method of introducing a nucleic acid to mitochondria. BACKGROUND ART Mitochondria play important roles in intracellular energy production, control of reactive oxygen species, and the like. Mitochondrial DNA (mtDNA), the genome of mitochondria themselves, encodes 13 kinds of proteins, which are important enzymes that function in oxidative phosphorylation in mitochondria. However, mtDNA is exposed to the oxidative environment in mitochondria and is not protected by histone proteins. Therefore, mtDNA is prone to undergo mutation, and as mutations accumulate with age, increased mitochondrial genetic variation may emerge within the same cell. This condition is called heteroplasmy, and is thought to cause a mitochondrial disease due to impaired normal expression of mitochondrial proteins encoded by mtDNA. Methods for efficiently introducing a gene to mitochondria in order to treat a mitochondrial disease have been studied for many years. A known classical mitochondrial delivery method uses lipophilic cations such as triphenylphosphine as a mitochondrial-targeting module. This method uses electrostatic interactions with highly negatively charged mitochondrial membranes, but is known to have a limited effect on mitochondria and is associated with cytotoxicity because lipophilic cations can also promote interactions with other negatively charged biomacromolecules. In recent years, mitochondrial gene therapy has been dominated by a technique of introducing a gene encoding a fusion protein, in which a protein of interest for treatment is fused with a mitochondrial-targeting sequence (MTS), to a nucleus, and transferring the expressed fusion protein to the mitochondria. This technique is called allotopic delivery, and clinical trials are underway for mitochondrial diseases. However, this technique is not a method of introducing a gene directly to mitochondria and is not sufficiently effective. As the most efficient method among conventional techniques, a method that uses an adeno-associated virus (AAV) is known. This method involves substituting the capsid protein of AAV with MTS to facilitate the transfer of AAV to the mitochondria. It has been revealed that the use of this method enables the introduction of the NADH subunit 4 gene to mitochondria, followed by sufficient expression in the mitochondria. However, there is a limit to the size of DNA that can be carried by AAV, with approximately 5 kbp being the upper limit. In gene therapy for mitochondrial diseases, it is desirable to introduce multiple genes to control complicated mitochondrial functions, and especially to improve the heteroplasmy: it is ideal to introduce the entire 16.6 kbp mtDNA. However, when AAV is used, it is not easy to manipulate AAV to carry multiple genes, including the entire mtDNA. Furthermore, the method using AAV is problematic in its complicated process of preparing AAV into which MTS has been introduced and cannot be easily carried out. Therefore, there is a need for new technologies to easily perform gene transfer to mitochondria. CITATION LIST Patent Literature Patent Literature 1: WO2013/129698 Non-Patent Literature Non-patent Literature 1: Chuah, J. A. et al., Biomacromolecules, 2016, 17 (11): 3547-3557. SUMMARY OF INVENTION Technical Problem An object of the present invention is to provide a method of easily introducing a gene to mitochondria and a method of introducing a relatively large nucleic acid to mitochondria. Solution to Problem In order to solve the above problems, the present inventors have prepared a carrier peptide/nucleic acid complex by: constructing a new carrier peptide as a fusion peptide prepared by combining a mitochondrial-targeting sequence comprising the transmembrane domain of a mitoNEET protein with a polycationic sequence; and then mixing the carrier peptide with a nucleic acid. The present inventors have discovered that through introduction of this carrier peptide/nucleic acid complex to a cell, the carrier peptide/nucleic acid complex can be efficiently transferred to mitochondria and the protein encoded by the nucleic acid can be expressed in the mitochondria. Furthermore, the present inventors have prepared a carrier peptide/mitochondrial DNA (mtDNA) complex by mixing the above carrier peptide with mtDNA, and then introduced this to cells with mtDNA-depletion, so that they have succeeded in restoring mitochondrial function. The present invention provides the following based on the above findings. (1) A carrier peptide for introducing a nucleic acid to mitochondria, comprising a mitochondrial-targeting sequence and a polycationic sequence, wherein said mitochondrial-targeting sequence consists of an N-terminal region