Search

EP-4736838-A2 - ENGINEERED MEGANUCLEASES THAT TARGET HUMAN MITOCHONDRIAL GENOMES

EP4736838A2EP 4736838 A2EP4736838 A2EP 4736838A2EP-4736838-A2

Abstract

Disclosed herein are recombinant meganucleases engineered to recognize and cleave a recognition sequence present in the human mitochondrial DNA (mtDNA). The disclosure further relates to the use of such recombinant meganucleases in methods for producing genetically-modified eukaryotic cells, and to a population of genetically-modified eukaryotic cells wherein the mtDNA has been having modified or edited.

Inventors

  • SMITH, JAMES JEFFERSON
  • TOMBERLIN, Ginger
  • MORRIS, JOHN
  • SHOOP, Wendy
  • MORAES, Carlos T.

Assignees

  • Precision Biosciences, Inc.
  • University of Miami

Dates

Publication Date
20260506
Application Date
20220422

Claims (10)

  1. A mitochondria-targeting engineered meganuclease (MTEM), or a polynucleotide comprising a nucleic acid sequence encoding an MTEM, for use in treating Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes (MELAS), Progressive External Ophthalmoplegia, maternally inherited diabetes, migraines, or ocular myopathy in a subject, wherein said MTEM produces a cleavage site in mutant mitochondrial genomes at a recognition sequence consisting of SEQ ID NO: 1, thereby degrading said mutant mitochondrial genomes, wherein said MTEM comprises an engineered meganuclease attached to a mitochondrial transit peptide (MTP), wherein said engineered meganuclease comprises a first subunit and a second subunit, wherein said first subunit binds to a first recognition half-site of said recognition sequence and comprises residues 7-153 of SEQ ID NO: 9, wherein said second subunit binds to a second recognition half-site of said recognition sequence and comprises residues 198-344 of SEQ ID NO: 9, and wherein said engineered meganuclease comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 9.
  2. The MTEM or the polynucleotide for the use of claim 1, wherein said engineered meganuclease comprises the amino acid sequence of SEQ ID NO: 9.
  3. The MTEM or the polynucleotide for use of claim 1 or claim 2, wherein said MTP comprises an amino acid sequence set forth in SEQ ID NO: 45, and wherein said MTP is attached at the N-terminus of said engineered meganuclease.
  4. The MTEM or the polynucleotide for use of any one of claims 1-3, wherein said MTEM is attached to a nuclear export sequence (NES) comprising the amino acid sequence of SEQ ID NO: 46, wherein said NES is attached at the C-terminus of said MTEM.
  5. The polynucleotide for the use of any one of claims 1-4, wherein said polynucleotide encoding said MTEM is comprised by a recombinant adeno-associated virus (AAV).
  6. The polynucleotide for the use of claim 5, wherein said recombinant AAV has an AAV9 capsid.
  7. The polynucleotide for the use of claim 5 or claim 6, wherein said polynucleotide comprises a promoter that is operably linked to said nucleic acid sequence encoding said MTEM.
  8. The polynucleotide for the use of claim 7, wherein said promoter is a ubiquitous promoter, or wherein said promoter is a muscle cell-specific promoter, a skeletal muscle-specific promoter, a myotube-specific promoter, a muscle satellite cell-specific promoter, a neuron-specific promoter, an astrocyte-specific promoter, a microglia-specific promoter, an eye cell-specific promoter, a retinal cell-specific promoter, a retinal ganglion cell-specific promoter, a retinal pigmentary epithelium-specific promoter, a pancreatic cell-specific promoter, or a pancreatic beta cell-specific promoter.
  9. The polynucleotide for the use of claim 7 or claim 8, wherein said promoter is a CAG promoter.
  10. The MTEM or the polynucleotide for use of use of any one of claims 1-8, wherein said MTEM targets an A3243G mutation of the mutant mitochondrial genomes.

Description

FIELD OF THE INVENTION The present disclosure relates to the field of molecular biology and recombinant nucleic acid technology. In particular, the present disclosure relates to recombinant meganucleases engineered to recognize and cleave recognition sequences found in the human mitochondrial genome. The present disclosure further relates to the use of such recombinant meganucleases in methods for producing genetically-modified eukaryotic cells, and to a population of genetically-modified eukaryotic cells wherein the mitochondrial DNA has been modified. REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on April 21, 2022 is named P89339_0139_5_SeqList_4-21-22.txt, and is 80.6 kb in size. BACKGROUND OF THE INVENTION In all organisms, mitochondria regulate cellular energy and metabolism under normal growth and development as well as in response to stress. Many of the proteins functioning in these roles are coded for in the mitochondrial genome. Thus, editing of the mitochondrial genome has diverse applications in both animals and plants. In humans, deleterious mitochondrial mutations are the source of a number of disorders for which gene editing therapies could be applied. Pathogenic mitochondrial DNA (mtDNA) mutations include large-scale rearrangements and point mutations in protein coding, transfer RNA (tRNA) or ribosomal RNA (rRNA) genes. Although the prevalence of mtDNA-related disease diagnosis is about 1 in 5,000, the population frequency of the ten most common pathogenic mtDNA mutations is much higher, approaching 1 in 200, implying that many "normal" individuals carry low levels of mutated genomes (Schon et al., Nat Rev Gen 13:878-890 (2012)). Mutated mtDNA, in most cases, co-exist with wild-type mtDNA in patients' cells (mtDNA heteroplasmy). Several studies showed that the wild-type mtDNA has a strong protective effect, and biochemical abnormalities were observed only when the levels of the mutated mtDNA were higher than 80-90% (Schon et al., Nature Reviews Genetics 13:878-890 (2012)). It has been shown that muscle fibers develop an OXPHOS defect only when the mutation load is above 80% (Sciacco et al., Hum Mol Genet 3:13-19 (1994)). Therefore, any approach that could shift this balance by even a small percentage towards the wild-type would have strong therapeutic potential. However, mtDNA manipulation remains an underexplored area of science because of the inability to target mtDNA at high efficiencies and generate precise edits. The mitochondrial genome is difficult to edit because it requires predictable repair mechanisms and delivery of an editing technology to this organelle. In view of the difficulty and unpredictability associated with mitochondrial genome editing, there is an unmet need for precise editing of mtDNA, which would open up an entire field of inquiry and opportunity in life sciences. The ability to target and edit a defined region (preferably limited to just one gene) of the mitochondrial genome in a more predictable manner would be a clear benefit over currently available systems. SUMMARY OF THE INVENTION Provided herein are compositions and methods for precise editing of mitochondrial genome. Up until now, attempts at mitochondrial genome editing have resulted in large and unpredictable deletions/rearrangements. The present invention demonstrates that engineered meganucleases can result in precise editing of mitochondrial DNA (mtDNA), thereby opening up an entire field of inquiry and opportunity in life sciences. The compositions and methods provided herein can be used for editing one specific mitochondrial gene without impacting surrounding regions. In one aspect, the invention provides a mitochondria-targeting engineered meganuclease (MTEM) that binds and cleaves a recognition sequence comprising SEQ ID NO: 1 in mitochondrial genomes of a eukaryotic cell, wherein the MTEM comprises an engineered meganuclease attached to a mitochondrial transit peptide (MTP), wherein the engineered meganuclease comprises a first subunit and a second subunit, wherein the first subunit binds to a first recognition half-site of the recognition sequence and comprises a first hypervariable (HVR1) region, and wherein the second subunit binds to a second recognition half-site of the recognition sequence and comprises a second hypervariable (HVR2) region. In some embodiments, the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of any one of SEQ ID NOs: 3-12. In some embodiments, the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any one of SEQ ID NOs: 3-12. In s