Engineered mitochondria

Abstract

The present invention provides an engineered mitochondrion comprising one or more exogenous protein binding motifs / peptides expressed on the outer membrane of the mitochondrion. Also disclosed is a polynucleotide, a vector, a host cell, a cell, a composition or pharmaceutical composition and methods of treatment thereof. Also disclosed is a method of improving uptake of mitochondria into a target cell comprising genetically modifying a host cell to express a modified mitochondrion, wherein the modified mitochondrion comprises one or more exogenous protein binding motifs / peptides expressed on the outer membrane of the mitochondrion.

Description

Technical Field

[0001] This disclosure generally relates to engineered organelles. Specifically, it relates to engineered mitochondria. This disclosure also relates to methods for improving / increasing mitochondrial uptake by cells of interest. Background Technology

[0002] Mitochondrial therapy, or mitochondrial transplantation, is a viable therapeutic approach for treating mitochondrial diseases and degenerative conditions. However, host cells currently exhibit low efficiency in mitochondrial uptake, and there are currently no methods to target mitochondria for uptake by specific cell types.

[0003] Cells can internalize extracellular mitochondria both in vitro and in vivo. However, the uptake efficiency is low. Therefore, there is a need to provide engineered mitochondria that can be easily taken up by specific cell types.

[0004] There is a need to provide alternative cell-based therapies. There is an urgent need to provide engineered organelles, such as engineered mitochondria. Summary of the Invention

[0005] In vivo, therapeutics benefit from the cell-specific internalization of the drug into cells of interest. One potential therapeutic approach utilizes exogenously generated healthy mitochondria to treat cells. However, mitochondrial uptake into cells is inefficient.

[0006] This disclosure improves mitochondrial uptake by increasing the duration or tendency of close proximity between mitochondria and host cells. The inventors achieve this by enhancing the interaction between the mitochondrial surface and the cell surface through the engineering of small protein motifs on the surface of donor mitochondria. Currently, no known small protein motifs have been shown to interact with the surface of cardiomyocytes.

[0007] Therefore, this paper discloses two methods for identifying potential motifs possessing this binding property. Using a Cas9-mediated genome editing strategy on wild-type induced pluripotent stem cells, this disclosure inserts the cell surface binding portion of a laminin-derived peptide sequence (IKVAV, SEQ ID NO:18) into the outer mitochondrial membrane to enhance its targeting and uptake by cells expressing β1-integrin. Using the same concept, the inventors genetically engineered human induced pluripotent stem cell (iPSC) cell lines using CRISPR to generate bioengineered mitochondria expressing the proposed cardiac peptide motif on their mitochondrial surface.

[0008] In one aspect, an engineered mitochondria is provided, comprising one or more exogenous protein-binding motifs / peptides expressed on the outer mitochondrial membrane.

[0009] In some instances, the exogenous protein binding motif / peptide is an extracellular matrix (ECM) protein binding motif / ECM-derived peptide, optionally located on the cytoplasmic surface of the outer mitochondrial membrane.

[0010] In some instances, the exogenous protein binding motif / peptide is an extracellular matrix (ECM) protein binding motif / ECM-derived peptide, which includes laminin-derived peptides, surface-labeled peptides, fibronectin-derived peptides, collagen-derived peptides, gelatin-derived peptides, synaptic glycan-derived peptides, and / or combinations thereof.

[0011] In some instances, the exogenous protein binding motif / peptide contains approximately 4 to 40 amino acid residues.

[0012] In some instances, the exogenous protein binding motif / peptide comprises laminin-derived peptides.

[0013] In some instances, the exogenous protein-binding motif / peptide comprises a laminin-derived peptide, which contains a peptide having the following sequence:

[0014] Ile-Lys-Val-Ala-Val (IKVAV, SEQ ID NO:17),

[0015] Tyr-Ile-Gly-Ser-Arg (YIGSR, SEQ ID NO:18),

[0016] Pro-Pro-Phe-Leu-Met-Leu-Leu-Lys-Gly-Ser-Thr-Arg (PPFLMLLKGSTR, SEQ IDNO:19),

[0017] Asp-Leu-Thr-Ile-Asp-Asp-Ser-Tyr-Trp-Tyr-Arg-Ile (DLTIDDSYWYRI, SEQ IDNO:20),

[0018] Asn-Ser-Ile-Lys-Val-Ser-Val-Ser-Ser (NSIKVSVSS, SEQ ID NO: 1-CP peptide 1),

[0019] Cys-Thr-Val-Ser-Pro-Gly-Val-Glu-Asp-Ser-Glu-Gly-Thr-Ile (CTVSPQVEDSEGTI, SEQ ID NO: 2-CP peptide 2),

[0020] Lys-Gly-Cys-Ser-Leu-Glu-Asn-Val-Tyr-Thr (KGCSLENVYT, SEQ ID NO: 3-CP peptide 3),

[0021] Ser-Gly-Gly-Thr-Pro-Ala-Pro-Pro-Arg-Arg-Lys-Arg-Arg-Glu-Thr-Gly-Glu-Ala (SGGTPAPPRRKRRQTGQA, SEQ ID NO:4-CP peptide 4), and / or combinations thereof.

[0022] In some instances, the exogenous protein binding motif / peptide comprises a heart-specific laminin that interacts with integrins on the surface of cardiomyocytes.

[0023] In some instances, the exogenous protein binding motif / peptide comprises one or more exposed regions on the interaction domains of laminin, including helices, loops, and laminin globular (LG) domains LG1, LG2, LG3, LG4, LG5, and / or combinations thereof. Optionally, the exogenous protein binding motif / peptide comprises an exposed region between a helix and LG1, an exposed region between LG1 and LG2, an exposed region between LG2 and LG3, and an exposed loop in LG3, or combinations thereof.

[0024] In some instances, the exogenous protein-binding motif / peptide comprises a laminin-derived peptide, which contains a peptide having the following sequence:

[0025] Asn-Ser-Ile-Lys-Val-Ser-Val-Ser-Ser (NSIKVSVSS, SEQ ID NO: 1-CP peptide 1),

[0026] Cys-Thr-Val-Ser-Pro-Gly-Val-Glu-Asp-Ser-Glu-Gly-Thr-Ile (CTVSPQVEDSEGTI, SEQ ID NO: 2-CP peptide 2),

[0027] Lys-Gly-Cys-Ser-Leu-Glu-Asn-Val-Tyr-Thr (KGCSLENVYT, SEQ ID NO: 3-CP peptide 3),

[0028] Ser-Gly-Gly-Thr-Pro-Ala-Pro-Pro-Arg-Arg-Lys-Arg-Arg-Glu-Thr-Gly-Glu-Ala (SGGTPAPPRRKRRQTGQA, SEQ ID NO:4-CP peptide 4), and / or combinations thereof.

[0029] In some instances, the exogenous protein binding motif / peptide comprises a surface-labeled peptide.

[0030] In some instances, the exogenous protein binding motif / peptide comprises a surface marker protein of cardiomyocytes.

[0031] In some instances, the exogenous protein binding motif / peptide contains SIRPα, a surface marker protein of cardiomyocytes.

[0032] In some instances, the exogenous protein-binding motif / peptide comprises a SIRPα surface marker protein, the surface marker protein comprising a peptide having the following sequence:

[0033] Thr-Lys-Ser-Val-Glu-Phe-Thr-Phe-Cys-Asn-Asp-Thr-Val-Val-Iso-Pro-Cys-Phe (TKSVEFTFCNDTVVIPCF, SEQ ID NO:7-Peptide 7),

[0034] Asn-Met-Glu-Ala-Gln-Asn-Thr-Thr-Glu-Val-Tyr (NMEAQNTTEVY, SEQ ID NO: 5-peptide 5),

[0035] Thr-Glu-Leu-Thr-Arg-Glu-Gly-Glu (TELTREGE, SEQ ID NO: 6-peptide 6), or combinations thereof.

[0036] In some instances, the exogenous protein-binding motif / peptide comprises a SIRPα surface marker protein, the surface marker protein comprising a peptide having the following sequence:

[0037] Val-Val-Iso-Pro-Cys-Phe-Val-Thr-Asn-Met-Glu-Ala (VVIPCFVTNMEA, SEQ IDNO:12-ABC peptide),

[0038] Cys-Asn-Asp-Thr-Val-Val-Iso-Pro-Cys-Phe (CNDTVVIPCF, SEQ ID NO:11-AB-rear),

[0039] Thr-Lys-Val-Glu-Phe-Thr-Phe-Cys-Asn-Asp-Thr-Val-Val-Iso-Pro-Cys-Phe(TKSVEFTFCNDTVVIPCF, SEQ ID NO:7-AB full length),

[0040] Glu-Phe-Thr-Phe-Cys-Asn-Asp-Thr-Val-Val (EFTFCNDTVV, SEQ ID NO:10-AB-Central),

[0041] Thr-Lys-Ser-Val-Glu-Phe-Thr-Phe-Cys-Asn (TKSVEFTFCN, SEQ ID NO:9-AB-front),

[0042] Asn-Met-Glu-Ala-Gln-Asn-Thr-Thr-Glu-Val-Tyr (NMEAQNTTEVY, SEQ ID NO: 5-BC peptide),

[0043] Or a combination thereof.

[0044] In some instances, the exogenous protein binding motif / peptide comprises a fibronectin-derived peptide, which comprises Arg-Gly-Asp (RGD, SEQ ID NO:13), Pro-His-Ser-Arg-Asn (PHSRN, SEQ ID NO:14), cyclic RGD, and / or combinations thereof.

[0045] In some instances, the exogenous protein binding motif / peptide comprises a collagen-derived peptide, which comprises Asp-Gly-Glu-Ala (DGEA, SEQ ID NO:16).

[0046] In some instances, the exogenous protein-binding motif / peptide is expressed on an exogenous mitochondrial outer membrane protein.

[0047] In some instances, the exogenous protein-binding motif / peptide is expressed on an exogenous extramitochondrial membrane translocation enzyme complex, optionally on exogenous TOM20, TOM70, TOM40, TOM22, TOM7, TOM6, TOM5, TOM70, SAM50, SAM35, SAM37, Mim1, Mim2, or combinations thereof.

[0048] In some instances, the exogenous protein-binding motif / peptide is expressed on exogenous TOM20, TOM22, TOM70, SAM50, porin, outer membrane protein 25 (OMP25), or combinations thereof.

[0049] In some instances, the exogenous protein-binding motif / peptide is expressed on the exogenous TOM20 protein.

[0050] In some instances, the mitochondria are derived from cells selected from induced pluripotent stem cells, mesenchymal stem cells, adipose-derived stem cells, and adult stem cells.

[0051] On the other hand, a polynucleotide is provided that encodes mitochondria as disclosed herein.

[0052] In some instances, the polynucleotide encodes an insert construct containing a sequence encoding a mitochondrial outer membrane protein and / or a mitochondrial protein-binding motif / peptide.

[0053] In some instances, the encoded polynucleotide encodes a foreign protein-binding motif / peptide having one or more sequences selected from the following:

[0054] Ile-Lys-Val-Ala-Val (IKVAV, SEQ ID NO:17),

[0055] Tyr-Ile-Gly-Ser-Arg (YIGSR, SEQ ID NO:18),

[0056] Pro-Pro-Phe-Leu-Met-Leu-Leu-Lys-Gly-Ser-Thr-Arg (PPFLMLLKGSTR, SEQ IDNO:19),

[0057] Asp-Leu-Thr-Ile-Asp-Asp-Ser-Tyr-Trp-Tyr-Arg-Ile (DLTIDDSYWYRI, SEQ IDNO:20),

[0058] Asn-Ser-Ile-Lys-Val-Ser-Val-Ser-Ser (NSIKVSVSS-CP peptide 1, SEQ ID NO: 1),

[0059] Cys-Thr-Val-Ser-Pro-Gly-Val-Glu-Asp-Ser-Glu-Gly-Thr-Iso (CTVSPQVEDSEGTI-CP peptide 2, SEQ ID NO: 2),

[0060] Lys-Gly-Cys-Ser-Leu-Glu-Asn-Val-Tyr-Thr (KGCSLENVYT-CP peptide 3, SEQ ID NO: 3),

[0061] Ser-Gly-Gly-Thr-Pro-Ala-Pro-Pro-Arg-Arg-Lys-Arg-Arg-Glu-Thr-Gly-Glu-Ala (SGGTPAPPRRKRRQTGQA-CP peptide 4, SEQ ID NO: 4),

[0062] Thr-Lys-Ser-Val-Glu-Phe-Thr-Phe-Cys-Asn-Asp-Thr-Val-Val-Iso-Pro-Cys-Phe (TKSVEFTFCNDTVVIPCF-peptide 7, SEQ ID NO: 7),

[0063] Asn-Met-Glu-Ala-Gln-Asn-Thr-Thr-Glu-Val-Tyr (NMEAQNTTEVY-peptide 5, SEQ ID NO: 5),

[0064] Thr-Glu-Leu-Thr-Arg-Glu-Gly-Glu (TELTREGE-peptide 6, SEQ ID NO: 6),

[0065] Val-Val-Iso-Pro-Cys-Phe-Val-Thr-Asn-Met-Glu-Ala (VVIPCFVTNMEA-ABC peptide, SEQ ID NO:12),

[0066] Cys-Asn-Asp-Thr-Val-Val-Iso-Pro-Cys-Phe (CNDTVVIPCF-AB rear, SEQ ID NO:11),

[0067] Thr-Lys-Val-Glu-Phe-Thr-Phe-Cys-Asn-Asp-Thr-Val-Val-Iso-Pro-Cys-Phe (TKSVEFTFCNDTVVIPCF-AB full length, SEQ ID NO:7)

[0068] Glu-Phe-Thr-Phe-Cys-Asn-Asp-Thr-Val-Val (EFTFCNDTVV-AB middle part, SEQ ID NO:10), Thr-Lys-Ser-Val-Glu-Phe-Thr-Phe-Cys-Asn (TKSVEFTFCN-AB front part),

[0069] Asn-Met-Glu-Ala-Gln-Asn-Thr-Thr-Glu-Val-Tyr (NMEAQNTTEVY-BC peptide, SEQ IDNO:5),

[0070] Or a combination thereof.

[0071] In some instances, the polynucleotide encodes an insert construct containing a sequence encoding TOMM20 and / or a cardiac peptide motif.

[0072] On the other hand, a vector is provided that contains a polynucleotide encoding mitochondria as disclosed herein or contains a polynucleotide as disclosed herein.

[0073] On the other hand, a host cell is provided which contains a vector as disclosed herein.

[0074] On the other hand, a cell is provided that contains mitochondria as disclosed herein.

[0075] In another aspect, a composition or pharmaceutical composition is provided that comprises engineered mitochondria or polynucleotides as disclosed herein.

[0076] On the other hand, compositions or pharmaceutical compositions as disclosed herein are provided for use in treatment / medicine.

[0077] On the other hand, a method for preventing and / or treating a disease in a subject in need is provided, the method comprising administering to the subject engineered mitochondria, polynucleotides, or compositions as disclosed herein.

[0078] In some cases, the disease is a mitochondrial disease or a proliferative disorder.

[0079] On the other hand, a method for improving mitochondrial uptake into target cells is provided, comprising genetically modifying a host cell to express modified mitochondria, wherein the modified mitochondria contain one or more exogenous protein-binding motifs / peptides expressed on the outer mitochondrial membrane.

[0080] In some instances, host cells are genetically modified via viral transduction or CRISPR gene modification systems.

[0081] definition

[0082] As used herein, the term “engineered organelle” or “bioengineered organelle” refers to organelles that have been modified by applying biotechnology (such as genome editing).

[0083] As used herein, the term "engineered mitochondria" or "bioengineered mitochondria" refers to mitochondria that have been modified by applying biotechnology (e.g., genome editing). In this disclosure, engineered mitochondria have improved / enhanced pinocytosis.

[0084] As used herein, the terms “motif,” “binding motif,” “binding motif,” “protein-binding motif,” “protein motif,” and “peptide” are understood to describe the same component and are therefore used interchangeably. The term “binding motif” refers to a protein amino acid sequence that exhibits increased affinity for binding or interaction with other cell surfaces as shown in this disclosure.

[0085] As used herein, the term "extracellular matrix / ECM" refers to a network of extracellular macromolecules and minerals (such as collagen, enzymes, glycoproteins, and hydroxyapatite) that provides structural and biochemical support to surrounding cells. The ECM is composed of an interwoven network of fibrin and glycosaminoglycans (GAGs).

[0086] As used herein, the term “cell-penetrating peptide” refers to a short peptide (5 to 12 amino acids) that penetrates the cell membrane, facilitates uptake in recipient cells, and allows escape from the endosome to the cytoplasm after endocytosis.

[0087] As used herein, the term "mitochondrial membrane protein" refers to mitochondrial membrane transport proteins / mitochondrial carrier proteins located in the mitochondrial membrane. The function of mitochondrial membrane proteins is to transport molecules and other factors (such as ions) into or out of the organelle. Mitochondria contain both an inner and an outer membrane, separated by an intermembrane space or inner boundary membrane.

[0088] As used herein, the term "extramitochondrial membrane proteins" refers to integrated proteins in the outer mitochondrial membrane, which consist of proteins with transmembrane β-barrels and proteins with one or more α-helical membrane anchors. The outer mitochondrial membrane forms the boundary between the mitochondria and their cellular environment. These outer membrane mitochondrial proteins perform functions in mitochondrial biogenesis and integration between mitochondria and the cellular system.

[0089] As used herein, the term "exogenous" refers to a substance originating outside an organism, tissue, or cell. This is in contrast to endogenous substances originating within a living system. In this disclosure, exogenous engineered mitochondria are introduced by promoting enhanced endocytosis in target cells. As disclosed herein, mitochondria are engineered to have an enhanced capacity for endocytosis by target cells.

[0090] As used herein, the term "insertion" refers to the addition of one or more nucleotide base pairs to a DNA sequence with the aim of introducing a peptide that is not naturally expressed. Therefore, as used herein, the term "insertion" enables engineered mitochondria to express one or more binding motifs that are not naturally expressed by wild-type mitochondria.

[0091] As used in this article, the term "death signal" refers to signals released during cell death, such as active or passive molecules. Death signals can communicate with recipient cells and regulate physiological or pathological events.

[0092] As described herein, a “vector” is any molecule or composition having the ability to deliver a nucleic acid sequence into a suitable host cell (where, for example, the synthesis of an encoded polypeptide can occur). Typically and preferably, a vector is a nucleic acid that has been engineered using recombinant DNA techniques known in the art to incorporate a desired nucleic acid sequence (e.g., the nucleic acid of this disclosure). Expression vectors typically contain one or more of the following components (if they are not already provided by a nucleic acid molecule): a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splicing sites, a leader sequence for secretion, a ribosome binding site, a polyadenylated sequence, a multi-connector region of the nucleic acid encoding the polypeptide to be expressed, and optional labeling elements.

[0093] Vectors are typically chosen to function in the host cells where they will be used (the vector is compatible with the host cell machinery, enabling gene amplification and / or gene expression to occur). Vectors as described herein can be expression vectors and / or cloning vectors.

[0094] As used herein, the term "host cell" is intended to refer to the cell into which the expression vector has been introduced. It should be understood that such a term is intended not only to refer to a specific subject cell, but also to the progeny of such cells. Due to mutations or environmental influences, certain modifications may occur in the progeny, and therefore the progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0095] In some instances, the terms “treating,” “treatment,” and “therapeutic,” and their synonyms, refer to therapeutic treatments and preventative or preventative measures aimed at preventing or alleviating (reducing) a medical condition, which includes, but is not limited to, disease, symptoms, and ailments. A medical condition also includes the body’s response to a disease or ailment, such as inflammation. People who require such treatment include those who already have a medical condition, those who are susceptible to a medical condition, or those who want to prevent a medical condition.

[0096] As used herein, the term "subject" includes both patients and non-patients. The term "patient" refers to an individual who has or is likely to have a medical condition, while "non-patient" refers to an individual who does not have and is unlikely to have a medical condition. "Non-patient" includes healthy individuals, non-ill individuals, and / or individuals without a medical condition. The term "subject" includes both humans and animals. Animals may include, but are not limited to, mammals (e.g., non-human primates, dogs, rodents, rabbits, etc.), etc. "Rodents" refers to any mammal from the family Muridae, such as mice, rats, etc. "Rabbits" refers to any mammal from the family Leporidae, such as hares, rabbits, etc.

[0097] As used herein, the terms “prevention” and / or “severity reduction” refer to the process of delaying onset, reducing the severity of symptoms, reducing and / or preventing weight loss, preventing death, inhibiting deterioration, inhibiting further deterioration, and / or improving at least one sign or symptom of the disease.

[0098] The term “and / or”, such as “X and / or Y”, is understood to mean “X and Y” or “X or Y”, and should be considered to provide explicit support for both meanings or either meaning.

[0099] Furthermore, in the description herein, whenever used, the word “substantially” is understood to include, but is not limited to, “completely” or “all”. Additionally, whenever used, terms such as “comprising” and “comprise” are intended to be unrestrictive descriptive language, meaning they broadly include the elements / components described after these terms, as well as other components not explicitly described. For example, when “comprising” is used, a reference to “one” feature is also intended to refer to “at least one” of that feature. In the appropriate context, terms such as “consisting” and “consist” can be considered as a subset of terms such as “comprising” and “comprise”. Therefore, in embodiments of the use of terms such as “comprising” and “comprise” herein, it will be appreciated that these embodiments provide instruction on corresponding embodiments using terms such as “consisting” and “consist”. In addition, whenever used, terms such as “about” or “approximately” generally refer to reasonable variation, such as a variation of + / - 5% of the disclosed value, or a variation of 4% of the disclosed value, or a variation of 3% of the disclosed value, a variation of 2% of the disclosed value, or a variation of 1% of the disclosed value.

[0100] Furthermore, certain values ​​may be disclosed as ranges in the description herein. Showing the values ​​at the endpoints of a range is intended to illustrate preferred ranges. Whenever a range is described, it is intended that the range encompasses and teaches all possible subranges within that range, as well as individual numerical values. That is, the endpoints of a range should not be interpreted as rigid limitations. For example, the description of a range of 1% to 5% is intended to specifically disclose subranges such as 1% to 2%, 1% to 3%, 1% to 4%, 2% to 3%, etc., and individual values ​​within that range, such as 1%, 2%, 3%, 4%, and 5%. It should be recognized that individual numerical values ​​within a range also include integers, fractions, and decimals. Furthermore, whenever a range is described, it is also intended that the range encompasses and teaches values ​​up to two additional decimal places or significant figures (where appropriate) from the endpoints of the indicated numerical values. For example, the description of a range of 1% to 5% is intended to specifically disclose ranges of 1.00% to 5.00% and 1.0% to 5.0%, as well as all intermediate values ​​across these ranges (e.g., 1.01%, 1.02%...4.98%, 4.99%, 5.00% and 1.1%, 1.2%...4.8%, 4.9%, 5.0%, etc.). The above specific disclosure is intended to apply to any range of depth / breadth. Detailed Implementation

[0101] This disclosure provides an engineered mitochondria (or organelle) comprising one or more binding motifs expressed on the outer membrane of the mitochondria (or organelle).

[0102] Therefore, in one respect, an engineered mitochondria is provided, which contains one or more exogenous protein-binding motifs / peptides expressed on the outer mitochondrial membrane.

[0103] In some instances, the mitochondria can be mammalian mitochondria. In some instances, the mitochondria can be from humans, non-human primates, mammals (e.g., pigs) and rodents (e.g., rats, mice, etc.). In some instances, the mouse background can include, but is not limited to, C57BL / 6, BALB / c, CD-1, SCID, etc. In some instances, the rat background can include, but is not limited to, A / J, Sprague Dawley, Wistar, etc. In some instances, the non-human primates can include, but are not limited to, rhesus monkeys, Japanese macaques, olive baboons, squirrel monkeys, capuchin monkeys, etc. In some instances, the mitochondria are human mitochondria.

[0104] In some instances, mitochondria can be non-mammalian mitochondria. Non-mammalian mitochondria may include yeast, etc.

[0105] In some instances, the binding motif / peptide is a cell surface binding motif / peptide.

[0106] In some instances, cell surface binding motifs / peptides may include, but are not limited to, extracellular matrix proteins, cell adhesion molecules, ligands, receptors, DNA aptamers, RNA aptamers, etc.

[0107] In some instances, the binding motif / peptide is an extracellular matrix (ECM) protein binding motif / ECM-derived peptide.

[0108] In some instances, the exogenous protein binding motif / peptide is an extracellular matrix (ECM) protein binding motif / ECM-derived peptide, optionally located on the cytoplasmic surface of the outer mitochondrial membrane.

[0109] In some instances, engineered mitochondria contain the insertion of ECM protein-binding motifs / ECM-derived peptides, or the insertion of two ECM protein-binding motifs / ECM-derived peptides, or the insertion of three ECM protein-binding motifs / ECM-derived peptides, or the insertion of four ECM protein-binding motifs / ECM-derived peptides, or the insertion of five ECM protein-binding motifs / ECM-derived peptides.

[0110] In some instances, ECM may include proteoglycans (e.g., heparan sulfate, chondroitin sulfate, keratin sulfate, etc.), non-proteoglycan polysaccharides (e.g., but not limited to hyaluronic acid, etc.), proteins (e.g., but not limited to collagen, elastin, etc.), extracellular vesicles, cell adhesion proteins (e.g., but not limited to fibronectin, laminin, etc.), and so on.

[0111] In some instances, the ECM protein-binding motif / ECM-derived peptide may include a protein tag. In some instances, the protein tag may include, but is not limited to, histidine / multihistidine tags, glutathione S-transferase tags (GST tags), hemagglutinin tags (HA tags), green fluorescent protein (GFP), streptavidin tags, V5 tags, and so on. In some instances, the protein tag includes three copies of the HA tag (i.e., 3xHA).

[0112] In some instances, the exogenous protein binding motif / peptide is an extracellular matrix (ECM) protein binding motif / ECM-derived peptide, which includes laminin-derived peptides, surface-labeled peptides, fibronectin-derived peptides, collagen-derived peptides, gelatin-derived peptides, synaptic glycan-derived peptides, and / or combinations thereof.

[0113] In some instances, extracellular matrix protein binding motifs / ECM-derived peptides may include, but are not limited to, laminin-derived peptides, fibronectin-derived peptides, collagen-derived peptides, gelatin-derived peptides, synaptic glycan-derived peptides, and / or combinations thereof.

[0114] In some instances, the exogenous protein binding motif / peptide contains approximately 4 to 40 amino acid residues. In some instances, the exogenous protein binding motif / peptide may contain 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acid residues. In some instances, the binding motif contains approximately 4 to 12 amino acid residues. In some instances, the exogenous protein binding motif / peptide may contain 5, 8, 9, 10, 11, 14, or 18 amino acid residues.

[0115] In some instances, the exogenous protein binding motif / peptide contains laminin-derived peptides.

[0116] In some instances, ECM protein binding motifs / ECM-derived peptides contain laminin-derived peptides.

[0117] In some instances, the exogenous protein-binding motif / peptide comprises a laminin-derived peptide, which contains a peptide having the following sequence:

[0118] Ile-Lys-Val-Ala-Val (IKVAV, SEQ ID NO:17),

[0119] Tyr-Ile-Gly-Ser-Arg (YIGSR, SEQ ID NO:18),

[0120] Pro-Pro-Phe-Leu-Met-Leu-Leu-Lys-Gly-Ser-Thr-Arg (PPFLMLLKGSTR, SEQ IDNO:19),

[0121] Asp-Leu-Thr-Ile-Asp-Asp-Ser-Tyr-Trp-Tyr-Arg-Ile (DLTIDDSYWYRI, SEQ IDNO:20),

[0122] Asn-Ser-Ile-Lys-Val-Ser-Val-Ser-Ser (NSIKVSVSS, SEQ ID NO: 1-CP peptide 1),

[0123] Cys-Thr-Val-Ser-Pro-Gly-Val-Glu-Asp-Ser-Glu-Gly-Thr-Ile (CTVSPQVEDSEGTI, SEQ ID NO: 2-CP peptide 2),

[0124] Lys-Gly-Cys-Ser-Leu-Glu-Asn-Val-Tyr-Thr (KGCSLENVYT, SEQ ID NO: 3-CP peptide 3),

[0125] Ser-Gly-Gly-Thr-Pro-Ala-Pro-Pro-Arg-Arg-Lys-Arg-Arg-Glu-Thr-Gly-Glu-Ala (SGGTPAPPRRKRRQTGQA, SEQ ID NO:4-CP peptide 4), and / or combinations thereof.

[0126] In some instances, laminin-derived peptides may include, but are not limited to, peptides having the following sequences: Ile-Lys-Val-Ala-Val (IKVAV), Tyr-Ile-Gly-Ser-Arg (YIGSR, SEQ ID NO:18), Pro-Pro-Phe-Leu-Met-Leu-Leu-Lys-Gly-Ser-Thr-Arg (PPFLMLLKGSTR, SEQ ID NO:19), Asp-Leu-Thr-Ile-Asp-Asp-Ser-Tyr-Trp-Tyr-Arg-Ile (DLTIDDSYWYRI, SEQ ID NO:20), and / or combinations thereof.

[0127] In some instances, the exogenous protein binding motif / peptide contains a heart-specific laminin that interacts with integrins on the surface of cardiomyocytes.

[0128] In some instances, the heart-specific laminin can be laminin-221 or α-lamin LAMA2. In some instances, the heart-specific laminin can be within LAMA1 or LAMA2.

[0129] In some instances, the exogenous protein binding motif / peptide comprises one or more exposed regions on the interaction domains of laminin, including helices, loops, and laminin globular (LG) domains LG1, LG2, LG3, LG4, LG5, and / or combinations thereof. Optionally, the exogenous protein binding motif / peptide comprises an exposed region between a helix and LG1, an exposed region between LG1 and LG2, an exposed region between LG2 and LG3, and an exposed loop in LG3, or combinations thereof.

[0130] In some instances, laminin-derived peptides contain the following localization and / or protein sequences:

[0131] In some instances, the exogenous protein-binding motif / peptide comprises a laminin-derived peptide, which contains a peptide having the following sequence:

[0132] Asn-Ser-Ile-Lys-Val-Ser-Val-Ser-Ser (NSIKVSVSS, SEQ ID NO: 1-CP peptide 1),

[0133] Cys-Thr-Val-Ser-Pro-Gly-Val-Glu-Asp-Ser-Glu-Gly-Thr-Ile (CTVSPQVEDSEGTI, SEQ ID NO: 2-CP peptide 2),

[0134] Lys-Gly-Cys-Ser-Leu-Glu-Asn-Val-Tyr-Thr (KGCSLENVYT, SEQ ID NO: 3-CP peptide 3),

[0135] Ser-Gly-Gly-Thr-Pro-Ala-Pro-Pro-Arg-Arg-Lys-Arg-Arg-Glu-Thr-Gly-Glu-Ala (SGGTPAPPRRKRRQTGQA, SEQ ID NO:4-CP peptide 4), and / or combinations thereof.

[0136] In some instances, the exogenous protein binding motif / peptide contains a surface-labeled peptide.

[0137] In some instances, surface-labeled peptides may include surface proteins that are specific to or interact with surface proteins of the cells of interest. As used herein, the cells of interest may be host cells used to generate engineered mitochondria and / or cells that will benefit from mitochondrial therapy (i.e., target cells to be treated). For example, the cells of interest may be any cells that require energy to function. Cells of interest may include, but are not limited to, cells of the cardiovascular system (e.g., cardiomyocytes), cells of the nervous system (e.g., but not limited to, cells of the central and / or peripheral nervous systems, such as nerve cells, glial cells, retinal glial cells, etc.), cells of the digestive system (e.g., but not limited to, hepatocytes, gastric cells, small intestinal cells, large intestinal cells, etc.), cells of the musculoskeletal system (e.g., but not limited to, muscle cells, chondrocytes, osteocytes, osteoblasts, etc.), cells of the endocrine system (e.g., but not limited to, cells from the adrenal glands, cells from the pancreas, including β cells, γ cells, α cells, etc.), cells of the reproductive system (e.g., but not limited to, eggs, sperm, etc.), immune cells (e.g., but not limited to, innate immune cells, including natural killer cells, macrophages, dendritic cells; adaptive immune cells, including T lymphocytes, NK T lymphocytes, B lymphocytes, etc.), epithelial cells (e.g., but not limited to, keratinocytes, hair follicles, etc.), etc. In some instances, cells of interest may be retinal cells, retinal glial cells, cardiomyocytes, etc. In some instances, the cells can be modified, recombinant, or engineered cells, such as, but not limited to, CAR cells, such as CAR T cells or CAR NK cells. Not wishing to be bound by theory, the cells can be CAR-T cells, as the transplanted mitochondria can provide energy for the cell division process. In some instances, the anticipated improvement in energy production is also expected to benefit the intervention, such as the in vitro fertilization (IVF) process.

[0138] In some instances, exogenous protein binding motifs / peptides contain surface marker proteins of cardiomyocytes.

[0139] In some instances, the exogenous protein binding motif / peptide contains a surface marker protein of cardiomyocytes, such as SIRPα.

[0140] In some instances, the surface marker protein is SIRPα, which interacts with surface marker proteins (such as CD47) expressed on immune cells to regulate immune responses near the heart. In some instances, the surface marker protein comprises amino acid elements between the A and B chains, the B and C chains (BC loop), and the F and G chains (FG loop) of SIRPα. In some instances, the surface marker protein contains the following localization and / or protein sequence:

[0141] In some instances, the exogenous protein binding motif / peptide comprises a SIRPα surface marker protein, which contains a peptide having the following sequence:

[0142] Thr-Lys-Ser-Val-Glu-Phe-Thr-Phe-Cys-Asn-Asp-Thr-Val-Val-Iso-Pro-Cys-Phe (TKSVEFTFCNDTVVIPCF, SEQ ID NO:7-Peptide 7),

[0143] Asn-Met-Glu-Ala-Gln-Asn-Thr-Thr-Glu-Val-Tyr (NMEAQNTTEVY, SEQ ID NO: 5-peptide 5),

[0144] Thr-Glu-Leu-Thr-Arg-Glu-Gly-Glu (TELTREGE, SEQ ID NO: 6-peptide 6), or combinations thereof.

[0145] In some instances, surface marker proteins may include the following alternative locations, combinations, and / or protein sequences:

[0146] In some instances, the exogenous protein binding motif / peptide comprises a SIRPα surface marker protein, which contains a peptide having the following sequence:

[0147] Val-Val-Iso-Pro-Cys-Phe-Val-Thr-Asn-Met-Glu-Ala (VVIPCFVTNMEA, SEQ IDNO:12-ABC peptide),

[0148] Cys-Asn-Asp-Thr-Val-Val-Iso-Pro-Cys-Phe (CNDTVVIPCF, SEQ ID NO:11-AB rear part),

[0149] Thr-Lys-Val-Glu-Phe-Thr-Phe-Cys-Asn-Asp-Thr-Val-Val-Iso-Pro-Cys-Phe(TKSVEFTFCNDTVVIPCF, SEQ ID NO:7-AB full length),

[0150] Glu-Phe-Thr-Phe-Cys-Asn-Asp-Thr-Val-Val (EFTFCNDTVV, SEQ ID NO:10-AB middle),

[0151] Thr-Lys-Ser-Val-Glu-Phe-Thr-Phe-Cys-Asn (TKSVEFTFCN, SEQ ID NO:9-AB anterior part),

[0152] Asn-Met-Glu-Ala-Gln-Asn-Thr-Thr-Glu-Val-Tyr (NMEAQNTTEVY, SEQ ID NO: 5-BC peptide),

[0153] Or a combination thereof.

[0154] In some instances, the exogenous protein binding motif / peptide comprises a fibronectin-derived peptide, which includes Arg-Gly-Asp (RGD, SEQ ID NO:13), Pro-His-Ser-Arg-Asn (PHSRN, SEQ ID NO:14), cyclic RGD, and / or combinations thereof.

[0155] In some instances, fibronectin-derived peptides may include, but are not limited to, (or selected from) Arg-Gly-Asp (RGD, SEQ ID NO:13), Pro-His-Ser-Arg-Asn (PHSRN, SEQ ID NO:14), cyclic RGD, and / or combinations thereof.

[0156] In some instances, the exogenous protein binding motif / peptide comprises a collagen-derived peptide, which comprises Asp-Gly-Glu-Ala (DGEA, SEQ ID NO:16).

[0157] In some instances, collagen-derived peptides may include, but are not limited to, Asp-Gly-Glu-Ala (DGEA, SEQ ID NO: 16).

[0158] In some instances, one or more binding motifs may include a combination of the same binding motifs or a combination of different binding motifs.

[0159] In some instances, combinations of the same binding motif may include multiple copies of the motif, such as, but not limited to, IKVAV (SEQ ID NO:17), RGD (SEQ ID NO:13), etc.

[0160] In some instances, combinations of different binding motifs may include IKVAV (SEQ ID NO:17) and RGD (SEQ ID NO:13), etc.

[0161] In some instances, engineered mitochondria may include the insertion of one binding motif / peptide, two binding motifs / peptides, three binding motifs / peptides, four binding motifs / peptides, five binding motifs / peptides, etc.

[0162] In some instances, engineered mitochondria can bind to one, two, three, four, or five targets of a target cell.

[0163] In some instances, engineered mitochondria include a binding motif / peptide.

[0164] In some instances, the peptide is a cell-penetrating peptide.

[0165] In some instances, exogenous protein-binding motifs / peptides are expressed on exogenous mitochondrial outer membrane proteins.

[0166] In some instances, mitochondrial membrane proteins are outer mitochondrial membrane proteins (e.g., proteins with transmembrane α-helices or β-barrels).

[0167] In some instances, outer mitochondrial membrane proteins may include β-barrel outer membrane proteins and / or α-helical outer membrane proteins, etc.

[0168] Examples of β-barrel outer membrane proteins may include, but are not limited to, outer membrane translocation enzyme (TOM) complexes (e.g., TOM20, TOM70, TOM40, TOM22, TOM7, TOM6, TOM5, TOM70, etc.), sorting and assembly machinery (SAM) complexes (e.g., SAM50, SAM35, SAM37, etc.), voltage-dependent anion channels (VDAC), porins, etc.

[0169] Examples of α-helical outer membrane proteins may include mitochondrial importation complexes (MIMs) (e.g., Mim1, Mim2, etc.).

[0170] In some instances, exogenous protein-binding motifs / peptides are expressed on exogenous extramitochondrial membrane translocation enzyme complexes, optionally on exogenous TOM20, TOM70, TOM40, TOM22, TOM7, TOM6, TOM5, TOM70, SAM50, SAM35, SAM37, Mim1, Mim2, or combinations thereof.

[0171] In some instances, exogenous protein-binding motifs / peptides are expressed on exogenous TOM20, TOM22, TOM70, SAM50, porins, outer membrane protein 25 (OMP25), or combinations thereof.

[0172] In some instances, outer mitochondrial membrane proteins may include TOM22, TOM70, SAM50, porins, outer membrane protein 25 (OMP25), etc.

[0173] In some instances, exogenous protein-binding motifs / peptides are expressed on exogenous TOM20 proteins.

[0174] In some instances, ECM-binding motifs / peptides are located on the cytoplasmic surface of outer mitochondrial membrane proteins.

[0175] To avoid being bound by theory, motifs are positioned on the cytoplasmic surface, allowing them to interact with targets (such as integrins) expressed on target cells to promote cellular uptake.

[0176] In some instances, the outer mitochondrial membrane protein is the outer mitochondrial membrane transloase complex subunit 20 (TOM20).

[0177] As used in this article, TOM20 (describing the TOM20 protein) and TOMM20 (describing the name of the TOM20 gene) are understood to describe the same component and are therefore used interchangeably.

[0178] In some instances, such as the engineered mitochondria disclosed herein, the fusion protein may contain an IKVAV targeting motif (tethered to the HA tag) inserted into the cytoplasmic surface of TOM20 (TOM20-1xIKVAV-3xHA).

[0179] As in this disclosure Figure 2 As shown, the expression of the TOM20 fusion protein was confirmed by methods known in the art (e.g., Western blotting using antibodies against HA and TOM20). As disclosed herein... Figure 3 As shown, mitochondria isolated from cells using antibody-based purification (using anti-HA microbeads / anti-TOM22 microbeads) exhibited similar size and particle size in flow cytometry analysis. This confirms that anti-HA microbeads can be used to purify mitochondria from cell lysates, and that the IKVAV-HA motif is located on the mitochondrial surface. Furthermore, as disclosed herein… Figure 4 As shown, compared with the use of anti-TOM22 microbeads, the use of anti-HA microbeads isolated a higher amount of mitochondrial protein, indicating that the binding efficiency of anti-HA microbeads to TOM20 fusion protein is higher than that of anti-TOM22 microbeads to TOM22 protein, resulting in a higher yield of mitochondria.

[0180] In some instances, outer mitochondrial membrane proteins may contain further genetic modifications. These modifications may include, but are not limited to, insertions, deletions, single amino acid substitutions, and amino acid modifications (e.g., acetylation, phosphorylation, etc.).

[0181] In some instances, mitochondria are derived from cells.

[0182] In some instances, cells may include, but are not limited to, pluripotent stem cells (e.g., induced pluripotent stem cells), mesenchymal stem cells, adipose-derived stem cells, adult stem cells, and cells carrying mitochondria.

[0183] In some instances, the cells are induced pluripotent stem cells.

[0184] On the other hand, a polynucleotide is provided that encodes an organelle (or mitochondria) as disclosed herein.

[0185] In some instances, the polynucleotide encodes an insert construct comprising a sequence encoding a foreign outer mitochondrial membrane protein and / or a foreign protein-binding motif / peptide. In some instances, the polynucleotide may comprise a polynucleotide encoding a fusion protein comprising a foreign outer mitochondrial membrane protein and a foreign protein-binding motif / peptide. In some instances, the fusion protein is configured to be expressed on the mitochondrial surface of a host cell. In some instances, the polynucleotide may encode a fusion protein, which typically comprises:

[0186] , or

[0187] .

[0188] In some instances, mitochondrial extracellular membrane proteins / mitochondrial membrane proteins are extramitochondrial membrane proteins, such as proteins with transmembrane α-helices or β-barrels.

[0189] In some instances, exogenous extramitochondrial membrane proteins may include β-barrel outer membrane proteins and / or α-helical outer membrane proteins, etc.

[0190] Examples of β-barrel outer membrane proteins may include, but are not limited to, outer membrane (TOM) complexes (e.g., TOM20, TOM70, TOM40, TOM22, TOM7, TOM6, TOM5, TOM70, etc.), sorting and assembly machinery (SAM) complexes (e.g., SAM50, SAM35, SAM37, etc.), voltage-dependent anion channels (VDAC), porins, etc.

[0191] Examples of α-helical outer membrane proteins may include mitochondrial importation complexes (MIMs) (e.g., Mim1, Mim2, etc.).

[0192] In some instances, exogenous protein-binding motifs / peptides are expressed on exogenous mitochondrial outer membrane translocation enzyme complexes, optionally on exogenous TOM20, TOM70, TOM40, TOM22, TOM7, TOM6, TOM5, TOM70, SAM50, SAM35, SAM37, Mim1, Mim2, or combinations thereof.

[0193] In some instances, exogenous protein-binding motifs / peptides are expressed on exogenous TOM20, TOM22, TOM70, SAM50, porins, outer membrane protein 25 (OMP25), or combinations thereof.

[0194] In some instances, outer mitochondrial membrane proteins may include TOM22, TOM70, SAM50, porins, outer membrane protein 25 (OMP25), etc.

[0195] In some instances, exogenous protein-binding motifs / peptides are expressed on exogenous TOM20 proteins.

[0196] In some instances, the exogenous protein binding motif / peptide is an extracellular matrix (ECM) protein binding motif / ECM-derived peptide, which includes laminin-derived peptides, surface-labeled peptides, fibronectin-derived peptides, collagen-derived peptides, gelatin-derived peptides, synaptic glycan-derived peptides, and / or combinations thereof.

[0197] In some instances, the encoded polynucleotide encodes a foreign protein-binding motif / peptide having one or more sequences selected from the following:

[0198] Ile-Lys-Val-Ala-Val (IKVAV, SEQ ID NO:17),

[0199] Tyr-Ile-Gly-Ser-Arg (YIGSR, SEQ ID NO:18),

[0200] Pro-Pro-Phe-Leu-Met-Leu-Leu-Lys-Gly-Ser-Thr-Arg (PPFLMLLKGSTR, SEQ IDNO:19),

[0201] Asp-Leu-Thr-Ile-Asp-Asp-Ser-Tyr-Trp-Tyr-Arg-Ile (DLTIDDSYWYRI, SEQ IDNO:20),

[0202] Asn-Ser-Ile-Lys-Val-Ser-Val-Ser-Ser (NSIKVSVSS-CP peptide 1, SEQ ID NO: 1),

[0203] Cys-Thr-Val-Ser-Pro-Gly-Val-Glu-Asp-Ser-Glu-Gly-Thr-Iso (CTVSPQVEDSEGTI-CP peptide 2, SEQ ID NO: 2),

[0204] Lys-Gly-Cys-Ser-Leu-Glu-Asn-Val-Tyr-Thr (KGCSLENVYT - CP peptide 3, SEQ ID NO:3),

[0205] Ser-Gly-Gly-Thr-Pro-Ala-Pro-Pro-Arg-Arg-Lys-Arg-Arg-Glu-Thr-Gly-Glu-Ala (SGGTPAPPRRKRRQTGQA - CP peptide 4, SEQ ID NO:4),

[0206] Thr-Lys-Ser-Val-Glu-Phe-Thr-Phe-Cys-Asn-Asp-Thr-Val-Val-Iso-Pro-Cys-Phe (TKSVEFTFCNDTVVIPCF - peptide 7, SEQ ID NO:7),

[0207] Asn-Met-Glu-Ala-Gln-Asn-Thr-Thr-Glu-Val-Tyr (NMEAQNTTEVY - peptide 5, SEQ IDNO:5),

[0208] Thr-Glu-Leu-Thr-Arg-Glu-Gly-Glu (TELTREGE - peptide 6, SEQ ID NO:6),

[0209] Val-Val-Iso-Pro-Cys-Phe-Val-Thr-Asn-Met-Glu-Ala (VVIPCFVTNMEA - ABC peptide, SEQ ID NO:12),

[0210] Cys-Asn-Asp-Thr-Val-Val-Iso-Pro-Cys-Phe (CNDTVVIPCF - AB - rear part, SEQ IDNO:11),

[0211] Thr-Lys-Val-Glu-Phe-Thr-Phe-Cys-Asn-Asp-Thr-Val-Val-Iso-Pro-Cys-Phe(TKSVEFTFCNDTVVIPCF - AB full length, SEQ ID NO:7),

[0212] Glu-Phe-Thr-Phe-Cys-Asn-Asp-Thr-Val-Val (EFTFCNDTVV-AB-Middle, SEQ IDNO:10), Thr-Lys-Ser-Val-Glu-Phe-Thr-Phe-Cys-Asn (TKSVEFTFCN-AB-Front),

[0213] Asn-Met-Glu-Ala-Gln-Asn-Thr-Thr-Glu-Val-Tyr (NMEAQNTTEVY-BC peptide, SEQ IDNO:5),

[0214] Or a combination thereof.

[0215] In some instances, the polynucleotide encodes an insert construct containing a sequence encoding TOMM20 and / or a cardiac peptide motif.

[0216] In some instances, the insert construct may also contain hemagglutinin tags, such as HA tags, 3xHA tags, etc.

[0217] In another aspect, a vector is provided that contains polynucleotides encoding organelles (or mitochondria) as disclosed herein.

[0218] In another aspect, a host cell is provided which contains a vector as disclosed herein.

[0219] In another aspect, a cell is provided that contains organelles (or mitochondria) as disclosed herein.

[0220] In some instances, the cells are pluripotent.

[0221] In some instances, the cells are pluripotent cells, optionally induced pluripotent cells.

[0222] In some instances, the cell is the cell of interest, such as a diseased cell whose function is improved when transplanted together with engineered mitochondria. For example, the cell of interest can be any cell that requires energy to function. Therefore, cells of interest may include, but are not limited to, cells of the cardiovascular system (e.g., cardiomyocytes), cells of the nervous system (e.g., but not limited to, cells of the central and / or peripheral nervous systems, such as nerve cells, glial cells, retinal glial cells, etc.), cells of the digestive system (e.g., but not limited to, hepatocytes, gastric cells, small intestinal cells, large intestinal cells, etc.), cells of the musculoskeletal system (e.g., but not limited to, muscle cells, chondrocytes, osteocytes, osteoblasts, etc.), cells of the endocrine system (e.g., but not limited to, cells from the adrenal glands, cells from the pancreas, including β cells, γ cells, α cells, etc.), cells of the reproductive system (e.g., but not limited to, eggs, sperm, etc.), immune cells (e.g., but not limited to, innate immune cells, including natural killer cells, macrophages, dendritic cells; adaptive immune cells, including T lymphocytes, NK T lymphocytes, B lymphocytes, etc.), epithelial cells (e.g., but not limited to, keratinocytes, hair follicles, etc.), etc. In some instances, cells of interest may be retinal cells, retinal glial cells, cardiomyocytes, etc. In some instances, the cells can be modified, recombinant, or engineered cells, such as, but not limited to, CAR cells, such as CAR T cells or CAR NK cells. Not wishing to be bound by theory, the cells can be CAR-T cells, as the transplanted mitochondria can provide energy for the cell division process. In some instances, the anticipated improvement in energy production is also expected to benefit the intervention, such as the in vitro fertilization (IVF) process.

[0223] On the other hand, methods for improving / enhancing / increasing mitochondrial uptake / targeting in target cells are provided, the methods including providing engineered mitochondria as disclosed herein.

[0224] On the other hand, a method for improving mitochondrial uptake into target cells is provided, the method comprising genetically modifying a host cell to express modified mitochondria, wherein the modified mitochondria contain one or more exogenous protein-binding motifs / peptides expressed on the outer mitochondrial membrane.

[0225] Unwilling to be bound by theory, mitochondrial transplantation / mitochondrial therapy (mitochondrial therapy) / mitochondrial transfer aims to transfer functional exogenous mitochondria into mitochondrial-deficient cells to restore cell viability and thus halt disease progression. Exogenous mitochondria can be directly introduced into mammalian cells for disease treatment after local and intravenous administration, where the transferred mitochondria perform their functions in the target cells, including energy production and maintaining cellular function. Methods known in the art for mitochondrial uptake by host cells are inefficient, and currently there are no methods to target mitochondria for uptake by specific cell types.

[0226] As disclosed herein Figure 8 As shown, IKVAV-labeled mitochondria were more significantly internalized by the mouse photoreceptor precursor cell line 661W compared to mitochondria without this motif. Furthermore, this disclosure… Figure 9 The study showed a dose-dependent increase in mitochondrial uptake of TOM20-HA and TOM20-IKVAV-HA mitochondria, with TOM20-IKVAV-HA mitochondria showing significantly higher internalization at 12 hours after treatment of human motor neurons (compared to 5 hours post-treatment).

[0227] In some instances, engineered mitochondria involve the insertion of one or more binding motifs on the outer mitochondrial membrane.

[0228] In some instances, one or more binding motifs can be inserted into the mitochondria of a host cell using methods known in the art, such as, but not limited to, CRISPR-Cas9 genome editing, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs).

[0229] In some instances, host cells can be genetically modified using viral transduction and / or CRISPR gene modification strategies. Those skilled in the art will recognize that viral transduction and CRISPR gene modification strategies are known in the art and will be readily adapted to implement the methods of this disclosure.

[0230] In some instances, host cells can be genetically modified via viral transduction or CRISPR gene modification systems.

[0231] In some instances, target cells may include, but are not limited to, immortalized cell lines, primary cells, cells with metabolic diseases (e.g., mitochondrial defects, mitochondrial DNA mutations), senescent cells (i.e., cells that tend to have increased mitochondrial DNA mutations), engineered mitochondria carrying death signals, etc.

[0232] In some instances, target cells may include, but are not limited to, β1-integrin-expressing cells, laminin receptor-expressing cells, collagen receptor-expressing cells, fibronectin receptor-expressing cells, etc.

[0233] In some instances, β1-integrin-expressing cells may include, but are not limited to, neurons (e.g., human motor neurons) and retinal cells (e.g., mouse photoreceptor precursor cell line 661W, retinal ganglion cells, etc.).

[0234] In some instances, the target cells can be mammalian cells. In some instances, the target cells can be derived from humans, non-human primates, pigs, mice, rats, etc. In some instances, the mouse background can include, but is not limited to, C57BL6, BALB / c, CD-1, SCID, etc. In some instances, the rat background can include, but is not limited to, A / J, Sprague Dawley, Wistar, etc. In some instances, non-human primates can include, but are not limited to, rhesus monkeys, Japanese macaques, olive baboons, squirrel monkeys, capuchin monkeys, etc.

[0235] In some instances, engineered mitochondria can be purified / isolated from cells using methods including: sonicating the cells; removing impurities by differential centrifugation; and / or filtering the mitochondrial-enriched precipitate to further reduce contaminants (e.g., other organelles).

[0236] Without being bound by theory, known methods in the art for deriving intact mitochondria (including ultracentrifugation, differential centrifugation, or bead-based sorting) are not well-suited for extracting intact mitochondria from induced pluripotent stem cells. For example, dounce homogenization (the standard method for mechanical cell lysis) is inefficient in lysing pluripotent stem cells (because they are smaller than typical cultured cells) and produces large batch-to-batch and user-to-user variability. Furthermore, the co-precipitation of microbeads with mitochondria and their inability to be effectively separated impacts downstream applications.

[0237] The inventors of this disclosure overcome the drawbacks of known methods in the art by using weak ultrasound to lyse cells while maintaining the integrity of mitochondria. This disclosure uses a combination of ultrasound treatment, differential centrifugation, and size selection (S+DC method) to separate highly enriched mitochondrial populations. Advantageously, the method of this disclosure can be completed within 60 minutes, which is faster than the microbead method known in the art. Furthermore, compared to differential centrifugation methods known in the art that exhibit significant nuclear fragment contamination, the method of this disclosure improves mitochondrial yield and purity. Figure 6 ).

[0238] As disclosed herein Figure 7As shown, the inventors of this disclosure demonstrate that mitochondria isolated by the S+DC method chelate tetramethylrhodamine perchlorate (TMRE) (a red fluorescent dye) and react with mitochondrial membrane depolarizing molecules (FCCP). This confirms that mitochondria isolated using the S+DC method of this disclosure are intact and biologically active.

[0239] In another aspect, a composition or pharmaceutical composition is provided comprising engineered mitochondria or nucleic acids as described herein.

[0240] In another aspect, a composition is provided comprising engineered mitochondria or nucleic acids as disclosed herein.

[0241] In another aspect, a pharmaceutical composition is provided comprising engineered mitochondria or nucleic acids as disclosed herein and suitable pharmaceutical compositions thereof.

[0242] On the other hand, a composition or pharmaceutical composition as described herein is provided for use in treatment / medicine.

[0243] In some instances, the composition is a preventative and / or therapeutic composition.

[0244] Pharmaceutically acceptable agents used in this pharmaceutical composition include carriers, excipients, diluents, antioxidants, preservatives, colorants, flavoring agents and diluents, emulsifiers, suspending agents, solvents, fillers, compatibilizers, buffers, delivery media, tensioning agents, solubilizers, wetting agents, complexing agents, buffers, antimicrobial agents and surfactants.

[0245] A composition as described herein is also disclosed for use in treatment / medicine.

[0246] In some instances, such compositions also contain excipients and / or stabilizers.

[0247] On the other hand, a method for preventing and / or treating a disease in a subject in need is provided, the method comprising administering engineered mitochondria or a composition as described herein to the subject.

[0248] On the other hand, a method is provided for preventing and / or treating diseased tissue in a subject in need, the method comprising administering to the subject engineered mitochondria or a composition as disclosed herein.

[0249] On the other hand, a method is provided for preventing and / or reducing the severity of symptoms caused by a disease in subjects in need, the method comprising administering to the subject engineered mitochondria or a composition as disclosed herein.

[0250] In some instances, the terms “prevention” and / or “reducing the severity of symptoms” refer to the process of delaying onset, reducing the severity of symptoms, reducing and / or preventing weight loss, preventing death, inhibiting deterioration, inhibiting further deterioration, and / or improving one or more signs or symptoms of the disease.

[0251] In some instances, the disease is a mitochondrial disease or a proliferative disease.

[0252] In some instances, diseased tissue may include, but is not limited to, tissue from mitochondrial diseases, proliferative diseases, etc.

[0253] In some instances, the disease may include, but is not limited to, mitochondrial diseases, proliferative diseases, etc.

[0254] In some instances, mitochondrial diseases may include, but are not limited to, mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome, Leber hereditary optic neuropathy (LHON), Leigh syndrome, Karns-Sell syndrome (KSS), myoclonic epilepsy with broken red fiber disease (MERRF), Friedrich's ataxia (FA), etc.

[0255] Proliferative diseases, as described in this article, include inflammatory / degenerative diseases.

[0256] In some instances, inflammatory / degenerative diseases can be neurodegenerative diseases, such as neuroinflammation that leads to neurodegeneration / brain damage / nerve injury. In some instances, the disease can be a neurodegenerative disease, such as dementia, Alzheimer's disease, Parkinson's disease, multiple sclerosis, Huntington's disease, etc. Other degenerative diseases may include, but are not limited to, arthritis, muscular dystrophy, amyotrophic lateral sclerosis (ALS), etc.

[0257] In some cases, inflammatory diseases may include, but are not limited to, fibrosis, muscle aging, etc.

[0258] In some instances, proliferative diseases can be inflammatory diseases, such as acute inflammatory diseases and / or chronic inflammatory diseases. In some instances, chronic inflammation / acute inflammation / chronic inflammation can include, but is not limited to, ulcerative colitis, Crohn's disease, infectious diseases, etc.

[0259] In some instances, infectious diseases can be caused by bacterial pathogens, viral pathogens, fungal pathogens, or parasites.

[0260] Examples of bacterial pathogens may include, but are not limited to, Escherichia coli, certain species of Mycobacteria spp, certain species of Salmonella spp, certain species of Staphylococcus spp, Clostridium difficile, Listeria monocytogenes, Group B streptococci, and vancomycin-resistant enterococci (VRE).

[0261] Examples of viral pathogens may include, but are not limited to, human papillomavirus, rhinovirus, human cytomegalovirus in HIV-1 positive patients, hepatitis virus, coronavirus (CoV), severe acute respiratory syndrome (SARS), monkeypox virus, enterovirus 71 (EV71), etc.

[0262] Examples of fungal pathogens include, but are not limited to, Botrytis cinerea, Pseudomonas syringae, and Fusarium oxysporum.

[0263] Examples of parasites may include, but are not limited to, Leishmania protozoan parasites, Giardia lamblia, Cryptosporidium, and Entamoeba histolytica.

[0264] On the other hand, the use of engineered mitochondria disclosed herein in the preparation of medicaments for the prevention and / or treatment of diseases is provided.

[0265] In some instances, the engineered mitochondria or compositions or pharmaceutical compositions or uses or methods described herein may be administered to subjects via one or more routes of administration (including but not limited to surface, intravascular, intravenous, oral, subcutaneous, intra-arterial, intrathecal, intraperitoneal, intranasal, intradermal, intramuscular, intravitreal, etc.).

[0266] The engineered mitochondria / methods described in this paper are disclosed.

[0267] The polynucleotide encoding the mitochondria described herein was also disclosed.

[0268] A vector containing the polynucleotides described herein is also disclosed.

[0269] Furthermore, when describing some embodiments, this disclosure may have already disclosed methods and / or processes in a specific sequence of steps. However, unless otherwise required, it should be understood that the method or process should not be limited to the specific sequence of steps disclosed. Other sequences of steps may be possible. The specific order of steps disclosed herein should not be construed as an undue limitation. Unless otherwise required, the methods and / or processes disclosed herein should not be limited to steps performed in the written order. The sequence of steps may vary and still remain within the scope of this disclosure.

[0270] Furthermore, it should be understood that although this disclosure provides embodiments having one or more of the features / characteristics discussed herein, one or more of these features / characteristics may also be waived in other alternative embodiments, and this disclosure provides support for such waivers and their associated alternative embodiments. Attached Figure Description

[0271] Exemplary embodiments of this disclosure will be better understood and will be apparent to those skilled in the art through the following discussion and, where applicable, in conjunction with the accompanying drawings. It should be understood that other modifications may be made without departing from the scope of the invention. Exemplary embodiments are not necessarily mutually exclusive, as some embodiments may be combined with one or more other embodiments to form new exemplary embodiments. Exemplary embodiments should not be construed as limiting the scope of this disclosure.

[0272] Figure 1 This paper illustrates a CRISPR-Cas9 cloning strategy for inserting an overexpression cassette into the TOMM20 fusion protein. First, the fusion protein cDNA sequence (1. to 3.) was constructed and cloned into the HDR donor plasmid. Co-transfection of the donor plasmid with the Cas9-gRNA expression plasmid resulted in a targeted double-strand break between exon 1 and exon 2 of the AAVS1 locus. The double-strand break was repaired using the host genome via homologous recombination with the HDR plasmid, leading to the insertion of the overexpression cassette into the host genomic DNA and subsequent overexpression of the TOMM20 fusion protein.

[0273] Figure 2 Immunostaining and Western blot data using specific antibodies against the HA epitope (top panel blot) and TOMM20 (bottom panel blot) are shown. As expected, both TOM20-HA and TOM20-1xIKVAV-HA clones showed positive signals when using anti-HA and anti-TOM20 antibody probes.

[0274] Figure 3 Flow cytometry analysis was performed to show that mitochondria extracted with anti-TOM22 microbeads and anti-HA microbeads were similar in size and particle size. The ability to purify mitochondria using anti-HA beads also confirmed the expression of HA epitopes on the outer surface of the mitochondrial membrane.

[0275] Figure 4 The relative protein mass was quantified by BCA determination after mitochondria were isolated using a microbead-based method. Three biological replicates (BR1 to BR3) were performed to compare the amount of mitochondria isolated using anti-TOM20 beads and anti-HA beads. In all three samples (prep), higher amounts of protein were isolated using anti-HA beads, indicating higher mitochondrial yield using this method.

[0276] Figure 5 This diagram illustrates a rapid protocol for obtaining high yields of mitochondria from iPSCs. First, the iPSCs are sonicated to lyse the cells, followed by a series of differential centrifugation steps to remove impurities. Subsequently, the highly mitochondrial-rich precipitate is passed through a 5 μm filter to further reduce contaminants from other organelles.

[0277] Figure 6 Western blot analysis of whole-cell lysates (lanes 1-3), S+DC mitochondrial samples (lanes 4-7), and conventional DC methods (lanes 8-9) is shown. While mitochondrial isolation via S+DC or conventional DC methods resulted in enrichment of the mitochondrial fraction (indicated by stronger TOM20 intensity), the conventional method was significantly affected by nuclear fragment contamination (indicated by the presence of histone 3 or H3 bands), whereas no observable histone 3 contamination was observed in the S+DC mitochondrial samples.

[0278] Figure 7 TMRE and oxygen consumption rate (OCR) measurements are shown to identify intact, functional mitochondria. TMRE accumulates in mitochondria with membrane potential differences, as shown by the rightward shift in the blue figure. Since FCCP is a mitochondrial uncoupling and depolarizing agent, incubation of mitochondrial samples with FCCP resulted in a leftward shift of TMRE-treated mitochondria, as shown in Figure (2) (see arrow), indicating biologically active and intact mitochondria. OCR was measured using a metabolic flux analyzer, where an increase in OCR was observed with increasing mitochondrial quantity.

[0279] Figure 8 Immunostaining and quantification of internalized engineered human mitochondria based on HA staining are shown. 10,000 661W cells were incubated with 1.5 μg of TOM20-IKVAV-HA or TOM20-HA mitochondria for 24 hours. In the control group, cells were not treated with mitochondria (far left). Immunostaining was performed using antibodies against HA and TOM20, while the cell nuclei were counterstained with DAPI. Quantification of HA intensity per cell showed higher intensity in 661W cells incubated with TOM20-IKVAV-HA mitochondria.

[0280] Figure 9This study demonstrated increased mitochondrial uptake of TOM20-IKVAV-HA by human motor neurons. Human motor neurons were incubated with increased amounts of TOM20-HA or TOM20-IKVAV-HA mitochondria. Quantification of HA intensity per cell showed that mitochondrial uptake increased in a dose-dependent manner with increasing mitochondrial quantity, and that TOM20-IKVAV-HA was taken up more efficiently than control TOM20-HA mitochondria.

[0281] Figure 10 This demonstrates that exogenous mitochondria can be internalized by RGCs. The efficiency of mitochondrial transfer in retinal ganglion cells. (A) 50 mg of human mitochondria or an equivalent volume of PBS was injected intravitreally into the eyes of WT mice. Tissue was harvested 1 week post-injection and serially sectioned at a sampling ratio of 1:32. (B) Representative immunofluorescence images from each section showing the density of Brn3a+ retinal ganglion cells and the presence of human mitochondria (HuMTs) in samples receiving mitochondria instead of the PBS control. (C) Treatment with mitochondria resulted in a significant increase in the presence of Brn3a+ / HuMT+ cells, while (D) the total number of Brn3a+ retinal ganglion cells remained similar between mitochondrial treatment and the PBS control.

[0282] Figure 11 This study demonstrated that mitochondrial therapy increased mitochondrial function in vivo. A) Retinal tissue from mice that received 50 mg of mitochondria or PBS as controls was collected one week after injection. Tissue was lysed and mitochondrial complex I activity was determined using enzyme-linked immunosorbent assay (ELISA). B) Mitochondrial-treated retinal tissue showed an approximately 20% increase in complex I activity after normalization using protein content as a substitute for cell number. C) Total protein levels obtained after lysis were similar between the mitochondrial-treated sample and the PBS control.

[0283] Figure 12 No significant retinal abnormalities were observed in the retina after mitochondrial therapy. Functional imaging of the mouse eyes showed no obvious abnormalities after mitochondrial therapy. Basement membrane imaging at weeks 0 and 6 (left) showed no observable differences in the retina of mouse eyes treated with mitochondria or PBS. OCT imaging at weeks 0 and 6 (right) also showed no retinal detachment. Quantification of retinal thickness from OCT images indicated no significant difference between the mitochondrial-treated and control eyes.

[0284] Figure 13Human BJ iPSCs engineered to express the exogenous TOM20-3xHA fusion protein. A) CRISPR strategy for expressing the exogenous TOM20-3xHA fusion protein driven by the CMV human overexpression promoter. (i) Insert construct for TOM20-3xHA fusion protein expression; (ii) Insert construct for TOM20-cardiac peptide motif-3xHA fusion protein expression. B) Immunofluorescence confirmation of TOM20-3xHA fusion protein expression on the mitochondrial surface in CRISPR-engineered TOM20-HA BJ iPSC lines.

[0285] Figure 14 Western blot analysis showing the purity of mitochondrial extracts. Lanes 1-3: Whole-cell controls before extraction. Lanes 4-5: Two cell line replicates using differential centrifugation. Lanes 6-7: Two cell line replicates using differential centrifugation and filtration. Lanes 8-9: Two cell line replicates using HA bead antibody column extraction. Mitochondrial load was labeled with TOM20, nuclear contamination with H3, endoplasmic reticulum contamination with PDI, and cytoplasmic contamination with GAPDH. DC: Differential centrifugation; BR1-BR2: Cell lines 1 and 2 expressing the TOM20 fusion protein with a 3xHA tag; ER: Endoplasmic reticulum.

[0286] Figure 15 The predicted structure of laminin α subunit 1 (LAMA1) protein is shown. A) Protein structure surrounding LG1 containing the IKVAV motif. B) Protein structure surrounding LG3 containing the RGD motif. The bottom figures (A' and B') show close-up views of the motifs of interest highlighted in green. CC: Coil-and-coil domain; LG1-5: Laminin globular domains 1 to 5; IKV: IKVAV protein motif; RGD: RGD protein motif.

[0287] Figure 16 The structure prediction of the laminin α subunit 2 (LAMA2) protein and the peptides of interest are shown. A) Peptide sequence 1 exposed between CC and LG1. B) Peptide sequences 2, 3, and 4 exposed between LG1 and LG2 (B'), between LG2 and LG3 (B''), and within LG3 (B'''), respectively. A', B', B'', and B''' show close-up views of the motifs of interest highlighted in green and labeled accordingly. Pep1-4: Peptide 1 to peptide 4.

[0288] Figure 17The protein structure of CD47-SIRPα elucidated by Cryo-EM is shown. A) Annotated amino acid sequence of CD47. Chains A through G form a β-sheet structure. B) Structure of CD47 (yellow band) complexed with SIRPα (blue band). The inset shows a close-up view of the interaction interface. The FG and BC loops of CD47 play a key role in the interaction with SIRPα. Figure courtesy of Hatherley et al.

[0289] Figure 18 Synthetic peptides exhibiting improved binding to the cardiomyocyte surface are shown. A) Treatment protocols for four different cell types with eight identified peptides. B) Quantification of fluorescence in the treated wells using a microplate fluorescence reader. Fluorescence data are normalized relative to untreated fluorescence intensity and expressed as mean ± SD (N = 3 replicates). ***, p < 0.0001; one-way ANOVA with Dunnett's multiple comparison test. Significance was only indicated for peptides showing significantly greater retention in CM compared to the other three cell types. CM: cardiomyocytes; Fib: fibroblasts; MN: motor neurons; ESC: embryonic stem cells.

[0290] Figure 19 Fluorescence imaging confirms a specific increase in binding to the CM surface. A) Representative fluorescence images of hPSC-derived cell types after washing two days following treatment with FITC-labeled synthetic peptides. Peptides 2, 3, and 7 described in this paper showed significantly increased retention on CMs compared to other cell types based on microplate fluorescence analysis. Figure 18 B). Peptides 1 and 4 described in this paper show a lack of retention on CM, similar to peptides 5, 6, and 8 (not shown). B). Bright-field images superimposed on fluorescence images show fluorescence colocalization with the cell surface. Scale bar: 200 μm.

[0291] Figure 20 The altered peptide sequences show that the latter part of peptide 7 and the former part of peptide 5 contain interacting peptides between CD47 and CM. Fluorescence of the processed wells was quantified using a microplate fluorescence reader. Peptide names are listed in Table 3. Fluorescence data were normalized to unprocessed fluorescence intensity and are expressed as mean ± SD (N = 3 replicates). *, p < 0.05; **, p < 0.01; ***, p < 0.0001; ns, p > 0.05; One-way ANOVA, using Dunnett's multiple comparison test. Only comparisons with AB-full-length (original full-length peptide 7) are shown.

[0292] Figure 21Western blotting confirms fusion protein expression in clonal donor cell lines. A) Top: Anti-HA blot shows expression of the exogenous fusion protein in CP2, but not CP3. Bottom: Anti-TOM20 blot shows the presence of the HA tag on the exogenous fusion TOM20. Furthermore, it confirms that although endogenous TOM20 protein was detected in CP3, the exogenous fusion protein was not expressed. B) Similar Western blot images depicting four clonal donor cell lines used for downstream analysis. C) Flow cytometry analysis of isolated mitochondria treated with TMRE (staining polarized mitochondria) and / or FCCP (depolarizing functional mitochondria).

[0293] Figure 22 shows immunofluorescence evidence indicating that CP2 and CP7 expression improves the efficiency of mitochondrial uptake into CM. A) Treatment protocols using mature CMs from mitochondria from four donor cell lines. Uptake by donor mitochondria into host cells was quantified by the presence of HA proteins within the host cells. B) Left: Quantification of cellular HA intensity, corresponding to total HA fluorescence detected in each CM. Right: Quantification of the number of HA spots, corresponding to the number of fluorescent spots detected in each CM. Student's t-test was performed between all treatment groups to determine significance, and only significant results are shown. UT: Untreated CM.

[0294] Figure 23 Fluorescent imaging shows increased uptake of exogenous mitochondria expressing CP2 or CP7 fusion proteins by CM. Representative images show cells treated with fixed mitochondria stained with CTNT, depicting >95% pure CM, successfully uptake of exogenous mitochondria expressing HA tags on the mitochondrial surface.

[0295] Figure 24 The use of exogenous mitochondria labeled with a mitotracker confirms that CP7 expression improves mitochondrial uptake into CMs. A) Treatment protocol for mature CMs using mitochondria from four donor cell lines. Donor mitochondria were pre-stained in donor cells with mitotracker red, which was used to quantify the uptake of donor mitochondria into host cells. B) Quantification of cellular mitotracker intensity, corresponding to the total red mitotracker fluorescence detected in each CM. Student's t-test was performed between all treatment groups to determine significance, and only significant results were shown. UT: Untreated CMs. C) Representative fluorescence images of live CMs, labeled with GCaMP expression. Mitotracker red (MT Red) carried by donor mitochondria was detected in live CMs.

[0296] Figure 25The treatment of aging CM models with donor mitochondria resulted in metabolic improvements and a reversal of the aging phenotype. A) Seahorse OCR assay to determine the respiratory capacity of aged CM. B) β-galactosidase staining (blue) to depict senescent and aged cells.

[0297] Experimental Section

[0298] Genetically modified induced pluripotent stem cell lines were generated with IKVAV-3xHA expressed on the cytoplasmic surface of the outer mitochondrial membrane.

[0299] Cloning methods for BJ iPSC lines expressing CMV-TOMM20-3xHA and CMV-TOMM20-1xIKVAV-3xHA

[0300] First, the fusion protein sequence was sequentially cloned into the MCS sequence of the pUC19 vector. First, TOMM20 cDNA was amplified from pMCherry N1TOMM20 (Addgene#157761) using the TOMM20_EcoRI_FP forward primer and the TOMM20-G-BamHI_RP reverse primer for sequences without 1xIKVAV; and the TOMM20-GSSG-1xIKV-G-BamHI_RP reverse primer for sequences containing 1xIKVAV. 3xHA cDNA was amplified from pGCS-N2 (3xHA) (Addgene#85719) using the HA_BamHI_FP forward primer and the HA_PstI_RP reverse primer. Using appropriate restriction enzymes, the two cDNA sequences were cloned into the MCS sequence of the pUC19 vector using EcoRI / BamHI / PstI. Finally, the entire fusion protein sequence was amplified using the TOMM20_SalI_FP forward primer and the HA_MluI_RP reverse primer, and then cloned into AAVS1-CAG-hrGFP (Addgene#52344) using SalI / MluI to serve as a homology-directed repair (HDR) template, thereby enabling the insertion of the expression cassette into the cell line using CRISPR. A gRNA targeting the AAVS1 locus was generated by cloning the gRNA sequence GAGCTGGCTGAAGATGATG into pSPCas9(BB)-2A-GFP (Addgene#48138), which will express the Cas9-gRNA fusion protein to enable CRISPR HDR. The cloning strategy is summarized below. Figure 1 The HDR template plasmid and Cas9-gRNA plasmid were then transfected into BJ iPSCs using Lipofectamine™ Stem, and cloned and amplified. The various DNA primers used in this gene cloning are listed in Table 4.

[0301] Table 4. Primer Names and Sequences

[0302] Validate the expression of the fusion TOM20 protein containing both a targeting motif and a tag motif.

[0303] To confirm the expression of the fusion TOM20 protein, the inventors performed Western blotting using antibodies against HA and TOM20. Using the anti-HA antibody, the inventors detected bands of approximately 20 kDa TOM20-HA and TOM20-1xIKVAV-HA. This corresponds to the genetically modified fusion protein. Next, using the anti-TOM20 antibody, endogenous TOM20 protein (approximately 15 kDa) was detected in the parental BJ-iPS wild-type (BJ-WT) line. For the engineered lines TOM20-HA and TOM20-IKVAV-HA, as expected, endogenous TOM20 protein (approximately 15 kDa) and genetically modified TOM20 (approximately 20 kDa) were detected. To increase the number of IKVAV (SEQ ID NO:18) motifs, the inventors also cloned 5 copies of IKVAV (5xIKVAV) using the same strategy described above, followed by the HA tag. However, the inventors noted that 5xIKVAV-HA could not be expressed as a fusion protein. Figure 2 ).

[0304] Verify the expression of IKVAV-HA on the cytoplasmic surface of the mitochondrial outer membrane.

[0305] The IKVAV targeting motif (which is tethered to the HA tag) must be inserted on the outer surface of the mitochondria, i.e., on the cytoplasmic surface, so that the motif on the purified mitochondria can interact with integrins expressed on the host cell to promote endocytosis. To confirm that the IKVAV-HA motif is located on the mitochondrial surface, the inventors performed antibody-based mitochondrial purification from iPSCs using anti-HA microbeads, using anti-TOM22 microbeads (Miltenyi Biotec Cat No. 130-094-872) as a baseline. First, the mitochondria purified by the microbeads were analyzed by flow cytometry, where the inventors found that the mitochondria isolated by the two methods had similar size and particle size (…). Figure 3 This confirms that anti-HA microbeads can be used to purify mitochondria from cell lysates, indicating that the IKVAV-HA motif is located on the mitochondrial surface. The inventors also performed BCA protein assays to determine protein concentration as a tool for quantifying mitochondrial yield, demonstrating that more mitochondria could be isolated using anti-HA microbeads compared to anti-TOM22 microbeads. Figure 4 ).

[0306] Methods for isolating intact and bioactive mitochondria from induced pluripotent stem cells

[0307] An improved method for deriving complete mitochondria (S+DC method)

[0308] While methods exist for deriving intact mitochondria via ultracentrifugation, differential centrifugation, or bead-based sorting, each method has specific advantages and disadvantages and is not well-suited for extracting intact mitochondria from induced pluripotent stem cells. For example, the inventors found that Dunns homogenization (the standard method for mechanical cell lysis) is inefficient in lysing pluripotent stem cells (because they are smaller than typical cultured cells) and produces large batch-to-batch and user-to-user variability. Ultimately, the inventors overcame this by using weak ultrasonic treatment to lyse cells while maintaining mitochondrial integrity. The inventors also found that microbeads co-precipitate with mitochondria and cannot be effectively separated, which is a significant limitation for downstream applications. Therefore, the inventors relied on a combination of ultrasonic treatment, differential centrifugation, and size selection (S+DC method) to separate highly enriched mitochondrial populations. Figure 5 The new method can be completed in 60 minutes, which is faster than the microbead method, and offers better mitochondrial yield and purity compared to traditional differential centrifugation. Figure 6 ).

[0309] Verification showed that the mitochondria isolated using the S+DC method are biologically active.

[0310] Intact, healthy, and biologically active mitochondria maintain a mitochondrial membrane potential supported by the activity of proton pumps (complexes I, III, and IV). Damaged and defective mitochondria, on the other hand, possess a depolarized membrane. Using TMRE (tetramethylrhodamine perchlorate methyl ester), a red fluorescent dye chelated by active mitochondria with a high mitochondrial membrane potential, the inventors evaluated the fluorescence of mitochondria isolated by the S+DC method, in which the inventors found that the isolated mitochondria chelated the TMRE dye (blue curve) and responded to FCCP (a well-characterized mitochondrial membrane depolarizing molecule) (orange curve). Figure 7 In summary, this indicates that the mitochondria isolated by the S+DC method are intact and biologically active.

[0311] In vitro evidence suggests that IKVAV-modified mitochondria are preferentially internalized by nerve cells and neurons.

[0312] Purified mitochondria with the IKVAV motif were more effectively internalized by control mitochondria in vitro.

[0313] Previous studies have shown that exogenous mitochondria can be internalized in cultured cells via a process called macropinocytosis. The inventors propose that mitochondria located closer to the cell membrane of the recipient cell are more efficiently internalized. β1-integrin is expressed in many cell types, most notably neurons in the central nervous system and retina, and the binding of the laminin-derived peptide IKVAV to β1-integrin has been well characterized. Therefore, the inventors compared the uptake efficiency on the surfaces of mitochondria with and without the IKVAV motif (TOM20-IKVAV-HA and TOM20-HA, respectively). Figure 8 In this study, the mouse photoreceptor precursor cell line 661W was used, in which 10,000 cells were seeded in a single well of a 96-well plate and incubated with 1.5 μg of mitochondria for 24 h. Subsequently, the cells were fixed and immunostained. TOM20 staining was performed to elucidate the host mitochondrial network within the 661W cells, while HA staining revealed internalized human mitochondria. Confocal imaging was performed, and the results are shown below. Figure 8 Individual imaging planes within the cytoplasmic layer of cells are shown. Quantification of HA intensity per cell also reveals that IKVAV-labeled mitochondria are more significantly internalized in 661W cells.

[0314] Building upon this, the inventors conducted similar mitochondrial uptake studies in human motor neurons. Different amounts of TOM20-HA or TOM20-IKVAV-HA mitochondria (0.25 μg to 1 μg) were incubated with 10,000 human motor neurons. Cells were fixed for immunostaining analysis at 5 or 12 hours post-treatment. Mitochondrial uptake was quantified based on the average intensity of the HA signal per cell. Using this method, the inventors demonstrated that mitochondrial uptake increased in a dose-dependent manner for both TOM20-HA and TOM20-IKVAV-HA mitochondria. More importantly, the inventors also demonstrated that TOM20-IKVAV-HA mitochondria showed significantly higher internalization at the 12-hour time point. Figure 9 ).

[0315] Knock in TOM20 expressing HA markers

[0316] The first step in achieving the overall project milestone was demonstrating that iPSC cells could be bioengineered to express protein motifs on their mitochondrial surface. The TOM20 protein, encoded by the TOMM20 genomic gene sequence, is an outer mitochondrial membrane protein with its C-terminus facing the cytoplasm. Previous work has shown that exogenous TOM20-3xHA fusion proteins can be transduced into cell lines via viral delivery to label endogenous mitochondria. However, viral modification of cell lines severely limits their practicality in disease modeling and therapeutic applications. Therefore, the inventors decided to employ a CRISPR gene modification strategy to introduce the TOM20-3xHA expression cassette into the AASV1 safe harbor locus to generate iPSC lines that serve as bioengineered mitochondrial factories. Figure 13 A, i). Gene modification was performed via plasmid transfection without exposing cells to viral material. Immunofluorescence analysis of successfully transfected clones amplified BJ iPSCs expressing the TOM20-3xHA fusion protein (referred to as TOM20-HA BJ iPSCs) showed co-expression of TOM20 and the HA tag ( Figure 13 B). Therefore, the inventors propose that an additional modification of the cardiac peptide motif (described below) can be added before the 3xHA motif on the TOM20-3xHA fusion protein expression cassette to produce bioengineered mitochondria that preferentially bind to cardiomyocytes. Figure 13 A, ii).

[0317] Demonstrates enhanced purity in the extraction of mitochondria from the iPSC line.

[0318] Extraction of functional mitochondria for therapeutic purposes involves several considerations, the most important being the purity of the mitochondrial extract. There are three main steps in extracting mitochondria for functional purposes: lysis of donor cells, purification of mitochondria from cell lysates, and quantification of the final mitochondrial product. The inventors first compared low-intensity sonication with the Durns homogenate lysis method, and found that the Durns homogenate had poor cell lysis efficiency, resulting in significant material loss during the initial whole-cell cleaning step. Subsequently, three purification methods were tested: 1. differential centrifugation; 2. differential centrifugation with a final 5 μm filter step; 3. HA microbead antibody for column-based extraction. Western blotting was used to compare equal volumes of mitochondrial extracts from these three methods (…). Figure 14 ).

[0319] The inventors discovered that an optimized differential centrifugation protocol achieved higher mitochondrial yields (TOM20 bands) than the whole-cell control, with virtually no nuclear contamination (H3 bands), endoplasmic reticulum contamination (PDI bands), and cytoplasmic contamination (GAPDH bands). Similar purity was achieved using the same differential centrifugation protocol with an additional filtration step, which the inventors deemed unnecessary. Surprisingly, extraction using HA microbead antibody columns yielded impure mitochondria. Despite the increased mitochondrial yield, significant nuclear contamination and the presence of endoplasmic reticulum and cytoplasmic contaminants were observed. Therefore, the inventors conclude that sonication and differential centrifugation protocols can produce pure mitochondria for functional and therapeutic purposes.

[0320] Identification of binding motifs from heart-specific laminin-221 that interact with integrins on the surface of cardiomyocytes

[0321] The extracellular matrix is ​​known to express specific components in different regions of the human body. One such variation occurs in the basement membrane, a thin extracellular structure immediately adjacent to the cell surface. Laminin is one such structural protein found on the basement membrane and is known to have multiple cell-specific isoforms. Transcriptomic analysis of the human left ventricle showed enhanced expression of the LAMA2, LAMB2, and LAMC1 genes, corresponding to the trimeric isoforms of laminin-221. 2 The laminin E8 fragment (which is a truncated C-terminal region of the trimer) contains five laminin globular (LG1-5) domains, of which LG1-3 are known to be domains through which the laminin trimer interacts with chaperone-binding integrin isoforms associated with its cell surface. In cardiomyocytes, this cell-specific integrin isoform is α7X2β1, with which laminin-221 has been shown to interact. Previous work has identified small peptide motifs (called cell adhesion peptides (CAPs), such as the peptide sequences RGD and IKVAV found on the laminin E8 fragment) as domains that interact with cellular integrins. These previous findings form the basis for the inventors' search for CAPs that preferentially interact with the cardiomyocyte surface.

[0322] First, the inventors identified previously studied known CAPs interacting with integrin, namely RGD and IKVAV, as residing on the α subunit of laminin, which contains the LG domain of the laminin trimer. The inventors used Alphafold protein prediction software to predict the structure surrounding the LG1-3 domain in the LAMA1 protein, which contains the RGD (SEQ ID NO:13) and IKVAV CAP motifs. Notably, the RGD and IKVAV (SEQ ID NO:18) motifs are not located within the globular domain of LG1-3. The RGD motif is located in the exposed region between the end of the coiled helical structure and the beginning of the LG1 domain. Figure 15 A), while the IKVAV motif is located in the exposed region on the LG3 domain ( Figure 15 B). These results support the hypothesis that the exposed regions on the LG domain found on the laminin α subunit will contain binding motifs that interact with cell-specific integrins.

[0323] Similarly, the inventors predicted the structures surrounding the LG1-3 domain of the LAMA2 protein, a major α-laminoid subunit found in the adult heart. From this prediction, the inventors identified four exposed peptide sequences as potential motifs with preferential binding to cardiomyocytes (Table 1). Figure 16 ).

[0324] Table 1. Exposed amino acids in the LAMA2 protein.

[0325] Identification of binding motifs from CD47 that interact with heart-specific SIRPα surface marker proteins.

[0326] An alternative approach to identifying cardiomyocyte-specific binding motifs is to identify the amino acid sequences of proteins known to interact with cardiomyocyte-specific surface proteins. One established cardiomyocyte surface marker protein is SIRPα. The primary known binding partner of SIRPα is CD47, a surface marker protein expressed on immune cells to regulate immune responses near the heart. Previous reports have elucidated the protein structures of SIRPα and CD47, as well as the protein sequences responsible for their protein-protein interactions—the amino acid element between the B and C chains (BC loop) and the element between the F and G chains (FG loop). Figure 17From these findings, the inventors identified peptide sequences for the BC and FG rings. The inventors also decided to investigate peptide sequences preceding the BC ring that enable the BC ring conformation to interact with SIRPα (Table 2). The comprehensive table of peptides covering the interaction between CD47 and SIRPα, provided by Hatherley et al., justifies the reasonable cutoff points for the peptide sequences identified in this work.

[0327] These peptide sequences, along with the peptide sequences identified in Table 1 and the 6x histidine control peptide sequence, were synthesized with small FITC fluorophore tags for downstream experiments to confirm their interaction and binding to the surface of cardiomyocytes.

[0328] Table 2. CD47 peptide sequence interacting with the cardiomyocyte surface marker SIRPα

[0329] The synthetic peptide sequence has improved cardiomyocyte surface binding efficiency.

[0330] Using human pluripotent stem cells (hPSCs), the inventors generated mature contractile cardiomyocytes (CM), fibroblasts (Fib), and motor neurons (MN) using a previously established laboratory protocol. These derived cells, along with the hPSCs, were seeded in a contiguous manner into 96-well plates and allowed to recover. The treatment protocol was followed as described. Figure 18 A) These cells were treated with each of the eight FITC-labeled synthetic peptides identified in Tables 1 and 2. Two days after treatment, the cells were thoroughly washed twice, and the residual peptides bound to the cells were quantified using a fluorescent microplate reader. Figure 18 B). Peptides 2, 3, and 7 showed significantly increased binding to CM compared to all other cell types. Analysis using fluorescence microscopy confirmed that these three peptides were retained in CM, while their levels were decreased compared to other cell types. Furthermore, no other peptides were detected. Figure 19 ).

[0331] Select peptide motifs for bioengineered expression on donor BJ hiPSCs.

[0332] For the source of the bioengineered mitochondria, the inventors selected the BJ human iPSC (hiPSC) line, which had previously been verified to possess genetically primitive mitochondria. For example... Figure 13 As outlined in section A, CRISPR gene modification was used to modify the donor BJ hiPSC line to express bioengineered mitochondria. Figure 18 As can be seen, the inventors have demonstrated that peptides 2, 3, and 7 have enhanced affinity for CM. Peptide 5 remains of interest because it is a known region where the SIRPα protein interacts with CD47, and it directly follows the peptide 7 motif.

[0333] Preliminary analyses from parallel projects were performed in the same manner to investigate the affinity of different regions of peptide 7 and peptide 5 for binding CM. Figure 18 A). The inventors demonstrated that although peptide 5 has a weak binding affinity, it is the region with the strongest binding affinity between peptides 7 and 5. Figure 20 (Table 3).

[0334] Table 3. Alternative peptide sequences tested to identify optimized binding motifs.

[0335] CRISPR gene modification to express the fusion TOM20 protein containing a targeting peptide and an HA tag.

[0336] For this project, the inventors decided to generate four additional donor systems expressing any one of the target peptides 2, 3, 5, or 7. Following successful CRISPR gene modification, clonal selection, and amplification, the inventors generated four BJ hiPSC lines expressing the fusion protein TOM20–cardiac peptide–3xHA, hereinafter referred to as CP2, CP3, CP5, and CP7, representing cardiac peptides 2, 3, 5, and 7, respectively. In addition, the inventors have a previously generated BJ hiPSC control line expressing the fusion protein TOM20–3xHA, hereinafter referred to as T20.

[0337] Genotyping was performed on four BJ hiPSC lines for homozygous insertion of the expression cassette, and Sanger sequencing was performed to confirm the absence of unplanned mutations. Western blot analysis showed that the CP3 line did not express the fusion protein ( Figure 21 A). CP2, CP5, and CP7 successfully expressed the fusion protein ( Figure 21 B). To confirm that the engineered mitochondria retained their function, TMRE / FCCP flow cytometry analysis of the isolated mitochondria was performed. Figure 21 C). The isolated engineered mitochondria were successfully stained with TMRE, indicating that the mitochondria were polarized. Furthermore, they were successfully depolarized by adding the mitochondrial uncoupling agent FCCP.

[0338] The peptide sequences expressed on the surface of bioengineered mitochondria demonstrate that they facilitate mitochondrial targeting and uptake in CM.

[0339] Mature cell lines (CMs) used in the following assays were magnetically sorted to obtain pure CMs. CMs were treated with mitochondria derived from four engineered cell lines (T20, CP2, CP5, and CP7) (Fig. 22A). After treatment, the cells were washed, fixed, permeabilized, and stained for CTNT and HA. Quantification of total HA staining intensity in CMs showed a significant increase in uptake from CP2 mitochondria compared to T20 control mitochondria (Fig. 22B, left). Interestingly, quantification of HA spots within each CM showed increased uptake by CP7 mitochondria compared to T20 (Fig. 22B, right).

[0340] Qualitatively, increased CP7 mitochondrial uptake can be observed from immunofluorescence images. Figure 23 The inventors hypothesize the role of unequal expression of exogenous fusion proteins. Figure 21 B-anti-HA) results in lower HA fluorescence intensity in each CP7 mitochondria that takes up HA.

[0341] To account for the uneven expression of exogenous fusion proteins, donor cell mitochondria were stained with red mitochondrial tracing agent prior to lysis and harvesting to ensure equal fluorescence levels in each donor mitochondria before host CM treatment. Figure 24 A). The inventors observed a significant increase in red mitochondrial tracer fluorescence per centimeter in the CP7 mitochondrial treatment well compared to the T20 mitochondrial treatment well, corresponding to a relatively increased uptake of CP7 mitochondria ( Figure 24 (B, C). These data support the hypothesis that CP7 engineered on the mitochondrial surface does indeed enhance CM's targeting and uptake.

[0342] Demonstrating mitochondrial function through improvements in cellular metabolism

[0343] The aging model CM was used to demonstrate the functional improvement of cells after mitochondrial treatment. The inventors previously generated hPSC lines capable of inducing SIRT6 protein knockdown, leading to the development of a rapid aging phenotype. Two hPSC lines (SIRT6C2 and SIRT6C3) were differentiated into mature CMs for the following assays. After SIRT6 knockdown was induced using doxycycline, CMs were then treated with mitochondria to reverse the aging phenotype. The metabolic capacity of CMs was assessed using the Seahorse oxygen consumption rate (OCR) assay. Compared with untreated control CMs, aged CMs showed significantly increased ATP production, basal respiration, and maximal respiration. Figure 25 A). β-galactosidase staining also confirmed that mitochondrial-treated CMs had less senescence staining ( Figure 25 B). Therefore, the mitochondrial treatment of CM using the harvest described in this paper successfully demonstrated a functional improvement in its metabolic capacity and reversed the aging-related phenotype.

[0344] References

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[0350] 6Huettner, N., Dargaville, TR & Forget, A. Discovering cell-adhesion peptides in tissue engineering: beyond RGD. Trends in biotechnology36, 372-383 (2018).

[0351] 7Jumper, J. et al. Highly accurate protein structure prediction withAlphaFold. Nature 596, 583-589 (2021).

[0352] 8Dubois, NC et al. SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells. NatBiotechnol 29, 1011-1018, doi:10.1038 / nbt.2005 (2011).

[0353] 9Hatherley, D. et al. Paired receptor specificity explained by structures of signal regulatory proteins alone and complexed with CD47. Molecular cell 31, 266-277 (2008).

[0354] application

[0355] The modified cell implementation schemes disclosed herein utilize the possibility of introducing exogenous outer membrane proteins onto the cytoplasmic surface of mitochondria. This disclosure establishes a protocol for harvesting and purifying engineered mitochondria from cell cultures (e.g., iPSC cultures). Using these bioengineered mitochondria, this disclosure demonstrates that cardiac peptides can be successfully expressed on the surface of these donor mitochondria, and that the expression of cardiac peptides enhances the uptake of engineered mitochondria into cardiomyocytes.

[0356] Furthermore, this disclosure also demonstrates in an aging cardiomyocyte model that the uptake of exogenous donor mitochondria is beneficial in enhancing the metabolic capacity of cardiomyocytes and reversing the accumulation of the aging biomarker β-galactosidase.

[0357] Those skilled in the art will understand that other changes and / or modifications can be made to the embodiments disclosed herein without departing from the spirit or scope of this disclosure. For example, features of different exemplary embodiments may be mixed, combined, interchanged, merged, adopted, modified, included, or similar among different exemplary embodiments in the description herein. Therefore, the present embodiments should be considered illustrative rather than restrictive in all respects.

Claims

1. An engineered mitochondria comprising: One or more foreign protein binding motifs / peptides expressed on the outer mitochondrial membrane.

2. The engineered mitochondria according to claim 1, wherein the exogenous protein binding motif / peptide is an extracellular matrix (ECM) protein binding motif / ECM-derived peptide, optionally wherein the ECM binding motif / peptide is located on the cytoplasmic surface of the outer mitochondrial membrane.

3. The engineered mitochondria according to claim 1 or 2, wherein the exogenous protein binding motif / peptide is an extracellular matrix (ECM) protein binding motif / ECM-derived peptide, comprising laminin-derived peptides, surface-labeled peptides, fibronectin-derived peptides, collagen-derived peptides, gelatin-derived peptides, synaptic glycan-derived peptides, and / or combinations thereof.

4. The engineered mitochondria according to any one of the preceding claims, wherein the exogenous protein binding motif / peptide comprises about 4 to 40 amino acid residues.

5. The engineered mitochondria according to any one of the preceding claims, wherein the exogenous protein binding motif / peptide comprises a laminin-derived peptide.

6. The engineered mitochondria according to any one of the preceding claims, wherein the exogenous protein binding motif / peptide comprises a laminin-derived peptide, the laminin-derived peptide comprising a peptide having the following sequence: Ile-Lys-Val-Ala-Val (IKVAV, SEQ ID NO:17), Tyr-Ile-Gly-Ser-Arg (YIGSR, SEQ ID NO:18), Pro-Pro-Phe-Leu-Met-Leu-Leu-Lys-Gly-Ser-Thr-Arg (PPFLMLLKGSTR, SEQ ID NO: 19), Asp-Leu-Thr-Ile-Asp-Asp-Ser-Tyr-Trp-Tyr-Arg-Ile (DLTIDDSYWYRI, SEQ ID NO: 20), Asn-Ser-Ile-Lys-Val-Ser-Val-Ser-Ser (NSIKVSVSS, SEQ ID NO: 1-CP peptide 1), Cys-Thr-Val-Ser-Pro-Gly-Val-Glu-Asp-Ser-Glu-Gly-Thr-Ile (CTVSPQVEDSEGTI, SEQ ID NO: 2-CP peptide 2), Lys-Gly-Cys-Ser-Leu-Glu-Asn-Val-Tyr-Thr (KGCSLENVYT, SEQ ID NO: 3-CP peptide 3), Ser-Gly-Gly-Thr-Pro-Ala-Pro-Pro-Arg-Arg-Lys-Arg-Arg-Glu-Thr-Gly-Glu-Ala (SGGTPAPPRRKRRQTGQA, SEQ ID NO:4-CP peptide 4), and / or combinations thereof.

7. The engineered mitochondria according to any one of the preceding claims, wherein the exogenous protein binding motif / peptide comprises a heart-specific laminin that interacts with integrins on the surface of cardiomyocytes.

8. The engineered mitochondria according to any one of the preceding claims, wherein the exogenous protein binding motif / peptide comprises one or more exposed regions on the interaction domains of laminin, including helices, loops, and laminin globular (LG) domains LG1, LG2, LG3, LG4, LG5, and / or combinations thereof, optionally, the exogenous protein binding motif / peptide comprises an exposed region between a helix and LG1, an exposed region between LG1 and LG2, an exposed region between LG2 and LG3, and an exposed loop in LG3, or combinations thereof.

9. The engineered mitochondria according to any one of the preceding claims, wherein the exogenous protein binding motif / peptide comprises a laminin-derived peptide, the laminin-derived peptide comprising a peptide having the following sequence: Asn-Ser-Ile-Lys-Val-Ser-Val-Ser-Ser (NSIKVSVSS, SEQ ID NO: 1-CP peptide 1), Cys-Thr-Val-Ser-Pro-Gly-Val-Glu-Asp-Ser-Glu-Gly-Thr-Ile (CTVSPQVEDSEGTI, SEQ ID NO: 2-CP peptide 2), Lys-Gly-Cys-Ser-Leu-Glu-Asn-Val-Tyr-Thr (KGCSLENVYT, SEQ ID NO: 3-CP peptide 3), Ser-Gly-Gly-Thr-Pro-Ala-Pro-Pro-Arg-Arg-Lys-Arg-Arg-Glu-Thr-Gly-Glu-Ala (SGGTPAPPRRKRRQTGQA, SEQ ID NO:4-CP peptide 4), and / or combinations thereof.

10. The engineered mitochondria according to any one of the preceding claims, wherein the exogenous protein binding motif / peptide comprises a surface-labeled peptide.

11. The engineered mitochondria according to any one of the preceding claims, wherein the exogenous protein binding motif / peptide comprises a surface marker protein of cardiomyocytes.

12. The engineered mitochondria according to any one of the preceding claims, wherein the exogenous protein binding motif / peptide comprises a surface marker protein of cardiomyocytes, wherein the surface marker protein is SIRPα.

13. The engineered mitochondria according to any one of the preceding claims, wherein the exogenous protein binding motif / peptide comprises a SIRPα surface marker protein, the surface marker protein comprising a peptide having the following sequence: Thr-Lys-Ser-Val-Glu-Phe-Thr-Phe-Cys-Asn-Asp-Thr-Val-Val-Iso-Pro-Cys-Phe (TKSVEFTFCNDTVVIPCF, SEQ ID NO: 7-peptide 7), Asn-Met-Glu-Ala-Gln-Asn-Thr-Thr-Glu-Val-Tyr (NMEAQNTTEVY, SEQ ID NO: 5-peptide 5), Thr-Glu-Leu-Thr-Arg-Glu-Gly-Glu (TELTREGE, SEQ ID NO: 6-peptide 6), or combinations thereof.

14. The engineered mitochondria according to any one of the preceding claims, wherein the exogenous protein binding motif / peptide comprises a SIRPα surface marker protein, the surface marker protein comprising a peptide having the following sequence: Val-Val-Iso-Pro-Cys-Phe-Val-Thr-Asn-Met-Glu-Ala (VVIPCFVTNMEA, SEQ ID NO:12-ABC peptide), Cys-Asn-Asp-Thr-Val-Val-Iso-Pro-Cys-Phe (CNDTVVIPCF, SEQ ID NO:11-AB-rear), Thr-Lys-Val-Glu-Phe-Thr-Phe-Cys-Asn-Asp-Thr-Val-Val-Iso-Pro-Cys-Phe(TKSVEFTFCNDTVVIPCF, SEQ ID NO:7-AB full length), Glu-Phe-Thr-Phe-Cys-Asn-Asp-Thr-Val-Val (EFTFCNDTVV, SEQ ID NO:10-AB-middle), Thr-Lys-Ser-Val-Glu-Phe-Thr-Phe-Cys-Asn (TKSVEFTFCN, SEQ ID NO:9-AB-front), Asn-Met-Glu-Ala-Gln-Asn-Thr-Thr-Glu-Val-Tyr (NMEAQNTTEVY, SEQ ID NO: 5-BC peptide), Or a combination thereof.

15. The engineered mitochondria according to any one of the preceding claims, wherein the exogenous protein binding motif / peptide comprises a fibronectin-derived peptide, the fibronectin-derived peptide comprising Arg-Gly-Asp (RGD, SEQ ID NO:13), Pro-His-Ser-Arg-Asn (PHSRN, SEQ ID NO:14), cyclic RGD and / or combinations thereof.

16. The engineered mitochondria according to any one of the preceding claims, wherein the exogenous protein binding motif / peptide comprises a collagen-derived peptide, the collagen-derived peptide comprising Asp-Gly-Glu-Ala (DGEA, SEQ ID NO:16).

17. The engineered mitochondria according to any one of the preceding claims, wherein the exogenous protein binding motif / peptide is expressed on an exogenous mitochondrial outer membrane protein.

18. The engineered mitochondria according to any one of the preceding claims, wherein the exogenous protein-binding motif / peptide is expressed on an exogenous mitochondrial membrane translocation enzyme complex, optionally on exogenous TOM20, TOM70, TOM40, TOM22, TOM7, TOM6, TOM5, TOM70, SAM50, SAM35, SAM37, Mim1, Mim2 or combinations thereof.

19. The engineered mitochondria according to any one of the preceding claims, wherein the exogenous protein binding motif / peptide is expressed on exogenous TOM20, TOM22, TOM70, SAM50, porin, outer membrane protein 25 (OMP25) or a combination thereof.

20. The engineered mitochondria according to any one of the preceding claims, wherein the exogenous protein binding motif / peptide is expressed on the exogenous TOM20 protein.

21. The engineered mitochondria according to any one of the preceding claims, wherein the mitochondria are derived from cells selected from: induced pluripotent stem cells, mesenchymal stem cells, adipose-derived stem cells, and adult stem cells.

22. A polynucleotide encoding mitochondria as described in any of the preceding claims.

23. The polynucleotide of claim 22, wherein the polynucleotide encodes an insert construct comprising a sequence encoding a mitochondrial outer membrane protein and / or a mitochondrial protein-binding motif / peptide.

24. The polynucleotide of claim 22 or 23, wherein the encoded polynucleotide encodes a foreign protein-binding motif / peptide having one or more sequences selected from: Ile-Lys-Val-Ala-Val (IKVAV, SEQ ID NO:18), Tyr-Ile-Gly-Ser-Arg (YIGSR, SEQ ID NO:19), Pro-Pro-Phe-Leu-Met-Leu-Leu-Lys-Gly-Ser-Thr-Arg (PPFLMLLKGSTR, SEQ ID NO: 19), Asp-Leu-Thr-Ile-Asp-Asp-Ser-Tyr-Trp-Tyr-Arg-Ile (DLTIDDSYWYRI, SEQ ID NO: 20), Asn-Ser-Ile-Lys-Val-Ser-Val-Ser-Ser (NSIKVSVSS-CP peptide 1, SEQ ID NO: 1), Cys-Thr-Val-Ser-Pro-Gly-Val-Glu-Asp-Ser-Glu-Gly-Thr-Iso (CTVSPQVEDSEGTI-CP peptide 2, SEQ ID NO: 2), Lys-Gly-Cys-Ser-Leu-Glu-Asn-Val-Tyr-Thr (KGCSLENVYT-CP peptide 3, SEQ ID NO: 3), Ser-Gly-Gly-Thr-Pro-Ala-Pro-Pro-Arg-Arg-Lys-Arg-Arg-Glu-Thr-Gly-Glu-Ala (SGGTPAPPRRKRRQTGQA-CP peptide 4, SEQ ID NO: 4), Thr-Lys-Ser-Val-Glu-Phe-Thr-Phe-Cys-Asn-Asp-Thr-Val-Val-Iso-Pro-Cys-Phe (TKSVEFTFCNDTVVIPCF-peptide 7, SEQ ID NO: 7), Asn-Met-Glu-Ala-Gln-Asn-Thr-Thr-Glu-Val-Tyr (NMEAQNTTEVY-peptide 5, SEQ ID NO: 5), Thr-Glu-Leu-Thr-Arg-Glu-Gly-Glu (TELTREGE-peptide 6, SEQ ID NO: 6), Val-Val-Iso-Pro-Cys-Phe-Val-Thr-Asn-Met-Glu-Ala (VVIPCFVTNMEA-ABC peptide, SEQ ID NO: 12), Cys-Asn-Asp-Thr-Val-Val-Iso-Pro-Cys-Phe (CNDTVVIPCF-AB-rear, SEQ ID NO:11), Thr-Lys-Val-Glu-Phe-Thr-Phe-Cys-Asn-Asp-Thr-Val-Val-Iso-Pro-Cys-Phe (TKSVEFTFCNDTVVIPCF-AB full length, SEQ ID NO:7) Glu-Phe-Thr-Phe-Cys-Asn-Asp-Thr-Val-Val (EFTFCNDTVV-AB-middle, SEQ ID NO:10), Thr-Lys-Ser-Val-Glu-Phe-Thr-Phe-Cys-Asn (TKSVEFTFCN-AB-front), Asn-Met-Glu-Ala-Gln-Asn-Thr-Thr-Glu-Val-Tyr (NMEAQNTTEVY-BC peptide, SEQ ID NO: 5), Or a combination thereof.

25. The polynucleotide according to any one of 22 to 24, wherein the polynucleotide encodes an insert construct comprising a sequence encoding TOMM20 and / or a cardiac peptide motif.

26. A vector comprising a polynucleotide encoding mitochondria as described in any one of claims 1 to 21 or comprising a polynucleotide as described in any one of claims 22 to 25.

27. A host cell comprising the vector of claim 26.

28. A cell comprising mitochondria according to any one of claims 1 to 21.

29. A composition or pharmaceutical composition comprising engineered mitochondria as described in any one of claims 1 to 21 or a polynucleotide as described in any one of claims 22 to 25.

30. The composition or pharmaceutical composition according to claim 29, for use in treatment / medicine.

31. A method for preventing and / or treating a disease in subjects in need, The method comprises administering to a subject an engineered mitochondrial cell as described in any one of claims 1 to 21, a polynucleotide as described in any one of claims 22 to 25, or a composition as described in claim 29.

32. The method of claim 31, wherein the disease is a mitochondrial disease or a proliferative disease.

33. A method for improving mitochondrial uptake into target cells, comprising: Genes modify host cells to express modified mitochondria. The modified mitochondria contain one or more exogenous protein-binding motifs / peptides expressed on the outer mitochondrial membrane.

34. The method of claim 33, wherein the host cell is genetically modified via viral transduction or a CRISPR gene modification system.

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