Transgenic mitochondrial delivery system

Abstract

The present disclosure provides a gene delivery system in which transgenic mitochondria are administered to an animal to permit expression of the transgene and secretion of a transgene- encoded protein to an animal.

Description

[0001] TRANSGENIC MITOCHONDRIAL DELIVERY SYSTEM

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No. 63 / 581,547 entitled “COMPOSITION AND METHODS FOR DELIVERY OF TRANSGENES INTO MITOCHONDRIA” filed on September 8, 2023 (Attorney Docket No. 123658-12201), which is hereby incorporated by reference in its entirety.

[0004] SEQUENCE LISTING

[0005] This application contains a Sequence Listing which has been submitted electronically as a WIPO Standard ST.26 XML file via Patent Center, created on September 6, 2024, is entitled “123658- 12202. xml” and is 22.8 KB in size. The sequence listing is incorporated herein by reference in its entirety.

[0006] BACKGROUND

[0007] Genetic mutations that result in loss or gain of function of a gene product can cause a diverse spectrum of hereditary and acquired diseases. Gene therapy has enormous potential for the treatment of hereditary disorders and chronic diseases. Although there are effective treatment approaches for some of these genetic diseases, for many others treatment and cure is not possible without correction of the defective gene. The transplantation of normal functional copies of genes into cells that contain defective or absent ones represents the basis of gene therapy approaches for treatment and cure of genetic diseases.

[0008] To date, the efficacy of gene therapy has been limited by inability to safely deliver and maintain transgene expression in all necessary types of cells and tissues. The lead approach for long-term therapeutic gene delivery to treat genetic disorders or chronic diseases uses adeno-associated virus (AAV) as the transgene vector. Presently, four such gene therapies have received FDA approval. These FDA-approved interventions use a virus, adeno-associated virus (AAV), as the vector for therapeutic transgene delivery. However, virus-vectored gene therapies such as AAV have limitations that restrict their broad applicability for treatment of all genetic diseases. SUMMARY

[0009] The disclosure provides a gene delivery system using mitochondria as a gene delivery vehicle.

[0010] The disclosure provides a method of delivering a heterologous protein to an animal by administering transgenic mitochondria to the animal, wherein the mitochondria comprise a transgene encoding a secretable heterologous protein, wherein expression of said transgene permits secretion of said protein from said mitochondrion, thereby delivering said heterologous protein to the animal.

[0011] The disclosure provides a synthetic minimal mitochondrial-DNA-based plasmid (“mitoDRTVE plasmid”') for delivery and expression of a transgene within mitochondria.

[0012] The disclosure provides a method of making a mitoDRIVE plasmid for delivery and expression of a transgene within mitochondria.

[0013] The disclosure provides a method of providing transgenic mitochondria, the method comprising contacting isolated mitochondria with a vector comprising a gene encoding a signal peptide fused to a heterologous protein, wherein said vector is capable of transfecting a mitochondrion, and expression of said gene permits secretion of said protein from said mitochondrion.

[0014] The disclosure provides a gene delivery system comprising a vector comprising a gene encoding a signal peptide fused to a heterologous protein, wherein said vector is capable of transfecting a mitochondrion, and expression of said gene permits secretion of said protein from said mitochondrion.

[0015] In one aspect, the disclosure provides a method of delivering a heterologous protein to an animal comprising administering transgenic mitochondria to an animal, said transgenic mitochondria comprising a transgene encoding a heterologous protein, said transgene comprising a sequence that permits secretion of said heterologous protein from said mitochondria; whereupon said administering permits expression of said transgene and secretion of said heterologous protein from said mitochondrion, thereby delivering said heterologous protein to said animal. In some embodiments of any of the aforesaid methods herein, the mitochondrion is not a megamitochondrion and / or said mitochondrion comprises a diameter of less than 2pm.

[0016] In some embodiments of any of the aforesaid methods herein, the sequence comprises a heterologous nucleic acid comprising first region encoding a signal peptide and a second region encoding said heterologous protein, and wherein said first region and said second region are operably linked.

[0017] In some embodiments of any of the aforesaid methods herein, the transgene further comprises one or more promoters that are operative in mitochondria and said promoter is capable of directing transcription of said nucleic acid.

[0018] In some embodiments of any of the aforesaid methods herein, the transgene further comprises one or more mitochondrial transcriptional terminators.

[0019] In some embodiments of any of the aforesaid methods herein, the transgene further comprises one or more transcript processing signals.

[0020] In some embodiments of any of the aforesaid methods herein, the transgene further comprises native replication signals capable of propagating said plasmid within mitochondria during mitogenesis.

[0021] In some embodiments of any of the aforesaid methods herein, the transgene further comprises mitochondrial export sequence and said mitochondrial export sequence is capable of improving and / or increasing mitochondrial export of said protein.

[0022] In some embodiments of any of the aforesaid methods herein, the transgene further comprises origins of strand (Ori) replication, tRNA genes and a displacement (D-loop) loop.

[0023] In some embodiments of any of the aforesaid methods herein, the heterologous nucleic acid further encodes one or more of cell-penetrating peptides or homeoprotein internalization- secretion domains to facilitate export of said heterologous protein from mitochondria.

[0024] In some embodiments of any of the aforesaid methods herein, the heterologous nucleic acid further comprises mitochondrial targeting sequences which facilitate nuclear import of said heterologous protein, or retention of said heterologous protein in the endoplasmic reticulum.

[0025] In some embodiments of any of the aforesaid methods herein, the heterologous nucleic acid encoding a plurality of heterologous proteins.

[0026] In some embodiments of any of the aforesaid methods herein, the size of the heterologous nucleic acid is less than or equal to 16 Kb.

[0027] In some embodiments of any of the aforesaid methods herein, the Ori are selected from the group consisting of OriL and OriH.

[0028] In some embodiments of any of the aforesaid methods herein, the D-Loop comprises regulatory elements for transcription and replication of mitochondrial DNA.

[0029] In some embodiments of any of the aforesaid methods herein, the nucleic acid is located downstream of heavy strand promoter (HSP1) and is optionally flanked by a mitochondrial t-RNA gene and / or flanked on one side by the OriL.

[0030] In some embodiments of any of the aforesaid methods herein, the promoter is a heavy strand promoter.

[0031] In some embodiments of any of the aforesaid methods herein, the promoter is a light strand promoter.

[0032] In some embodiments of any of the aforesaid methods herein, the heavy strand promoter is selected from the group consisting of HSP1 and HSP2.

[0033] In some embodiments of any of the aforesaid methods herein, the light strand promoter is LSP1 or LSP2.

[0034] In another aspect, the disclosure provides a method of delivering a heterologous protein to a cell comprising administering transgenic mitochondria to a cell, said transgenic mitochondria comprising a transgene encoding a heterologous protein, said transgene comprising a sequence that permits secretion of said heterologous protein from said mitochondria; whereupon said administering permits expression of said transgene and secretion of said heterologous protein from said mitochondrion, thereby delivering said heterologous protein to said cell. In some embodiments of any of the aforesaid methods herein, the mitochondrion is not a megamitochondrion and / or said mitochondrion comprises a diameter of less than 2pm.

[0035] In some embodiments of any of the aforesaid methods herein, the transgene comprises a heterologous nucleic acid comprising first region encoding a signal peptide and a second region encoding said heterologous protein, and wherein said first region and said second region are operably linked.

[0036] In some embodiments of any of the aforesaid methods herein, the transgene further comprises one or more promoters that are operative in mitochondria and said promoter is capable of directing transcription of said nucleic acid.

[0037] In some embodiments of any of the aforesaid methods herein, the transgene further comprises one or more mitochondrial transcriptional terminators.

[0038] In some embodiments of any of the aforesaid methods herein, the transgene further comprises one or more transcript processing signals.

[0039] In some embodiments of any of the aforesaid methods herein, the transgene further comprises native replication signals capable of propagating said plasmid within mitochondria during mitogenesis.

[0040] In some embodiments of any of the aforesaid methods herein, the transgene further comprises mitochondrial export sequence and said sequence is capable of improving and / or increasing mitochondrial export of said protein.

[0041] In some embodiments of any of the aforesaid methods herein, the transgene further comprises origins of strand (Ori) replication, tRNA genes and a displacement (D-loop) loop.

[0042] In some embodiments of any of the aforesaid methods herein, the heterologous nucleic acid further encodes one or more of cell-penetrating peptides or homeoprotein internalization- secretion domains to facilitate export of said heterologous protein from mitochondria.

[0043] In some embodiments of any of the aforesaid methods herein, the heterologous nucleic acid further comprises mitochondrial targeting sequences which facilitate nuclear import of said heterologous protein, or retention of said heterologous protein in the endoplasmic reticulum.

[0044] In some embodiments of any of the aforesaid methods herein, the heterologous nucleic acid encoding a plurality of heterologous proteins.

[0045] In some embodiments of any of the aforesaid methods herein, the size of said heterologous nucleic acid is less than or equal to 16 Kb.

[0046] In some embodiments of any of the aforesaid methods herein, the Ori are selected from the group consisting of OriL and OriH.

[0047] In some embodiments of any of the aforesaid methods herein, the D-Loop comprises regulatory elements for transcription and replication of mitochondrial DNA.

[0048] In some embodiments of any of the aforesaid methods herein, the nucleic acid is located downstream of heavy strand promoter (HSP1) and is optionally flanked by a mitochondrial t-RNA gene and / or flanked on one side by the OriL.

[0049] In some embodiments of any of the aforesaid methods herein, the promoter is a heavy strand promoter.

[0050] In some embodiments of any of the aforesaid methods herein, the promoter is a light strand promoter.

[0051] In some embodiments of any of the aforesaid methods herein, the heavy strand promoter is selected from the group consisting of HSP1 and HSP2.

[0052] In some embodiments of any of the aforesaid methods herein, the light strand promoter is LSP1 or LSP2.

[0053] In some embodiments of any of the aforesaid methods herein, the heterologous nucleic acid further comprises a signal sequence which facilitate nuclear import of said heterologous protein, or retention of said heterologous protein in the endoplasmic reticulum.

[0054] In some embodiments of any of the aforesaid methods herein, the signal peptide is selected from the group consisting of IL-6 signal peptide, IL-2 signal peptide, Human OSM peptide, VSV-G peptide, Mouse Ig Kappa signal peptide, Mouse Ig Heavy signal peptide, BM40 signal peptide, Secrecon signal peptide, Human IgKVIII signal peptide, CD33 signal peptide, tPA signal peptide, Lipoic Acid Signal Peptide, Glycosylphosphatidylinositol (GPI) Anchor Signal Sequence, Mitochondrial Targeting Sequence (MTS), Bacterial Sec System Signal Sequence, Fibroblast Growth Factor (FGF) Signal Sequence, Serine Protease Inhibitor (SERPIN) Signal Sequence, Chitinase Signal Sequence and Erythropoietin (EPO) Signal Sequence.

[0055] In some embodiments of any of the aforesaid methods herein, the heterologous protein is selected from the group consisting of insulin, erythropoietin, human growth hormone, Factor VIII, Factor IX, luciferase, eGFP, interferons (such as IFN-a, IFN-P, IFN-y), Tumor Necrosis Factor (TNF) inhibitors, tissue plasminogen activator (tPA), dystrophin, and CFTR protein.

[0056] In one aspect, the disclosure provides a transgenic mitochondrion comprising a heterologous nucleic acid comprising a first region encoding a signal peptide and a second region encoding a heterologous protein, wherein the first region and the second region are operably linked to permit expression of the nucleic acid, wherein expression of the nucleic acid permits secretion of the protein from the mitochondrion, and wherein the mitochondrion is not a megamitochondrion and / or the mitochondrion comprises a diameter of less than 2pm.

[0057] In some embodiments, the mitochondrion comprises a heterologous plasmid comprising the nucleic acid.

[0058] In some embodiments of any of the aforesaid mitochondrion, the plasmid comprises a promoter operative in mitochondria and the promoter is capable of directing transcription of the nucleic acid. In some embodiments, the plasmid comprises a mitochondrial transcription terminator. In some embodiments, the plasmid comprises a transcript processing signal. In some embodiments, the plasmid comprises native replication signals capable of propagating the plasmid within mitochondria during mitogenesis. In some embodiments, the plasmid further comprises a mitochondrial export sequence and the export sequence is capable of improving and / or increasing mitochondrial export of the protein.

[0059] In some embodiments of any of the aforesaid mitochondrion, the promoter is a heavy strand promoter. In some embodiments, the promoter is a light strand promoter. In some embodiments, the heavy strand promoter is selected from the group consisting of HSP1 and HSP2. In some embodiments, the light strand promoter is selected from the group consisting of LSPl and LSP2.

[0060] In some embodiments of any of the aforesaid mitochondrion, the plasmid comprises origins of strand (Ori) replication, tRNA genes and displacement (D-loop) loop. In some embodiments, the Ori are selected from the group consisting of OriL and OriH. In some embodiments, the D-Loop comprises regulatory elements for transcription and replication of mitochondrial DNA.

[0061] In some embodiments of any of the aforesaid mitochondrion, the heterologous nucleic acid is located downstream of heavy strand promoter (HSP1) and is optionally flanked by a mitochondrial t-RNA gene and / or flanked on one side by the OriL. Optionally the t-RNA gene is t-RNA-Val gene. In some embodiments, the heterologous nucleic acid further encodes one or more of cell-penetrating peptides or homeoprotein internalization- secretion domains to facilitate export of the heterologous protein from mitochondria. In some embodiments, the heterologous nucleic acid further comprises a signal sequence which facilitate nuclear import of the heterologous protein, or retention of the heterologous protein in the endoplasmic reticulum.

[0062] In some embodiments of any of the aforesaid mitochondrion, the plasmid comprises heterologous nucleic acid encoding a plurality of heterologous proteins. In some embodiments, the total size of the heterologous nucleic acid is less than or equal to 16 kb.

[0063] In some embodiments, the mitochondrion comprises a vector comprising the nucleic acid. In some embodiments, the vector comprises one or more promoters operative in mitochondria and the promoter is capable of directing transcription of the nucleic acid. In some embodiments, the vector comprises one or more mitochondrial transcriptional terminators. In some embodiments, the vector comprises one or more transcript processing signals. In some embodiments, the vector comprises native mitochondrial replication signals capable of propagating the plasmid within mitochondria during mitogenesis. In some embodiments, the vector comprises a mitochondrial export sequence and the export sequence is capable of improving and / or increasing mitochondrial export of the protein.

[0064] In some embodiments of any of the aforesaid mitochondrion, the promoter is a heavy strand promoter. In some embodiments, the promoter is a light strand promoter. In some embodiments, the heavy strand promoter is selected from the group consisting of HSP1 and HSP2. In some embodiments, the light strand promoter is LSP1 or LSP2.

[0065] In some embodiments of any of the aforesaid mitochondrion, the vector comprises origins of strand (Ori) replication, tRNA genes and displacement (D-loop) loop. In some embodiments, the Ori are selected from the group consisting of OriL and OriH. In some embodiments, the D-Loop comprises regulatory elements for transcription and replication of mitochondrial DNA.

[0066] In some embodiments of any of the aforesaid mitochondrion, the heterologous nucleic acid is located downstream of heavy strand promoter (HSP1) and is optionally flanked by a mitochondrial t-RNA gene and / or flanked on one side by the OriL. Optionally the mitochondrial t-RNA gene is a mitochondrial t-RNA-Val gene. In some embodiments, the heterologous nucleic acid further encodes one or more of cell-penetrating peptides or homeoprotein internalization- secretion domains to facilitate export of the heterologous protein from mitochondria. In some embodiments, the heterologous nucleic acid further comprises mitochondrial targeting sequences which facilitate nuclear import of the heterologous protein, or retention of the heterologous protein in the endoplasmic reticulum.

[0067] In some embodiments of any of the aforesaid mitochondrion, the signal peptide is 8- 50 amino acids in length. In some embodiments, the signal peptide is selected from the group consisting of IL-6 signal peptide, IL-2 signal peptide, Human OSM peptide, VSV-G peptide, Mouse Ig Kappa signal peptide, Mouse Ig Heavy signal peptide, BM40 signal peptide, Secrecon signal peptide, Human IgKVIII signal peptide, CD33 signal peptide, tPA signal peptide, Lipoic Acid Signal Peptide, Glycosylphosphatidylinositol (GPI) Anchor Signal Sequence, Mitochondrial Targeting Sequence (MTS), Bacterial Sec System Signal Sequence, Fibroblast Growth Factor (FGF) Signal Sequence, Serine Protease Inhibitor (SERPIN) Signal Sequence, Chitinase Signal Sequence and Erythropoietin (EPO) Signal Sequence.

[0068] In another aspect, the disclosure provides a composition comprising a transgenic mitochondrion comprising a heterologous nucleic acid comprising a first region encoding a signal peptide and a second region encoding a heterologous protein, wherein the first region and the second region are operably linked to permit expression of the nucleic acid, wherein expression of the nucleic acid permits secretion of the protein from the mitochondrion, and wherein the mitochondrion is not a megamitochondrion and / or the mitochondrion comprises a diameter of less than 2|jm. In some embodiments, the composition comprises a plurality of transgenic mitochondrion.

[0069] In another aspect, the disclosure provides a vector comprising a mitochondrial promoter directing a heterologous nucleic acid comprising a first region encoding a signal peptide and second region encoding a heterologous protein, wherein the first region is operably linked to the second region such that the vector upon transfection to a mitochondrion permits secretion of the protein from the mitochondrion through the mitochondrial promoter.

[0070] In some embodiments of any of the aforesaid vector, the vector comprises one or more mitochondrial transcriptional promoters. In some embodiments of any of the aforesaid vector, the vector comprises one or more mitochondrial transcriptional terminators. In some embodiments of any of the aforesaid vector, the vector comprises one or more transcript processing signals.

[0071] In some embodiments of any of the aforesaid vector, the vector comprises native mitochondrial replication signals capable of propagating the plasmid within mitochondria during mitogenesis.

[0072] In some embodiments of any of the aforesaid vector, the vector further comprises a mitochondrial export sequence and is capable of improving and / or increasing mitochondrial export of the protein.

[0073] In some embodiments of any of the aforesaid vector, the promoter is a heavy strand promoter. In some embodiments of any of the aforesaid vector, the promoter is a light strand promoter. In some embodiments of any of the aforesaid vector, the heavy strand promoter is selected from the group consisting of HSP1 and HSP2. In some embodiments of any of the aforesaid vector, the light strand promoter is LSP1 or LSP2.

[0074] In some embodiments of any of the aforesaid vector, the vector comprises origins of strand (Ori) replication, tRNA genes and displacement (D-loop) loop. In some embodiments, the Ori are selected from the group consisting of OriL and OriH. In some embodiments of any of the aforesaid vector, the D-Loop comprises regulatory elements for transcription and replication of mitochondrial DNA. In some embodiments of any of the aforesaid vector, the heterologous nucleic acid is located downstream of heavy strand promoter (HSP1) and flanked by mitochondrial t-RNA- Val gene.

[0075] In some embodiments of any of the aforesaid vector, the heterologous nucleic acid further encodes one or more of cell-penetrating peptides or homeoprotein internalizationsecretion domains to facilitate export of the heterologous protein from mitochondria.

[0076] In some embodiments of any of the aforesaid vector, the heterologous nucleic acid further comprises mitochondrial targeting sequences which facilitate nuclear import of the heterologous protein, or retention of the heterologous protein in the endoplasmic reticulum.

[0077] In some embodiments of any of the aforesaid vector, the vector comprises heterologous nucleic encoding a plurality of heterologous proteins. In some embodiments of any of the aforesaid vector, the size of the heterologous nucleic acid is less than or equal to 16 Kb.

[0078] In some embodiments of any of the aforesaid vector, the signal peptide is 8-50 amino acids in length.

[0079] In some embodiments of any of the aforesaid vector, the signal peptide is selected from the group consisting of IL-6 signal peptide, IL-2 signal peptide, Human OSM peptide, VSV-G peptide, Mouse Ig Kappa signal peptide, Mouse Ig Heavy signal peptide, BM40 signal peptide, Secrecon signal peptide, Human IgKVIII signal peptide, CD33 signal peptide, tPA signal peptide, Lipoic Acid Signal Peptide, Glycosylphosphatidylinositol (GPI) Anchor Signal Sequence, Mitochondrial Targeting Sequence (MTS), Bacterial Sec System Signal Sequence, Fibroblast Growth Factor (FGF) Signal Sequence, Serine Protease Inhibitor (SERPIN) Signal Sequence, Chitinase Signal Sequence and Erythropoietin (EPO) Signal Sequence.

[0080] In another aspect, the disclosure provides a method of gene delivery comprising administering to an organism a transgenic mitochondrion, wherein the transgenic mitochondrion comprises a heterologous nucleic acid comprising a first region encoding a signal peptide fused to a second region encoding a heterologous protein, wherein upon the administration the nucleic acid are expressed as a secretable heterologous protein and the heterologous protein is secreted from the transgenic mitochondrion. In some embodiments of any of the aforesaid method, the mitochondrion comprises a vector comprising the heterologous nucleic acid encoding the signal peptide fused to the heterologous protein.

[0081] In some embodiments of any of the aforesaid method, the vector further comprises one or more promoters that are operative in mitochondria and the promoter is capable of directing transcription of the nucleic acid.

[0082] In some embodiments of any of the aforesaid method, the vector further comprises one or more mitochondrial transcriptional terminators. In some embodiments of any of the aforesaid method, the vector further comprises one or more transcript processing signals. In some embodiments of any of the aforesaid method, the vector further comprises native replication signals capable of propagating the plasmid within mitochondria during mitogenesis.

[0083] In some embodiments of any of the aforesaid method, the vector further comprises mitochondrial export sequence and the sequence is capable of improving and / or increasing mitochondrial export of the protein.

[0084] In some embodiments of any of the aforesaid method, the promoter is a heavy strand promoter. In some embodiments of any of the aforesaid method, the promoter is a light strand promoter. In some embodiments of any of the aforesaid method, the heavy strand promoter is selected from the group consisting of HSP1 and HSP2. In some embodiments of any of the aforesaid method, the light strand promoter is LSP1 or LSP2.

[0085] In some embodiments of any of the aforesaid method, the vector further comprises origins of strand (Ori) replication, tRNA genes and a displacement (D-loop) loop. In some embodiments of any of the aforesaid method, the Ori are selected from the group consisting of OriL and OriH. In some embodiments of any of the aforesaid method, the D-Loop comprises regulatory elements for transcription and replication of mitochondrial DNA.

[0086] In some embodiments of any of the aforesaid method, the nucleic acid is located downstream of heavy strand promoter (HSP1) and flanked by mitochondrial t-RNA-Val gene. In some embodiments of any of the aforesaid method, the heterologous nucleic acid further encodes one or more of cell-penetrating peptides or homeoprotein internalizationsecretion domains to facilitate export of the heterologous protein from mitochondria.

[0087] In some embodiments of any of the aforesaid method, the heterologous nucleic acid further comprises mitochondrial targeting sequences which facilitate nuclear import of the heterologous protein, or retention of the heterologous protein in the endoplasmic reticulum.

[0088] In some embodiments of any of the aforesaid method, the vector comprises heterologous nucleic acid encoding a plurality of heterologous proteins. In some embodiments of any of the aforesaid method, the size of the heterologous nucleic acid is less than or equal to 16 Kb.

[0089] In another aspect, the disclosure provides a method of providing transgenic mitochondria, the method comprising contacting one or more isolated mitochondrion with a vector capable of transfecting a mitochondrion and a transfection agent, the vector comprising a heterologous nucleic acid comprising a first region encoding a signal peptide fused to a second region encoding a heterologous protein, wherein the contacting provides one or more mitochondrion comprising the vector, thereby forming a transgenic mitochondrion having a diameter of less than 2pm, and expression of the nucleic acid permits secretion of the protein from the mitochondrion.

[0090] In some embodiments of any of the aforesaid method, vector further comprises one or more promoters operative in mitochondria and the transfection agent is selected from the group consisting of Lipofectamine, DOTAP, MITO-porter, protamine DNA complex, electroporation and Stearylamine-based liposomes.

[0091] In some embodiments of any of the aforesaid method, the vector further comprises one or more mitochondrial transcriptional terminators. In some embodiments of any of the aforesaid method, the vector further comprises one or more transcript processing signals. In some embodiments of any of the aforesaid method, the vector further comprises native replication signals capable of propagating the plasmid within mitochondria during mitogenesis. In some embodiments of any of the aforesaid method, the vector further comprises mitochondrial export sequence and the sequence is capable of improving and / or increasing mitochondrial export of the protein. In some embodiments of any of the aforesaid method, the promoter is a heavy strand promoter. In some embodiments of any of the aforesaid method, the promoter is a light strand promoter. In some embodiments of any of the aforesaid method, the heavy strand promoter is selected from the group consisting of HSP1 and HSP2. In some embodiments of any of the aforesaid method, the light strand promoter is LSP1 or LSP2.

[0092] In some embodiments of any of the aforesaid method, the vector further comprises origins of strand (Ori) replication, tRNA genes and displacement (D-loop) loop.

[0093] In some embodiments of any of the aforesaid method, the Ori are selected from the group consisting of OriL and OriH.

[0094] In some embodiments of any of the aforesaid method, the D-Loop comprises regulatory elements for transcription and replication of mitochondrial DNA. In some embodiments of any of the aforesaid method, the nucleic acid is located downstream of heavy strand promoter (HSP1) and flanked by mitochondrial t-RNA-Val gene.

[0095] In some embodiments of any of the aforesaid method, the heterologous nucleic acid further encodes one or more of cell-penetrating peptides or homeoprotein internalizationsecretion domains to facilitate export of the heterologous protein from mitochondria.

[0096] In some embodiments of any of the aforesaid method, the heterologous nucleic acid further comprises mitochondrial targeting sequences which facilitate nuclear import of the heterologous protein, or retention of the heterologous protein in the endoplasmic reticulum.

[0097] In another aspect, the disclosure provides a method of making an allogenic mitochondria comprising a heterologous nucleic acid comprising a first region encoding a signal peptide operably linked to a second region encoding a heterologous protein, wherein expression of the nucleic acid permits secretion of the protein from the allogenic mitochondrion, and wherein the method comprises contacting a mitochondria devoid of the heterologous protein with a heterologous plasmid comprising the heterologous nucleic acid under conditions that enable uptake of the heterologous plasmid by the mitochondrion to produce the allogenic mitochondrion.

[0098] In another aspect, the disclosure provides a method of expressing a heterologous protein in a cell using a mitochondria comprising: a) contacting a mitochondria with a heterologous plasmid comprising a heterologous nucleic acid comprising a first region encoding a signal peptide operably linked to a second region encoding the heterologous protein; b) isolating the mitochondria comprising the plasmid, c) contacting the isolated mitochondria with the cell under conditions that promote uptake of the isolated mitochondria by the cell, and d) culturing the cell under conditions that promote expression of the heterologous nucleic acid, and wherein expression of the heterologous nucleic acid permits secretion of the heterologous protein from the mitochondrion.

[0099] In another aspect, the disclosure provides a method of making transgenic mitochondrion expressing a secretable heterologous protein comprising, a) providing a heterologous nucleic acid comprising a first region encoding a signal peptide operably linked to a second region encoding the heterologous protein, b) inserting the heterologous nucleic acid into a mitochondrial plasmid, c) contacting the mitochondrial plasmid comprising the heterologous nucleic acid with a mitochondrion of size less than 2pm in presence of a transfection agent to form a transgenic mitochondrion, d) isolating the transgenic mitochondrion comprising the mitochondrial plasmid and wherein the transgenic mitochondrion upon insertion into a cell is capable of expressing and secreting the heterologous protein.

[0100] In some embodiments of any of the aforesaid method, the plasmid comprises mitochondrial DNA.

[0101] In some embodiments of any of the aforesaid method, the plasmid comprises one or more promoters operative in mitochondria and the promoter is capable of directing transcription of the nucleic acid.

[0102] In some embodiments of any of the aforesaid method, the plasmid comprises one or more mitochondrial transcriptional terminators. In some embodiments of any of the aforesaid method, the plasmid comprises one or more transcript processing signals.

[0103] In some embodiments of any of the aforesaid method, the plasmid comprises native replication signals capable of propagating the plasmid within mitochondria during mitogenesis.

[0104] In some embodiments of any of the aforesaid method, the plasmid comprises mitochondrial export sequence and the export sequence is capable of improving and / or increasing mitochondrial export of the protein.

[0105] In some embodiments of any of the aforesaid method, the promoter is a heavy strand promoter.

[0106] In some embodiments of any of the aforesaid method, the promoter is a light strand promoter.

[0107] In some embodiments of any of the aforesaid method, the heavy strand promoter is selected from the group consisting of HSP1 and HSP2.

[0108] In some embodiments of any of the aforesaid method, the light strand promoter is LSP1 or LSP2.

[0109] In some embodiments of any of the aforesaid method, the plasmid comprises origins of strand (Ori) replication, tRNA genes and displacement (D-loop) loop.

[0110] In some embodiments of any of the aforesaid method, the Ori are selected from the group consisting of OriL and OriH.

[0111] In some embodiments of any of the aforesaid method, the D-Loop comprises regulatory elements for transcription and replication of mitochondrial DNA.

[0112] In some embodiments of any of the aforesaid method, the heterologous nucleic acid is located downstream of heavy strand promoter (HSP1) and flanked by mitochondrial t-RNA- Val gene. In some embodiments of any of the aforesaid method, the heterologous nucleic acid further encodes one or more of cell-penetrating peptides or homeoprotein internalizationsecretion domains to facilitate export of the heterologous protein from mitochondria.

[0113] In some embodiments of any of the aforesaid method, the heterologous nucleic acid further comprises mitochondrial targeting sequences which facilitate nuclear import of the heterologous protein, or retention of the heterologous protein in the endoplasmic reticulum.

[0114] In some embodiments of any of the aforesaid method, the heterologous nucleic acid encodes a plurality of heterologous protein.

[0115] In some embodiments of any of the aforesaid method, the total size of the heterologous nucleic acid is less than or equal to 16 kb.

[0116] In some embodiments of any of the aforesaid method, the mitochondrion comprises a vector comprising the nucleic acid.

[0117] In some embodiments of any of the aforesaid method, the vector comprises one or more promoters operative in mitochondria and the promoter is capable of directing transcription of the nucleic acid.

[0118] In some embodiments of any of the aforesaid method, the vector comprises one or more mitochondrial transcriptional terminators.

[0119] In some embodiments of any of the aforesaid method, the vector comprises one or more transcript processing signals.

[0120] In some embodiments of any of the aforesaid method, the vector comprises native replication signals capable of propagating the plasmid within mitochondria during mitogenesis.

[0121] In some embodiments of any of the aforesaid method, the vector comprises mitochondria export sequences and the export sequence is capable of improving and / or increasing mitochondrial export of the protein.

[0122] In some embodiments of any of the aforesaid method, the promoter is a heavy strand promoter. In some embodiments of any of the aforesaid method, the promoter is a light strand promoter.

[0123] In some embodiments of any of the aforesaid method, the heavy strand promoter is selected from the group consisting of HSP1 and HSP2.

[0124] In some embodiments of any of the aforesaid method, the light strand promoter is LSP1 or LSP2.

[0125] In some embodiments of any of the aforesaid method, the vector comprises origins of strand (Ori) replication, tRNA genes and displacement (D-loop) loop.

[0126] In some embodiments of any of the aforesaid method, the Ori are selected from the group consisting of OriL and OriH.

[0127] In some embodiments of any of the aforesaid method, the D-Loop comprises regulatory elements for transcription and replication of mitochondrial DNA.

[0128] In some embodiments of any of the aforesaid method, the heterologous nucleic acid is located downstream of heavy strand promoter (HSP1) and flanked by mitochondrial t-RNA- Val gene.

[0129] In some embodiments of any of the aforesaid method, the heterologous nucleic acid further encodes one or more of cell-penetrating peptides or homeoprotein internalizationsecretion domains to facilitate export of the heterologous protein from mitochondria.

[0130] In some embodiments of any of the aforesaid method, the heterologous nucleic acid further comprises mitochondrial targeting sequences which facilitate nuclear import of the heterologous protein, or retention of the heterologous protein in the endoplasmic reticulum.

[0131] In some embodiments of any of the aforesaid method, the vector comprises heterologous nucleic acid encoding a plurality of heterologous proteins.

[0132] In some embodiments of any of the aforesaid method, the size of the heterologous nucleic acid is less than or equal to 16 Kb. BRIEF DESCRIPTION OF THE DRAWINGS

[0133] FIG. 1 is a schematic overview of the mitoDRIVE plasmid for transgene expression.

[0134] FIG. 2 is a schematic version of mitoDRIVE plasmid, including D-loop, origins of strand replication (OriL and OriH) and promoters for heavy (HSP1, HSP2) and light strand (LSP1). The transgene (eGFP) is terminated by mitochondrial-tRNA- Valine.

[0135] FIG. 3 discloses examples of mitoDRIVE plasmid constructs. FIG. 3 A illustrates mitoDRIVE plasmid construct that is capable of expressing eGFP protein when transfected to a mitochondrial cell. FIG. 3B illustrates mitoDRIVE plasmid construct that is capable of expressing luciferase protein when transfected to a mitochondria.

[0136] FIG. 4 discloses the ability of mitoDRIVE plasmid to express transgenes in mitochondria upon transfection. FIG. 4A illustrates expression of luciferase protein in presence of controls upon transfection. 293T cells were transfected with mitoDRIVE platform encoding a mitochondria-restricted and optimized firefly luciferase transgene [flue]. No DNA transfection, and transfection with mitoDRIVE encoding mitochondria-restricted GFP, were included as negative controls. Luciferase activity was measured after 48 hours incubation and reported as relative light units (RLU). FIG. 4B illustrates expression of luciferase protein having FLAG tag inside mitochondria. The expressed luciferase protein has a FLAG tag which facilitates ease of identification using anti-FLAG antibodies. FIG.4B illustrates the immunofluorescent microscopy of 293T cells 4-days after loading with control and mitoDRIVE transfected mitochondria and stained with a FITC-conjugated anti-FLAG antibody to visualize expression and intracellular localization of the FLAG-tagged mitoDRIVE-encoded luciferase transgene.

[0137] FIG. 5 illustrates immunofluorescent microscopy of 293T cells 4-days after loading with control and mitoDRIVE-eGFP transfected mitochondria. Fig. 5(A) illustrates that no florescence in the gfp channel was observed in control cells, but punctate eGFP signal was observed in cells transfected with mitoDRIVE-eGFP plasmid (See Fig 5B). To confirm this result, cells were stained with PE-anti-eGFP antibody. Fig. 5(C) illustrates diffuse, nonspecific background PE staining was observed in control cells while bright and more punctate staining was observed in mitoDRIVE-eGFP cells (See Fig. 5D). Fig. 5(E) is an enlarged field of view of the punctate staining observed in mitoDRIVE-eGFP cells which indicates that much of the eGFP was localized in spherical structures consistent with mitochondria, with some accumulation of protein outside of these structures as well. The results show that eGFP transgene is being expressed from the mitochondria as cytoplasmic expression of the transgene is not possible. Cytoplasmic expression of plasmid is prevented because the mitoDRIVE plasmid lacks a cytoplasmic gene expression promoter, and the conversion to mitochondrial anticodon usage introduces a canonical cytoplasmic gene expression stop codon.

[0138] FIG. 6 illustrates the ability of mitochondria loaded with mitoDRIVE to enter into different cell types and express heterologous proteins such as luciferase and eGFP. FIG. 6A schematically illustrates the ability of mitoDRIVE plasmid to transfect mitochondria. Fig. 6B illustrates that the transfected mitochondria containing the mitoDRIVE are successfully taken up by cells of different types such as 293T cells, HeEa cells and arising retinal pigment epithelia (ARPE) cells. The cells containing mitochondria having the mitoDRIVE plasmid are able to express the transgene protein (luciferase).

[0139] FIGS. 7 A and 7B are schematic representations of mitoDRIVE plasmids containing luciferase transgene. FIG. 7A illustrates a mitoDRIVE plasmid that expresses luciferase protein inside mitochondria whereas FIG. 7B illustrates a mitoDRIVE plasmid that enables the mitochondria to secrete luciferase protein into cytoplasm of the cell. FIGS. 7C and 7D illustrate that n-Luc mitoDRIVE plasmid expresses luciferase protein inside mitochondria and is localized within the cells whereas sec-n-Luc mitoDRIVE plasmid allows for accumulation of luciferase protein in the supernatant indicating that the secreted transgene protein is transported out of the mitochondria into the cytoplasm of the cell containing the mitochondria having the mitoDRIVE plasmid.

[0140] FIG. 8 illustrates that allogenic mitochondria containing mitoDRIVE plasmid do not induce inflammatory innate immune responses in human immune cells. Mitochondria (MITO) do not induce an innate immune response as observed by a lack of Fig. 8(A) reactive oxygen species (ROS) production from primary blood leukocytes, and absence of Fig. 8(B) TNFa and Fig. 8(C) IL-6 accumulation in THP macrophage or monocyte culture supernatant after 24-hour stimulation. NEG=unstimulated, Positive controls are LPS, and n-Formyl- peptides (NFP).

[0141] FIG. 9 illustrates that mitochondrial expression of heterologous protein is persistent even under in vivo conditions. FIG. 9A illustrates a schematic construct of the mitoDRIVE plasmid. FIG. 9B illustrates that the mitochondrial in vivo expression of transgenes is persistent, and a steady state concentration of luciferase protein is observed for over 12 days. The levels of the expressed protein remain consistent even after the cell culture is split once the culture becomes confluent.

[0142] FIG. 10 illustrates that the amount of transgene expressed by mitochondria upon transfection with mitoDRIVE plasmid is increased when the construct is codon optimized.

[0143] FIG. 11 illustrates a schematic of safety and tolerability studies in mice to determine the amount of transfected allogenic mitochondria that can be administered without causing immune responses. The schematic also shows testing different administrations such as intravenous (IV), intramuscular (IM), Intraperitoneal (IP) and Intranasal (IN) injection to determine the best way to ensure proper anatomical distribution.

[0144] FIG. 12 illustrates a schematic of different mitoDRIVE plasmids. Fig. 12A shows a mitoDRIVE plasmid that is bicistronic and has the ability to express two different transgenes upon transfection. Fig. 12B shows a mitoDRIVE plasmid that has two promoters that are capable of independently controlling the expression of two different transgenes. Fig. 12C shows mitoDRIVE plasmid having different sizes of transgene to determine the carrying capacity of construct.

[0145] FIG. 13 illustrates temperature and weight curves of different cohorts of mice. Fig. 13A illustrates temperature curve for various cohorts dosed with one of saline or LPS or syngenic mitochondria or mitochondria that contains a mitodrive plasmid at 0 hours, 6 hours, 24 hours, and 48 hours post administration. The cohorts include male and female mice dosed with 5 mg / kg of LPS, male and female mice dosed with saline, male and female mice dosed with Img / kg of syngenic mitochondria, and male and female mice dosed with 1 mg / kg of mitochondria that comprises a mitodrive plasmid.

[0146] Fig. 13B illustrates weight curve for various cohorts dosed with one of saline or LPS or syngenic mitochondria or mitochondria that contains a mitodrive plasmid at 0 hours, 6 hours, 24 hours, and 48 hours post administration. The cohorts include male and female mice dosed with 5 mg / kg of LPS, male and female mice dosed with saline, male and female mice dosed with 1 mg / kg of syngenic mitochondria, and male and female mice dosed with 1 mg / kg of mitochondria that comprises a mitodrive plasmid. Fig. 13C illustrates levels of interferon gamma (IFN-y) for various mice cohorts 48 hours post administration of one of saline or LPS or syngenic mitochondria or mitochondria that contains mitodrive plasmid. The cohorts include male and female mice dosed with 5 mg / kg of LPS, male and female mice dosed with saline, male and female mice dosed with Img / kg of syngenic mitochondria, and male and female mice dosed with 1 mg / kg of mitochondria that comprises a mitodrive plasmid. Statistics: Ordinary One-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05; ** p < 0.01; **** p < 0.0001. LLOD: Lower limit of detection.

[0147] Fig. 13D illustrates levels of TNF-a for various mice cohorts 48 hours post administration of one of saline or LPS or syngenic mitochondria or mitochondria that contains a mitodrive plasmid. The cohorts include male and female mice dosed with 5 mg / kg of LPS, male and female mice dosed with saline, male and female mice dosed with Img / kg of syngenic mitochondria, and male and female mice dosed with 1 mg / kg of mitochondria that comprises a mitodrive plasmid. Statistics: Ordinary One-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05; ** p < 0.01; **** p < 0.0001. LLOD: Lower limit of detection.

[0148] Fig. 13E illustrates levels of interleukin- 10 (IL- 10) for various mice cohorts 48 hours post administration of one of saline or LPS or syngenic mitochondria or mitochondria that contains a mitodrive plasmid. The cohorts include male and female mice dosed with 5 mg / kg of LPS, male and female mice dosed with saline, male and female mice dosed with Img / kg of syngenic mitochondria, and male and female mice dosed with 1 mg / kg of mitochondria that comprises a mitodrive plasmid. Statistics: Ordinary One-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05; ** p < 0.01; **** p < 0.0001. LLOD: Lower limit of detection.

[0149] Fig. 13F illustrates levels of interleukin-6 (IL-6) for various mice cohorts 48 hours post administration of one of saline or LPS or syngenic mitochondria or mitochondria that contains a mitodrive plasmid. The cohorts include male and female mice dosed with 5 mg / kg of LPS, male and female mice dosed with saline, male and female mice dosed with Img / kg of syngenic mitochondria, and male and female mice dosed with 1 mg / kg of mitochondria that comprises a mitodrive plasmid. Statistics: Ordinary One-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05; ** p < 0.01; **** p < 0.0001. LLOD: Lower limit of detection.

[0150] Fig. 14A illustrates the schematic of dosage administration of one of syngenic mitochondria (1 mg.kg) , mitochondria with mitodrive plasmid (1 mg / kg) or saline solution. The mice cohorts are administered with respective doses and sacrificed after two weeks or four weeks. The weight and temperature of the cohorts were measure before sacrificing the mice to measure the amount of mitochondrial antibodies.

[0151] Fig. 14B illustrates the weight and temperature curves of female and male mice cohorts two weeks post administration with one of syngenic mitochondria (1 mg.kg), mitochondria with mitodrive plasmid (1 mg / kg) or saline solution.

[0152] Fig. 14C illustrates the temperature curves of female and male mice cohorts two weeks post administration with one of syngenic mitochondria (1 mg.kg), mitochondria with mitodrive plasmid (1 mg / kg) or saline solution.

[0153] Fig. 14D illustrates the weight curves of female and male mice cohorts four weeks post administration with one of syngenic mitochondria (1 mg.kg), mitochondria with mitodrive plasmid (1 mg / kg) or saline solution.

[0154] Fig. 14E illustrates the temperature curves of female and male mice cohorts four weeks post administration with one of syngenic mitochondria (1 mg.kg), mitochondria with mitodrive plasmid (1 mg / kg) or saline solution.

[0155] Fig. 14F illustrates the amount of antimitochondrial antibody in mice cohorts dosed with one of syngenic mitochondria (1 mg.kg), mitochondria with mitodrive plasmid (1 mg / kg) or saline solution determined by ELISA. (N=6 mice per group, n=3 mice per sex)

[0156] Fig. 14G illustrates a comparison in the amount of antimitochondrial antibody in mice cohorts dosed with either saline or mitochondria with mitodrive plasmid (1 mg / kg) and were sacrificed two weeks post dosage.

[0157] Fig. 14H illustrates a comparison (2Way ANOVA with multiple comparisons; unpaired t-test for 2wk saline vs mitos and 4wk saline vs mitos) in the amount of antimitochondrial antibody in mice cohorts dosed with either saline or mitochondria with mitodrive plasmid (1 mg / kg) and were sacrificed four weeks post dosage.

[0158] Fig. 15A illustrates a direct transfection of nuclear encoded nanoluc with lipofectamine 3000 in 293t cells and CEM. NKR CCR5 T cells. Fig. 15B illustrates a direct transfection of mitoDRIVE plasmid with lipofectamine

[0159] 3000.

[0160] Fig. 15C illustrates luciferase expression levels in relative light units (RLU) after CEM.NKR cells were given mitoDRIVE laden mitochondria derived from 293t cells at different amounts.

[0161] Fig. 15D illustrates correlation between relative gene expression of mitoDRIVE nanoluc and nanoluc RLU (relative light units)

[0162] Fig. 15E illustrates a schematic for mitoDRIVE delivery. (N = 2 independent experiments, n =3 wells per experiment).

[0163] Fig. 16 illustrates a graphical correlation between mean fluorescence intensity (MFI) and the amount of with fluorescently-labeled plasmid indicating that plasmid DNA can be introduced into mitochondria via electroporation.

[0164] Fig. 17 illustrates an in vivo mitoDRIVE-based transgene expression delivered via mitochondrial transplant in model mice. Fig. 17A illustrates an Administration Schema for in vivo luciferase expression. Fig 17B illustrates Max RLU fold change normalized to the saline control, n=2 independent experiments. Fig. 17C illustrates an representative image of maximum RLU. Statistics: One-way ANOVA with Tukey multiple comparisons test; **, p < 0.01.

[0165] DETAILED DESCRIPTION

[0166] Mitochondria are semi-autonomous organelles present in the cytoplasm of all eukaryotic cells save erythrocytes. Mitochondria contain their own circular genome, and gene transcription and translation machinery that is separate from those used for the expression of genes encoded in nuclear DNA. Mitochondria are freely shared between all types of cells in the body, and given their endemic state, they do not elicit immune responses during homeostasis.

[0167] The disclosure provides a gene delivery system based on mitochondria. The gene delivery system comprises a mt-DNA plasmid engineered to encode a transgene, and upon transfection of the plasmid to mitochondria, the encoded product is expressed and produced by the protein expression system native to mitochondria. The disclosure also provides delivery of the plasmid-loaded mitochondria to different cells, tissues, or organisms. The disclosure also provides for the delivery and expression of a gene that encodes a gene editing protein thereby enabling genetic modification of nuclear DNA. Common gene editing proteins include but not limited to proteins from classes as Cas (Cas9, Casl2, Casl3, Casl4) (Hillary VE, Ceasar SA. A Review on the Mechanism and. Applications of CRISPR / Cas9 / Casl2 / Casl3 / Casl4 Proteins Utilized for Genome Engineering. Mol Biotechnol. 2023 Mar;65(3):311 -325), Zinc finger nucleases, Transcription activator-like effector nucleases (TALENs), and meganucleases (Sayed N, Allawadhi P, Khurana A, Singh V, Navik U, Pasumarthi SK, Khurana I, Banothu AK, Weiskirchen R, Bharani KK. Gene therapy: Comprehensive overview and therapeutic applications. Life Sci. 2022 Apr 1;294: 120375).

[0168] In humans and other mammals, the mt-DNA genome is approximately 16.5 kbp in size, encoding a total of 37 genes required for mitochondrial function (Scheffler IE. 2008. Mitochondria, 2nd ed. Wiley-Liss, Hoboken, N.J; Kotrys AV, Szczesny RJ. 2019. Mitochondrial Gene Expression and Beyond-Novel Aspects of Cellular Physiology. Cells). Only 22 tRNAs are utilized by the mitochondria, and the mitochondrial genetic code partially differs from that used for cytosolic translation. Mitochondria genes are expressed as polycistronic transcripts from both DNA strands, with the majority of genes expressed from the guanine-rich heavy strand. Heavy stand transcription is regulated by two promoters located in a non-coding region within a displacement loop (D-loop) structure (Kotrys AV, Szczesny RJ. 2019. Mitochondrial Gene Expression and Beyond-Novel Aspects of Cellular Physiology. Cells; Bouda E, Stapon A, Garcia-Diaz M. 2019. Mechanisms of mammalian mitochondrial transcription. Protein Sci 28:1594-1605; PfannerN, Warscheid B, Wiedemann N. 2019. Mitochondrial proteins: from biogenesis to functional networks. Nat Rev Mol Cell Biol 20:267-284). This D-loop region contains essential regulatory elements for transcription and mt-DNA replication (Nicholls TJ, Minczuk M. 2014. In D-loop: 40 years of mitochondrial 7S DNA. Exp Gerontol 56:175-81; Falkenberg M. 2018. Mitochondrial DNA replication in mammalian cells: overview of the pathway. Essays Biochem 62:287-296). Expression of the individual genes that encode protein requires processing of the polycistronic transcript by excision of mt-tRNAs via endonucleolytic cleavage, and further processing to generate individual mt-mRNA transcripts (9, 14, 15). Although all mammals have similar mtDNA size and composition, there is extensive diversity in other eukaryotes which is best exemplified among unicellular eukaryotes, with mtDNA genome sizes of less than 6 kbp encoding 5 genes for Plasmodium species, 75-150 kbp encoding 35 genes for Saccharomyces species, and over 69 kbp encoding 97 genes for Reclinomonas americana. (See, FEMS Yeast Research, Volume 18, Issue 5, August 2018, foy040)

[0169] The disclosure relates to a synthetic minimal mt-DNA-based plasmid (herein referred to as a “mitoDRIVE” plasmid) for delivery and expression of a transgene within mitochondria. The mitoDRIVE plasmid comprises several elements: a) native mitochondrial promoters for transcribing the heavy and light strands, b) the mitochondrial D-loop, c) tRNA genes for mitochondrial transcript processing, and d) the origins of replication for replication of mtDNA during mitogenesis, in many cases, these elements will be the element native to mitochondria, however, elements non-native to mitochondria are also contemplated according to the invention. For example, the promoters, D-loop, tRNA genes, and / or origins of replication may have a nucleotide sequence different from the native element, as long as the element is functional in mitochondria. The mitoDRIVE plasmid further comprises a transgene of interest inserted downstream of the heavy strand promoter 1 (HSP 1) or heavy strand promoter 2 (HSP 2) and flanked by a mt-tRNA-Val gene. In some instances, the transgene of interest is inserted downstream of the light strand promoter (LSP). The mt- tRNA-Val serves as a transcript processing signal for the transgene. Other tRNAs or alternative endogenous sequences present in the mtDNA can be used for transcript processing signals.

[0170] In one aspect, the disclosure provides a synthetic minimal mitochondrial-DNA-based plasmid (“mitoDRIVE plasmid”) for delivery and expression of a transgene within mitochondria. In another aspect, the disclosure provides a method of making a mitoDRIVE plasmid for delivery and expression of a transgene within mitochondria.

[0171] In another aspect, the disclosure provides a method of delivering a transgenic mitochondria comprising a heterologous gene encoding a signal peptide fused to a heterologous protein, wherein expression of said gene permits secretion of said protein from said mitochondrion.

[0172] In another aspect, the disclosure provides a method of providing transgenic mitochondria, the method comprising contacting isolated mitochondria with a vector comprising a gene encoding a signal peptide fused to a heterologous protein, wherein said vector is capable of transfecting a mitochondrion, and expression of said gene permits secretion of said protein from said mitochondrion. In another aspect, the disclosure provides a gene delivery system comprising a vector comprising a gene encoding a signal peptide fused to a heterologous protein, wherein said vector is capable of transfecting a mitochondrion, and expression of said gene permits secretion of said protein from said mitochondrion.

[0173] In some embodiments, the plasmid comprises natural promoters for the heavy and light strands, the D-loop, tRNA genes needed for transcript processing, and the origins of replication needed for replication of mtDNA during mitogenesis.

[0174] In some embodiments, the plasmid (mitoDRIVE plasmid) comprises a transgene of interest is inserted downstream of the heavy strand promoter 1 and flanked by mt-tRNA-Val gene. The mt-tRNA-Val serves as a transcript processing signal for the transgene.

[0175] In some embodiments, the mitoDRIVE plasmid comprises multiple transgenes between native mt- tRNA, driven by any of the 3 endogenous mitochondrial promoters. In some embodiments, the mitoDRIVE plasmid is ~2.3 kbp in size and comprises only essential elements of mitochondrial DNA and hence exhibits higher capacity to transport multiple transgenes. In some embodiments, transfection of purified mitochondria with mitoDRIVE results in expression of transgene such as enhanced green fluorescent protein (eGFP) embedded in the mitoDRIVE after internalization of said transfected mitochondria by allogeneic host cell. Expression of encoded fluorescent protein shows that mitoDRIVE can successfully express transgene inside a said transfected mitochondria residing in a host cell.

[0176] DESCRIPTION

[0177] Definitions

[0178] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described.

[0179] "About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of + 20% or + 10%, more 1 preferably + 5%, even more preferably + 1%, and still more preferably + 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0180] As used herein, where an element is “native” with respect to mitochondria means that the element is naturally found (i.e., endogenous to) the mitochondria.

[0181] As used herein, where a gene or a segment of DNA or RNA or protein is “heterologous ” to mitochondria, it means that it originates from outside the mitochondria and therefore is not endogenous to the mitochondria. Heterologous is an equivalent term to exogeneous, i.e., as used herein, not occurring within mitochondria in nature. A heterologous protein is one that is not native to mitochondria.

[0182] As used herein, the term “secretable heterologous protein” refers to a heterologous protein that is capable of being expressed by a mitochondrion and secreted out of the mitochondrion into the cell in which the mitochondria resides in, onto the cell surface, or out of the cell into the extracellular space.

[0183] As used herein, the term “heterologous protein” refers to any protein that is expressed in a different species or cell type than the one it naturally originates from. This involves the insertion of foreign genetic material (DNA or RNA) into the host, leading to transcription, translation, and expression of the protein in a non-native environment. Heterologous expression is commonly employed in molecular biology and biotechnology for protein characterization, therapeutic protein production, and the study of protein function.

[0184] As used herein, the term “heterologous nucleic acid” refers to a segment of DNA or RNA that originates from one organism or species and is introduced into a different host organism or cell system to express a protein of interest. This non-native nucleic acid is typically introduced via recombinant DNA technology, genetic engineering, or transfection, allowing the host to replicate or express genes that it does not naturally possess.

[0185] As used herein, the term "polypeptide” refers to an amino acid polymer, including both naturally occurring amino acids as well as synthetic non-naturally occurring analogs thereof linked via peptide bonds.

[0186] As used here the term "isolated' means removed from the natural state. For example, a nucleic acid or a polypeptide naturally present in a living animal is not "isolated," but the same nucleic acid or polypeptide partially or completely separated from that animal is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form or can exist in a non-native environment such as, for example, a host cell.

[0187] As used herein, "substantially purified" refers to being essentially free of other components. For example, a substantially purified polypeptide is a polypeptide that has been separated from other components with which it is normally associated in its naturally occurring state. Non-limiting embodiments include 95% purity, 99% purity, 99.5% purity, 99.9% purity and 100% purity.

[0188] As used herein the term "oligonucleotide" or "polynucleotide" is a nucleic acid ranging from at least 2, in certain embodiments at least 8, 15 or 25 nucleotides in length, but may be up to 50, 100, 1000, or 5000 nucleotides long or a compound that specifically hybridizes to a polynucleotide.

[0189] As used herein, the term "pharmaceutical composition" or "composition" refers to a mixture of at least one compound useful within the disclosure with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a patient. Multiple techniques of administering a compound exist in the art including, but not limited to, subcutaneous, intravenous, oral, aerosol, inhalational, rectal, vaginal, transdermal, intranasal, buccal, sublingual, parenteral, intrathecal, intragastrical, ophthalmic, pulmonary, and topical administration.

[0190] As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained; for example, phosphate- buff ered saline (PBS).

[0191] As used herein, the term "vector" means a nucleic acid molecule that carries and is covalently linked to exogenous nucleic acids. In some embodiments, a vector is a plasmid, i.e., a circular double stranded DNA into which additional DNA segments may be ligated. Types of vectors include a viral vector; wherein additional nucleotide sequences may be ligated into the viral genome. A vector may replicate autonomously in a host cell into which it is introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). A vector (e.g., non-episomal mammalian vectors) may integrate into the genome of a host cell upon introduction into the host cell and is thus replicated along with the host genome. Moreover, certain vectors (expression vectors) are capable of directing the expression of genes to which they are operatively linked.

[0192] As used herein, the term "recombinant host cell" (or simply "host cell"), means a cell into which an exogenous nucleic acid and / or recombinant vector has been introduced. It should be understood that "recombinant host cell" and "host cell" mean not only the particular subject cell but also the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0193] As used herein, the term "transcriptional regulatory region", refers to a portion or segment or fragment of nucleic acid containing one or more regulatory elements that control transcription. The transcriptional regulatory region according to the disclosure includes one or more promoters and, optionally, one or more enhancers.

[0194] As used herein, the term "promoter", refers to a portion of a nucleic acid that is located 5’ to and functions to control the transcription of one or more polynucleotides to which it is linked, and which is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites, and any other DNA sequences including, but not limited to, transcription factor binding sites, repressor, and activator protein binding sites, and any other sequences of nucleotides known in the art to act directly or indirectly to regulate the amount of transcription from the promoter. Promoters include inducible promoters, constitutive promoters and tissue- specific promoters.

[0195] As used herein, the term "inducible promoter", refers to a promoter that is physiologically or developmentally regulated, e.g., by the application of a chemical inducer. For example, it can be a tetracycline-inducible promoter, a mifepristone (RU-486)-inducible promoter and the like.

[0196] As used herein, the term "constitutive promoter", refers to a promoter whose activity is maintained at a relatively constant level in all cells of an organism, or during most developmental stages, with little or no regard to cell environmental conditions. Examples of constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), the SV40 promoter, the dihydrofolate reductase promoter, the (3-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter (Boshart M, et al., Cell 1985; 41:521-530).

[0197] As used herein, the term "enhancer" , refers to a DNA sequence element to which transcription factors bind to increase gene transcription. Examples of enhancers may be, without limitation, RSV enhancer, CMV enhancer, HCR enhancer, etc.

[0198] As used herein, the term " polyadenylation signal", relates to portion of a nucleic acid that mediates the attachment of a polyadenine stretch to the 3' terminus of the mRNA. Suitable poly adenylation signals include, without limitation, the SV40 early poly adenylation signal, the SV40 late poly adenylation signal, the HSV thymidine kinase poly adenylation signal, the protamine gene polyadenylation signal, the adenovirus 5 Elb polyadenylation signal, the bovine growth hormone polyadenylation signal, the human variant growth hormone polyadenylation signal, all of which are well known in the art.

[0199] As used herein, the term “mitochondrial terminator” refers to a portion of a nucleic acid usually positioned 3’ to a coding sequence that cause transcription to stop. Transcription of the mitochondrial genome originates in the major non-coding region containing the L- strand (LSP) and H-strand (HSP) promoters. The light strand promoter controls the transcription of eight of the tRNAs and the MT-ND6 gene. There are several transcription termination factors known in art, for example, the mammalian mitochondrial transcription termination factor mTERF binds with high affinity to a site within the tRNALeu(UUR) gene and regulates the amount of read through transcription from the ribosomal DNA into the remaining genes of the major coding strand of mitochondrial DNA (mtDNA).

[0200] As used herein, the term "signal peptide”, (also referred to as signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence or leader peptide) is a short peptide, for example, 8-50 amino acids in length, 10-45 amino acids in length, 12-40 amino acids in length, 14-35 amino acids in length, or 16-30 amino acids in length or 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 or 50 amino acids in length present at the N-terminus (or occasionally non-classically at the C- terminus or internally) of most newly synthesized proteins that are destined toward the secretory pathway. These proteins include those that reside either inside certain organelles (the endoplasmic reticulum, Golgi or endosomes), secreted from the cell, or inserted into most cellular membranes. Although most type I membrane-bound proteins have signal peptides, the majority of type II and multi-spanning membrane-bound proteins are targeted to the secretory pathway by their transmembrane domain, which biochemically resembles a signal sequence except that it is not cleaved.

[0201] Signal peptides function to prompt a cell to translocate the protein, usually to the cellular membrane. In prokaryotes, signal peptides direct the newly synthesized protein to the SecYEG protein-conducting channel, which is present in the plasma membrane. A homologous system exists in eukaryotes, where the signal peptide directs the newly synthesized protein to the Sec61 channel, which shares structural and sequence homology with SecYEG, but is present in the endoplasmic reticulum. Both the SecYEG and Sec61 channels are commonly referred to as the translocon, and transit through this channel is known as translocation. While secreted proteins are threaded through the channel, transmembrane domains may diffuse across a lateral gate in the translocon to partition into the surrounding membrane. (Lodish et al., 2000, Molecular Cell Biology, 4th edition).

[0202] In both prokaryotes and eukaryotes signal sequences may act co-translationally or post-translationally. The co-translational pathway is initiated when the signal peptide emerges from the ribosome and is recognized by the signal-recognition particle (SRP). SRP then halts further translation (translational arrest only occurs in Eukaryotes) and directs the signal sequence-ribosome-mRNA complex to the SRP receptor, which is present on the surface of either the plasma membrane (in prokaryotes) or the ER (in eukaryotes). Once membrane-targeting is completed, the signal sequence is inserted into the translocon. Ribosomes are then physically docked onto the cytoplasmic face of the translocon and protein synthesis resumes.

[0203] The post-translational pathway is initiated after protein synthesis is completed. In prokaryotes, the signal sequence of post-translational substrates is recognized by the SecB chaperone protein that transfers the protein to the SecA ATPase, which in turn pumps the protein through the translocon. Although post-translational translocation is known to occur in eukaryotes, it is poorly understood. It is however known that in yeast post-translational translocation requires the translocon and two additional membrane-bound proteins, Sec62 and Sec63. Signal peptides are extremely heterogeneous, and many prokaryotic and eukaryotic signal peptides are functionally interchangeable even between different species however the efficiency of protein secretion is determined by the nature of signal peptide.

[0204] Some non-limiting examples of signal peptide that are known in art are shown below:

[0205] As used herein, the term "operatively linked" , refers to the functional relation and location of a promoter sequence with respect to a polynucleotide of interest (e.g. a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence). Generally, a promoter operatively linked is contiguous to the sequence of interest. However, an enhancer does not have to be contiguous to the sequence of interest to control its expression. As used herein, the term “Megamitochondria ” refers to mitochondria which are considerably larger than normal mitochondria, as a result of which they can be seen under a light microscope without staining They are also known as "giant mitochondria". Megamitochondria have a diameter of >2 pm, or of 2-10 pm, preferably of 2, 3, 4, 5, 6, 7, 8, 9 or 10 pm, most preferably of 4-10 pm or of 4, 5, 6, 7 or 10 nm. The megamitochondria are generally reversibly induced prior to the physical transfection of the mitochondria. Megamitochondria can also be reversibly induced either by the administration of active substances and / or chemicals into the culture medium, such as valinomycin, chloramphenicol, H2O2, hydrazine or by the incubation of the cells in an acidic culture medium, wherein the culture medium is acidified by the addition or creation of pH-lowering substances, e.g., acids or the salts thereof such as lactic acid, lactate, acetic acid and / or sodium acetate or mixtures thereof. Generally normal mitochondria have a diameter ranging from 0.5 to 2 pm.

[0206] As used herein, the term “mitogenesis” refers to the process of induction of mitosis in cells.

[0207] As used herein, the term “mitochondrial export” refers to the process of transporting proteins expressed in the mitochondria out into the cytosol of cells containing the mitochondria.

[0208] As used herein, the term “heavy strand promoter” refers to the two promoters on the heavy strand of mitochondrial DNA: heavy-strand promoter 1 (HSP1) and heavy-strand promoter 2 (HSP2).

[0209] As used herein, the term “light strand promoter” refers to the two promoters on the light strand of mitochondrial DNA: light-strand promoter 1 (LSP1) and light-strand promoter 2 (LSP-2).

[0210] As used herein, the term “mitochondrial tRNA genes” refers to 22 tRNAs present in the mitochondrial genome. In addition to being used for translation, these tRNAs are signals for processing of polycistronic mitochondrial transcripts to be cleaved into multiple transcripts.

[0211] As used herein, the term “nuclear import” refers to the process of transporting proteins into the nucleus. This can be done by the binding of a nuclear signal peptide to a carrier protein for import into the nucleus through a nuclear pore complex. As used herein, the term “transcript processing signals” refers to sequences of nucleic acids, not limited to, but including mitochondrial tRNA genes. Their function include, but are not limited to, cleavage of a polycistronic transcript into daughter transcripts and recruitment of polyadenylation machinery for transcript maturation.

[0212] As used herein, the term “homeoprotein internalization- secretion domains” refers to a polypeptide sequence that allows for noncanonical secretion of proteins independent of a vesicle mediated secretion pathway. These sequences allow for noncanonical internalization of the whole polypeptide into a cell for further translocation into a respective subcellular compartment. These polypeptide sequences are denoted by their DNA binding domain, termed ‘homeodomain.’ Examples of homeoproteins are Engrailed, Fibroblast growth factor 2, and the human immunodeficiency virus transactivator of transcription (JoliotA, Prochiantz A. Unconventional Secretion, Gate to Homeoprotein Intercellular Transfer. Front Cell Dev Biol. 2022 Jun 28; 10:926421).

[0213] As used herein, the term “D-Loop” or “displacement loop” refers to a DNA structure where the two strands of a double- stranded DNA molecule are separated for a stretch and held apart by a third strand of DNA. An R-loop is similar to a D-loop, but in this case the third strand is RNA rather than DNA. The third strand has a base sequence which is complementary to one of the main strands and pairs with it, thus displacing the other complementary main strand in the region. Within that region the structure is thus a form of triple- stranded DNA. The mitochondrial D-loop is a triple-stranded region found in the major non-coding region (NCR) of many mitochondrial genomes, and is formed by stable incorporation of a third, short DNA strand known as 7S DNA. A D-loop as it occurs in a mitoDRIVE plasmid, as used herein, has a minimum length of 650 bp (Jemt E, Persson O, Shi Y, Mehmedovic M, Uhler JP, Davila Lopez M, Freyer C, Gustafsson CM, Samuelsson T, Falkenberg M. Regulation of DNA replication at the end of the mitochondrial D-loop involves the helicase TWINKLE and a conserved sequence element. Nucleic Acids Res. 2015 Oct 30;43(19):9262-75.) and a maximum length of 1200 bp. A typical human D loop is about 1124 bp in length and present at positions -16024-576 of the human mitochondrial genome, as numbered according to Sharma et al. (See Sharma H, Singh A, Sharma C, Jain SK, Singh N. Mutations in the mitochondrial DNA D-loop region are frequent in cervical cancer. Cancer Cell Int. 2005 Dec 16;5:34.) As used herein, the term “cell-penetrating peptide" (CPP) refers to a short peptide (5- 30 amino acids) that have a positive charge, which facilitates its penetration into a cell across the cell membrane. A CPP can enhance the delivery of its cargo into a cell through endocytosis. Based on its origins, a CPP can be divided into (1) a protein derived CPP, (2) a synthetic CPP, and (3) a chimeric CPP. Protein-derived CPPs, including the TAT protein and penetrating can enter the cell because they have a specific motifs or helical structures. (2). Synthetic CPPs. In this group, polyarginine 8-10-mers are most widely studied because of their high efficiency in cellular uptake. (3). Chimeric CPPs. This group of CPPs is generally recognized as a transition from natural to synthetic CPPs since they contain sequences from two or more different naturally occurring proteins. Examples include amphipathic peptide (CADY) (20 amino acids) which combines aromatic residue (Tryptophan, W) and cationic residue (Arginine, R).

[0214] As used herein, the term “canonical signal sequence” refers to a signal peptide having a sequence that reflects the most common choice of amino acid at each position. Despite the diversity of existing secretory pathways, signal peptides have conserved common structural features across the species. Three regions, termed N-, H- and C-region, have been studied and characterized. N-region is a short (1-5 residues) positive-charged domain. Its basic amino acids (Arg or Lys) are preferentially encoded by high-translational-rate codons. The charges are involved in the interaction with signal recognition particle (SRPs) as well with the phosphate of the lipid layer. H-region is a hydrophobic helix (7-15 residues) that will be anchored in the membrane. Leucines are predominant in such domain and 3-5 residues of leucine have been shown to positively affect secretory efficiency of the signal peptide. C- region is a beta-sheet folded uncharged region (3-7 residues) that serves as substrate for the signal peptidase (SPase). SPases are the enzymes that will cleave the SP and release the protein in the secretory pathway. A motif V / A-X-A is highly conserved in eukaryotes just before the cleavage site, while eukaryotes SPases seem to tolerate a wider X-X-A motif.

[0215] As used herein, the term “transgenic” refers to organisms or cells that have been genetically modified to carry one or more exogenous genes or DNA sequences. These foreign genes may originate from the same species or from different species altogether. The purpose of creating transgenic organisms or cells is often to study gene function, investigate the effects of specific genes or DNA sequences, or introduce new traits into an organism for various purposes, such as improving agricultural crops, producing therapeutic proteins, or studying disease mechanisms.

[0216] As used herein, the term “allogenic” or "allogeneic” refers to a biological context in which cells, tissues, or organs are derived from a genetically different individual of the same species. In allogenic situations, the donor and recipient of the biological material are not genetically identical, and there are differences in the genetic makeup between them. Allogenicity is commonly discussed in the context of transplantation, where organs or tissues or cells are transferred from one individual (the donor) to another (the recipient) for therapeutic purposes.

[0217] As used herein, the term “allogenic mitochondria” refers to mitochondria that are derived from a genetically different individual of the same species, cultured and / or manipulated ex vivo and administered to a different individual of the same species. An allogenic mitochondria may or may not have a transgene in them.

[0218] As used herein, the term “autogenic mitochondria” refers to mitochondria that are derived from an individual, cultured and / or manipulated ex vivo and administered back to the same individual. Autogenic mitochondria may or may not have a transgene in them.

[0219] As used herein, the term “permissive cell lines” refers to cell lines that are highly amenable to introducing exogenous DNA or RNA into their cellular machinery. These cell lines possess certain properties that make them suitable for efficient and successful transfection, allowing the delivery and expression of foreign genetic material. Some nonlimiting examples include permissive cell lines for transfection include HEK293 (Human Embryonic Kidney 293), CHO (Chinese Hamster Ovary) cells, Jurkat (Human T-cell Leukemia) cells, HeLa (Human Cervical Carcinoma) cells, and Cos-7 (African Green Monkey Kidney) cells.

[0220] As used herein, the term “organism” encompasses a fungus, plant, or an animal such as a mammal or a human that possesses mitochondria.

[0221] As used herein, the term “mammal” is a vertebrate animal of the class Mammalia. Mammals are characterized by the presence of milk-producing mammary glands for feeding their young, a neocortex region of the brain, fur or hair, and three middle ear bones. Mammals include rodents such as mice, rats, and guinea pigs, as well as large animals such as cows and pigs, Mammals include humans.

[0222] As defined herein, the term ''subject'', “individual" or "patient” refers to mammal preferably a human.

[0223] As used herein, the term "treatment" or "treating" is defined as the application or administration of a therapeutic agent such as a transgene (alone or in combination with another agent), to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient (e.g., for diagnosis or ex vivo applications), who has a disease or disorder, a symptom of a disease or disorder or the potential to develop a disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, the symptoms of the disease or disorder, or the potential to develop the disease or disorder. Such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.

[0224] As used herein, the term "prevent" or "prevention" or "reduce" means no disorder or disease development if none had occurred, or no further disorder or disease development if there had already been the development of the disorder or disease. Also considered is the ability of one to prevent some or all of the symptoms associated with the disorder or disease.

[0225] As used herein, the term " effective amount" refers to an amount of an agent (e.g., a transgene or a polypeptide encoded by a transgene) which, as compared to a corresponding subject who has not received such an amount, sufficient to provide improvement of a condition, disorder, disease, or to provide a decrease in progression or advancement of a condition, disorder, or disease. An effective amount also may result in treating, healing, preventing or ameliorating a condition, disease, or disorder. The term also includes within its scope amounts effective to enhance normal physiological function.

[0226] Ranges: throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0227] DETAILED DESCRIPTION

[0228] Design of mitoDRIVE Trans genes

[0229] A transgene of interest such as eGFP or Luciferase is selected and then the mRNA sequence of the chosen transgene is taken from the reference source such as NCBI, Genbank or Uniprot databases. The nuclear encoded translation is compared to the consensus sequence to verify the coding is accurate. The target species into which the transgene would be expressed is identified and the mitochondrial codon frequency for that species is gathered. The heterologous peptide sequence, which would be encoded by the transgene, is then back translated first using an ATA start, and then with the codons with the highest usage in the mitochondria for the selected species of interest.

[0230] Once the first pass is complete, the GC content is assessed and if it differs by more than 5% from the selected species’ mitochondrial GC content, then the codons are then changed with codons that have the least difference between levels of prevalence in the codon frequency table to minimize the effect of using a rare codon. All tryptophans can be optionally encoded as nuclear stops in the mitochondrial coding. The transgene of interest is then translated into both a nuclear decoded form and a mitochondrial decoded form to assess for nuclear stop codons. Mitochondrial optimization strives to maximize amino acid decoherence between the nuclear encoding and the mitochondrial encoding while maintaining the amino acid sequence in the mitochondrial encoding.

[0231] Generation of mitoDRIVE plasmids mitoDRIVE plasmids are produced using any number or combination of standard molecular biology approaches including gene synthesis, PCR, restriction digestions, site- directed mutagenesis, subcloning, and Gibson assembly or variants thereof. Plasmids are initially made using a variety of standard cloning vectors such as pUC57, pCCl, pcDNA3.1, and pUC57-mini, and any of these plasmids can be used for both in vitro and in vivo mitoDRIVE studies.

[0232] The production of mitoDRIVE plasmids typically involves several steps. Initially, a desirable plasmid backbone such as pUC57, pCCl, pcDNA3.1, and pUC57 is chosen, which often contains essential genetic elements such as a replication origin, selectable markers for screening and maintenance, and regulatory sequences. This backbone provides the necessary framework for gene cloning and expression.

[0233] The next step involves the introduction of desired genetic sequences, such as genes of interest, into the plasmid backbone. This can be achieved through various methods, including restriction enzyme digestion and ligation, polymerase chain reaction (PCR)-based amplification, or recombinant DNA technologies. The gene of interest is inserted into the plasmid backbone, resulting in a recombinant plasmid.

[0234] Once the recombinant mitoDRIVE plasmid is obtained, it needs to be propagated and amplified to generate sufficient quantities for downstream applications. This is typically achieved through transformation, where the recombinant plasmid is introduced into host organisms capable of replicating and maintaining the plasmid DNA. Common hosts include bacteria such as Escherichia coli, yeast, or mammalian cell lines. A simple schematic of mitoDRIVE plasmid with representative key elements are shown in Figure 1.

[0235] Components of mitoDRIVE plasmid

[0236] 1) The D-loop region of mitochondrial DNA from human NCBI mitochondrial DNA reference sequence (NC_012920) for mitoDRIVE for use in humans, and from mouse NCBI mitochondrial DNA reference sequence (NCBI NC_05089) for mitoDRIVE for use in mice models. For other animal models, species specific sequences can be used. The D-loop, also known as the displacement loop, is a non-coding control region of mitochondria that contains key genetic elements needed for mitochondrial DNA replication and gene expression.

[0237] 2) Natural mitochondrial transcription promoters. The D-loop region included in mitoDRIVE constructs contains a promoter for the mitochondria heavy strand, heavy strand promoter 1 (HSP1), and light strand promoters 1 and 2 (LSP1, LSP2).

[0238] 3) Origin of replication for the heavy strand. Contained in the D-loop region that is included in mitoDRIVE constructs. Origins of replication are sequences of mitochondrial DNA required for replication during the process of mitogenesis and are therefore included to promote replication and persistence of mitoDRIVE DNA in host cells. 4) A portion of the coding region for the first mitochondrial ribosome that contains the second heavy strand mitochondrial transcription promoter, heavy strand promoter 2 (HSP2).

[0239] 5) The transgene of interest. The transgene must be in mitochondrial genetic code, which has several key differences compared to the genetic code used to express genes from nuclear DNA. The transgene can be optimized for improved expression and restricted to mitochondrial expression. The start codon used can be either ATG or ATA. In some embodiments, the mitoDRIVE plasmid comprises a transgene that is expressed from the heavy strand.

[0240] 6) The region containing the light strand origin of replication which also contains the coding sequence for tRNA asparagine (tRNA-Asn) and tRNA cysteine (tRNA-Cys), preserving the order found in native mitochondrial DNA: tRNA-Asn, OriL, tRNA-Cys.

[0241] In some embodiments, the mitoDRIVE plasmid further comprises a region encoding mitochondria phenylalanine tRNA (tRNA-Phe). This tRNA is present in this location, adjacent to the D-loop, in normal mitochondrial DNA and is believed to play a role in transcript processing.

[0242] In some embodiments, the mitoDRIVE plasmid further comprises two coding regions for mitochondrial tRNAs needed for transcriptional termination and transcriptional punctuation, valine tRNA (tRNA- Vai) and leucine tRNA (tRNA-Leu).

[0243] In some embodiments, the mitoDRIVE plasmid comprises a transgene that is expressed from the light strand. In some embodiments, the mitoDRIVE plasmid comprises a transgene that is expressed from the heavy strand and from the light strand.

[0244] In some embodiments, the mitoDRIVE plasmid comprises only one heavy strand promoter HSP1.

[0245] In some embodiments, the mitoDRIVE plasmid comprises only one heavy strand promoter HSP2.

[0246] In some embodiments, the mitoDRIVE plasmid comprises both heavy strand promoters HSP1 and HSP2. In some embodiments, the mitoDRIVE plasmid comprises only one light strand promoter LSP1.

[0247] In some embodiments, the mitoDRIVE plasmid comprises only one light strand promoter LSP2.

[0248] In some embodiments, the mitoDRIVE plasmid comprises both light strand promoters LSP1 and LSP2.

[0249] In some embodiments, the mitoDRIVE plasmid comprises one heavy strand promoter and one light strand promoter.

[0250] In some embodiments, the mitoDRIVE plasmid comprises signal peptide sequence to direct expressed transgene to different cellular compartments or for secretion outside the cell.

[0251] In some embodiments, the signal sequence is selected from the group consisting of IL-6 secretion signal, native secretion signal of Factor IX, native secretion signal of secreted embryonic alkaline phosphatase (SEAP) gene and secretion signals capable of directing the expressed transgene to other cellular compartments, ER, golgi, nucleus, cell surface / plasma membrane, or retention in the mitochondria.

[0252] In some embodiments, the mitoDRIVE plasmid comprises cell penetrating peptides and / or homeoprotein domains to direct translocation of expressed transgene to desired cellular compartments.

[0253] Production of mitoDRIVE plasmid DNA

[0254] After successful transformation, the host cells carrying the recombinant mitoDRIVE plasmid are selected using markers, such as antibiotic resistance genes, that are present on the plasmid. The selected cells are then grown in culture under appropriate conditions, allowing the plasmid to replicate along with the host genome.

[0255] The plasmids are purified from the host cells using various methods, such as alkaline lysis, chromatography, or commercial purification kits. Purified plasmids can be analyzed for their integrity, concentration, and quality using techniques like agarose gel electrophoresis or spectrophotometry . Large-scale production of mitoDRIVE plasmids involves the generation of significant quantities of plasmid DNA for various applications, such as gene therapy, vaccine development, or biopharmaceutical production. The process typically involves several stages, including plasmid amplification, purification, and quality control.

[0256] To initiate plasmid production at a large scale, a suitable host organism, often Escherichia coli, is selected for cultivation. The host cells are transformed with the mitoDRIVE plasmid of interest, typically using a high-efficiency transformation method, and allowed to grow in a controlled fermentation system. This system provides optimal conditions for cell growth, such as temperature, pH, dissolved oxygen, and nutrient supply.

[0257] During the fermentation process, the host cells multiply and propagate the plasmid DNA along with their own genomic DNA. Various strategies known in art can be employed to enhance plasmid yield, such as the use of high-copy plasmids, optimization of culture conditions, and genetic modifications of the host strain.

[0258] After reaching the desired cell density, the cells are harvested by centrifugation or filtration, and the plasmid DNA is extracted from the cell pellets. Different purification methods can be employed, including alkaline lysis, column chromatography, precipitation, or membrane filtration, depending on the desired level of purity and the specific requirements of the downstream applications.

[0259] Once the plasmid DNA is purified, it undergoes rigorous quality control procedures. This involves assessing parameters such as DNA concentration, purity, integrity, and endotoxin levels. Analytical techniques such as spectrophotometry, gel electrophoresis, and high-performance liquid chromatography (HPLC) are commonly used for quality assessment.

[0260] For preclinical testing and clinical testing, DNA sequences that encode antibiotic restriction genes and bacterial plasmid replication elements of the plasmid are removed using the minicircle DNA system described below.

[0261] Production of mitoDRIVE minicircle DNA

[0262] Minicircle DNA vectors consisting of a circular expression cassette devoid of the bacterial plasmid DNA backbone provides several advantages including sustained transgene expression in quiescent cells / tissues. Minicircle production is accomplished in vivo in Escherichia coli via induction of intramolecular recombination of a parental plasmid. This process relies on the expression of a site-specific recombinase that acts upon two recognition sequences. These sites are strategically placed in the backbone of the parental plasmid, flanking the regions dedicated to replication in E. coli (prokaryotic backbone harboring the replication origin and antibiotic resistance) and the eukaryotic cassette with the gene of interest. During the event, the recombinase mediates the recombination between its recognition sequences, originating two circular, covalently closed molecules (i) the minicircle containing the eukaryotic cassette and (ii) a mini plasmid comprised of the prokaryotic backbone. (See Alves et al., Minicircle Biopharmaceuticals-An Overview of Purification Strategies, Front. Chem. Eng., 13 January 2021) The production of minicircle DNA involves the generation of circular DNA molecules devoid of bacterial backbone sequences, resulting in highly pure and streamlined genetic constructs. Minicircle method offers advantages over traditional plasmid DNA production methods, as they eliminate non-essential sequences, minimize immune responses, and enhance transgene expression.

[0263] The production of minicircle DNA begins with the design and construction of a parental plasmid (such as pMC.BESPX-MCS2 from System Biosciences) which is then modified to contain the desired genetic elements, such as gene of interest, mitoDRIVE cassette, promoter, and other regulatory sequences. This plasmid typically incorporates a bacterial backbone and a site-specific recombinase recognition site.

[0264] To generate minicircles, a site-specific recombinase, such as Cre recombinase or PhiC31 integrase, is introduced to the parental plasmid. The recombinase recognizes and mediates recombination between the two recognition sites, resulting in excision of the bacterial backbone and generation of a minicircle DNA molecule. This process can be performed in vivo within bacterial cells or in vitro using purified plasmid DNA and recombinase enzyme. Alternatively, plasmids can be digested using specific restriction enzymes to remove the bacterial backbone and ligated with DNA ligase to re-circularize and generate the mitoDRIVE minicircle DNA.

[0265] After minicircle production, purification steps are employed to isolate and enrich the minicircle DNA. These purification methods commonly include endonuclease digestion to remove any remaining parental plasmid, followed by size-based separation techniques such as agarose gel electrophoresis or column chromatography. The aim is to separate and collect the smaller minicircle DNA molecules from larger residual DNA fragments. Quality control procedures are performed to ensuring the purity and integrity of the minicircle DNA product. Analytical techniques such as gel electrophoresis, spectrophotometry, and PCR amplification are employed to verify the presence of the desired genetic elements, absence of bacterial backbone sequences, and overall integrity of the minicircle DNA.

[0266] The resulting purified minicircle DNA can be further characterized and quantified using advanced analytical methods like next-generation sequencing or qPCR. This allows for accurate assessment of the minicircle DNA population, confirmation of genetic content, and determination of copy number.

[0267] Delivery of mitoDRIVE DNA to mitochondria

[0268] Delivery of mitoDRIVE DNA into mitochondria is accomplished by transfection. Several methods have been developed to facilitate the transfection of DNA into mitochondria. Here are a few approaches:

[0269] (a) Liposome-based transfection'. Liposomes can be used as carriers to encapsulate DNA and facilitate its delivery into mitochondria. These liposomes are typically composed of cationic lipids or lipids with mitochondrial targeting sequences. The liposomes interact with the mitochondrial membrane, facilitating the transfer of mitoDRIVE DNA into the mitochondria. Some non-limiting examples of liposome transfecting agents are shown below:

[0270] Lipofectamine: Lipofectamine is a commercially available liposome-based transfection reagent commonly used for DNA delivery. It forms lipoplexes by combining cationic lipids with DNA molecules. Lipofectamine-mediated transfection has been utilized in mitochondrial transfection studies to deliver DNA into mitochondria or target genes involved in mitochondrial function.

[0271] DOTAP: N-[l-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP) is a cationic lipid commonly used in liposome-based transfection. It forms lipoplexes with DNA, facilitating efficient cellular uptake. DOTAP-based liposomes have been used for delivering DNA into mitochondria in various research studies.

[0272] MITO-Porter: MITO-Porter is a liposome-based delivery system specifically designed for mitochondrial transfection. It utilizes a combination of fusogenic lipids and a mitochondrial targeting peptide to facilitate the delivery of cargo, including DNA, into mitochondria. MITO-Porter has been employed to successfully deliver DNA into mitochondria for functional studies and potential therapeutic applications.

[0273] Protamine-DNA complexes: Protamine, a small positively charged protein, can be complexed with DNA to form nanoparticles that facilitate cellular uptake. These complexes can be further encapsulated within liposomes to improve stability and enhance delivery. Protamine-DNA lipoplexes have been used for mitochondrial transfection in various experimental settings.

[0274] Stearylamine-based liposomes: Stearylamine, a cationic lipid, can be incorporated into liposomes to form lipoplexes with DNA. These lipoplexes have been utilized for mitochondrial transfection, targeting specific genes or genetic elements within mitochondria.

[0275] (b) Mitochondrial targeting peptides'. Specific mitochondrial targeting peptides can be coupled to the DNA or associated with delivery vectors to direct DNA molecules into mitochondria. These peptides contain sequences that are recognized by mitochondrial import machinery, allowing them to efficiently deliver DNA into the mitochondria.

[0276] (c)Electroporation'. Electroporation involves the application of brief electric pulses to create temporary pores in the mitochondrial membrane. This temporary disruption allows DNA molecules to pass through and enter the mitochondria.

[0277] (d) Microinjection'. Microinjection involves direct injection of DNA into individual mitochondria using a fine microneedle. This technique allows precise delivery of DNA into mitochondria.

[0278] Isolation of mitoDRIVE loaded mitochondria

[0279] The isolation and purification of transfected mitochondria involve a series of steps to separate and obtain a purified fraction enriched with mitochondria carrying the transfected mitoDRIVE plasmid DNA. The process typically begins with the collection of transfected cells, followed by gentle cell lysis to release the cellular contents.

[0280] Following cell lysis, the lysate undergoes differential centrifugation to separate different cellular components based on their size, density, and sedimentation properties. This involves a series of centrifugation steps at gradually increasing speeds. The initial low-speed spin removes cellular debris and nuclei, while the subsequent high-speed spins pellet mitochondria along with other organelles.

[0281] To enrich the mitochondrial fraction further, additional purification steps can be employed. Density gradient centrifugation is commonly used to separate mitochondria from other organelles and contaminants. The lysate is layered onto a density gradient medium, and centrifugation is performed to separate the different organelles based on their buoyant densities. This technique allows for the collection of a purified fraction enriched in mitochondria.

[0282] After density gradient centrifugation, the purified mitochondrial fraction can undergo further washing steps to remove residual contaminants and unwanted components. Mitochondria are carefully resuspended in a suitable buffer and subjected to additional centrifugation or filtration steps to improve purity. These washing steps aid in minimizing contamination and ensure the isolation of highly pure transfected mitochondria.

[0283] To optimize purity, additional washing steps can be performed to remove any remaining impurities or cellular debris. The isolated and purified transfected mitochondria can then be characterized by assessing their morphology, functional activity such as expression of transgenes that encode a detectable protein such as GFP or luciferase and confirming the presence of the transfected DNA.

[0284] Microscopic examination allows for the evaluation of mitochondrial morphology, confirming intact and functional organelles. Functional assays, such as measuring mitochondrial respiration or ATP production, provide insights into mitochondrial activity and functionality. Additionally, the presence of the transfected DNA can be verified using techniques such as PCR or fluorescence-based methods specific to the transfected gene or reporter.

[0285] Assessing the expression of transgene by mitochondria using reporter genes

[0286] The ability and the extent of mitoDRIVE plasmid to express transgene of interest within mitochondria can be assessed by using reporter genes such as GFP or Luciferase.

[0287] A mitoDRIVE plasmid comprising either luciferase transgene or eGFP transgene is generated following the protocols described above. The mitoDRIVE plasmid comprising the luciferase transgene (mitoDRIVE-nLuc) or mitoDRIVE plasmid comprising eGFP transgene (mitoDRIVE-eGFP) is transfected to the mitochondria using lipofectamine as described above. The cells containing loaded mitochondria comprising the mitoDRIVE plasmid encoding luciferase or eGFP is cultured. mitoDRIVE-loaded mitochondria can deliver and express functional transgene after transplantation to new cells. mitoDRIVE DNA is loaded into donor cells, the mitochondria are then isolated from these cells and co-incubated with new cells. The mitoDRIVE-loaded mitochondria are imported via natural uptake systems, and the transgene is expressed in the new cells.

[0288] To measure the expression of luciferase, a luminescence assay is often employed. The cells containing mitochondria expressing the luciferase reporter gene are lysed, and a luciferase substrate, such as luciferin, is added to the lysate. Luciferase catalyzes a lightemitting reaction with the luciferin substrate, resulting in the production of luminescence. This luminescent signal can be quantified using a luminometer or a plate reader equipped with a luminescence detection module. The intensity of the luminescent signal correlates with the expression level of the luciferase reporter gene, providing a quantitative measure of gene expression.

[0289] The emission wavelength for the oxidation to D-luciferin to oxyluciferin by firefly luciferase is typically around 560 nanometers (nm). Other types of luciferase enzymes, such as Renilla luciferase and NanoLuc luciferase, oxidize other substrates with different emission characteristics. For example, coelenterazine emits light at 480 nm when oxidized by Renilla luciferase and furimazine emits light at 460 nanometers (nm) after oxidation by NanoLuc luciferase.

[0290] In the case of GFP, measuring expression of transgene typically involves fluorescence-based techniques. Cells comprising mitochondria expressing the GFP transgene can be visualized using a fluorescence microscope equipped with appropriate excitation and emission filters. The GFP fluorophore is excited with specific wavelengths of light, and the resulting green fluorescence emitted by GFP is captured and visualized. The intensity and distribution of the green fluorescence indicate the expression level and localization of the GFP reporter gene. Alternatively, flow cytometry can be employed to quantify GFP expression. Cells expressing GFP are analyzed using a flow cytometer, which detects and measures the fluorescence emitted by GFP. The percentage of GFP-positive cells and the mean fluorescence intensity (MFI) provide quantitative measures of GFP expression.

[0291] Excitation Wavelength: The optimal excitation wavelength for GFP is around 488 nanometers (nm). This means that GFP absorbs light most efficiently when it is excited with light at or near 488 nm.

[0292] Emission Wavelength: The emission peak of GFP occurs around 509 nanometers (nm). This is the wavelength at which GFP emits light after it has been excited by the appropriate excitation wavelength.

[0293] Colocalization Assay

[0294] Colocalization analysis using DAPI dye and mitochondrial staining is a valuable technique in cell biology and microscopy to investigate the spatial relationship between DNA and mitochondria within cells. DAPI (4',6-diamidino-2-phenylindole) is a fluorescent dye that binds specifically to DNA, while mitochondrial staining allows visualization of the mitochondria.

[0295] In this colocalization analysis, cells are typically treated with a mitochondrial dye, such as MitoTracker, to label the mitochondria with a specific fluorescent signal. DAPI is then used to stain the DNA within the cells. The cells are fixed, permeabilized, and subjected to fluorescent microscopy.

[0296] Using appropriate excitation and emission filters, images of the DAPI-labeled DNA and the mitochondrial staining are captured separately. The acquired images are then analyzed using colocalization software or image analysis tools to determine the degree of colocalization between the two signals.

[0297] Colocalization analysis involves quantifying the spatial overlap or correlation between the DAPI and mitochondrial signals within individual cells. This can be achieved by calculating metrics such as Pearson's correlation coefficient, Manders' coefficients, or colocalization coefficients. These measurements provide quantitative information about the degree of colocalization between DNA and mitochondria within cells, indicating the extent to which the two signals occupy the same subcellular space.

[0298] Codon Optimization for improved expression of transgene Codon optimization is a well-known molecular biology technique used to enhance the expression of recombinant genes or transgenes by modifying the nucleotide sequence of the gene to optimize codon usage for the host organism. Codon optimization can also be applied specifically for improving the expression of recombinant genes in mitochondria, which have distinct codon usage patterns compared to the nuclear genome.

[0299] Mitochondria have their own specific codon usage patterns, which differ from those of the nuclear genome. While the exact codon preferences can vary between species, here are some commonly observed codons that are frequently used in mitochondrial genes:

[0300] Leucine (Leu): UUG, UUA, CUU, CUC, CUA, CUG

[0301] Isoleucine (He): AUU, AUC,

[0302] Methionine (Met, initiation codon): AUG, AU A

[0303] Phenylalanine (Phe): UUU, UUC

[0304] Valine (Vai): GUU, GUC, GUA, GUG

[0305] Serine (Ser): UCU, UCC, UCA, UCG, AGU, AGC

[0306] Proline (Pro): CCU, CCC, CCA, CCG

[0307] Threonine (Thr): ACU, ACC, ACA, ACG

[0308] Alanine (Ala): GCU, GCC, GCA, GCG

[0309] Tyrosine (Tyr): UAU, UAC

[0310] Tryptophan (Trp): UGG, UGA

[0311] Cysteine (Cys): UGU, UGC

[0312] Glutamine (Gin): CAA, CAG

[0313] Asparagine (Asn): AAU, AAC

[0314] Histidine (His): CAU, CAC

[0315] These codons represent some of the frequently observed codons in mitochondrial genes, and their usage can vary across different mitochondrial genomes and species. It is important to note that the codon preferences can be influenced by various factors, including the availability of tRNA molecules specific to mitochondria. Therefore, codon optimization for mitochondrial expression involves adapting the recombinant gene sequence to match the specific codon preferences and tRNA availability in the target mitochondria.

[0316] Mitochondria possess unique features in terms of genetic code and stop codon usage compared to the nuclear genome. While the nuclear genetic code utilizes three stop codons (UAA, UAG, and UGA), mitochondria exhibit variations in their codon assignments, particularly for termination signals.

[0317] In most organisms, mitochondria primarily utilize UAA and UAG as stop codons, with UGA being used for tryptophan (Trp) incorporation instead of termination. This phenomenon is known as the "universal genetic code deviation" in mitochondria.

[0318] Additionally, the presence of mitochondrial release factors plays a crucial role in stop codon recognition and translation termination within mitochondria. Mitochondrial release factors are distinct from their nuclear counterparts and have evolved to recognize and interact with the specific stop codons used in mitochondria.

[0319] Furthermore, mitochondrial stop codon usage can differ between nuclear-encoded mitochondrial genes and genes encoded within the mitochondrial genome itself. Nuclear- encoded mitochondrial genes often follow the nuclear genetic code, utilizing UAA, UAG, and UGA as stop codons.

[0320] This process involves replacing codons that are rare or inefficiently recognized by the host's translational machinery with codons that are more frequently used and efficiently translated.

[0321] Codon optimization begins with the analysis of the nucleotide sequence of the gene of interest and the identification of codons that may be suboptimal for the host organism. Factors such as codon usage bias, tRNA availability, and the presence of rare codons are considered in the optimization process. Computational algorithms and bioinformatics tools known in art are commonly used to analyze and design codon-optimized sequences based on the specific host organism's codon usage patterns. To optimize the expression of recombinant genes in mitochondria, the codon usage patterns of the mitochondrial genome and the corresponding tRNA availability are considered.

[0322] Bioinformatics tools and computational algorithms are utilized to analyze the codon usage preferences of mitochondrial genes in the specific organism of interest. Subsequently, the codons in the recombinant gene sequence are modified to match the preferred codons used by the mitochondrial translation machinery. Rare codons or codons that are inefficiently recognized by mitochondrial tRNA are replaced with synonymous codons that are frequently used in mitochondrial genes. The identified suboptimal codons are then replaced with synonymous codons that are more frequently used in the host organism. The selection of synonymous codons takes into account their frequency of occurrence in highly expressed genes of the host organism and the availability of corresponding tRNAs. This process aims to increase the efficiency of translation by providing a more favorable codon usage pattern.

[0323] For instance, tools such as the Codon Usage Database (CUD), the JCat tool, and the OptimumGene™ tool employ algorithms and reference databases to assess codon usage bias and propose optimized codon sequences. These programs analyze factors like codon frequency, tRNA availability, GC content, and mRNA secondary structure to generate codon- optimized sequences that enhance translation efficiency and protein expression levels.

[0324] In addition to codon optimization, other sequence modifications may be incorporated to further enhance gene expression. These modifications can include the removal of mRNA secondary structures or cis-acting regulatory elements that could interfere with translation initiation or elongation. The introduction of Kozak consensus sequences around the translation start site can also enhance translation efficiency.

[0325] For instance, one can enhance the efficiency of translation by providing codons that are more readily recognized by the mitochondrial ribosomes and are compatible with the available mitochondrial tRNA population. Additionally, the removal of mRNA secondary structures or cis-acting regulatory elements that could hinder translation initiation or elongation may also be considered to further optimize gene expression. The codon-optimized gene sequence can then be synthesized or cloned into an appropriate vector for subsequent mitochondrial transfection or transformation.

[0326] Key elements of mitochondrial codon optimization of trans gene i. Transgene protein sequence is converted into DNA sequence using the codons that occur most frequently in mitochondrial DNA ii. GC content of the DNA sequence is verified and adjusted if needed to be comparable to that of mitochondrial DNA (Bauer AP, Leikam D, Krinner S, Notka F, Ludwig C, Langst G, Wagner R. The impact of intragenic CpG content on gene expression. Nucleic Acids Res. 2010 Jul;38(12):3891 -908; Kudla G, Lipinski L, Caffin F, Helwak A, Zylicz M. High guanine and cytosine content increases mRNA levels in mammalian cells. PLoS Biol. 2006 Jun;4(6):el80.) iii. Presence of intragenic mitochondrial transcription factor A (TFAM) sites is verified by screening sequences for G residues separated by 10 nucleotides. (Choi WS, Garcia-Diaz M. A minimal motif for sequence recognition by mitochondrial transcription factor A (TFAM). Nucleic Acids Res. 2022 Jan ll;50(l):322-332.) iv. Codon sequences of the desired transgene is adjusted to best match native mitochondrial DNA GC content and to eliminate intragenic TFAM sites. v. Transgene expression is regulated by a mitochondrial DNA-derived promoter (HSP1, HSP2, LSP1, LSP2) so transgene cannot be expressed by extraneous cytoplasmic promoter. vi. mitoDRIVE transgene start sites can use ATA as initiation codon instead of the ATG used for nuclear DNA-based gene expression. vii. No polyadenylation site is included in mitoDRIVE to ensure transgene expression only in mitochondira. viii. Canonical nuclear “stop” codons can be optionally introduced into the transgene at tryptophan positions during mitochondrial codon optimization. This is achieved by using the nucleotide sequence TGA, which in the mitochondrial genetic code is tryptophan, but in the nuclear DNA code is a stop codon. This feature serves as another control to ensure transgene expression occurs only with mitochondrial machinery.

[0327] Optimization of secretion and targeted export of transgenic protein from mitochondria

[0328] Optimizing the export of recombinant proteins encoded by the transgene in the mitoDRIVE plasmid from mitochondria involves enhancing the efficiency of protein translocation across the mitochondrial membranes. Some non-limiting examples for optimizing the export of recombinant proteins from mitochondria are shown below:

[0329] Signal Peptide Optimization: The addition of a suitable signal peptide at the N- terminus of the recombinant protein can facilitate its targeting and translocation into mitochondria. Optimization of the signal peptide sequence can enhance its recognition and interaction with the mitochondrial import machinery, improving translocation efficiency.

[0330] Targeting Sequences: Mitochondria possess specific targeting sequences that direct proteins to different compartments within the organelle. By incorporating the appropriate targeting sequences into the recombinant protein, efficient and specific localization to the desired mitochondrial sub compartment can be achieved, facilitating export.

[0331] Chaperones and Translocases: Mitochondrial chaperones and translocases play crucial roles in protein translocation. Optimizing the expression levels or activity of these factors can improve the efficiency of protein export. Overexpression of specific chaperones or modulation of translocase levels can enhance the translocation capacity and aid in the export of recombinant proteins.

[0332] Post-Translational Modifications: Some recombinant proteins may require specific post-translational modifications, such as proper folding or maturation, to be efficiently exported from mitochondria. Optimization of these modifications through the introduction of relevant enzymes or co-factors can enhance the export efficiency.

[0333] Co-expression of Helper Proteins: The co-expression of specific helper proteins or factors involved in mitochondrial protein translocation can improve the export of recombinant proteins. These proteins can assist in guiding the recombinant protein through the translocation process, enhancing efficiency and accuracy.

[0334] One can incorporate a specific mitochondrial export signals or sequences at the C- terminus of the recombinant protein encoded by the transgene. These export signals facilitate the recognition and translocation of the protein across the inner mitochondrial membrane. Optimization of the export signal sequence, such as through modifications or the addition of specific amino acid motifs, can enhance the efficiency of translocation.

[0335] One can also optimize the transgene such that the encoded protein is secreted out of the mitochondria. For instance, one can fuse the recombinant protein of interest encoded by the transgene with a signal peptide that directs it to the secretory pathway of the cell. The signal peptide can be derived from a protein that naturally undergoes secretion or can be designed based on known signal peptide motifs. Optimization of the signal peptide sequence, such as by introducing specific amino acid modifications, can enhance its efficiency in guiding the recombinant protein towards extracellular secretion.

[0336] Additionally, the incorporation of specific motifs or domains that aid in translocation across the endoplasmic reticulum (ER) membrane can also facilitate the subsequent secretion of the recombinant protein. Depending on the protein characteristics and intended destination, the recombinant protein can be targeted for secretion via the ER-Golgi pathway or unconventional secretion pathways, such as exosome-mediated release. By employing strategies such as incorporating signal peptides, disrupting native mitochondrial targeting signals, modulating co-factors, and ensuring proper protein folding, one can redirect the secretion of recombinant proteins and achieve higher yields for various biotechnological and therapeutic applications.

[0337] Signal sequences

[0338] The disclosure contemplates the use of various signal sequences known in the art for secretion of protein in prokaryotic and eukaryotic cells in mitoDRIVE to promote the secretion of proteins from mitochondria. A non-limiting example is the N-terminal mitochondrial targeting sequence also known as mitochondrial presequence. Presequences typically consist of 15-70 amino acids and possess a characteristic positively charged N- terminus, followed by a hydrophobic region. Presequences direct the translocation of proteins across the mitochondrial membranes via the translocase of the outer mitochondrial membrane (TOM) and the translocase of the inner mitochondrial membrane (TIM) complexes. Upon translocation, the presequence is cleaved by the mitochondrial processing peptidase (MPP), resulting in the release of the mature protein into the mitochondrial matrix.

[0339] Another example of signal sequence for protein secretion is the mitochondrial signalanchor sequence (MAS). MAS sequences are typically located near the N-terminus of the protein and contain both hydrophobic and positively charged regions. MAS sequences direct the insertion of the protein into the mitochondrial inner membrane, allowing a portion of the protein to reside in the intermembrane space while the rest remains exposed in the cytosol or other cellular compartments.

[0340] Several signal sequences enable the secretion of proteins from cells, targeting them to the extracellular space or specific cellular compartments. Some more non-limiting examples of signal sequences commonly used for protein secretion and contemplated by the disclosure for use in mitoDRIVE plasmid to enable secretion of protein from the transgene include:

[0341] Signal Peptide (SP): The signal peptide is a classic example used for protein secretion. It is typically located at the N-terminus of the protein and directs the nascent polypeptide chain to the endoplasmic reticulum (ER) for further processing and secretion. Examples of signal peptides include the SP sequence from the human growth hormone (hGH) and the immunoglobulin heavy chain (IgH) signal peptide.

[0342] Lipoic Acid Signal Peptide: This signal sequence is involved in the secretion of lipoylated proteins, which are essential for various cellular processes. The lipoic acid signal peptide mediates the attachment of a lipoyl moiety to specific lysine residues and directs the secretion of lipoylated proteins. An example of this signal sequence is found in the E2 subunit of pyruvate dehydrogenase complex.

[0343] Glyco sy Ipho sphatidy lino sitol ( GPI) Anchor Signal Sequence: GPI anchor signal sequences are responsible for attaching proteins to the outer leaflet of the plasma membrane through a glycosylphosphatidylinositol anchor. This signal sequence facilitates the secretion of GPI-anchored proteins, allowing them to be presented on the cell surface. An example is the GPI anchor signal sequence from CD59, a membrane regulatory protein.

[0344] Mitochondrial Targeting Sequence (MTS): While most mitochondrial targeting sequences direct proteins into mitochondria, some can also enable protein secretion. Modified or hybrid mitochondrial targeting sequences have been designed to enable the secretion of proteins. Examples include the MTS from cytochrome c oxidase subunit VIII and the hybrid signal sequence combining elements from mitochondrial and secretory signal sequences.

[0345] Bacterial Sec System Signal Sequence: The bacterial Sec secretion system is utilized for the secretion of proteins across the bacterial inner membrane. Signal sequences recognized by the Sec system can be employed for efficient secretion in bacterial expression systems. Examples include the Sec signal sequences from E. coli outer membrane proteins, such as OmpA or PhoA.

[0346] Fibroblast Growth Factor (FGF) Signal Sequence: The FGF signal sequence is responsible for the secretion of fibroblast growth factors, which play crucial roles in cell proliferation, differentiation, and tissue repair. Examples include the signal sequences from FGF1 and FGF2.

[0347] Interleukin (IL) Signal Sequences: ILs are important cytokines involved in immune responses and inflammation. Signal sequences derived from ILs are used to direct the secretion of recombinant ILs or other proteins of interest. Examples include the signal sequences from IL-2, IL-4, and IL-6. Interleukin-2 (IL-2) Signal Peptide: The signal peptide from interleukin-2, a cytokine involved in immune responses, is employed for the secretion of recombinant IL-2 or other proteins of interest.

[0348] Interleukin-6 (IL-6) Signal Peptide: The signal peptide derived from interleukin- 6, a cytokine involved in immune responses and inflammation, is employed for the secretion of recombinant IL-6 or other proteins of interest.

[0349] Tissue Plasminogen Activator (tPA) Signal Sequence: The tPA signal sequence is employed for the secretion of recombinant tPA, a protein involved in blood clot dissolution. This signal sequence directs the secretion of tPA to facilitate its therapeutic use in managing thrombotic disorders.

[0350] Immunoglobulin (Ig) Signal Sequences: Ig signal sequences are utilized for the secretion of immunoglobulins, also known as antibodies. Signal sequences from various Ig isotypes, such as IgG, IgM, and IgA, enable the secretion of recombinant antibody fragments or full-length antibodies.

[0351] Serine Protease Inhibitor (SERPIN) Signal Sequence: SERPIN signal sequences are employed for the secretion of serine protease inhibitors, which regulate protease activity in various physiological processes. Examples include the signal sequences from al-antitrypsin and al-antichymotrypsin.

[0352] Chitinase Signal Sequence: Chitinases are enzymes involved in the breakdown of chitin, a major component of fungal cell walls and insect exoskeletons. The signal sequences from chitinases are used for the secretion of recombinant chitinases or other chitin-binding proteins.

[0353] Erythropoietin (EPO) Signal Sequence: The EPO signal sequence is utilized for the secretion of recombinant erythropoietin, a hormone involved in the regulation of red blood cell production. This signal sequence enables the efficient secretion of therapeutic EPO.

[0354] Alpha-Fetoprotein (AFP) Signal Peptide: The signal peptide from alpha-fetoprotein, a glycoprotein primarily synthesized in the liver, is used to direct the secretion of recombinant proteins of interest. Insulin Signal Peptide: The signal peptide derived from insulin is utilized for the secretion of recombinant proteins in various expression systems. It efficiently guides the protein of interest through the secretory pathway.

[0355] Tumor Necrosis Factor (TNF) Signal Peptide: The signal peptide derived from tumor necrosis factor, a cytokine involved in immune responses and inflammation, is used for the secretion of recombinant TNF or other proteins.

[0356] Epidermal Growth Factor (EGF) Signal Peptide: The signal peptide from epidermal growth factor, a growth factor involved in cell proliferation and tissue regeneration, is employed for the secretion of recombinant proteins.

[0357] Interferon- Gamma (IFN-Y) Signal Peptide: The signal peptide from interferongamma, a cytokine involved in immune responses, is used for the secretion of recombinant IFN-y or other proteins of interest.

[0358] Transforming Growth Factor-Beta (TGF-0) Signal Peptide: The signal peptide derived from transforming growth factor-beta, a multifunctional cytokine involved in cell growth and differentiation, is employed for the secretion of recombinant TGF-P or other proteins.

[0359] Platelet-Derived Growth Factor (PDGF) Signal Peptide: The signal peptide from platelet-derived growth factor, a growth factor involved in cell proliferation and tissue repair, is used for the secretion of recombinant proteins.

[0360] Granulocyte Colony-Stimulating Factor (G-CSF) Signal Peptide: The signal peptide from granulocyte colony- stimulating factor, a growth factor involved in the production of white blood cells, is utilized for the secretion of recombinant G-CSF or other proteins.

[0361] Growth Hormone Signal Peptide: The signal peptide derived from growth hormone is employed for the secretion of recombinant proteins in various expression systems. It efficiently guides the protein of interest through the secretory pathway.

[0362] Vascular Endothelial Growth Factor (VEGF) Signal Peptide: The signal peptide from vascular endothelial growth factor, a protein involved in angiogenesis and tissue vascularization, is used for the secretion of recombinant VEGF or other proteins. Insulin-Like Growth Factor (IGF) Signal Peptide: The signal peptide derived from insulin-like growth factor is utilized for the secretion of recombinant proteins in various expression systems. It efficiently directs the protein of interest for secretion.

[0363] Hepatocyte Growth Factor (HGF) Signal Peptide: The signal peptide from hepatocyte growth factor, a protein involved in tissue repair and regeneration, is employed for the secretion of recombinant HGF or other proteins.

[0364] Safety and toxicology assays of mitochondrial insertion into human immune cells.

[0365] Safety and toxicology of mitochondrial insertion into human immune cells in vitro, several assays can be employed to assess potential adverse effects. Some commonly used in vitro safety and toxicology assays for evaluating mitochondrial insertion into human immune cells are shown below:

[0366] Viability Assays'. Cell viability assays, such as the MTT (3-(4,5-Dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide) or resazurin assays, can be performed to evaluate the cytotoxicity of mitochondrial insertion. These assays measure cell metabolic activity and determine if the presence of exogenous mitochondria affects cell viability.

[0367] Apoptosis and Cell Death Assays: Annexin V / propidium iodide staining or TUNEL (Terminal deoxynucleotidyl transferase dUTP nick-end labeling) assays can be utilized to assess the induction of apoptosis or cell death in immune cells following mitochondrial insertion. These assays detect DNA fragmentation or phosphatidylserine extemalization, indicating apoptotic processes.

[0368] Reactive Oxygen Species (ROS) Assays: Mitochondrial insertion can potentially impact intracellular redox balance and induce oxidative stress. ROS assays, such as DCFDA (2',7'-Dichlorofluorescein diacetate) or DHE (Dihydroethidium) staining, can be employed to measure the generation of reactive oxygen species in immune cells.

[0369] Mitochondrial Membrane Potential Assays: Changes in mitochondrial membrane potential can signify mitochondrial dysfunction or perturbations due to inserted mitochondria. Assays utilizing JC-1 (5,5',6,6'-Tetrachloro-l,r,3,3'-tetraethylbenzimidazolylcarbocyanine iodide) or TMRM (tetramethylrhodamine methyl ester) can assess alterations in mitochondrial membrane potential. Cytokine Release Assays: Immune cells play a critical role in cytokine production and immune responses. Measurement of cytokine release, such as IL-ip (interleukin- 1 beta), TNF-a (tumor necrosis factor-alpha), or IL-6 (interleukin-6), using ELISA or multiplex assays, can determine if mitochondrial insertion induces excessive inflammatory responses or immune activation.

[0370] Immunophenotyping: Flow cytometry analysis can be utilized to assess changes in immune cell populations and surface marker expression following mitochondrial insertion. Specific immune cell subsets can be analyzed using fluorescently labeled antibodies against markers such as CD3, CD4, CD8, CD14, CD19, etc.

[0371] Functional Assays: Functional assays, such as phagocytosis assays or lymphocyte proliferation assays, can evaluate the impact of mitochondrial insertion on immune cell activities and responses.

[0372] These in vitro safety and toxicology assays provide insights into the potential effects of mitochondrial insertion on human immune cells. By assessing cell viability, apoptosis, oxidative stress, cytokine release, immunophenotyping, and functional activities, one can gain a comprehensive understanding of the safety profile and potential toxicological impacts of mitochondrial insertion into immune cells.

[0373] Disease Treatment

[0374] The disclosure contemplates delivering one or more genetically modified mitochondria, wherein a genetically modified mitochondrion contains a transgene encoding a therapeutic protein to a mammal and expressing the therapeutic transgene in the mitochondria such that the encoded protein is secreted from the one or more delivered genetically modified mitochondria, the secreted protein having a therapeutic effect on the mammal. The disclosure sets forth a mitoDRIVE plasmid containing a transgene encoding a therapeutic protein, which is transfected to the mitochondria. The genetically modified mitochondria therefore are a means for delivering a therapeutic agent to a mammal.

[0375] Many diseases are caused by a genetic deficiency whereby a protein encoded by a gene is deficient in an organism. Genetic deficiencies may be caused by one or more mutations in a single gene which results in nonfunctional protein encoded by the defective gene, by a deficiency in regulation of a gene, at the level of transcription or translation, resulting in a deficit in the amount of a protein in an organism, and / or by a deficit in the protein’s activity, for example, as a result of shorter half-life. The disclosure treats such diseases by providing a mitoDRIVE plasmid that contains the transgene of interest in order to replace or supplement the genetic deficiency. For example, a transgene may encode a functional protein lacking a disease-causing mutation which is then transfected to a mitochondrion, and upon administration to an organism is then carried in a cell. One or more genetically modified mitochondria (containing a mitoDRIVE plasmid) is administered to an organism. A mitochondrion containing one or more mitoDRIVE plasmids is carried in one or more cells of the organism, where the mitochondrion expresses the protein of interest, thereby providing the deficient protein to the organism, and thereby treating a disease.

[0376] Diseases caused by a genetic mutation(s) are well-known in the art. The disclosure contemplates treating a such a disease by providing a non-defective gene of interest and expressing the non-defective gene in a mammal (an organism) by delivering one or more mitochondria containing a mitoDRIVE plasmid.

[0377] Expression of multiple transgenes from a single mitoDRIVE plasmid

[0378] The disclosure also contemplates expressing two or more transgenes from the mitochondria comprising a mitoDRIVE plasmid. The transgenes can be expressed sequentially or simultaneously if desired.

[0379] Bicistronic Expression

[0380] Mitochondrial bicistronic expression typically involves the use of internal ribosome entry sites (IRES) or the incorporation of 2A peptides. Figure 12A shows a schematic of a mitoDRIVE construct capable of bicistronic expression.

[0381] Internal Ribosome Entry Sites (IRES): Similar to cytoplasmic bicistronic expression, an IRES element can be incorporated into the mitochondrial mRNA to allow ribosomes to initiate translation at a downstream coding sequence independently of the upstream one. This enables the simultaneous translation of two or more proteins from a single mitochondrial mRNA molecule.

[0382] 2A Peptides: In mitochondria, 2A peptides derived from viruses can be utilized to achieve bicistronic expression. These 2A peptides are engineered to facilitate a ribosome skipping event, resulting in the independent translation of multiple proteins. The use of 2A peptides allows for the production of distinct proteins from a single mitochondrial mRNA.

[0383] Bicistronic expression in mitochondria offers several advantages, including the coordinated production of multiple therapeutic proteins within the mitochondria, the investigation of gene interactions, and the study of complex mitochondrial processes and functions.

[0384] Dual Promoter Expression

[0385] The disclosure also contemplates he expression of two transgenes using separate promoters in mitochondria using a single mitoDRIVE plasmid construct. Figure 12B shows a schematic of a mitoDRIVE construct capable of expressing two transgenes using two separate mitochondrial promoters.

[0386] The expression of two transgenes using separate promoters in mitochondria can be achieved by designing a mitoDRIVE plasmid construct that incorporates multiple expression cassettes, each containing a distinct promoter and coding sequence for the transgene of interest.

[0387] Assessing the carrying capacity of a mitoDRIVE plasmid.

[0388] Assessing the carrying capacity of a mitoDRIVE plasmid involves determining the maximum size of DNA that the plasmid can stably accommodate. The carrying capacity depends on several factors, including the size of the plasmid, the replication origin, and the host organism. One can use stuff er DNA of varying sizes ranging from 2Kb to 16Kb to determine the maximum size of a transgene that can be accommodated by the mitoDRIVE plasmid. One can perform one or more of the following methods to determine the carrying capacity of the mitoDRIVE plasmid.

[0389] Stability Studies'. Stability studies provide insights into the ability of the mitoDRIVE plasmid to be maintained within the host cells. This can be assessed by monitoring plasmid retention rates through cell passaging or subculturing experiments. Comparing the stability of plasmids with different sizes allows researchers to determine if larger plasmids are more prone to loss or instability. Copy Number Analysis'. Analyzing mitoDRIVE plasmid copy number within host cells provides information about the carrying capacity. Techniques such as quantitative PCR (qPCR) or plasmid-specific probes can be used to quantify the number of plasmid copies per cell. Comparing the copy numbers of plasmids with varying sizes can help identify any changes associated with plasmid size.

[0390] Gene Expression Analysis'. Expression analysis is also crucial in assessing carrying capacity. The introduction of larger DNA inserts may impact gene expression levels. Evaluating the expression levels of genes carried by the mitoDRIVE plasmid can indicate if larger plasmids affect transcription and translation efficiency.

[0391] Generally, mitoDRIVE plasmid comprises a transgene having size ranging from 0.5 kb to 20 kb. In some embodiments, mitoDRIVE plasmid comprises a transgene having size greater than 20kb but less than 100 Kb. In some embodiments, mitoDRIVE plasmid comprises a transgene having size greater than 0.1 kb but less than 0.5 Kb.

[0392] In some embodiments, the mitoDRIVE plasmid comprises a plurality of transgenes, wherein each transgene has a size ranging from 0.5 kb to 20 Kb. The size of the transgene that is compatible with mitoDRIVE plasmid can be determined by following the protocols taught below in Example 18.

[0393] Briefly stuffer fragments are used to generate mitoDRIVE plasmids containing transgene sizes ranging from 0.5 kb to 20 kb. The mitoDRIVE constructs are then loaded onto mitochondria by transfection and the loaded mitochondria are exposed to cells which then take up the loaded mitochondria and express the transgene.

[0394] The in vitro luminescent expression assays can be used to quantify luciferase expression with a multimode plate reader. Loss of luciferase activity (return to background levels observed for cells transfected with a control plasmid lacking the luciferase reporter) at a given size of stuffer DNA would indicate the upper limit of carrying capacity.

[0395] In some embodiments, the mitoDRIVE plasmid comprises a transgene having a size ranging from 0.5 kb, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 16 kb, 17 kb, 18 kb, 19 kb or 20 kb. Dosage

[0396] Administering mitochondria as a therapeutic intervention involves delivering exogenous mitochondria to target cells or tissues to restore or enhance mitochondrial function. The dosage amount of mitochondria to be administered depends on several factors, including the specific therapeutic objective, target tissue, and the delivery method employed.

[0397] Actual dosage of transgenic mitochondria containing the active ingredient (e.g., protein expressed by the transgene of interest) may be varied so as to obtain a sufficient level or therapeutic amount of transgenic protein that is effective to achieve the desired response for a particular patient, disease state, and mode of administration, without being toxic to the patient. A medical doctor, e.g., physician, having ordinary skill in the art may readily determine and prescribe the effective amount of the transgenic mitochondrial compositions as required. For example, the physician or veterinarian could start doses of transgenic mitochondria disclosed herein employed in the at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0398] In some embodiments, the novel mitochondrial compositions disclosed herein are administered to the patient in dosages such that upon administration of the transgenic mitochondria, the transgenic protein of interest is expressed in desired amounts. In some embodiments, the transgenic mitochondrion is administered in sufficient amounts such that it generates expression of transgenic protein resulting in concentration of Img / kg to 20 mg / kg. In some embodiments, the transgenic mitochondrion is administered in sufficient amounts such that it generates expression of transgenic protein resulting in concentration of 1 mg / kg, 2mg / kg, 3mg / kg, 4mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg or 20 mg / kg.

[0399] In some embodiments, the transgenic mitochondrion is administered in sufficient amounts such that it generates expression of transgenic protein resulting in concentration of 5-100 pg per ml of cell culture. In some embodiments, the transgenic mitochondrion is administered in sufficient amounts such that it generates expression of transgenic protein resulting in concentration of 5-25 pg per ml of cell culture. In some embodiments, the transgenic mitochondrion is administered in sufficient amounts such that it generates expression of transgenic protein resulting in concentration of 5 pg / ml , 10 pg / ml, 15 pg / ml, 20 pg / ml, 25 pg / ml , 30 pg / ml, 35 pg / ml, 40 pg / ml, 45 pg / ml, 50 pg / ml, 55 pg / ml, 60 pg / ml, 65 pg / ml, 70 pg / ml, 75 pg / ml, 80 pg / ml, 85 pg / ml, 90 pg / ml, 95 pg / ml and 100 pg / ml of cell culture.

[0400] In some embodiments, the compositions disclosed herein are administered to the patient based on the titer of the mitoDRIVE plasmid which is determined based on the copy number.

[0401] Routes of Administration

[0402] Routes of administration of any of the compositions of the disclosure include inhalational, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal, and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.

[0403] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. The formulations and compositions that would be useful in the present disclosure are not limited to the particular formulations and compositions that are described herein.

[0404] Parenteral administration of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intravenous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic infusion techniques. Animal model

[0405] Mouse models have been instrumental in the study of single gene defect diseases, providing valuable insights into disease mechanisms and potential therapeutic interventions. For instance, mouse models have been extensively used to study diseases caused by single gene defects, such as cystic fibrosis, hemophilia, Duchenne muscular dystrophy, Huntington's disease, and many others. The disclosure contemplates the use of mouse models for diseases caused by gene mutations.

[0406] Briefly, a mitoDRIVE plasmid comprising a normal copy of a gene (for example DMD gene or factor IX gene) whose mutation causes the disease state is devised using the protocols mentioned above. The mitoDRIVE plasmid containing the transgene of interest (for example DMD gene or factor IX gene or CFTR gene) that expresses a functional protein that can treat the disease state is produced in large scale by using host cells in vitro.

[0407] The mitoDRIVE plasmid is then purified and transfected to a mitochondria. The loaded mitochondria are produced in large amounts by cell culture of host cells and purified. The purified mitochondria loaded with mitoDRIVE plasmid comprising the transgene expressing the functional protein is then administered to a specific mouse model (for example mdx mouse (DMD) or FIX mouse (Hemophilia B) or CFTR Knockout mice (Cystic fibrosis). A null mitochondria containing mitoDRIVE without the transgene is administered to a group of control mice. Therapeutic effects of the mitoDRIVE plasmid comprising the transgene can be observed by examining the test mice and control mice. The test mice receiving mitochondria loaded with mitoDRIVE comprising the transgene of interest is expected to show improved health when compared with that of control mice receiving null mitochondria with mitoDRIVE having no transgene.

[0408] Mitochondrial Transplant

[0409] Mitochondrial transplantation is a process by which exogenous mitochondria loaded with mitoDRIVE comprising a transgene is introduced into cells or tissues or animals to enable expression of transgene thereby treating genetic diseases and / or mitochondrial dysfunction.

[0410] The process of mitochondrial transplantation involves isolating mitochondria from a donor source, which can be from the same species or a different species. In some instances, the mitochondria is isolated , manipulated and administered back to the same individual (autogenic mitochondrial transplant). The isolated mitochondria are manipulated ex vivo such as introducing mitoDRIVE plasmid comprising transgene and are then introduced into the target cells or tissues of the recipient. Delivery methods for mitochondrial transplantation can include direct injection, systemic infusion, or localized application, depending on the desired target and experimental setup.

[0411] The transplanted mitochondria can integrate with the recipient cells and contribute to their energy production and overall mitochondrial function. They may fuse with resident mitochondria, leading to a mixed population of donor and recipient mitochondria in the recipient cells.

[0412] EXAMPLES

[0413] The present disclosure is further exemplified by the following examples. The examples are for illustrative purpose only and are not intended, nor should they be construed as limiting the disclosure in any manner.

[0414] Generally, a mitoDRIVE plasmid, mitoDRIVE plasmid DNA, or mitoDRIVE minicircle DNA is prepared using a combination of standard molecular biology approaches. The mitoDRIVE DNA is then loaded into mitochondria by transfection of a permissive cell lines. Mitochondria are then purified from these cells and are then used as gene therapy vectors.

[0415] Example 1 - Production of mitoDRIVE minicircle DNA

[0416] In order to make the mitoDRIVE minicircle, the full mitoDRIVE plasmid was inserted into plasmid pMC.BESPX-MCS2 using any variety of subcloning techniques such as restriction digestion and ligation or Gibson assembly. Alternatively, minicircle can be generated following the protocols disclosed in Minicircle Biopharmaceuticals-An Overview of Purification Strategies, Front. Chem. Eng., 13 January 2021.

[0417] The mitoDRIVE plasmid includes cassette includes the D-loop, the promoter, the gene and signal sequence, termination sequences, and the origins of replication . Minicircle DNA preparation was done by transformation of ZYCY10P3S2T with the mitoDRIVE containing pMC.BESPX-MCS2. Cells were then selected with antibiotic resistance. Cells were then grown with commercially available broth and then induced with arabinose to cleave the mitoDRIVE construct from the parental plasmid.

[0418] This process produced two plasmids, one with mitoDRIVE and the other is the bacterial backbone. The backbone plasmid was then targeted by an induced restriction enzyme, such as the homing endonuclease I-Scel, which linearizes and degrades the backbone, leaving just the mitoDRIVE product. The mitoDRIVE product was then isolated and purified. The process of excising mitoDRIVE plasmid from the mitoDRIVE cassette was accomplished by alternate plasmid trimming systems that are known in art besides minicircle DNA. A schematic construct of mitoDRIVE plasmid with eGFP transgene is shown in Figure 2 (also in Figure 3A) and mitoDRIVE plasmid with luciferase transgene is shown in Figure 3B. (Dre recombinase, like Cre, is a highly efficient site-specific recombinase in E. coli, mammalian cells and mice, Dis Model Meeh (2009) 2 (9-10): 508-515)

[0419] Example 2 - Delivery of mitoDRIVE DNA to mitochondria mitoDRIVE plasmids or minicircle DNA was delivered to mitochondria using transfection. Predominantly supercoiled plasmid DNA, containing the mitochondrially encoded transgene of interest, was complexed with Lipofectamine 3000 according to manufacturer recommendations. 2 pg of DNA was added to each well in a 24- well plate seeded with 150,000 cells. Cells were incubated with the DNA / lipofectamine for 3 days and then 50,000 cells were taken for analysis by reporter assay. For firefly luciferase reporter assays, BrightGlo reagent (Promega) was used, and for experiments involving nanoLuc containing mitoDRIVE plasmid, Nanoluc substrate (Promega) was used to determine the amount of luciferase being expressed upon transfection.

[0420] Luminescence readings were taken on a Perkin Elmer nivo instrument. An empty vector (Lipofectamine alone) was used as a negative control for the experiment. In addition to transfection, mitoDRIVE plasmids or minicircle DNA can be delivered to mitochondria using electroporation, nucleofection, and passive DNA uptake (import buffer).

[0421] Example 3 - Isolation of mitoDRIVE loaded mitochondria

[0422] Predominantly supercoiled plasmid DNA, containing the mitochondrially encoded transgene of interest, was complexed with Lipofectamine 3000 according to manufacturer recommendations. For mitochondrial repass, 5 ug of DNA in a T25 flask containing 60-70% confluent 293t cells was used. Cells were incubated with the reagent for 3 days, at which point the mitochondria were isolated from the cells using a commercially available kit (abeam, ab288084) using the douncing method. Cells were dounced for 3 times in an isolation buffer. Homogenate was spun down at 600 xg for 10 minutes and the supernatant was collected. (Dounce, A. L. (1955). "A Method for Isolating Intact Mitochondria and Nuclei from the Same Homogenate, and the Influence of Mitochondrial Destruction on the Properties of Cell Nuclei”. The Journal of Cell Biology. 1 (2): 139-153)

[0423] The mitochondrial fraction was spun down from this fraction at 7000xg for 10 minutes. The resulting pellet was washed with isolation buffer and then pelleted again at 7000xg for 10 minutes. The pellet was resuspended in storage buffer for further analysis and for repass. The mitochondria were quantitated by protein amount using the Quick Start Bradford Protein Assay Kit (Biorad, #5000201). After protein determination, an aliquot of mitochondria (20 ug) was assessed for the presence of reporter protein (firefly or nanoluc) and processed according to those reporter assays. Mitochondria were then placed onto recipient cells (20 pg of DNA for 50,000 cells). The cells were incubated for up to 14 days and then 50,000 cells were taken for analysis via reporter assay to determine the presence of functional amounts of plasmid containing the mitochondrially encoded transgene.

[0424] Example 4 - Scale-up of mitoDRIVE loaded mitochondria

[0425] In vivo use of the mitoDRIVE technology for gene therapy requires scalable sources of mitoDRIVE-loaded mitochondria. To enrich for cells containing mitoDRIVE-loaded mitochondria, hygromycin B selection is utilized. Hygromycin is a protein translation inhibitor was used to apply selection pressure in eukaryotic cell culture. Resistance to hygromycin B is conferred by the resistance gene (HygR ). The product of this gene (hygromycin B-phosphotransferase, HBP) is targeted to the mitochondria by fusion with a targeting peptide (Yoon YG, Koob MD. 2008. Selection by drug resistance proteins located in the mitochondria of mammalian cells. Mitochondrion 8:345-51). The mitochondria targeted HBP confers resistance to hygromycin B selection (Yoon et al.).

[0426] Mitochondria from cells expressing mitochondria targeted HBP is isolated and transfected with desired mitoDRIVE plasmid. The transfected mitochondria is then transferred to new host cells and hygromycin is applied to select (enrich) cells that incorporated the donated mitochondria. In some instances, the purification of cell lines containing mitoDRIVE- loaded mitochondria is generated by allowing the cells to grow and passaged. The cells are then plated at 1 cell per well and allow for clonal expansion after hygromycin selection. Clones are screened for the presence and copy number of the mitoDRIVE encoded transgene using quantitative real-time PCR. The highest copy number clones were selected and further expanded until a pure high mitoDRIVE copy number cell line was isolated. These cells are then used as a scalable source of transgene-expressing mitochondria for preclinical in vivo studies.

[0427] The expansion of mitoDRIVE-loaded cell clones is dependent on persistent replication of the mitoDRIVE plasmid in host mitochondria. The mitoDRIVE plasmid comprises known native mt-DNA elements needed for replication and maintenance of the plasmid during mitogenesis. Optionally, if needed the mitoDRIVE plasmid can be engineered to iteratively add back regions of native mt-DNA into the mitoDRIVE plasmid to identify any additional regions needed for mitochondrial DNA replication to further improve persistent levels of mitoDRIVE-loaded mitochondria in clonal cells.

[0428] Example 5 - Mitochondria-based gene expression using mitoDRIVE platform mitoDRIVE plasmids encoding two different reporter transgenes -eGFP and nanoluciferase (nLuc) were generated following the protocols described in Examples 1-4. (See Figures 2 and 3).

[0429] To ensure transgene expression was mitochondria-restricted, the mitochondria ATA start codon, and TGA codon for tryptophan was utilized to generate an in-frame nuclear DNA stop that would result in truncated non-functional protein if translated by cytoplasmic ribosomes. ATA encoding for methionine was used and that would result in isoleucine mutations if translated by cytoplasmic ribosomes. Collectively, this approach when combined with the use of unique mitochondria transcriptional promoters, ensures that mitoDRIVE can only be expressed by mitochondrial transcription and translation systems.

[0430] Transfection of mitoDRIVE-fLuc into the transfection-permissive 293T human cell line resulted in the exhibition of robust luciferase activity 48 hours post transfection (See Fig. 4A). The mitoDRIVE-encoded fLuc to include a C-terminal FLAG®-tag so that protein expression could be confirmed using an anti-FLAG antibody. Immunofluorescent staining was performed using a FITC-conjugated anti-FLAG antibody (See Fig. 4B) and observed strong staining only in mitoDRIVE-transfected cells. FITC staining was denser in perinuclear regions, consistent with observations of mitochondrial networks.

[0431] Example 6 - Assessing and optimizing the persistence of transgene expression mitoDRIVE plasmids encoding two different reporter transgenes -eGFP and nanoluciferase (nLuc) were generated following the protocols described in Examples 1-4. (See Figures 2 and 3)

[0432] Expression of these transgenes (eGFP or nLuc) using mitoDRIVE plasmids can be monitored by fluorescence microscopy or luminescence detection, respectively. The mitoDRIVE plasmids containing eGFP (mitoDRIVE-eGFP) or nLuc (mitoDRIVE-nEuc) were loaded onto the mitochondria by following the mitochondria-transfection protocol of Examples 5-6.The loaded mitochondria containing mitoDRIVE-eGFP or mitoDRIVE-nEuc are then introduced into common cell lines representing different types of cells such as fibroblasts, epithelial cells, and immune cells. Transgene expression is monitored over time by both detection of the gene products and with reverse-transcription quantitative real-time PCR (RT-qPCR) detection and quantification of transgene RNA within the mitochondrial RNA pool. Even in different cell lines (293T, HeLa, ARPE) and high level of luciferase activity was observed indicating that mitoDRIVE loaded mitochondria are successful in expressing the transgene in all cell types. (See Figure 6).

[0433] FIG. 6A schematically illustrates the ability of mitoDRIVE plasmid to transfect mitochondria. Fig. 6B illustrates that the transfected mitochondria containing the mitoDRIVE are successfully taken up by cells of different types such as 293T cells, HeLa cells and arising retinal pigment epithelia (ARPE) cells. The cells containing mitochondria having the mitoDRIVE plasmid are able to express the transgene protein (luciferase).

[0434] The following experiment also demonstrates that mitoDRIVE-loaded mitochondria can be transplanted into new host cells and maintain transgene expression. 293T cells were first transfected with mitoDRIVE-fLuc and observed strong luciferase activity (Fig. 6). The loaded mitochondria containing mitoDRIVE-fLuc was isolated from these donor cells and transplanted (by co -incubation) donor mitochondria into new recipient host cells (Fig. 6). As shown in Fig. 6, strong luciferase activity was observed even 3 days later. The results thus show that loaded mitochondria can be isolated from donor cells and then transplanted into recipient cells, the donor mitochondria are able to express transgenes in the recipient cells as well.

[0435] Because the mitoDRIVE plasmid has been engineered with the native mitochondrial DNA elements needed for replication and maintenance of the plasmid during mitogenesis, qPCR can be used to track the persistence of mitoDRIVE plasmid DNA within the mt-DNA pool. The cells containing allogenic mitochondria which contain the mitoDRIVE plasmid are grown, passaged and cultured. Samples of cell culture are taken every day and analyzed to see if the expression of the transgene is consistent over a period of time. (See Figure 9).

[0436] FIG. 9A illustrates a schematic construct of the mitoDRIVE plasmid. FIG. 9B illustrates that the mitochondrial in vivo expression of transgenes is persistent, and a steady state concentration of luciferase protein is observed for over 12 days. The levels of the expressed protein remain consistent even after the cell culture is split once the culture becomes confluent.

[0437] Example 7 - Assessment of localization of mitochondrial-expressed trans gene products into other cellular compartments

[0438] To determine whether the eGFP protein produced in the mitochondria is getting localized in other cellular compartments, mitoDRIVE-eGFP or mitoDRIVE-nLuc plasmid was used to evaluate the movement of mitochondria-expressed transgenes in vitro. eGFP protein localization was studied by fluorescence microscopy using the native eGFP signal, and after staining with PE-conjugated anti-eGFP antibody. By using specific dye stains, the regions in which the expressed transgenic protein from the mitochondria gets accumulated was evaluated.

[0439] Established protocols known in the art (Chazotte B. Labeling mitochondria with MitoTracker dyes. Cold Spring Harb Protoc. 2011 Aug l;2011(8):990-2. ) to were used to evaluate co-localization with other subcellular structures such as the nucleus (DAPI staining), mitochondria (MitoTracker dye), lysosomes (LysoTracker dye), and the endoplasmic reticulum (ER-Tracker dye). Microscopy was performed at multiple time intervals post mitochondrial transfer to assess protein localization over time. Nano-Gio luciferase assay and mitoDRIVE-nLuc plasmid was used to quantify transgene-encoded protein expression (luminescence units) in cell culture supernatant, within whole cells, and within different cytoplasmic fractions after differential centrifugation. (See Figure 5A-E). Results show that the expression of transgene from the cells containing allogenic mitochondria exhibits eGFP accumulation in both dense, spherical, punctate formations within cells, and more diffusely across the cellular cytoplasm.

[0440] Example 8 - Assessment of secretion of mitochondrial-expressed trans gene products from the cell

[0441] Expression of transgene from the cells containing allogenic mitochondria shows mitoDRIVE-eGFP accumulation in both dense, spherical, punctate formations within cells, and more diffusely across the cellular cytoplasm. (See Figure 5). FIG. 5 illustrates immunofluorescent microscopy of 293T cells 4-days after loading with control and mitoDRIVE-eGFP transfected mitochondria. Fig. 5(A) illustrates that no florescence in the gfp channel was observed in control cells, but punctate eGFP signal was observed in cells transfected with mitoDRIVE-eGFP plasmid (See Fig 5B). To confirm this result, cells were stained with PE-anti-eGFP antibody. Fig. 5(C) illustrates diffuse, non-specific background PE staining was observed in control cells while bright and more punctate staining was observed in mitoDRIVE-eGFP cells (See Fig. 5D). Fig. 5(E) is an enlarged field of view of the punctate staining observed in mitoDRIVE-eGFP cells which indicates that much of the eGFP was localized in spherical structures consistent with mitochondria, with some accumulation of protein outside of these structures as well. This data suggests that eGFP is present not only within the mitochondria where it was produced but has also been shuttled into the cytoplasm.

[0442] Given the fact that the mitoDRIVE plasmid by itself cannot express the transgenic protein in the cytoplasm because the mitoDRIVE plasmid lacks a cytoplasmic gene expression promoter. Furthermore, the conversion to mitochondrial anticodon usage introduces a canonical cytoplasmic gene expression stop codon. Therefore, the diffuse fluorescence across the cellular cytoplasm must be the eGFP protein produced in the mitochondria using the mitoDRIVE plasmid that has gotten shuttled into the cytoplasm.

[0443] To confirm the secretion of eGFP protein produced in the mitochondria and secreted into the cytoplasm, mitoDRIVE-eGFP or mitoDRIVE-nLuc plasmids are used to evaluate the movement of mitochondria-expressed transgenes in vitro, and to evaluate approaches to modify expression levels and subcellular movement of transgene products. mitoDRIVE-nLuc plasmid and mitoDRIVE-sec-nLuc plasmid were generated following the protocols outlined in Examples 1-4. mitoDRIVE-sec-nLuc plasmid is similar to mitoDRIVE-Luc plasmid but contains a secretory signal fused to the transgene. Various secretory signals are known in the art, as a non-limiting example, IL-6 secretion signal peptide was used. A schematic of generated constructs mitoDRIVE-nLuc and mitoDRIVE- sec-nLuc are shown in Figures 7 A and 7B. The plasmids were then loaded onto mitochondria following the protocols of Examples 5 and 6.

[0444] The loaded mitochondria containing either the mitoDRIVE-nLuc or mitoDRIVE-sec- nLuc was introduced into human 293 T cells. Mitochondria loaded with null vector (base) was used as a base-level control. The amount of luciferase protein generated by each mitoDRIVE construct in cells was measured and the same measurements were done in the supernatant. Results show that the mitoDRIVE construct containing the secretion signal (mitoDRIVE-sec-nLuc) exhibited higher levels of luciferase protein in the supernatant confirming that mitoDRIVE plasmid is able to express the protein in mitochondria and export it out into cytoplasm of the cell containing said mitochondria. (See Figure 7C). The same experiment was repeated with mouse Neuro 2A cells. The results also show that mitoDRIVE construct containing the secretion signal (mitoDRIVE-sec-nLuc) exhibited higher levels of luciferase protein in the supernatant of mouse neuro 2A cells (See Figure 7D).

[0445] Example 9 - Optimization of expression of transgene by codon optimization.

[0446] The following experiment aims to perform codon and promoter optimization to improve transgene expression. The process of codon optimization involves the use of alternative start codons, incorporating nuclear & cytoplasmic stop codons, removing negative regulatory elements and optimizing frequency of natural codon usage. The genetic code used by mitochondria has several differences from the standard genetic code used by eukaryotes to express gene products encoded within nuclear DNA as shown below. Codon optimization of mitoDRIVE plasmid with mitochondrion specific codon usage and higher frequency of natural codon usage results in increased in the expression of transgene of interest. (See Figure 10).

[0447] Example 10 - Optimization of targeted export of expressed transgenic protein from mitochondria.

[0448] The transgenic protein produced by mitoDRIVE plasmid inside a mitochondria can be more efficiently exported out of the mitochondria by using cell penetrating peptides or homeoprotein internalization- secretion domains which enable efficient export of protein out of mitochondria.

[0449] MitoDRIVE plasmids containing transgene fused with cell penetrating peptides and / or homeoprotein internalization- secretion domains are made following the protocols outlined in examples 1-4. Heterologous protein fused with cell penetrating peptide (Derakhshankhah H, Jafari S. 2018. Cell penetrating peptides: A concise review with emphasis on biomedical applications. Biomed Pharmacother 108: 1090-1096.) or homeoprotein internalization- secretion domains (Spatazza J, Di Lullo E, Joliot A, Dupont E, Moya KL, Prochiantz A. 2013. Homeoprotein signaling in development, health, and disease: a shaking of dogmas offers challenges and promises from bench to bed. Pharmacol Rev 65:90-104) encoded by transgene in the mitochondria is therefore expected to improve protein export from the mitochondria.

[0450] The same experiments are repeated with mitoDRIVE plasmids containing transgene fused with signal sequences for cell secretion or retention in the ER (Owji H, Nezafat N, Negahdaripour M, Hajiebrahimi A, Ghasemi Y. 2018. A comprehensive review of signal peptides: Structure, roles, and applications. Eur J Cell Biol 97:422-441; Stomaiuolo M, Lotti LV, Borgese N, Torrisi MR, Mottola G, Martire G, Bonatti S. 2003. KDEL and KKXX retrieval signals appended to the same reporter protein determine different trafficking between endoplasmic reticulum, intermediate compartment, and Golgi complex. Mol Biol Cell 14:889-902). Heterologous protein fused with signal sequences for cell secretion or retention encoded by transgene in the mitochondria is expected to improve protein secretion and export from the mitochondria. Example 11 - In vitro safety and toxicology assays of mitochondrial insertion into human immune cells.

[0451] A major drawback of AAV gene therapy is that AAV administration induces undesirable immune responses that can cause adverse safety events and / or reduce the efficacy of transgene delivery (Wang L, Zoppe M, Hackeng TM, Griffin JH, Lee KF, Verma IM. 1997. A factor IX-deficient mouse model for hemophilia B gene therapy. Proc Natl Acad Sci U SA 94:11563-6). This is especially problematic for repeated administrations. Mitochondrial gene therapy using mitoDRIVE plasmids carrying transgene eliminates this hurdle since results show that administration of allogenic mitochondria under in vitro conditions do not trigger immune response.

[0452] Mitochondria loaded with mitoDRIVE plasmids were exposed to human immune cells. Inflammatory markers such as reactive oxygen species (ROS), TNFa and IL-6 accumulation were measured after exposing the immune cells with allogenic mitochondria. Lipopolysaccharides (LPS) and n-Formyl-peptides (NFP) were used as positive controls for immune response. Immune cells not exposed to a foreign agent (unstimulated immune cells) were used as negative control. Results show that uptake of allogenic mitochondria (MITO) does not induce an innate immune response as observed by a lack of A) reactive oxygen species (ROS) production from primary blood leukocytes, and absence of B) TNFa or C) IL- 6 accumulation in THP macrophage or monocyte culture supernatant after 24-hour stimulation. (See Figure 8). It was observed that allogenic mitochondria containing mitoDRIVE plasmid do not induce inflammatory innate immune responses in human immune cells. Mitochondria (MITO) do not induce an innate immune response as observed by a lack of Fig. 8(A) reactive oxygen species (ROS) production from primary blood leukocytes, and absence of Fig. 8(B) TNFa and Fig. 8(C) IL-6 accumulation in THP macrophage or monocyte culture supernatant after 24-hour stimulation. NEG=unstimulated, Positive controls are LPS, and n-Formyl-peptides (NFP).

[0453] No immune response was observed with unstimulated human immune cells whereas severe immune response was observed when immune cells were exposed to LPS or NFP. The results show that mitochondrial gene therapy does not elicit immune reaction unlike conventional gene therapy involving viral vectors. Example 12 - In vivo Safety and Toxicological assays.

[0454] Mouse models were used to generate in vivo safety and anti-drug responses, distribution and persistence of transgene expression from mitoDRIVE-transfected mitochondria, and the functionality of a therapeutic transgene expressed from mitoDRIVE- transfected mitochondria in a clinically relevant mouse model.

[0455] The following experiment aims to assess overall health, inflammation, and the generation of anti-drug antibodies after repeated administration of mitoDRIVE-transfected mitochondrial in a mouse model. MitoDRIVE-Luc (firefly luciferase) loaded mitochondria is introduced into BALB / c mice (Jackson Labs) via tail vein injection.

[0456] Safety Studies

[0457] First, a short-term in vivo experiment focused on the first 48 hours post mitochondria administration was performed to look for evidence of inflammation or allergic response. For these experiments, the mitoDRIVE-fLuc plasmid was modified to encode native mouse mitochondria sequences for use in mouse cells. This plasmid was then transfected into mouse Neuro-2a cells. Similar to human 293T cells, murine Neuro-2a cells are highly transfection permissive, which ensures maximal mitoDRIVE-loading of mitochondria. The sterile mitoDRIVE-loaded mitochondria was isolated 2 days later using protocols described in prior examples above. The isolated mitochondria was then resuspended in sterile PBS lacking Ca2+and Mg2+and introduced into the circulation of BALB / c mice via the IV route (tail vein) using a dose titration of -0.25 to 1.0 mg / kg body weight (6.25 pg, 12.5 pg and 25 pg) (n=6 mice) as previously described (Shi X, Zhao M, Fu C, Fu A. 2017. Intravenous administration of mitochondria for treating experimental Parkinson's disease. Mitochondrion 34:91-100 ).

[0458] The same experiment was repeated with a control group (n=3 mice) that receives volume-matched buffered saline solutions only. Peripheral blood samples were collected prior to mitochondria introduction and 24 hours post introduction via the submandibular or submental vein.

[0459] Observations of overall health including activity level / response to touch, and body weight were monitored and documented each day. Body temperature was measured at 0 hr, 6, 24, and 48 hours post mitochondria administration using non-contact infrared thermometry of the perianal region as described (Mei J, Riedel N, Grittner U, Endres M, Banneke S, Emmrich JV. 2018. Body temperature measurement in mice during acute illness: implantable temperature transponder versus surface infrared thermometry. Sci Rep 8:3526.). Mice were euthanized at 48 hours post mitochondria administration for the collection of blood by cardiac puncture for measurement of serum levels of TNFa, IL-6, IL- lb and IL-8 using AlphaLISA® (Perkin Elmer).

[0460] Tolerability Studies

[0461] To establish mitoDRIVE toleration, mouse experiments lasting for 2 weeks post mitochondrial administration were performed. Groups of 6 mice were administered mitoDRIVE-fLuc-loaded mitochondria via IV injection as described above, either one single administration, or with two administrations spaced 2 weeks apart. The highest tolerated dose from the safety study was then used for tolerability experiments.

[0462] Administrations of mitochondria and blood collection are repeated every 2 weeks for a total of 3 administrations (Week 0, 2, and 4). Final blood collection for immune evaluation is done in Week 6.

[0463] Observations of overall health including behavior, activity level, food and water consumption are monitored daily, and body weight were recorded weekly. Body temperature is measured at 6, 24, and 48 hours post mitochondria administration using non-contact infrared thermometry of the perianal region as described (Mei J et al, Sci Rep 8:3526.). Serum levels of TNF-a, IL-6, IL-ip and IFN-y were assessed by AlphaLISA (Perkin Elmer). To evaluate anti-drug responses, anti-mitochondrial antibodies will be assessed by ELISA (MBS Kit 262592) 2 weeks after each mitochondria administration. The schematic process that describes the experiments is shown in Figure 11.

[0464] Success criteria:

[0465] • Lack of evidence of impacts on overall mouse health (activity levels / response to touch, less than 15% weight loss, normal body temperature)

[0466] • Insignificant levels of proinflammatory cytokines (IL-6, IL-8, TNFa, IL- lb) in serum when compared to control animals that did not receive mitoDRIVE using Wilcoxon rank sum test. • Lack of significant anti-drug immune responses when compared to control animals that did not receive mitoDRIVE.

[0467] Power estimates

[0468] Each experiment addressing safety and / or tolerability will compare a control group to a test group. With n=5 there is an 80% power at the 0.05 significance level to detect a difference of 2 standard deviations between the control and experimental group using the Wilcoxon rank sum test. Group sizes of 6 are used to maintain power in the event of unforeseen loss.

[0469] The experiment is expected to show that there is little to no activation of immune cells even with multiple administrations of allogenic mitochondria having mitoDRIVE plasmid.

[0470] Safety results of mitoDRIVE technology in a mouse model

[0471] Balb / c mice aged 8-12 weeks (n=6 per group, n=3 per sex) were sourced from Jackson Labs (Bar Harbor, Maine). To generate a positive control, LPS (O5:B55, sigma chemicals) was administered at 5mg / kg via tail vein injection. As per the Institutional Animal Care and Use Committee (IACUC) protocol, if temperature deviation from baseline exceeds 4°C in the mice, then the mice will be sacrificed as a humane endpoint. Temperature and weight were evaluated at 0 hours, 6 hours, 24 hours, and 48 hours post administration. Serum was collected at time of sacrifice (humane 6h endpoint for LPS group, planned 48h endpoint for the rest of the mice). (See Figures 13A & 13B)

[0472] Syngeneic mitochondria were sourced from the liver of a Balb / c female aged 8-12 weeks. Allogeneic mitochondria were sourced from the Neuro2a cell line that was loaded with the mitoDRIVE construct expressing luciferase gene. No significant change in temperature was found between saline negative controls and mitochondrial administered groups (see FIG. ure 13A). No significant difference in weight found between the saline negative control and the mitochondrial groups (FIG. 13B).

[0473] The presence and quantitation of pro-inflammatory cytokines was assessed via Luminex using the Mouse Luminex Discovery Assay (LXSAMSM, R&D systems). Serum was diluted to 1:2 with RD6-52 calibrator diluent according to manufacturer specifications. A standard curve was generated using the supplied recombinant proteins in the kit to compare test serum cytokine levels according to manufacturer specifications. Diluted serum was tested in duplicate as suggested by the manufacturer. Only the positive control group (LPS) elicited cytokine production above the lower limit of detection (FIG. 13C-13F). Post procedural observations also found that all mice remained bright, alert, and responsive to touch with normal food and water intake and waste production. Collectively these data suggest that mitochondrial administration does not elicit a systemic pro-inflammatory response when administered at our highest dose (Img / kg).

[0474] Luminex Assay Procedure

[0475] 1. 50 ul of the diluted sample or standard were added to individual wells, and each sample or standard was plated in duplicate.

[0476] 2. 50 ul of the custom diluted microparticle cocktail was added to each well and incubated at room temperature for 2 hours with shaking at 800 rpm.

[0477] 3. The wells were washed by removing the liquid from each well and then filling with 100 uL of wash buffer and then the wash buffer was removed. This was performed 3 times.

[0478] 4. 50 ul of diluted biotin antibody cocktail was added to each well and then incubated for 1 hour at room temperature with shaking at 800 rpm.

[0479] 5. The wash step (step 3) was repeated.

[0480] 6. 50 uL of diluted Streptavidin-PE was added to each well and incubated at room temperature for 30 minutes with shaking at 800 rpm.

[0481] 7. The wash step (step 3) was repeated.

[0482] 8. 100 uL of wash buffer was added to each well and incubated for 2 minutes at room temperature with shaking 800 rpm.

[0483] 9. The plate was read within 90 minutes using a Luminex Analyzer.

[0484] 10. A standard curve was generated from the diluted standards and used to interpolate the values of the samples.

[0485] Calculation of Mitochondrial Protein Assay

[0486] 1. Mitochondria were isolated as previously described.

[0487] 2. Mitochondria were diluted 1:150 and 1:30 in Dulbeco’s PBS (Gibco, 14190144).

[0488] 3. A standard curve was generated for protein using the Pierce Bovine Serum Albumin Standard Ampules (23209, Thermo Scientific) by diluting the standards with Dulbeco’s PBS, ranging from 40 ug / ml to 0 ug / ml 4. Quick Start Bradford Protein Dye Reagent (Biorad) was used to generate a chromogenic signal for detection with a plate reader.

[0489] 5. The Dye reagent was added 1:1 with the sample and standards and incubated for 5 minutes at room temperature.

[0490] 6. The amount of absorbance at 595 nm was used to generate a standard curve from the standards and used to interpolate the mitochondrial protein samples.

[0491] Tolerability results of mitoDRIVE technology in a mouse model

[0492] After short-term safety was demonstrated, the repeated dosing of mitochondria was evaluated. Syngeneic mitochondria were sourced from the liver of a Balb / c female aged 8-12 weeks, while allogeneic mitochondria were sourced from Neuro2a cells loaded with the mitoDRIVE construct. The dosing schedule, shown in FIG. 14A, was designed to assess whether exposure to mitochondria would elicit an antimitochondrial adaptive immune response. Mice received either one dose or two doses of mitochondria (1 mg / kg), spaced 2 weeks apart. Temperature and weight changes were monitored at 6 hours, 24 hours, and 48 hours after dosing to observe any acute exposure effects. For the second dose, these parameters were again evaluated at the same time points. Forty-eight hours after the final dosing with mitochondria or saline vehicle, the mice were sacrificed, and serum was collected. No differences in temperature or weight were observed between the saline negative control and the mitochondria-exposed groups (See FIGs. 14B, 14C, 14D and 14E).

[0493] The potential development of antibodies in mitochondria-exposed mice after one or two doses was also evaluated, revealing no difference between the saline-treated negative control group and the mitochondria-dosed groups (FIGs. 14 F, 14G and 14H). Postprocedural observations indicated that all mice remained bright, alert, and responsive to touch, with normal food and water intake and waste production.

[0494] Anti-mitochondrial Antibody Assay

[0495] 1. Sample serums were prepared according to manufacturer’s recommendations.

[0496] 2. A standard curve was generated using the recombinant protein supplied at the following concentrations: 5000,2500, 1250, 625, 312, 156, 78, and 0 U / mE.

[0497] 3. 100 ul of sample or standard was added in duplicate wells to the plate. The wells were incubated at 37C for 90 minutes. 4. The liquid was removed from each well and then washed using 350 ul of wash buffer, allowing the wash buffer to incubate for at least 30 seconds at room temperature. The liquid was completely removed and then washed again for a total of 2 times.

[0498] 5. The antigen solution was prepared according to manufacturer specification and then added to each well (100 ul). The plate was incubated at 37C for 60 minutes.

[0499] 6. The plate was washed as previously described 3 more times.

[0500] 7. The enzyme conjugate solution was prepared according to manufacturer specification and then added to each well (100 ul). The plate was then incubated at 37 C for 30 minutes.

[0501] 8. The plate was washed for another 5 times.

[0502] 9. 100 ul of the prepared color reagent was added to each well and incubated at 37C protected from light.

[0503] 10. Once the highest standards become dark, the chromogenic reaction was stopped by adding 100 ul of the stop color reagent to each well.

[0504] 11. The absorbance of each well at 450 nm was used to generate a standard curve with the standard wells and then used to interpolate the samples.

[0505] The results of the experiments demonstrate that dosing of mice with mitochondria does not elicit a pro-inflammatory response, and the use of third-party sourced mitochondria also does not show any evidence of inflammation. Furthermore, the use of mitoDRIVE-laden mitochondria did not elicit a response different from that of syngeneic mitochondria or the saline negative control. Repeated dosing was well tolerated, with no evidence of anti- mitochondrial antibodies being induced in any group. In total, mitochondria were administered to a total of 60 mice, and no adverse events of any kind were observed.

[0506] Example 13 - Assessment of distribution and persistence of transgene expression (luciferase) using whole animal bioluminescent imaging in a mouse model.

[0507] The following experiment demonstrates that mitoDRIVE-based transgene expression delivered via mitochondrial transplant is robust and widely distributed in vivo.

[0508] Mitochondria isolated from cells transfected with mitoDRIVE-fLuc plasmid are introduced into BALB / c mice via one of the intramuscular (IM), intraperitoneal (IP) and intranasal (IN) and intravenous (IV) route using a single administration, and with a repeated administration. The dosage determined by tolerability studies in example 12 is used for the instant experiment.

[0509] In some instances, the loaded allogenic mitochondria containing the mitoDRIVE-fLuc plasmid is resuspended in sterile PBS lacking Ca2+and Mg2+and introduced into BALB / c mice via the IV, IN, IM, and IP route (n=6 mice each with 6 matched control mice that receive cell-line-derived mitochondria without mitoDRIVE platform by each route) either using one single administration, or with two administrations spaced 2 weeks apart.

[0510] Bioluminescent imaging of the whole mouse is used to assess transgene expression levels and anatomical distribution. Whole animal bioluminescent imaging is performed on a mouse model using the mitoDRIVE-Luc and control mice and on an additional group of n=6 BALB / c mice that receive a single administration of mitoDRIVE-Luc mitochondria (0.5 mg / kg body weight via tail vein injection) at week 0.

[0511] Imaging is performed at day 1 and 3, then weekly, after each mitochondria administration. After the final administration of mitochondria imaging will be performed once per week for an additional 8 weeks if transgene expression remains detectable as expected. Imaging is performed using the IVS Lumina small animal imaging system after intraperitoneal injection of D-Luciferin (150 mg / kg). Mice are be sedated with isoflurane and kept warm with the use of a heated stage during image acquisition. Optionally, the same experiment can be repeated in SKH1 hairless mice (Charles River Labs) to further minimize interference of hair with the imaging signals.

[0512] Persistence of mitodDRIVE-nfLuc expression after a single administration of mitoDRIVE and after a repeat dose administered 2 weeks after the first administration is measured. Whole body immunofluorescence is measured weekly for up to 8 weeks post mitochondria administration to determine if transgene expression remains detectable when compared to background levels in control mice. Experimental group sizes of 6 mice are used, and control mice are a group of mice (n=6) that received cell-line-derived mitochondria without mitoDRIVE platform.

[0513] Method: Mitochondria (8 mg / kg) or mitochondria laden with mitoDRIVE (8 mg / kg) were administered to male and female balb / c mice (age 8 to 16 weeks) R.O. 24 hours later, 1.5 umoles of Nano-Gio® Fluorofurimazine In Vivo Substrate (Promega) were administered to the mice via intra peritoneal injection, and luminescence was recorded for 1 minute. Based on the success in transgene expression from mitoDRIVE-loaded mitochondria in vitro, combined with positive results from safety and tolerability studies, an in vivo study using a mouse model was performed to evaluate mitoDRIVE-based transgene expression in vivo. The same method for mitochondrial transplant as in the in vitro model was utilized, wherein mitochondria laden with mitoDRIVE-luciferase plasmid were isolated and administered intravenously into BALB / c mice via retroorbital (RO) injection. Luciferase activity was then assessed on a kinetic in vivo imaging system (IVIS) following intraperitoneal (i.p.) injection of the luciferase substrate at an equivalent dose to all animals, the schematic of the experimental protocol is shown in FIG. 17A.

[0514] A viable signal was produced 24 hours after administration of the transgenic mitochondria encoding and secreting luciferase to mice, but not after administration of mitochondria alone or a saline control (See Fig 17B, and 17C). Some localized expression of the luciferase signal may be caused by poor distribution of the luciferase substrate after IP administration. Importantly, luciferase activity was observed distal to the site of injection, and therefore the mitoDRIVE-loaded mitochondria effectively spread throughout the mouse with IV administration.

[0515] The results of the experiment show that robust transgene expression is achieved using transgenic mitochondria as a vehicle for delivering heterologous protein in mice, and that it is effectively distributed in the mice.

[0516] Example 14 - Assessment of in vitro expression of two trans genes using mitoDRIVE platform

[0517] A schematic overview of in vitro studies and plasmid maps demonstrating the approaches for assessing the ability of mitoDRIVE to express more than one transgene are shown in Figure 12. mitoDRIVE platforms designed to express two transgenes can be devised using two different methods. The first method is designed to express two transgenes from a single mitochondrial promoter, (bicistronic approach, See Fig.l2A). Mitochondria genes are expressed as polycistronic transcripts from both the heavy strand and the light strand, with the majority of mitochondrial genes expressed from the guanine-rich heavy strand. Expression of the individual genes that encode protein requires processing of the polycistronic transcript by excision of mt-tRNAs via endonucleolytic cleavage, and further processing to generate individual mt-mRNA transcripts (Kotrys AV, Szczesny RJ. 2019. Mitochondrial Gene Expression and Beyond-Novel Aspects of Cellular Physiology. Cells; Bouda E, Stapon A, Garcia-Diaz M. 2019. Mechanisms of mammalian mitochondrial transcription. Protein Sci 28:1594-1605; Pfanner N, Warscheid B, Wiedemann N. 2019. Mitochondrial proteins: from biogenesis to functional networks. Nat Rev Mol Cell Biol 20:267-284.). The bicistronic approach uses native promoter / regulation systems for heavy strand expression and the endogenous tRNA excision system is used to process the generated bicistronic transcript. The second method is focused on expressing the second transgene from the light strand under control of the canonical light stand promoter (dual promoter approach, see FIG. 12B).

[0518] For both methods, luciferase (fLuc) and fluorescent (eGFP) reporter genes are used to allow for in vitro quantification of transgene expression. Fluorescence (eGFP) and luminescence (fLuc) are measured in triplicate wells using our multi-mode plate reader.

[0519] The experiment is expected to show confirmed expression of both transgenes in vitro as indicated by fluorescence and luminescence beyond that of cells transfected with a mitoDRIVE plasmid lacking both reporter genes.

[0520] Example 15 - Assessment of mitoDRIVE transgene carrying capacity

[0521] The following experiment aims to demonstrate that the carrying capacity of mitoDRIVE will be higher than that of AAV. “ staffer DNA” (Grieger JC, Samulski RJ. 2005. Packaging capacity of adeno-associated virus serotypes: impact of larger genomes on infectivity and postentry steps. J Virol 79:9933-44; Parks RJ, Bramson JL, Wan Y, Addison CL, Graham FL. 1999. Effects of staffer DN A on trans gene expression from helperdependent adenovirus vectors. J Virol 73:8027-34; Sweeney NP, Vink CA. 2021. The impact oflentiviral vector genome size and producer cell genomic to gag-pol mRNA ratios on packaging efficiency and titre. Mol Ther Methods Clin Dev 21:574-584) from lambda phage or human intronic regions, of various sizes is introduced into the mitoDRIVE-fLuc plasmid. A schematic overview of the process of generating mitoDRIVE-fLuc plasmid with varying sizes of transgene is shown in Figure 12C.

[0522] Normal human and mouse mtDNA is -16.2 kb. Loaded mitoDRIVE platforms of this size may retain functionality. Stuff er fragments are used to generate mitoDRIVE plasmids containing transgene sizes ranging from 2kb to 16 kb. The mitoDRIVE constructs are then loaded onto mitochondria by transfection and the loaded mitochondria are exposed to cells which then take up the loaded mitochondria and express the transgene.

[0523] The in vitro assays disclosed in prior examples is used to quantify luciferase expression with a multimode plate reader. Loss of luciferase activity (return to background levels observed for cells transfected with a control plasmid lacking the luciferase reporter) at a given size of stuffer DNA would indicate the upper limit of carrying capacity.

[0524] The experiment is expected to show persistent expression of luciferase transgene even when the size of transgene is over 5kb which is greater than the upper limit observed for AAV vectors.

[0525] Example 16 - Assessment of efficacy of transgene expression in disease model of factor IX deficient (Type B hemophilic) mice.

[0526] Hemophilia is an X-linked bleeding disorder caused by mutations in blood coagulation factors. The two most common forms of hemophilia, hemophilia A and hemophilia B, are inherited rare diseases caused by deficiencies in factor VIII or factor IX (FIX), respectively. The incidence of hemophilia B is approximately 1 in 19,300 male births, with a prevalence of 3.7 cases per 100,000 U.S. males (Soucie JM, Miller CH, Dupervil B, Le B, Buckner TW. 2020. Occurrence rates of haemophilia among males in the United. States based on surveillance conducted in specialized haemophilia treatment centres. Haemophilia 26:487-493). Although treatment options exist, gene therapy offers the only path to cure hemophilia B. mitoDRIVE-FIX plasmid is generated to evaluate the efficacy of mitochondria- delivered therapeutic transgene delivery in FIXKO mice. mitoDRIVE-FIX loaded mitochondria is administered into male FIXKO mice (Jackson Labs Strain #004303, test group, n=5) via tail vein injection. Control male FIXKO mice (n=3) are administered with mitoDRIVE-eGFP loaded mitochondria. Plasma samples are collected weekly for control and test mice groups.

[0527] Eevels of FIX will be assessed by quantitative EEISA (ESBio ES-F30594) and activated partial thromboplastin time (aPTT) will be measured using the Roche CoaguChek aPTT Test. Mice are evaluated for 8 weeks post mitochondria infusion. A terminal blood draw is collected and used to check for the presence of anti-mitochondria antibodies (MBS ELISA Kit 262592) and anti-FIX antibodies (custom ELISA).

[0528] Example 17 - Assessment of efficacy of transgene (DMD) expression in disease model of DMD deficient (mdx) mice.

[0529] Duchenne muscular dystrophy (DMD) is a severe genetic disorder characterized by progressive muscle degeneration and weakness. It is caused by mutations in the dystrophin gene, which leads to the absence or deficiency of the dystrophin protein in muscle cells. DMD primarily affects males, with symptoms typically manifesting in early childhood.

[0530] The absence of dystrophin disrupts the structural integrity of muscle fibers, making them more susceptible to damage during muscle contraction and relaxation. Over time, repeated cycles of muscle degeneration and regeneration result in progressive muscle weakness and loss of function. DMD primarily affects skeletal and cardiac muscles, leading to difficulties in walking, muscle wasting, respiratory complications, and eventual heart failure. mitoDRIVE-DMD plasmid is generated to evaluate the efficacy of mitochondria- delivered therapeutic transgene delivery in mdx mice. mitoDRIVE-DMD loaded mitochondria is administered into male mdx mice (Jackson Labs Strain #001801, test group, n=5) via tail vein injection. Control male mdx mice (n=3) are administered with mitoDRIVE- eGFP loaded mitochondria. Plasma samples are collected weekly for control and test mice groups.

[0531] Levels of DMD protein will be assessed by quantitative ELISA. Mice are evaluated for 8 weeks post mitochondria infusion. A terminal blood draw is collected and used to check for the presence of anti-mitochondria antibodies (MBS ELISA Kit 262592) and anti-DMD antibodies (custom ELISA).

[0532] Example 18 - Assessment of efficacy of transgene (CFTR) expression in disease model of CFTR knockout mice.

[0533] Cystic fibrosis (CF) is a progressive, genetic disorder primarily affecting the respiratory and digestive systems. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, leading to defective CFTR protein function. This dysfunction results in the production of thick, sticky mucus that clogs the airways, impairs lung function, and affects various organs throughout the body.

[0534] In individuals with CF, the abnormal CFTR protein disrupts the balance of ions and fluid in epithelial cells, leading to reduced hydration and altered mucus properties. The accumulation of thickened mucus in the airways obstructs the flow of air, providing an environment conducive to bacterial growth and recurrent infections. Chronic inflammation and progressive lung damage can occur, leading to respiratory complications and declining lung function over time. mitoDRIVE-CFTR plasmid is generated to evaluate the efficacy of mitochondria-delivered therapeutic transgene delivery in CFTR mice. mitoDRIVE-CFTR loaded mitochondria is administered into male CFTR KO mice (Jackson Labs Strain #002364, test group, n=5) via tail vein injection. Control male CFTR KO mice (n=3) are administered with mitoDRIVE-eGFP loaded mitochondria. Plasma samples are collected weekly for control and test mice groups.

[0535] Levels of CFTR protein will be assessed by quantitative ELISA. Mice are evaluated for 8 weeks post mitochondria infusion. A terminal blood draw is collected and used to check for the presence of anti-mitochondria antibodies (MBS ELISA Kit 262592) and anti-CFTR antibodies (custom ELISA).

[0536] Example 19 - Administration of mitoDRIVE- loaded mitochondria to allow for successful transfection of poorly transfectable cells.

[0537] The following experiment is designed to assess the versatility of the mitochondrial delivery platform in enabling gene expression in cells that are refractory to traditional transfection methods.

[0538] Direct transfection of two different cell lines, the human 293T kidney cell line and the human CEM.NKR CCR5 T cell line, was assessed using a standard expression plasmid with nuclear-encoded luciferase under the control of a CMV promoter.

[0539] The CEM.NKR CCR5 T cells are known in the literature to be refractory to traditional transfection methods, whereas the 293T cells are not refractory (Olden BR , Cheng E , Cheng Y , Pun SH . Identifying key barriers in cationic polymer gene delivery to human T cells. Biomater Sci. 2019 Feb 26;7(3):789-797). To demonstrate this, the plasmid pcDNA3.1- NL was directly transfected into the two cell lines using Lipofectamine 3000 according to the manufacturer's specifications, and luciferase activity was assessed after 3 days.

[0540] Method: 293t cells (ATCC# CRL-3216) and CEM.NKRCCR5 (BEI# ARP-4376) cells were transfected with pcDNA3.1-NL using lipofectamine 3000 according to manufacturer’s protocol. 293t cells and CEM.NKR cells were transfected with mitoDRIVE- nluc using lipofectamine 3000 according to manufacturer’s protocol. 293t cells were transfected with mitoDRIVE-nluc and then mitochondria were isolated.

[0541] Mitochondria were placed on CEM.NKR CCR5 cells and transgene protein expression was assessed using Promega NanoGio assay 2 days later and is reported as relative luminescence units (RLU). Gene transcript expression data was gathered by isolating RNA from CEM.NKR CCR5 cell pellets using the Qiagen RNeasy isolation kit according to manufacturer specifications. Ambion TURBO DNA-free DNase was used to clean the extracted nucleic acids of any residual DNA according to manufacturer specifications. cDNA was generated using the Ambicon High-Capacity cDNA Reverse transcriptase kit according to manufacturer specifications. Transgene specific mRNA was assessed using nanoLuc specific primers with the SsoAdvanced Universal SYBR Green Supermix on a BioRad CFX Connect. Linear regression of relative gene expression of mitoDRIVE nluc compared to amount of mitochondria given to CEM.NKR cells. N = 2 independent experiments.

[0542] As shown in FIG. 15A, the CEM.NKR CCR5 cells were found to be poorly transfectable, as evidenced by the 5-log lower luciferase activity when exposed to the luciferase substrate compared to the 293T cells. This is consistent with findings in the literature about these two cell lines.

[0543] Next, the ability of the mitoDRIVE plasmid to express luciferase in these two cell lines was assessed. Similar to the conventional nuclear-encoded luciferase plasmid, direct transfection of the mitoDRIVE plasmid containing a mitochondrial-encoded luciferase gene also resulted in poor expression in the CEM.NKR CCR5 cell line (FIG. 15 B).

[0544] A distinct advantage of the mitochondrial delivery system comprising the mitoDRIVE plasmid disclosed here is its versatility. The mitoDRIVE plasmid can be directly transfected into the cells of interest. Additionally, the mitoDRIVE plasmid can be loaded into a first cell type, allowing it to be incorporated into the mitochondria. These mitochondria, now loaded with the mitoDRIVE plasmid, can then be isolated from the first cell type and administered to a second cell type for cellular uptake. This method enables the second cell type to internalize the mitochondria from the first cell type, which are loaded with mitoDRIVE, and subsequently express the transgene present in the mitoDRIVE plasmid.

[0545] The mitochondrial delivery system is designed to allow for the transfection of difficult-to-transfect cells by utilizing previously loaded mitochondria from a transfected cell line as the vehicle for delivery.

[0546] To demonstrate this capability, the 293T cells were first loaded with the mitoDRIVE luciferase plasmid, the mitochondria were isolated 1 day later, and these mitochondria were administered to the CEM.NKR CCR5 cells to allow for cellular uptake and utilization of the mitoDRIVE-laden mitochondria.

[0547] With this approach, a luciferase signal in the CEM.NKR CCR5 cells comparable to that observed with direct transfection of the highly transfectable 293T cells was observed (FIG. 15C). Thus, the refractory nature of the CEM.NKR CCR5 cells to transfection was overcome by utilizing mitochondria as the delivery vehicle.

[0548] Furthermore, the amount of luciferase activity was compared to the amount of RNA transcripts present in the cells, and it was observed that, as expected, 1) the transcript level and the amount of luciferase activity were dependent on the dose of mitoDRIVE-loaded mitochondria administered to the cells, and 2) these two signals correlated strongly (FIG. 15D). These data demonstrate that the mitoDRIVE-loaded mitochondria were internalized by the CEM.NKR CCR5 cells and the luciferase transgene was expressed. Thus, the results of these experiments show that the mitoDRIVE system allows for gene expression in cells that are refractory to transfection by conventional approaches (FIG. 15E).

[0549] The results of the experiments demonstrate that: 1) proteins of interest (from transgene located in mitoDRIVE plasmid) are expressed from the mitochondria by mitoDRIVE, and 2) mitoDRIVE-laden mitochondria can be used to deliver mitoDRIVE plasmid to difficult-to-transfect cell lines where other methods fail to do so.

[0550] Example 20 - Introduction of Plasmid DNA into mitochondria by Electroporation

[0551] Plasmid DNA can be introduced into mitochondria via electroporation following the protocols outlined herein. Mitochondrial Preparation and Electroporation

[0552] 1. Mitochondria Suspension: 200 pg of mitochondria were taken and suspended in 1% glycerol (v / v).

[0553] 2. Centrifugation: The mixture was spun at 7000 x g for 10 minutes.

[0554] 3. Supernatant Removal: The supernatant was removed, and the mitochondria were resuspended in 1% glycerol (v / v).

[0555] 4. Second Centrifugation: The mixture was spun again at 7000 x g for 10 minutes.

[0556] 5. Supernatant Removal and Resuspension: The supernatant was removed, and the mitochondria were resuspended in 10% glycerol (v / v).

[0557] 6. Electroporation Setup: The mitochondria were added to an electroporation cuvette with a 0.1 cm gap.

[0558] 7. DNA Addition: 5 pg of DNA (ensuring the volume was less than 20 pL) was added.

[0559] 8. Electroporation: The cuvette was placed into the electroporator and electroporated at 1.4 kV for at least 500 ms.

[0560] 9. Immediate Cooling: The cuvette was immediately placed on ice.

[0561] 10. Mitochondria Retrieval: The mitochondria were removed using a transfer pipette.

[0562] 11. Cuvette Wash: The cuvette was washed with 50 pL of PBS, and this was added to the mitochondria.

[0563] 12. Dilution and Centrifugation: The mixture was diluted to 1% glycerol with PBS and spun at 13000 x g for 10 minutes.

[0564] 13. Final Resuspension: The mitochondria were resuspended in a buffer containing 225 mM sucrose, 44 mM KH2PO4, 12.5 mM magnesium acetate, and 6 mM EDTA.

[0565] DNA Labeling (Using Label IT Nucleic Acid Labeling Kit, MIR7100 Mirus Bio)

[0566] 1. Labeling Preparation: The plasmid DNA was labeled according to the manufacturer's recommendations.

[0567] 2. Reagent Reconstitution: The labeling reagent was prepared by reconstituting it in the reconstitution solution provided. 3. Labeling Reaction: The DNA was labeled using a ratio between 1 : 1 and 0.1:1 (volume (|jL) of labeling reagent to mass of nucleic acid (pg)) .

[0568] 4. Incubation: The labeling reaction was incubated at 37°C for 1 hour.

[0569] 5. Sample Purification: The samples were purified using G50 microspin purification columns (Cytiva) according to the manufacturer's recommendations .

[0570] This detailed protocol ensures the efficient introduction of plasmid DNA into mitochondria via electroporation, followed by proper labeling and purification of the DNA to facilitate subsequent experimental applications.

[0571] Mitochondria were thus electroporated above with fluorescently-labeled plasmid DNA at 2.5 ug, 0.25 ug, 0.025 ug, and 0.0025 ug or with sham electroporation. Mitochondria were then assessed for fluorescence by flow cytometry to measure the amount of internalized DNA compared to the sham control. (See Figure 16). The results demonstrate that plasmid DNA can be introduced into mitochondria by electroporation.

[0572] INCORPORATION BY REFERENCE

[0573] The disclosure of each and every U.S. and foreign patent and pending patent application and publication referred to herein is specifically incorporated herein by reference in its entirety, as are the contents of Sequence Listing and Figures.

[0574] EQUIVALENTS

[0575] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims. Any combination of the embodiments disclosed in the any plurality of the dependent claims or Examples is contemplated to be within the scope of the disclosure.

[0576] OTHER EMBODIMENTS

[0577] From the foregoing description, it will be apparent that variations and modifications may be made to the disclosure described herein to adopt it to various usages and conditions. Recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or sub combination) of listed elements. Recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0578] All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0579] Other embodiments are within the following claims.

Claims

What is claimed is:

1. A method of delivering a heterologous protein to an animal comprising: administering transgenic mitochondria to an animal, said transgenic mitochondrion comprising a transgene encoding a heterologous protein, said transgene comprising a sequence that permits secretion of said heterologous protein from said mitochondrion; whereupon said administering permits expression of said transgene and secretion of said heterologous protein from said mitochondrion, thereby delivering said heterologous protein to said animal.

2. The method of claim 1, wherein said mitochondrion is not a megamitochondrion and / or said mitochondrion comprises a diameter of less than 2pm.

3. The method of claim 1, wherein said sequence comprises a heterologous nucleic acid comprising first region encoding a signal peptide and a second region encoding said heterologous protein, and wherein said first region and said second region are operably linked.

4. The method of claim 3, wherein said transgene further comprises one or more promoters that are operative in mitochondrion and said promoter is capable of directing transcription of said nucleic acid.

5. The method of claim 3, wherein said transgene further comprises one or more mitochondrial transcriptional terminators.

6. The method of claim 3, wherein said transgene further comprises one or more transcript processing signals.

7. The method of claim 3, wherein said transgene further comprises native replication signals capable of propagating said transgene within mitochondrion during mitogenesis.

8. The method of claim 3, wherein said transgene further comprises mitochondrial export sequence and said mitochondrial export sequence is capable of improving and / or increasing mitochondrial export of said heterologous protein.

9. The method of claim 3, wherein said transgene further comprises origins of strand (Ori) replication, tRNA genes and a displacement (D-loop) loop.

10. The method of claim 3, wherein said heterologous nucleic acid further encodes one or more of cell-penetrating peptides or homeoprotein internalization- secretion domains to facilitate export of said heterologous protein from mitochondrion.

11. The method of claim 3, wherein said heterologous nucleic acid further comprises mitochondrial targeting sequences which facilitate nuclear import of said heterologous protein, or retention of said heterologous protein in the endoplasmic reticulum.

12. The method of claim 3, wherein said heterologous nucleic acid encoding a plurality of heterologous proteins.

13. The method of claim 12, wherein the size of said heterologous nucleic acid is less than or equal to 16 Kb.

14. The method of claim 9, wherein said Ori are selected from the group consisting of OriL and OriH.

15. The method of claim 9, wherein said D-Loop comprises regulatory elements for transcription and replication of mitochondrial DNA.

16. The method of claim 9, wherein said nucleic acid is located downstream of heavy strand promoter (HSP1) and is optionally flanked by a mitochondrial t-RNA gene and / or flanked on one side by the OriL.

17. The method of claim 4, wherein said promoter is a heavy strand promoter.

18. The method of claim 4, wherein said promoter is a light strand promoter.

19. The method of claim 17, wherein said heavy strand promoter is selected from the group consisting of HSP1 and HSP2.

20. The method of claim 18, wherein said light strand promoter is LSP1 or LSP2.

21. A method of delivering a heterologous protein to a cell comprising: administering transgenic mitochondrion to a cell, said transgenic mitochondrion comprising a transgene encoding a heterologous protein, said transgene comprising a sequence that permits secretion of said heterologous protein from said mitochondrion;whereupon said administering permits expression of said transgene and secretion of said heterologous protein from said mitochondrion, thereby delivering said heterologous protein to said cell.

22. The method of claim 21, wherein said mitochondrion is not a megamitochondrion and / or said mitochondrion comprises a diameter of less than 2pm.

23. The method of claim 21, wherein said transgene comprises a heterologous nucleic acid comprising first region encoding a signal peptide and a second region encoding said heterologous protein, and wherein said first region and said second region are operably linked.

24. The method of claim 23, wherein said transgene further comprises one or more promoters that are operative in mitochondrion and said promoter is capable of directing transcription of said nucleic acid.

25. The method of claim 23, wherein said transgene further comprises one or more mitochondrial transcriptional terminators.

26. The method of claim 23, wherein said transgene further comprises one or more transcript processing signals.

27. The method of claim 23, wherein said transgene further comprises native replication signals capable of propagating said transgene within mitochondrion during mitogenesis.

28. The method of claim 23, wherein said transgene further comprises mitochondrial export sequence and said sequence is capable of improving and / or increasing mitochondrial export of said protein.

29. The method of claim 23, wherein said transgene further comprises origins of strand (Ori) replication, tRNA genes and a displacement (D-loop) loop.

30. The method of claim 23, wherein said heterologous nucleic acid further encodes one or more of cell-penetrating peptides or homeoprotein internalization- secretion domains to facilitate export of said heterologous protein from mitochondrion.

31. The method of claim 23, wherein said heterologous nucleic acid further comprises mitochondrial targeting sequences which facilitate nuclear import of said heterologous protein, or retention of said heterologous protein in the endoplasmic reticulum.

32. The method of claim 23, wherein said heterologous nucleic acid encoding a plurality of heterologous proteins.

33. The method of claim 32, wherein the size of said heterologous nucleic acid is less than or equal to 16 Kb.

34. The method of claim 29, wherein said Ori are selected from the group consisting of OriL and OriH.

35. The method of claim 29, wherein said D-Loop comprises regulatory elements for transcription and replication of mitochondrial DNA.

36. The method of claim 29, wherein said nucleic acid is located downstream of heavy strand promoter (HSP1) and is optionally flanked by a mitochondrial t-RNA gene and / or flanked on one side by the OriL.

37. The method of claim 24, wherein said promoter is a heavy strand promoter.

38. The method of claim 24, wherein said promoter is a light strand promoter.

39. The method of claim 37, wherein said heavy strand promoter is selected from the group consisting of HSP1 and HSP2.

40. The method of claim 38, wherein said light strand promoter is LSP1 or LSP2.

41. The method of claims 3 or 23, wherein said heterologous nucleic acid further comprises a signal sequence which facilitate nuclear import of said heterologous protein, or retention of said heterologous protein in the endoplasmic reticulum.

42. The method of claims 3 or 23, wherein said signal peptide is selected from the group consisting of IL-6 signal peptide, IL-2 signal peptide, Human OSM peptide, VSV-G peptide, Mouse Ig Kappa signal peptide, Mouse Ig Heavy signal peptide, BM40 signal peptide, Secrecon signal peptide, Human IgKVIII signal peptide, CD33 signal peptide, tPA signal peptide, Lipoic Acid Signal Peptide, Glycosylphosphatidylinositol (GPI) AnchorSignal Sequence, Mitochondrial Targeting Sequence (MTS), Bacterial Sec System Signal Sequence, Fibroblast Growth Factor (FGF) Signal Sequence, Serine Protease Inhibitor (SERPIN) Signal Sequence, Chitinase Signal Sequence and Erythropoietin (EPO) Signal Sequence.

43. The method of claim 1 or claim 20, wherein said heterologous protein is selected from the group consisting of insulin, erythropoietin, human growth hormone, Factor VIII, Factor IX, luciferase, eGFP, interferons (such as IFN-a, IFN-P, IFN-y), Tumor Necrosis Factor (TNF) inhibitors, tissue plasminogen activator (tPA), dystrophin, and CFTR protein.

44. A transgenic mitochondrion comprising a heterologous nucleic acid comprising a first region encoding a signal peptide and a second region encoding a heterologous protein, wherein said first region and said second region are operably linked to permit expression of said nucleic acid, wherein expression of said nucleic acid permits secretion of said heterologous protein from said mitochondrion, and wherein said mitochondrion is not a megamitochondrion and / or said mitochondrion comprises a diameter of less than 2pm.

45. The transgenic mitochondrion of claim 44, wherein said mitochondrion comprises a heterologous plasmid comprising said nucleic acid.

46. The transgenic mitochondrion of claim 45, wherein said plasmid comprises a promoter operative in mitochondrion and said promoter is capable of directing transcription of said nucleic acid.

47. The transgenic mitochondrion of claim 45, wherein said plasmid comprises a mitochondrial transcription terminator.

48. The transgenic mitochondrion of claim 45, wherein said plasmid comprises a transcript processing signal.

49. The transgenic mitochondrion of claim 45, wherein said plasmid comprises native replication signals capable of propagating said plasmid within mitochondrion during mitogenesis.

50. The transgenic mitochondrion of claim 45, wherein said plasmid further comprises a mitochondrial export sequence and said export sequence is capable of improving and / or increasing mitochondrial export of said protein.

51. The transgenic mitochondrion of claim 46, wherein said promoter is a heavy strand promoter.

52. The transgenic mitochondrion of claim 46, wherein said promoter is a light strand promoter.

53. The transgenic mitochondrion of claim 51, wherein said heavy strand promoter is selected from the group consisting of HSP1 and HSP2.

54. The transgenic mitochondrion of claim 52, wherein said light strand promoter is selected from the group consisting of LSP1 and LSP2.

55. The transgenic mitochondrion of claim 45, wherein said plasmid comprises origins of strand (Ori) replication, tRNA genes and displacement (D-loop) loop.

56. The transgenic mitochondrion of claim 55, wherein said Ori are selected from the group consisting of OriL and OriH.

57. The transgenic mitochondrion of claim 55, wherein said D-Loop comprises regulatory elements for transcription and replication of mitochondrial DNA.

58. The transgenic mitochondrion of claim 55, wherein said heterologous nucleic acid is located downstream of heavy strand promoter (HSP1) and is optionally flanked by a mitochondrial t-RNA gene and / or flanked on one side by the OriL.

59. The transgenic mitochondrion of claim 45, wherein said heterologous nucleic acid further encodes one or more of cell-penetrating peptides or homeoprotein internalizationsecretion domains to facilitate export of said heterologous protein from mitochondrion.

60. The transgenic mitochondrion of claim 45, wherein said heterologous nucleic acid further comprises a signal sequence which facilitate nuclear import of said heterologous protein, or retention of said heterologous protein in the endoplasmic reticulum.

61. The transgenic mitochondrion of claim 45, wherein said plasmid comprises heterologous nucleic acid encoding a plurality of heterologous proteins.

62. The transgenic mitochondrion of claim 61, wherein the total size of said heterologous nucleic acid is less than or equal to 16 kb.

63. The transgenic mitochondrion of claim 44, wherein said mitochondrion comprises a vector comprising said nucleic acid.

64. The transgenic mitochondrion of claim 63, wherein said vector comprises one or more promoters operative in mitochondrion and said promoter is capable of directing transcription of said nucleic acid.

65. The transgenic mitochondrion of claim 63, wherein said vector comprises one or more mitochondrial transcriptional terminators.

66. The transgenic mitochondrion of claim 63, wherein said vector comprises one or more transcript processing signals.

67. The transgenic mitochondrion of claim 63, wherein said vector comprises native mitochondrial replication signals capable of propagating said plasmid within mitochondrion during mitogenesis.

68. The transgenic mitochondrion of claim 63, wherein said vector comprises a mitochondrial export sequence and said export sequence is capable of improving and / or increasing mitochondrial export of said protein.

69. The transgenic mitochondrion of claim 64, wherein said promoter is a heavy strand promoter.

70. The transgenic mitochondrion of claim 64, wherein said promoter is a light strand promoter.

71. The transgenic mitochondrion of claim 69, wherein said heavy strand promoter is selected from the group consisting of HSP1 and HSP2.

72. The transgenic mitochondrion of claim 70, wherein said light strand promoter is LSP1 or LSP2.

73. The transgenic mitochondrion of claim 63, wherein said vector comprises origins of strand (Ori) replication, tRNA genes and displacement (D-loop) loop.

74. The transgenic mitochondrion of claim 73, wherein said Ori are selected from the group consisting of OriL and OriH.

75. The transgenic mitochondrion of claim 73, wherein said D-Loop comprises regulatory elements for transcription and replication of mitochondrial DNA.

76. The transgenic mitochondrion of claim 73, wherein said heterologous nucleic acid is located downstream of heavy strand promoter (HSP1) and flanked by mitochondrial t- RNA-Val gene.

77. The transgenic mitochondrion of claim 63, wherein said heterologous nucleic acid further encodes one or more of cell-penetrating peptides or homeoprotein internalizationsecretion domains to facilitate export of said heterologous protein from mitochondrion.

78. The transgenic mitochondrion of claim 63, wherein said heterologous nucleic acid further comprises mitochondrial targeting sequences which facilitate nuclear import of said heterologous protein, or retention of said heterologous protein in the endoplasmic reticulum.

79. A composition comprising a transgenic mitochondrion of claim 44.

80. A composition comprising a plurality of transgenic mitochondrion of claim 44.

81. A vector comprising a mitochondrial promoter directing a heterologous nucleic acid comprising a first region encoding a signal peptide and second region encoding a heterologous protein, wherein said first region is operably linked to said second region such that said vector upon transfection to a mitochondrion permits secretion of said protein from said mitochondrion through said mitochondrial promoter.

82. The vector of claim 81, wherein said vector comprises one or more mitochondrial transcriptional promoters.

83. The vector of claim 81, wherein said vector comprises one or more mitochondrial transcriptional terminators.

84. The vector of claim 81, wherein said vector comprises one or more transcript processing signals.

85. The vector of claim 81, wherein said vector comprises native mitochondrial replication signals capable of propagating said plasmid within mitochondrion during mitogenesis.

86. The vector of claim 81, wherein said vector further comprises a mitochondrial export sequence and is capable of improving and / or increasing mitochondrial export of said protein.

87. The vector of claim 81, wherein said promoter is a heavy strand promoter.

88. The vector of claim 81, wherein said promoter is a light strand promoter.

89. The vector of claim 87, wherein said heavy strand promoter is selected from the group consisting of HSP1 and HSP2.

90. The vector of claim 88, wherein said light strand promoter is LSP1 or LSP2.

91. The vector of claim 81, wherein said vector comprises origins of strand (Ori) replication, tRNA genes and displacement (D-loop) loop.

92. The vector of claim 91, wherein said Ori are selected from the group consisting of OriL and OriH.

93. The vector of claim 91, wherein said D-Loop comprises regulatory elements for transcription and replication of mitochondrial DNA.

94. The vector of claim 91, wherein said heterologous nucleic acid is located downstream of heavy strand promoter (HSP1) and is optionally flanked by a mitochondrial t- RNA gene and / or flanked on one side by the OriL.

95. The vector of claim 81, wherein said heterologous nucleic acid further encodes one or more of cell-penetrating peptides or homeoprotein internalization- secretion domains to facilitate export of said heterologous protein from mitochondrion.

96. The vector of claim 81, wherein said heterologous nucleic acid further comprises mitochondrial targeting sequences which facilitate nuclear import of said heterologous protein, or retention of said heterologous protein in the endoplasmic reticulum.

97. The vector of claim 81, wherein said vector comprises heterologous nucleic acid encoding a plurality of heterologous proteins.

98. The vector of claim 97, wherein the size of said heterologous nucleic acid is less than or equal to 16 Kb.

99. A method of gene delivery comprising administering to an organism a transgenic mitochondrion, wherein said transgenic mitochondrion comprises a heterologous nucleic acid comprising a first region encoding a signal peptide fused to a second region encoding a heterologous protein, wherein upon said administration said nucleic acid are expressed as a secretable heterologous protein and said heterologous protein is secreted from said transgenic mitochondrion.

100. The method of claim 99, wherein said mitochondrion comprises a vector comprising said heterologous nucleic acid encoding said signal peptide fused to said heterologous protein.

101. The method of claim 100, wherein said vector further comprises one or more promoters that are operative in mitochondrion and said promoter is capable of directing transcription of said nucleic acid.

102. The method of claim 100, wherein said vector further comprises one or more mitochondrial transcriptional terminators.

103. The method of claim 100, wherein said vector further comprises one or more transcript processing signals.

104. The method of claim 100, wherein said vector further comprises native replication signals capable of propagating said plasmid within mitochondrion during mitogenesis.

105. The method of claim 100, wherein said vector further comprises mitochondrial export sequence and said sequence is capable of improving and / or increasing mitochondrial export of said protein.

106. The method of claim 101, wherein said promoter is a heavy strand promoter.

107. The method of claim 101, wherein said promoter is a light strand promoter.

108. The method of claim 106, wherein said heavy strand promoter is selected from the group consisting of HSP1 and HSP2.

109. The method of claim 107, wherein said light strand promoter is LSP1 or LSP2.

110. The method of claim 100, wherein said vector further comprises origins of strand (Ori) replication, tRNA genes and a displacement (D-loop) loop.

111. The method of claim 110, wherein said Ori are selected from the group consisting of OriL and OriH.

112. The method of claim 110, wherein said D-Loop comprises regulatory elements for transcription and replication of mitochondrial DNA.

113. The method of claim 110, wherein said nucleic acid is located downstream of heavy strand promoter (HSP1) and is optionally flanked by a mitochondrial t-RNA gene and / or flanked on one side by the OriL.

114. The method of claim 100, wherein said heterologous nucleic acid further encodes one or more of cell-penetrating peptides or homeoprotein internalization- secretion domains to facilitate export of said heterologous protein from mitochondrion.

115. The method of claim 100, wherein said heterologous nucleic acid further comprises mitochondrial targeting sequences which facilitate nuclear import of said heterologous protein, or retention of said heterologous protein in the endoplasmic reticulum.

116. The method of claim 100, wherein said vector comprises heterologous nucleic acid encoding a plurality of heterologous proteins.

117. The method of claim 116, wherein the size of said heterologous nucleic acid is less than or equal to 16 Kb.

118. A method of providing transgenic mitochondrion, the method comprising contacting one or more isolated mitochondrion with a vector capable of transfecting a mitochondrion and a transfection agent, said vector comprising a heterologous nucleic acid comprising a first region encoding a signal peptide fused to a second region encoding a heterologous protein, wherein said contacting provides one or more mitochondrion comprising said vector, thereby forming a transgenic mitochondrion having a diameter of less than 2pm, and expression of said nucleic acid permits secretion of said protein from said mitochondrion.

119. The method of claim 118, wherein said vector further comprises one or more promoters operative in mitochondrion and said transfection agent is selected from the group consisting of Lipofectamine, DOTAP, MITO-porter, protamine DNA complex, electroporation and Stearylamine-based liposomes.

120. The method of claim 118, wherein said vector further comprises one or more mitochondrial transcriptional terminators.

121. The method of claim 118, wherein said vector further comprises one or more transcript processing signals.

122. The method of claim 118, wherein said vector further comprises native replication signals capable of propagating said plasmid within mitochondrion during mitogenesis.

123. The method of claim 118, wherein said vector further comprises mitochondrial export sequence and said sequence is capable of improving and / or increasing mitochondrial export of said protein.

124. The method of claim 119, wherein said promoter is a heavy strand promoter.

125. The method of claim 119, wherein said promoter is a light strand promoter.

126. The method of claim 124, wherein said heavy strand promoter is selected from the group consisting of HSP1 and HSP2.

127. The method of claim 125, wherein said light strand promoter is LSP1 or LSP2.

128. The method of claim 118, wherein said vector further comprises origins of strand (Ori) replication, tRNA genes and displacement (D-loop) loop.

129. The method of claim 126, wherein said Ori are selected from the group consisting of OriL and OriH.

130. The method of claim 128, wherein said D-Loop comprises regulatory elements for transcription and replication of mitochondrial DNA.

131. The method of claim 128, wherein said nucleic acid is located downstream of heavy strand promoter (HSP1) and flanked by mitochondrial t-RNA-Val gene.

132. The method of claim 128, wherein said heterologous nucleic acid further encodes one or more of cell-penetrating peptides or homeoprotein internalization- secretion domains to facilitate export of said heterologous protein from mitochondrion.

133. The method of claim 128, wherein said heterologous nucleic acid further comprises mitochondrial targeting sequences which facilitate nuclear import of said heterologous protein, or retention of said heterologous protein in the endoplasmic reticulum.

134. A method of making an allogenic mitochondria comprising a heterologous nucleic acid comprising a first region encoding a signal peptide operably linked to a second region encoding a heterologous protein, wherein expression of said nucleic acid permits secretion of said protein from said allogenic mitochondrion, and wherein said method comprises contacting a mitochondrion devoid of said heterologous protein with a heterologous plasmid comprising said heterologous nucleic acid under conditions that enable uptake of said heterologous plasmid by said mitochondrion to produce said allogenic mitochondrion.

135. A method of expressing a heterologous protein in a cell comprising: a) contacting a mitochondrion with a heterologous plasmid comprising a transgene comprising a first region encoding a signal peptide operably linked to a second region encoding said heterologous protein; b) isolating said mitochondrion comprising said plasmid, c) contacting said isolated mitochondrion with said cell under conditions that promote uptake of said isolated mitochondrion and expression of said transgene and secretion of said heterologous protein from said mitochondrion to said cell.

136. A method of making transgenic mitochondrion expressing a secretable heterologous protein comprising, a) providing a heterologous nucleic acid comprising a first region encoding a signal peptide operably linked to a second region encoding said heterologous protein, b) inserting said heterologous nucleic acid into a mitochondrial plasmid, c) contacting said mitochondrial plasmid comprising said heterologous nucleic acid with a mitochondrion of size less than 2pm in presence of a transfection agent to form a transgenic mitochondrion,d) isolating said transgenic mitochondrion comprising said mitochondrial plasmid and wherein said transgenic mitochondrion upon insertion into a cell is capable of expressing and secreting said heterologous protein.

137. The method of anyone of claims 135-136, wherein said plasmid comprises mitochondrial DNA.

138. The method of claim 137, wherein said plasmid comprises one or more promoters operative in mitochondrion and said promoter is capable of directing transcription of said nucleic acid.

139. The method of claim 137, wherein said plasmid comprises one or more mitochondrial transcriptional terminators.

140. The method of claim 137, wherein said plasmid comprises one or more transcript processing signals.

141. The method of claim 137, wherein said plasmid comprises native replication signals capable of propagating said plasmid within mitochondrion during mitogenesis.

142. The method of claim 137, wherein said plasmid comprises mitochondrial export sequence and said export sequence is capable of improving and / or increasing mitochondrial export of said protein.

143. The method of claim 138, wherein said promoter is a heavy strand promoter.

144. The method of claim 138, wherein said promoter is a light strand promoter.

145. The method of claim 143, wherein said heavy strand promoter is selected from the group consisting of HSP1 and HSP2.

146. The method of claim 144, wherein said light strand promoter is LSP1 or LSP2.

147. The method of claim 137, wherein said plasmid comprises origins of strand (Ori) replication, tRNA genes and displacement (D-loop) loop.

148. The method of claim 147, wherein said Ori are selected from the group consisting of OriL and OriH.

149. The method of claim 147, wherein said D-Loop comprises regulatory elements for transcription and replication of mitochondrial DNA.

150. The method of claim 147, wherein said heterologous nucleic acid is located downstream of heavy strand promoter (HSP1) and flanked by mitochondrial t-RNA-Val gene.

151. The method of claim 137, wherein said heterologous nucleic acid further encodes one or more of cell-penetrating peptides or homeoprotein internalization- secretion domains to facilitate export of said heterologous protein from mitochondrion.

152. The method of claim 137, wherein said heterologous nucleic acid further comprises mitochondrial targeting sequences which facilitate nuclear import of said heterologous protein, or retention of said heterologous protein in the endoplasmic reticulum.

153. The method of claim 137, wherein said heterologous nucleic acid encodes a plurality of heterologous protein.

154. The method of claim 153, wherein the total size of said heterologous nucleic acid is less than or equal to 16 kb.

155. The method of claim 137, wherein said mitochondrion comprises a vector comprising said nucleic acid.

156. The method of claim 155, wherein said vector comprises one or more promoters operative in mitochondrion and said promoter is capable of directing transcription of said nucleic acid.

157. The method of claim 155, wherein said vector comprises one or more mitochondrial transcriptional terminators.

158. The method of claim 155, wherein said vector comprises one or more transcript processing signals.

159. The method of claim 155, wherein said vector comprises native replication signals capable of propagating said plasmid within mitochondrion during mitogenesis.

160. The method of claim 155, wherein said vector comprises mitochondrion export sequences and said export sequence is capable of improving and / or increasing mitochondrial export of said protein.

161. The method of claim 156, wherein said promoter is a heavy strand promoter.

162. The method of claim 156, wherein said promoter is a light strand promoter.

163. The method of claim 161, wherein said heavy strand promoter is selected from the group consisting of HSP1 and HSP2.

164. The method of claim 162, wherein said light strand promoter is LSP1 or LSP2.

165. The method of claim 155, wherein said vector comprises origins of strand (Ori) replication, tRNA genes and displacement (D-loop) loop.

166. The method of claim 165, wherein said Ori are selected from the group consisting of OriL and OriH.

167. The method of claim 165, wherein said D-Loop comprises regulatory elements for transcription and replication of mitochondrial DNA.

168. The method of claim 165, wherein said heterologous nucleic acid is located downstream of heavy strand promoter (HSP1) and is optionally flanked by a mitochondrial t- RNA gene and / or flanked on one side by the OriL.

169. The method of claim 155, wherein said heterologous nucleic acid further encodes one or more of cell-penetrating peptides or homeoprotein internalization- secretion domains to facilitate export of said heterologous protein from mitochondrion.

170. The method of claim 155, wherein said heterologous nucleic acid further comprises mitochondrial targeting sequences which facilitate nuclear import of said heterologous protein, or retention of said heterologous protein in the endoplasmic reticulum.

171. The method of claim 155, wherein said vector comprises heterologous nucleic acid encoding a plurality of heterologous proteins.

172. The method of claim 156, wherein the size of said heterologous nucleic acid is less than or equal to 16 Kb.

173. The transgenic mitochondrion of claim 44, wherein said signal peptide is 8-50 amino acids in length.

174. The transgenic mitochondrion of claim 44, wherein said signal peptide is selected from the group consisting of IL-6 signal peptide, IL-2 signal peptide, Human OSM peptide, VSV-G peptide, Mouse Ig Kappa signal peptide, Mouse Ig Heavy signal peptide, BM40 signal peptide, Secrecon signal peptide, Human IgKVIII signal peptide, CD33 signal peptide, tPA signal peptide, Lipoic Acid Signal Peptide, Glycosylphosphatidylinositol (GPI) Anchor Signal Sequence, Mitochondrial Targeting Sequence (MTS), Bacterial Sec System Signal Sequence, Fibroblast Growth Factor (FGF) Signal Sequence, Serine Protease Inhibitor (SERPIN) Signal Sequence, Chitinase Signal Sequence and Erythropoietin (EPO) Signal Sequence.

175. The vector of claim 91, wherein said signal peptide is 8-50 amino acids in length.

176. The vector of claim 83, wherein said signal peptide is selected from the group consisting of IL-6 signal peptide, IL-2 signal peptide, Human OSM peptide, VSV-G peptide, Mouse Ig Kappa signal peptide, Mouse Ig Heavy signal peptide, BM40 signal peptide, Secrecon signal peptide, Human IgKVIII signal peptide, CD33 signal peptide, tPA signal peptide, Lipoic Acid Signal Peptide, Glycosylphosphatidylinositol (GPI) Anchor Signal Sequence, Mitochondrial Targeting Sequence (MTS), Bacterial Sec System Signal Sequence, Fibroblast Growth Factor (FGF) Signal Sequence, Serine Protease Inhibitor (SERPIN) Signal Sequence, Chitinase Signal Sequence and Erythropoietin (EPO) Signal Sequence.

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