Patent Publication Number: US-8541550-B2

Title: Methods and compositions for delivering polynucleotides

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of prior application U.S. Ser. No. 10/972,963 filed on Oct. 25, 2004, entitled “Methods and Compositions for Delivering Polynucleotides”, by Shaharyar Khan, now allowed U.S. Pat. No. 8,039,587, which claims benefit of and priority to U.S. Provisional Patent Application No. 60/513,983 filed on Oct. 24, 2003, and U.S. Provisional Patent Application No. 60/568,436 filed on May 5, 2004, all of which are herein incorporated by reference in their entirety. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
     This invention was made with Government Support under Agreement No. AG022780 awarded by the National Institutes of Health-National Institute on Aging Small Business Innovation Research (NIH-NIA SBIR). The Government has certain rights in the invention. 
    
    
     INCORPORATION BY REFERENCE 
     This application incorporates by reference in its entirety the sequence listing, filename 120701-2030.txt, on the accompanying computer readable medium. The file was created on or about Oct. 14, 2004, and is about 696 Kb. 
     BACKGROUND 
     1. Technical Field 
     The present disclosure is generally directed to compositions and methods for the delivery of polynucleotides, more particularly to compositions and methods for transfection, for example transfection of organelles. 
     2. Related Art 
     Many mitochondrial diseases have been described that arise from single homoplasmic mutations in mitochondrial DNA (mtDNA). These diseases typically affect non-mitotic tissues (brain, retina, muscle), present with variable phenotypes, can appear sporadically and are untreatable. Evolving evidence implicates mtDNA abnormalities in diseases such as Parkinson&#39;s and type II diabetes, but specific causal mutations for these conditions remain to be defined. Understanding the mtDNA genotype-phenotype relationships and developing specific treatment for mtDNA-based diseases is hampered by inability to manipulate the mitochondrial genome. 
     In the course of evolution, many organisms tackled the task of introducing macromolecules into living cells. Aside from the cell-specific, usually receptor-mediated or active uptake mechanisms, the general solution that has independently emerged in many lineages relies on peptides specifically evolved to interact with, and insert into lipid bilayer membranes. Thus, bacterial colicins, human porins, and protein transduction domains (PTD&#39;s) from diverse species share the motif of a positively charged alpha-helix, frequently with an amphipathic structure, which is capable of inserting into lipid membranes, and delivering larger cargoes intracellularly. Recent research reports confirm the successful use of PTD&#39;s fused to proteins for their delivery across biological boundaries, including the blood-brain barrier, and the placenta. 
     Another issue of great importance in the delivery of macromolecules in organisms is the need to protect them from proteolytic, nucleolytic and immune degradation and removal while traversing extracellular spaces. An often used approach is coating DNA with proteins capable of surviving the harsh journey to the target. Viral capsid proteins have been quite successful, yet for the purpose of DNA delivery in humans they suffer from a significant drawback—immunogenicity, the capacity to evoke a strong immune reaction greatly reducing the effectiveness of gene therapy. 
     Thus, there is a need for improved compositions and methods for the delivery of polynucleotides to the interior of cell. 
     SUMMARY 
     Non-viral polynucleotide delivery vehicles and methods of their use are provided. In general, the disclosure provides modified polynucleotide-binding proteins comprising a protein transduction domain operably linked to a targeting signal, for example a non-nuclear organelle targeting signal. One aspect provides a polypeptide comprising at least one HMG box domain, more typically at least two HMG box domains and at least one protein transduction domain. The polypeptide can associate with a polynucleotide causing the polynucleotide to condense. The polypeptide can also coat the polynucleotide. Coating and/or condensing the polynucleotide helps protect the polynucleotide from degradation. The protein transduction domain helps the polypeptide-polynucleotide complex cross membranes and enter the interior of cell or an organelle. The targeting signal helps direct the complex to a site of interest and thereby deliver the polynucleotide. 
     The disclosed compositions can be used to deliver polynucleotides to specific locations within a cell, including but not limited to mitochondria and chloroplasts. In some aspects, the polynucleotides encode a therapeutic protein or a protein that compensates for non-functional proteins or the absence of proteins. Accordingly, some aspects provide methods for treating diseases, for example diseases related to mitochondria or chloroplasts. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The patent or application file contains at least one drawing executed in color. Copies of this patent or application with color drawings will be provided by the Patent and Trademark Office upon request and payment of the appropriate fee. 
         FIG. 1  Is an diagram of one exemplary plasmid design (left) and exemplary protein structure (right) for TFAM with a PTD domain followed by a MLS. 
         FIG. 2  is a panel of fluorescence micrographs showing the time course of Alexa 488 Labeled mtDNA (green) complexed with PTD-MLS-TFAM added to Sy5y cells. Red=MitoTracker Red. 
         FIGS. 3A-C  are fluorescence micrographs showing MtRed and BrdU (FITC) staining of rho0 (A), normal SY5Y (B) and rho0 cells 16 hrs after transfection with mtDNA complexed with PTD-MLS-TFAM (C). 
         FIG. 4  is an agarose gel of PCR products amplifying region around LHON 11778A mutation after SfaN1. Lane 1-LHON Cybrid; Lane 2-Sy5y; Lane 3-Rho0; Lane 4-LHON mtDNA Transfected Rho0; Lane 5-Transfected Rho0 no DNA. 
         FIG. 5A  is fluorescence micrographs showing normal SY5Y cell 24 hours after transfection with mtDNA-MtEGFP cloned as a fusion gene with ND6 into the BamHI site of mtDNA and costained with MiitoTracker Red. 
         FIG. 5B  is an immunoblot for EGFP using JL-8 (Clontech) antibody showing a 45 kDa protein fusion product between ND6 and EGFP (top arrow) as well as the 33 kDa EGFP (bottom arrow). 
         FIG. 6  is an immunoblot of mtEGFP upon knockdown with siRNA to PoIG over five days. Final lane shows failure of EGFP siRNA to achieve knockdown in a similar time frame. 
         FIG. 7A  is an exemplary schematic of mtEGFP containing a nuclear localization signal (NLS) and PTD cloned into mtDNA containing novel restriction sites, BgIII and NotI. 
         FIGS. 7B-C  are fluorescence micrographs showing Sy5y cells expressing NLS-mtEGFP-PTD which localizes to mitochondria and the nucleus. (C) Cells are counterstained with Mito Tracker Red. 
         FIG. 8A  is an exemplary schematic of BamHI site abolished in mtDNA and BamHI cDNA cloned into mtDNA. 
         FIG. 8B  is a gel 24 hours after transfection showing PCR fragment of mtDNA from cells was digested with BamHI. Lane 1 failed to be digested and lane 2 control mtDNA was digested. 
         FIG. 8C  is a gel showing mitochondrial lysates from cells that were incubated with DNA containing a BamHI site. Control cells (lane 1) do not show BamHI activity whereas (lane 2) transfected cells possess BamHI activity. 
         FIG. 9  is a gel showing recombinant PTD-BgIII targeted to mitochondria caused the rapid removal of mtEGFP protein in cells expressing from a BgIII restriction site carrying mtDNA-mtEGFP over a 5 day time period. 
     
    
    
     DETAILED DESCRIPTION 
     1. Definitions 
     In describing and claiming the disclosed subject matter, the following terminology will be used in accordance with the definitions set forth below. 
     The term “polypeptides” includes proteins and fragments thereof. Polypeptides are disclosed herein as amino acid residue sequences. Those sequences are written left to right in the direction from the amino to the carboxy terminus. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gln, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Val, V). 
     “Variant” refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide, but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. 
     Modifications and changes can be made in the structure of the polypeptides of in disclosure and still obtain a molecule having similar characteristics as the polypeptide (e.g., a conservative amino acid substitution). For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide&#39;s biological functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence and nevertheless obtain a polypeptide with like properties. 
     In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a polypeptide is generally understood in the art. It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still result in a polypeptide with similar biological activity. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. Those indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5). 
     It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant polypeptide, which in turn defines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and the like. It is known in the art that an amino acid can be substituted by another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. 
     Substitution of like amino acids can also be made on the basis of hydrophilicity, particularly, where the biological functional equivalent polypeptide or peptide thereby created is intended for use in immunological embodiments. The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamnine (+0.2); glycine (0); proline (−0.5±1); threonine (−0.4); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent polypeptide. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. 
     As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gln, His), (Asp: Glu, Cys, Ser), (Gln: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gln), (Ile: Leu, Val), (Leu: Ile, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Val: Ile, Leu). Embodiments of this disclosure thus contemplate functional or biological equivalents of a polypeptide as set forth above. In particular, embodiments of the polypeptides can include variants having about 50%, 60%, 70%, 80%, 90%, and 95% sequence identity to the polypeptide of interest. 
     “Identity,” as known in the art, is a relationship between two or more polypeptide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide as determined by the match between strings of such sequences. “Identity” and “similarity” can be readily calculated by known methods, including, but not limited to, those described in (Computational Molecular Biology, Lesk, A. M., Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., Ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., Eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., Eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J Applied Math., 48: 1073 (1988). 
     Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. The percent identity between two sequences can be determined by using analysis software (i.e., Sequence Analysis Software Package of the Genetics Computer Group, Madison Wis.) that incorporates the Needelman and Wunsch, (J. Mol. Biol., 48: 443-453, 1970) algorithm (e.g., NBLAST, and XBLAST). The default parameters are used to determine the identity for the polypeptides of the present disclosure. 
     By way of example, a polypeptide sequence may be identical to the reference sequence, that is be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the % identity is less than 100%. Such alterations are selected from: at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence. The number of amino acid alterations for a given % identity is determined by multiplying the total number of amino acids in the reference polypeptide by the numerical percent of the respective percent identity (divided by 100) and then subtracting that product from said total number of amino acids in the reference polypeptide. 
     As used herein, the term “low stringency” refers to conditions that permit a polynucleotide or polypeptide to bind to another substance with little or no sequence specificity. 
     As used herein, the term “purified” and like terms relate to the isolation of a molecule or compound in a form that is substantially free (at least 60% free, preferably 75% free, and most preferably 90% free) from other components normally associated with the molecule or compound in a native environment. 
     As used herein, the term “pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water and emulsions such as an oil/water or water/oil emulsion, and various types of wetting agents. 
     As used herein, the term “treating” includes alleviating the symptoms associated with a specific disorder or condition and/or preventing or eliminating said symptoms. 
     “Operably linked” refers to a juxtaposition wherein the components are configured so as to perform their usual function. For example, control sequences or promoters operably linked to a coding sequence are capable of effecting the expression of the coding sequence, and an organelle localization sequence operably linked to protein will direct the linked protein to be localized at the specific organelle. 
     “Localization Signal or Sequence or Domain” or “Targeting Signal or Sequence or Domain” are used interchangeably and refer to a signal that directs a molecule to a specific cell, tissue, organelle, or intracellular region. The signal can be polynucleotide, polypeptide, or carbohydrate moiety or can be an organic or inorganic compound sufficient to direct an attached molecule to a desired location. Exemplary organelle localization signals include nuclear localization signals known in the art and other organelle localization signals known in the art such as those provided in Tables 1 and 2 and described in Emanuelson et al., Predicting Subcellular Localization of Proteins Based on Their N-terminal Amino Acid Sequence.  Journal of Molecular Biology.  300(4):1005-16, 2000 Jul. 21, and in Cline and Henry, Import and Routing of Nucleus-encoded Chloroplast Proteins.  Annual Review of Cell  &amp;  Developmental Biology.  12:1-26, 1996, the disclosures of which are incorporated herein by reference in their entirety. It will be appreciated that the entire sequence listed in Tables 1 and 2 need not be included, and modifications including truncations of these sequences are within the scope of the disclosure provided the sequences operate to direct a linked molecule to a specific organelle. Organelle localization signals of the present disclosure can have 80 to 100% homology to the sequences in Tables 1 and 2. One class of suitable organelle localization signals include those that do not interact with the targeted organelle in a receptor:ligand mechanism. For example, organelle localization signals include signals having or conferring a net charge, for example a positive charge. Positively charged signals can be used to target negatively charged organelles such as the mitochondria. Negatively charged signals can be used to target positively charged organelles. 
     “Protein Transduction Domain” or PTD refers to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compounds that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD attached to another molecule facilitates the molecule traversing membranes, for example going from extracellular space to intracellular space, or cytosol to within an organelle. Exemplary PTDs include but are not limited to HIV TAT YGRKKRRQRRR (SEQ. ID NO. 1) or RKKRRQRRR (SEQ. ID NO. 2); 11 Arginine residues, or positively charged polypeptides or polynucleotides having 8-15 residues, preferably 9-11 residues. 
     As used herein, the term “exogenous DNA” or “exogenous nucleic acid sequence” or “exogenous polynucleotide” refers to a nucleic acid sequence that was introduced into a cell or organelle from an external source. Typically the introduced exogenous sequence is a recombinant sequence. 
     As used herein, the term “transfection” refers to the introduction of a nucleic acid sequence into the interior of a membrane enclosed space of a living cell, including introduction of the nucleic acid sequence into the cytosol of a cell as well as the interior space of a mitochondria, nucleus or chloroplast. The nucleic acid may be in the form of naked DNA or RNA, associated with various proteins or the nucleic acid may be incorporated into a vector. 
     As used herein, the term “vector” is used in reference to a vehicle used to introduce a nucleic acid sequence into a cell. A viral vector is virus that has been modified to allow recombinant DNA sequences to be introduced into host cells or cell organelles. 
     As used herein, the term “organelle” refers to cellular membrane bound structures such as the chloroplast, mitochondrion, and nucleus. The term “organelle” includes natural and synthetic organelles. 
     As used herein, the term “non-nuclear organelle” refers to any cellular membrane bound structure present in a cell, except the nucleus. 
     As used herein, the term “polynucleotide” generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Thus, for instance, polynucleotides as used herein refers to, among others, single-and double-stranded DNA, DNA that is a mixture of single-and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. The term “nucleic acid” or “nucleic acid sequence” also encompasses a polynucleotide as defined above. 
     In addition, polynucleotide as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. 
     As used herein, the term polynucleotide includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are “polynucleotides” as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. 
     It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia. 
     “Oligonucleotide(s)” refers to relatively short polynucleotides. Often the term refers to single-stranded deoxyribonucleotides, but it can refer as well to single-or double-stranded ribonucleotides, RNA:DNA hybrids and double-stranded DNAs, among others. 
     2. Modified Polynucleotide Binding or Polynucleotide-Packaging Polypeptides 
     A. Polynucleotide Binding Domain 
     The compositions and methods for the delivery of a polynucleotide provided herein include polynucleotide-binding polypeptides or polynucleotide-packaging polypeptides having a PTD and optionally a targeting signal or domain. The modified or recombinant polypeptide can be any polypeptide known to bind or package a polynucleotide. The recombinant polypeptide can be used as therapeutic agent either alone or in combination with a polynucleotide. In one embodiment, the polynucleotide-binding polypeptide includes at least a portion of a member of the high mobility group (HMG) of proteins, in particular at least one HMG box domain . Generally, the HMG domain includes a global fold of three helices stabilized in an ‘L-shaped’ configuration by two hydrophobic cores. The high mobility group chromosomal proteins HMG1 or HMG2, which are common to all eukaryotes, bind DNA in a non-sequence-specific fashion, for example to promote chromatin function and gene regulation. They can interact directly with nucleosomes and are believed to be modulators of chromatin structure. They are also important in activating a number of regulators of gene expression, including p53, Hox transcription factors and steroid hormone receptors, by increasing their affinity for DNA. HMG proteins include HMG-1/2, HMG-I(Y) and HMG-14/17. 
     The HMG-1/2-box proteins can be further distinguished into three subfamilies according to the number of HMG domains present in the protein, their specific of sequence recognition and their evolutionary relationship. The first group contains chromosomal proteins bound to DNA with no sequence specificity (class I, HMG1 and HMG2), the second ribosomal and mitochondrial transcription factors which show sequence specificity in the presence of another associating factor when bound with DNA (class II, yeast ARS binding protein ABF-2, UBF and mitochondrial transcription factor mtTF-1), and the third gene-specific transcription factors which show sequence specific DNA binding (class III, lymphoid enhancer-binding factors LEF-1 and TCF-1; the mammalian sex-determining factor SRY, and the closely related SOX proteins; and the fungal regulatory proteins Mat-MC, Mat-a1, Ste11 and Rox1). The HMG1/2-box DNA binding domain is about 75 amino acid and contains highly conserved proline, aromatic and basic residues. Common properties of HMG domain proteins include interaction with the minor groove of the DNA helix, binding to irregular DNA structure, and the capacity to modulate DNA structure by bending. 
     SOX (SRY-type HMG box) proteins have critical functions in a number of developmental processes, including sex determination, skeleton formation, pre-B and T cell development and neural induction. SOX9 plays a direct role during chondrogenesis by binding and activating the chondrocyte-spacific enhancer of the Col2a1 gene. Loss of SOX9 gene function leads to the genetic condition known as Campomelic Dysplsia (CD), a form of dwarfism characterized by extreme skeletal malformation, and one in which three-quarters of XY individual are either intersexes or exhibit male to female sex reversal. There are more than 20 members cloned in SOX family. All of which contain an HMG domain, which can bind specifically to the double strand DNA motif and shares &gt;50% identify with the HMG domain of SRY, the human testis-determining factor. The preferred DNA-binding site of SOX9 have been defined to be AGAACAATGG, which contains the SOX core-binding element (SCBE), AACAAT, flanking 5′ AG and 3′ GG nucleotides enhance binding by SOX9. 
     In one embodiment, the recombinant polynucleotide-binding protein has at least one HMG box domain, generally at least two, more particularly 2-5 HMG box domains. The HMG box domain can bind to an AT rich DNA sequence, for example, using a large surface on the concave face of the protein, to bind the minor groove of the DNA. This binding bends the DNA helix axis away from the site of contact. The first and second helices contact the DNA, their N-termini fitting into the minor groove whereas helix 3 is primarily exposed to solvent. Partial intercalation of aliphatic and aromatic residues in helix 2 occurs in the minor groove. 
     In other embodiments, the polynucleotide binding polypeptide can have at least one polynucleotide binding domain, typically two or more polynucleotide binding domains. Representative polynucleotide binding domains known in the art include, but are not limited to helix-turn-helix motifs including homeodomains and POU domains; zinc finger domains such as C 2 H 2  and C 2 C 2 ; amphipathic helix domains such as leucine zipper and helix-loop-helix domains; and histone folds. The polynucleotide binding domain can be specific for a specific polynucleotide sequence, or preferably non-specifically binds to a polynucleotide. Alternatively, the polynucleotide-binding domain can have more than one type of polynucleotide binding domain. 
     Certain embodiments provide modified polynucleotide-binding polypeptides having a helix-turn-helix motif or at least a polynucleotide binding region of a helix-turn-helix protein. Helix-turn-helix proteins have a similar structure to bacterial regulatory proteins such as the I repressor and cro proteins, the lac repressor and so on which bind as dimers and their binding sites are palindromic. They contain 3 a helical regions separated by short turns which is why they are called helix-turn-helix proteins. One protein helix (helix 3) in each subunit of the dimer occupies the major groove of two successive turns of the DNA helix. Thus, in another embodiment, the disclosed polynucleotide-binding polypeptides can form dimers or other multi-component complexes, and have 1 to 3 helices. 
     In yet another embodiment, the modified polynucleotide-binding polypeptide includes a homeodomain or a portion of a homeodomain protein. Homeodomain proteins bind to a sequence of 180 base pairs initially identified in a group of genes called homeotic genes. Accordingly, the sequence was called the homeobox. The 180 by corresponds to 60 amino acids in the corresponding protein. This protein domain is called the homeodomain. Homeodomain-containing proteins have since been identified in a wide range of organisms including vertebrates and plants. The homeodomain shows a high degree of sequence conservation. The homeodomain contains 4 α helical regions. Helices II and III are connected by 3 amino acids comprising a turn. This region has a very similar structure to helices II and III of bacterial DNA binding proteins. 
     Yet another embodiment provides a modified polynucleotide-binding polypeptide having a zinc finger domain or at least a portion of a zinc finger protein. Zinc finger proteins have a domain with the general structure: Phe (sometimes Tyr)-Cys-2 to 4 amino acids-Cys-3 amino acids-Phe (sometimes Tyr)-5 amino acids-Leu-2 amino acids-His-3 amino acids-His. The phenylalanine or tyrosine residues which occur at invariant positions are required for DNA binding. Similar sequences have been found in a range of other DNA binding proteins though the number of fingers varies. For example, the SP1 transcription factor which binds to the GC box found in the promoter proximal region of a number of genes has 3 fingers. This type of zinc finger which has 2 cysteines and 2 histidines is called a C 2 H 2  zinc finger. 
     Another type of zinc finger which binds zinc between 2 pairs of cysteines has been found in a range of DNA binding proteins. The general structure of this type of zinc finger is: Cys-2 amino acids-Cys-13 amino acids-Cys-2 amino acids-Cys. This is called a C 2 C 2  zinc finger. It is found in a group of proteins known as the steroid receptor superfamily, each of which has 2 C 2 C 2  zinc fingers. 
     Another embodiment provides a modified polynucleotide-binding polypeptide having a leucine zipper or at least a portion of a leucine zipper protein. The first leucine zipper protein was identified from extracts of liver cells, and it was called C/EBP because it is an enhancer binding protein and it was originally thought to bind to the CAAT promoter proximal sequence. C/EBP will only bind to DNA as a dimer. The region of the protein where the two monomers join to make the dimer is called the dimerization domain. This lies towards the C-terminal end of the protein. When the amino acid sequence was examined it was found that a leucine residue occurs every seventh amino acid over a stretch of 35 amino acids. If this region were to form an a helix then all of these leucines would align on one face of the helix. 
     Because leucine has a hydrophobic side chain, one face of the helix is very hydrophobic. The opposite face has amino acids with charged side chains which are hydrophilic. The combination of hydrophobic and hydrophilic characteristics gives the molecule is amphipathic moniker. Adjacent to the leucine zipper region is a region of 20-30 amino acids which is rich in the basic (positively charged) amino acids lysine and arginine. This is the DNA binding domain—often referred to as the bZIP domain—the basic region of the leucine zipper. C/EBP is thought to bind to DNA by these bZIP regions wrapping round the DNA helix 
     The leucine zipper—bZIP structure has been found in a range of other proteins including the products of the jun and fos oncogenes. Whereas C/EBP binds to DNA as a homodimer of identical subunits, fos cannot form homodimers at all and jun/jun homodimers tend to be unstable. However fos/jun heterodimers are much more stable. These fos/jun heterodimers correspond to a general transcription factor called AP1 which binds to a variety of promoters and enhancers and activates transcription. The consensus AP1 binding site is TGACTCA (SEQ. ID. NO.: 3) which is palindromic. 
     Another embodiment provides a modified polynucleotide-binding polypeptide having helix-loop-helix domain or a polynucleotide binding portion of a helix-loop-helix protein. Helix-loop-helix proteins are similar to leucine zippers in that they form dimers via amphipathic helices. They were first discovered as a class of proteins when a region of similarity was noticed between two enhancer binding proteins called E47 and E12. This conserved region has the potential to form two amphipathic separated by a loop hence helix-loop-helix. Next to the dimerization domain is a DNA binding domain, again rich in basic amino acids and referred to as the bHLH domain. These structures are also found in a number of genes required for development of the Drosophila nervous system—the Achaete-scute complex, and in a protein called MyoD which is required for mammalian muscle differentiation. 
     In still another embodiment, the modified polynucleotide binding polypeptide includes a histone polypeptide, a fragment of a histone polypeptide, or at least one histone fold. Histone folds exist in histone polypeptides monomers assembled into dimers. Histone polypeptides include H2A, H2B, H3, and H4 which can form heterodimers H2A-2B and H3-H4. It will be appreciated that histone-like polypeptides can also be used in the disclosed compositions and methods. Histone-like polypeptides include, but are not limited to, HMf or the histone from  Methanothermous fervidus , other archaeal histones known in the art, and histone-fold containing polypeptides such as MJ1647, CBF, TAFII or transcription factor IID, SPT3, and Dr1-DRAP (Sanderman, K. et al. (1998) CMLS. Cell. Mol. Life Sci. 54:1350-1364, which is incorporated by reference in its entirety). 
     One embodiment, among others, provides a modified mitochondrial transcription factor A (TFAM) polypeptide. TFAM is a member of the high mobility group (HMG) of proteins having two HMG-box domains. TFAM as well as other HMG proteins bind, wrap, bend, and unwind DNA. Thus, embodiments of the present disclosure include polypeptides that induce a structural change in the polynucleotide when the polypeptide binds or becomes associated with the polynucleotide. By inducing a conformational change in the polynucleotide, the polypeptide packages the polynucleotide. It has been reported that TFAM binds to mitochondrial DNA in a ratio of 900:1 (Alam, T. I. et al. (2003) Nucleic Acid Res. 31(6):1640-1645). It will be appreciated that the amount of polynucleotide-binding polypeptide used in the compositions and methods disclosed herein can vary depending on the size and amount of the polynucleotide to be delivered. Suitable ratios of polynucleotide-binding polypeptide to base pairs of polynucleotide to be delivered include, but are not limited to, about 1:1 to 1:1,000; more preferably 1:100; even more preferably 1: about 10 to about 20 base pairs of polynucleotide to be delivered. It will also be appreciated that TFAM, another polynucleotide-binding polypeptide, or a combination of two or more polynucleotide-binding polypeptides can be added to a polynucleotide to wrap or cover the polynucleotide, and thereby package the polynucleotide and protected it from degradation. 
     TFAM can be modified to include a PTD and optionally a targeting signal. The targeting signal can include a sequence of monomers that facilitates the localization of the molecule to a specific tissue, cell, or organelle. The monomers can be amino acids, nucleotide or nucleoside bases, or sugar groups such as glucose, galactose, and the like which form carbohydrate targeting signals. 
     B. Protein Transduction Domain 
     The polynucleotide-binding polypeptide can be modified to include a protein transduction domain (PTD), also known as cell penetrating peptides (CPPS). PTDs are known in the art, and include but are not limited to small regions of proteins that are able to cross a cell membrane in a receptor-independent mechanism (Kabouridis, P. (2003) Trends in Biotechnology (11):498-503). Although several of PTDs have been documented, the two most commonly employed PTDs are derived from TAT (Frankel and Pabo, 1988) protein of HIV and Antennapedia transcription factor from Drosophila, whose PTD is known as Penetratin (Derossi et al., (1994) J Biol Chem. 269(14):10444-50). 
     The Antennapedia homeodomain is 68 amino acid residues long and contains four alpha helices (SEQ. ID NO. 4). Penetratin is an active domain of this protein which consists of a 16 amino acid sequence derived from the third helix of Antennapedia.(Fenton et al., 1998) TAT protein (SEQ. ID NO. 5) consists of 86 amino acids and is involved in the replication of HIV-1. The TAT PTD consists of an 11 amino acid sequence domain (residues 47 to 57; YGRKKRRQRRR (SEQ. ID. NO. 1)) of the parent protein that appears to be critical for uptake (Vives et al., 1997). Additionally, the basic domain Tat(49-57) or RKKRRQRRR (SEQ. ID NO. 2) (Wender et al. 2000) has been shown to be a PTD. In the current literature TAT has been favored for fusion to proteins of interest for cellular import. Several modifications to TAT, including substitutions of Glutatmine to Alanine, i.e., Q→A, have demonstrated an increase in cellular uptake anywhere from 90% (Wender et al. 2000) to up to 33 fold in mammalian cells. (Ho et al. (2001) Cancer Res. 61(2):474-7) The most efficient uptake of modified proteins was revealed by mutagenesis experiments of TAT-PTD, showing that an 11 arginine stretch was several orders of magnitude more efficient as an intercellular delivery vehicle. Thus, some embodiments include PTDs that are cationic or amphipathic. Additionally exemplary PTDs include but are not limited to poly-Arg-RRRRRRR (SEQ. ID. NO.: 6); PTD-5-RRQRRTSKLMKR (SEQ. ID. NO.: 7); Transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ. ID. NO.: 8); KALA-WEAKLAKALAKALAKHLAKALAKALKCEA (SEQ. ID. NO.: 9); and RQIKIWFQNRRMKWKK (SEQ. ID. NO.: 207). 
     In some embodiments, the protein transduction domain is YGRKKRRQRRR (SEQ. ID. NO.: 1), RKKRRQRRR (SEQ. ID. NO. 2), amino acids 48-60 of the HIV-1 TAT protein, the third helix of Antennapedia homeodomain, RQIKIWFQNRRMKWKK (SEQ. ID. NO.: 207), a poly-arginine region, about 7 arginine residues, RRQRRTSKLMKR (SEQ. ID. NO.: 7), GWTLNSAGYLLGKINLKALAALAKKIL (SEQ. ID. NO.: 8), WEAKLAKALAKALAKHLAKALAKALKACEA (SEQ. ID. NO.: 9), a fragment thereof, or a combination thereof. 
     C. Targeting Signal or Domain 
     In still other embodiments, the modified polynucleotide-binding polypeptide is optionally modified to include a targeting signal or domain. The targeting signal or sequence can be specific for a tissue, organ, cell, organelle, non-nuclear organelle, or cellular compartment. For example, the compositions disclosed herein can be modified with galactosyl-terminating macromolecules to target the compositions to the liver or to liver cells. The modified compositions selectively enter hepatocytes after interaction of the carrier galactose residues with the asialoglycoprotein receptor present in large amounts and high affinity only on these cells. Moreover, the compositions disclosed here can be targeted to specific intracellular regions, compartments, or organelles. 
     Additional embodiments of the present disclosure are directed to specifically delivering polynucleotides to intracellular compartments or organelles. The polynucleotides can encode a polypeptide or interfere with the expression of a different polynucleotide. Eukaryotic cells contain membrane bound structures or organelles. Organelles can have single or multiple membranes and exist in both plant and animal cells. Depending on the function of the organelle, the organelle can consist of specific components such as proteins and cofactors. The polynucleotides delivered to the organelle can encode polypeptides that can enhance or contribute to the functioning of the organelle. Some organelles, such as mitochondria and chloroplasts, contain their own genome. Nucleic acids are replicated, transcribed, and translated within these organelles. Proteins are imported and metabolites are exported. Thus, there is an exchange of material across the membranes of organelles. In some embodiments, polynucleotides encoding mitochondrial polypeptides are specifically delivered to mitochondria. 
     Exemplary organelles include the nucleus, mitochondrion, chloroplast, lysosome, peroxisome, Golgi, endoplasmic reticulum, and nucleolus. Synthetic organelles can be formed from lipids and can contain specific proteins within the lipid membranes. Additionally, the content of synthetic organelles can be manipulated to contain components for the translation of nucleic acids. 
     1. Nuclear Localization Signals 
     Compositions disclosed herein can include one or more nuclear localization signals. Nuclear localization signals (NLS) or domains are known in the art and include for example, SV 40 T antigen or a fragment thereof, such as PKKKRKV (SEQ. ID. NO.: 10). The NLS can be simple cationic sequences of about 4 to about 8 amino acids, or can be bipartite having two interdependent positively charged clusters separated by a mutation resistant linker region of about 10-12 amino acids. Additional representative NLS include but are not limited to GKKRSKV (SEQ. ID. NO.: 11); KSRKRKL (SEQ. ID. NO.: 12); KRPAATKKAGQAKKKKLDK (SEQ. ID. NO.: 13); RKKRKTEEESPLKDKAKKSK (SEQ. ID. NO.: 14); KDCVMNKHHRNRCQYCRLQR (SEQ. ID. NO.: 15); PAAKRVKLD (SEQ. ID. NO.: 16); and KKYENWIKRSPRKRGRPRK (SEQ. ID. NO.: 17). 
     2. Mitochondria Targeting 
     In other embodiments of the present disclosure, modified polynucleotide-binding polypeptides are disclosed that specifically deliver polynucleotides to mitochondria. Mitochondria contain the molecular machinery for the conversion of energy from the breakdown of glucose into adenosine triphosphate (ATP). The energy stored in the high energy phosphate bonds of ATP is then available to power cellular functions. Mitochondria are mostly protein, but some lipid, DNA and RNA are present. These generally spherical organelles have an outer membrane surrounding an inner membrane that folds (cristae) into a scaffolding for oxidative phosphorylation and electron transport enzymes. Most mitochondria have flat shelf-like cristae, but those in steroid secreting cells may have tubular cristae. The mitochondrial matrix contains the enzymes of the citric acid cycle, fatty acid oxidation and mitochondrial nucleic acids. 
     Mitochondrial DNA is double stranded and circular. Mitochondrial RNA comes in the three standard varieties; ribosomal, messenger and transfer, but each is specific to the mitochondria. Some protein synthesis occurs in the mitochondria on mitochondrial ribosomes that are different than cytoplasmic ribosomes. Other mitochondrial proteins are made on cytoplasmic ribosomes with a signal peptide that directs them to the mitochondria. The metabolic activity of the cell is related to the number of cristae and the number of mitochondria within a cell. Cells with high metabolic activity, such as heart muscle, have many well developed mitochondria. New mitochondria are formed from preexisting mitochondria when they grow and divide. 
     The inner membranes of mitochondria contain a family of proteins of related sequence and structure that transport various metabolites across the membrane. Their amino acid sequences have a tripartite structure, made up of three related sequences about 100 amino acids in length. The repeats of one carrier are related to those present in the others and several characteristic sequence features are conserved throughout the family. 
     Targeting to specific polynucleotides to organelles can be accomplished by modifying the disclosed compositions to express specific organelle targeting signals. These sequences target specific organelles, but in some embodiments the interaction of the targeting signal with the organelle does not occur through a traditional receptor:ligand interaction. The eukaryotic cell comprises a number of discrete membrane bound compartments, or organelles. The structure and function of each organelle is largely determined by its unique complement of constituent polypeptides. However, the vast majority of these polypeptides begin their synthesis in the cytoplasm. Thus organelle biogenesis and upkeep require that newly synthesized proteins can be accurately targeted to their appropriate compartment. This is often accomplished by amino-terminal signaling sequences, as well as post-translational modifications and secondary structure. For mitochondria, several amino-terminal targeting signals have been deduced. Genes and proteins having mitochondrial localization signals are included, in part, in Table 1. 
     In one embodiment, the organelle targeting signal can contain at least two, preferably 5-15, most preferably about 11 charged groups, causing the targeting signal to be drawn to organelles having a net opposite charge. In another embodiment, the targeting signal can contain a series of charged groups that cause the targeting signal to be transported into an organelle either against or down an electromagnetic potential gradient. Suitable charged groups are groups that are charged under intracellular conditions such as amino acids with charged functional groups, amino groups, nucleic acids, and the like. Mitochondrial localization/targeting signals generally consist of a leader sequence of highly positively charged amino acids. This allows the protein to be targeted to the highly negatively charged mitochondria. Unlike receptor:ligand approaches that rely upon stochastic Brownian motion for the ligand to approach the receptor, the mitochondrial localization signal of some embodiments is drawn to mitochondria because of charge. 
     In order to enter the mitochondria, a protein generally must interact with the mitochondrial import machinery, consisting of the Tim and Tom complexes (Translocase of the Inner/Outer Mitochondrial Membrane). With regard to the mitochondrial targeting signal, the positive charge draws the linked protein to the complexes and continues to draw the protein into the mitochondria. The Tim and Tom complexes allow the proteins to cross the membranes. Accordingly, one embodiment of the present disclosure delivers compositions of the present disclosure to the inner mitochondrial space utilizing a positively charged targeting signal and the mitochondrial import machinery. In another embodiment, PTD-linked polypeptides containing a mitochondrial localization signal do not seem to utilize the TOM/TIM complex for entry into the mitochondrial matrix, see Del Gaizo et al. (2003) Mol Genet Metab. 80(1-2):170-80. 
     Given the importance of mitochondria in human disease, cell proliferation, cell death, and aging, embodiments of the present disclosure also encompasses the manipulation of the mitochondrial genome to supply the means by which known mitochondrial diseases (LHON, MELAS, etc.) and putative mitochondrial diseases (aging, Alzheimer&#39;s, Parkinson&#39;s, Diabetes, Heart Disease) can be treated. 
     3. Chloroplast Targeting 
     In another embodiment, modified compositions disclosed herein specifically deliver polynucleotides to chloroplasts by including a chloroplast localization signal or domain. For chloroplasts, several amino-terminal targeting signals have been deduced and are included, in part, in Table 2. The chloroplast is a photosynthetic organelle in eukaryotes with a double surrounding membrane. The fluid inside the double-membrane is called the stroma. The chloroplast has a nucleoid region to house its circular, naked DNA. The stroma is also the site of the Calvin Cycle. The Calvin Cycle is the series of enzyme-catalyzed chemical reactions that produce carbohydrates and other compounds from carbon dioxide. 
     Within the stroma are tiny membrane sacs called thylakoids. The sacs are stacked in groups. Each group is called a granum. There are many grana in each chloroplast. The thylakoid membranes are the site of photosynthetic light reactions. The thylakoids have intrinsic and extrinsic proteins, some with special prosthetic groups, allowing for electrons to be moved from protein complex to protein complex. These proteins constitute an electron transport system sometimes known as the Z-scheme. 
     The prosthetic group for two critical membrane proteins (P680 and P700) is a chlorophyll a pigment molecule. These chlorophyll-binding proteins give the thylakoids an intense green color. The many thylakoids in a chloroplast give the chloroplast a green color. The many chloroplasts in a leaf mesophyll cell give that cell a green color. The many mesophyll cells in a leaf give the leaf a green color. The chlorophyll molecule absorbs light energy and an electron is boosted within the electron cloud in a resonating chemical structure surrounding a magnesium ion. This excited electron is removed by the surrounding electron transport proteins in the membrane. The movement of these electrons, and accompanying protons, results ultimately in the trapping of energy in a phosphate bond in ATP. The thylakoid is thus the location for light absorption and ATP synthesis. The stroma uses the ATP to store the trapped energy in carbon-carbon bonds of carbohydrates. Some chloroplasts show developing starch grains. These represent complex polymers of carbohydrates for long-term storage. 
     Given the bioenergetic functions of chloroplasts, the ability to introduce exogenous genes may lead to plants with increased viability in otherwise hostile environments and increased efficiency of photosynthesis. Furthermore, the expression of exogenous genes within the chloroplasts is believed to be significantly more efficient in chloroplasts relative the expression of exogenous genes introduced into the nucleus of the cell. Thus, other embodiments are directed to the transfection of chloroplasts for more effective biosynthesis strategies for commercial compounds. 
     3. Modified Polynucleotide-Binding Polypeptide: Polynucleotide Complexes 
     Modified polynucleotide-binding polypeptides having a protein transduction domain, and optionally, a targeting signal can be combined with a polynucleotide of interest to form a polypeptide-polynucleotide complex. For example, the modified polypeptide can reversibly bind the polynucleotide of interest. The binding or interaction between the modified polypeptide and the polynucleotide of interest is strong enough to protect the polynucleotide from degradation but reversible so that the polynucleotide maintains its biological activity once it has been delivered to the cell or organelle. The biological activity of the polynucleotide can include expressing the polypeptide encoded by the polynucleotide or the enzymatic activity of the polynucleotide if it is a ribozyme or DNAzyme. 
     Another embodiment provides a method for transfecting a non-nuclear organelle by combining a polynucleotide-binding polypeptide, for example TFAM, with a polynucleotide to be delivered and an amount of a lipid and/or polyamine to form a complex and contacting a cell, for example a mammalian cell, with the complex . The polynucleotide-binding protein optionally includes a PTD and optionally a targeting signal. The lipid and/or polyamine can be branched or unbranched, saturated or unsaturated, and typically has a carbon chain length of about 6 to about 50 carbons, more typically about 10 to about 30 carbons, even more typically about 15 to about 20 carbons. A nuclease can also be delivered to the non-nuclear organelle. The nuclease can be selected so that it cleaves endogenous nucleic acids, but does not cleave the heterologous nucleic acids that are introduced into the non-nuclear organelle. Alternatively, a transfected non-nuclear organelle, for example a mitochondrion, can be have a nuclease delivered to it wherein the nuclease is selected so that it cleaves the transfected nucleic acids or the heterologous nucleic acids in the non-nuclear organelle. 
     In one embodiment, the polynucleotide of interest is operably linked to a promoter or other regulatory elements known in the art. Thus, the polynucleotide can be a vector such as an expression vector. The engineering of polynucleotides for expression in a prokaryotic or eukaryotic system may be performed by techniques generally known to those of skill in recombinant expression. It is believed that virtually any expression system may be employed in the expression of the disclosed nucleic and amino sequences. 
     An expression vector typically comprises one of the disclosed compositions under the control of one or more promoters. To bring a coding sequence “under the control of ”a promoter, one positions the 5′ end of the translational initiation site of the reading frame generally between about 1 and 50 nucleotides “downstream” of (i.e., 3′ of) the chosen promoter. The “upstream” promoter stimulates transcription of the inserted DNA and promotes expression of the encoded recombinant protein. This is the meaning of “recombinant expression” in the context used here. 
     Many standard techniques are available to construct expression vectors containing the appropriate nucleic acids and transcriptional/translational control sequences in order to achieve protein or peptide expression in a variety of host-expression systems. Cell types available for expression include, but are not limited to, bacteria, such as  E. coli  and  B. subtilis  transformed with recombinant phage DNA, plasmid DNA or cosmid DNA expression vectors. It will be appreciated that any of these vectors may be packaged and delivered using one or more of the disclosed polynucleotide packaging polypeptides. 
     Expression vectors for use in mammalian cells ordinarily include an origin of replication (as necessary), a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences. The origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV) source, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient. 
     The promoters may be derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Further, it is also possible, and may be desirable, to utilize promoter or control sequences normally associated with the desired gene sequence, provided such control sequences are compatible with the host cell systems. 
     A number of viral based expression systems may be utilized, for example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40 (SV40). The early and late promoters of SV40 virus are useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication. Smaller or larger SV40 fragments may also be used, provided there is included the approximately 250 by sequence extending from the Hindil site toward the BgII site located in the viral origin of replication. 
     In cases where an adenovirus is used as an expression vector, the coding sequences may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing proteins in infected hosts. 
     Specific initiation signals may also be required for efficient translation of the disclosed compositions. These signals include the ATG initiation codon and adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may additionally need to be provided. One of ordinary skill in the art would readily be capable of determining this need and providing the necessary signals. It is well known that the initiation codon must be in-frame (or in-phase) with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements or transcription terminators. 
     In eukaryotic expression, one will also typically desire to incorporate into the transcriptional unit an appropriate polyadenylation site if one was not contained within the original cloned segment. Typically, the poly A addition site is placed about 30 to 2000 nucleotides “downstream” of the termination site of the protein at a position prior to transcription termination. 
     For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express constructs encoding proteins may be engineered. Rather than using expression vectors that contain viral origins of replication, host cells can be transformed with vectors controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched medium, and then are switched to a selective medium. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci, which in turn can be cloned and expanded into cell lines. 
     One embodiment provides a modified TFAM polypeptide having at least one PTD, and optionally, at least one targeting signal, for example, a nuclear localization signal or mitochondrial localization signal. The modified TFAM can be associated with a polynucleotide of interest. The association can be accomplished in vitro or in vivo. TFAM can be mixed in amounts sufficient to wrap or bind the polynucleotide of interest. Typically, one molecule of TFAM wraps about 15 base pairs of a target polynucleotide. Enough modified TFAM can be added to a polynucleotide of interest to completely coat the exterior of the polynucleotide and/or to condense the polynucleotide. The polynucleotide is packaged so that the PTD and the optional targeting signal are displayed on the surface of the packaged polynucleotide. It will be appreciated that more than one polynucleotide can be packaged into a single complex using more than one modified polynucleotide-binding or packaging polypeptides. 
     The polynucleotide generally encodes a functional polypeptide, an antisense polynucleotide, or an inhibitory RNA and is packaged with the modified polynucleotide-binding polypeptide. At least one cell is contacted with the resulting complex either in vitro or in vivo. The protein transduction domain facilitates crossing the cell&#39;s outer membrane and delivers the polynucleotide to the interior of the cell. Once in the cytoplasm, an optional targeting signal or domain facilitates the localization of the polynucleotide of interest to the a region of interest, for example to the mitochondrion or nucleus. Once the polynucleotide of interest is delivered to its destination, it can be transcribed and ultimately translated. Alternatively, if the polynucleotide of interest is an antisense polynucleotide or enzymatic polynucleotide, the polynucleotide of interest can act at or near the deliver site, for example in the cytosol or in an organelle. 
     It has been reported that inhibitory polynucleotides are unstable in vivo, in part, because endogenous enzymes and immune responses actively degrade inhibitory polynucleotides, for example small inhibitory RNA (siRNA). siRNA technology is known in the art, and any siRNA, including single or multi-stranded siRNAs, can be used with the present disclosure. Thus, one embodiment of the present disclosure provides compositions and methods for delivering intact inhibitory RNA, for example siRNA, to a cell, tissue, or organ of interest. An siRNA can be combined with a polynucleotide-binding polypeptide having a protein transduction domain, and optionally, a targeting signal to form a complex. The modified polynucleotide-binding polypeptide can associate with the siRNA so that the siRNA is wrapped, covered, condensed, or bound by the modified polypeptide thereby protecting the siRNA from enzymatic degradation. The association is reversible such that upon delivery of the siRNA to the desired destination, the siRNA can function to inhibit the transcription or translation of its target polynucleotide. 
     Another exemplary embodiment provides a method for transfecting a host, a host&#39;s cell, or a host&#39;s cellular organelle, for example the nucleus, mitochondria, or chloroplasts, including the steps of contacting a host&#39;s cell with a complex including a modified polynucleotide-binding polypeptide having at least one PTD, and optionally, at least one targeting signal, in combination with a polynucleotide of interest. In one embodiment, the polynucleotide-polypeptide complex acts as a non-viral vector. Cells from one host can be transfected and administered to a second host, or a host&#39;s cells can be transfected and administered to the host. The transfection can occur in vivo or in vitro. 
     Suitable cells for transfection include cells capable of being transfected, for example eukaryotic or prokaryotic cells. The cells can be somatic, quiescent, embryonic, mitotic, stem cells, progenitor cells, germ line cells, pluripotent cells, totipotent cells, embryonic stem cells, heterologous cells, undifferentiated, partially differentiated, endoderm, mesoderm, ectoderm, immortalized, or primary cultures. Organelle targeting signals of the present disclosure include polypeptides having a net positive charge, an NLS, and those listed in Tables 1 and 2. Suitable PTDs include but are not limited to HIV TAT YGRKKRRQRRR (SEQ. ID NO. 1) or RKKRRQRRR (SEQ. ID NO. 2); 11 Arginine residues, or positively charged polypeptides or polynucleotides having 8-15 residues, preferably 9-11 residues. The term non-nuclear organelle is intended to encompass all organelles other than the nucleus. It will be appreciated the disclosed compositions include a targeting signal, for example an organelle targeting signal, which causes the complex to associate with the organelle, typically to an organelle having a net negative charge or a region having a negative charge. In one embodiment, the association of the targeting signal with the organelle does not occur through a receptor:ligand interaction. The association of the organelle and complex can be ionic, non-covalent, covalent, reversible or irreversible. Exemplary complex:organelle associations include but are not limited to protein-protein, protein-carbohydrate, protein-nucleic acid, nucleic acid-nucleic acid, protein-lipid, lipid-carbohydrate, antibody-antigen, or avidin-biotin. The organelle targeting signal of the complex can be a protein, peptide, antibody, antibody fragment, lipid, carbohydrate, biotin, avidin, steptavidin, chemical group, or other ligand that causes specific association between the organelle and complex, preferably an electromagnetic association as between oppositely charged moieties. 
     The specific interaction between the introduced complex and its target, for example a specific type of cell or an organelle, can be accomplished by at least two methods. In one exemplary method a recombinant non-viral complex can include a recombinant polypeptide that expresses a targeting signal that interacts with the targeted the organelle. Preferably, the complex expresses a outer polypeptide that is specific to the target organelle. In another method the complex is modified to incorporate an exogenous targeting protein to which an organelle binds. Alternatively, a complex can include a modified recombinant polypeptide that specifically interacts with a desired cell, tissue, organ, or organelle, for example by expressing a amino acid sequence that interacts with the specific cell or organelle. It will be appreciated by those of skill in the art that the complex can be chemically modified to have a net positive or negative charge depending on the modification agent. For example, the complex can be coated with polylysine or other agents containing a primary amino group. Additionally, amino groups can be linked to the complex or compound containing amino groups can be linked to the complex. The linkage can be reversible or irreversible, covalent or non-covalent. Other charged groups for conferring a charge to a compound are known in the art and can be incorporated into the complex. 
     Nucleic acids including but not limited to polynucleotides, anti-sense nucleic acids, peptide nucleic acids, natural or synthetic nucleic acids, nucleic acids with chemically modified bases, RNA, DNA, RNA-DNA hybrids, enzymatic nucleic acids such as ribozymes and DNAzymes, native/endogenous genes and non-native/exogenous genes and fragments or combinations thereof, can be introduced into a cell or organelle of a host cell, in particular cells or organelles that can transcribe and or translate nucleic acids into proteins such as the nucleus, mitochondria and chloroplasts. In one embodiment of the present disclosure, all or part of the mitochondrial or chloroplastic genome can be introduced into an organelle. The nucleic acids can be introduced into the organelle with the complex when the complex crosses the organelle membrane via protein transduction domains. 
     Another embodiment provides a method for transfecting cellular organelles, for example eukaryotic organelles, by contacting the cell with a complex including a polynucleotide-binding polypeptide in combination with a polynucleotide, wherein the polynucleotide-binding polypeptide includes a PTD and optionally, a targeting signal or domain. The targeting signal can be a polypeptide, modified or unmodified, displayed on the surface of the complex which enables the complex to specifically associate with the target cell or organelle. Exemplary targeting signals include, nuclear localization signals, mitochondrial targeting signals including the targeting signals of proteins and genes listed in TABLE 1 and other signals having a net positive charge. Contacting a cell with the complex in a manner that introduces the complex or the polynucleotide into the cytosol of said cell. The complex can further associates with its specific target organelle or intracellular region of the cell and the polynucleotide can be introduced into the organelle. Introduction of the polynucleotide into the organelle can be accomplished by transducing the polynucleotide across organelle membranes via a protein transduction domain expressed on a surface of the complex. 
     Introduction of a polynucleotide into the cytosol of a eukaryotic cell, in an intact functional form, can be accomplished using standard techniques known to those skilled in the art or through modification of the recombinant polynucleotide-binding polypeptide with a protein transduction domains. Such transfection procedures include but are not limited to microinjection, electroporation, calcium chloride premeablization, polyethylene glycol permeabilization, protoplast fusion or cationic lipid premeablization. In one embodiment a polynucleotide-binding polypeptide is modified to include a Protein Transduction Domain that enables the polypeptide bound to a polynucleotide to be transduced across a lipid bilayer including a cellular membrane, organelle membrane, or plasma membrane. Suitable PTDs include but are not limited to an 11 Arginine PTD or Tat-PTD (SEQ. ID NOs. 3 or 4) and poly-Arg-RRRRRRR (SEQ. ID. NO.: 6); PTD-5-RRQRRTSKLMKR (SEQ. ID. NO.: 7); Transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ. ID. NO.: 8); and KALA-WEAKLAKALAKALAKHLAKALAKALKCEA (SEQ. ID. NO.: 9). 
     In accordance with one embodiment a method is provided for introducing exogenous nucleic acid sequences into a mitochondrion of a mammalian cell. Any mitochondrial transfection technique should ensure that a nucleic acid crosses three membranes (the plasma membrane and the outer and inner mitochondrial membranes), addresses the high copy of mtDNA molecules, and utilizes a minimal, circular mitochondrial replicon. In one embodiment of the present disclosure a recombinant polynucleotide-binding polypeptide is used as a delivery vehicle for introducing nucleic acid sequences into an organelle, for example the mitochondrion. The recombinant polypeptide packages the polynucleotides to prevent them from being degraded, and to condense the polynucleotides for delivery. Condensation of polynucleotides includes the ordered structure of polynucleotides under concentrated conditions. 
     In accordance with another embodiment a recombinant polynucleotide-binding polypeptide having a PTD and a mitochondrial targeting signal is used for mitochondrial transfection. This approach allows for direct manipulation of mtDNA and introduction of the circular genome at high-copy number. In one embodiment this method is used to manipulate or replace mtDNA. In another embodiment the entire human mitochondrial genome (SEQ ID NO. 218) can be replaced by introduced sequences. For example, Rho 0  cells can be first generated to remove endogenous mtDNA, followed by mitochondrial transfection, resulting in the entire mitochondrial genome of cells being replaced. Alternatively, mitochondria can be transfected without first proceeding with the generation of Rho 0  cells. In this case the introduced nucleic acid will be incorporated (recombined) with the existing endogenous mtDNA sequences resulting in the manipulation of the mtDNA sequences. Either method can be used to restore full functionality to damaged mitochondria. 
     Another embodiment provides a method for modifying the genome of a non-nuclear organelle, for example a mitochondrion, comprising transfecting the non-nuclear organelle with a polynucleotide encoding an enzyme that specifically cleaves the non-nuclear organelle&#39;s genome but does not cleave the polynucleotide. An exemplary enzyme includes, but is not limited to, a nuclease such as BamH1. It will be appreciated that any enzyme can be used that selectively cleaves nucleic acids endogenous to the non-nuclear organelle without cleaving heterologous nucleic acids. Heterologous nucleic acids refers to nucleic acids introduced from another organism or from a source other than the mitochondrion or host organism of the mitochondrion. The polynucleotide encoding the enzyme can be delivered alone or in combination with second polynucleotide using the methods described herein. The second polynucleotide can encode a second polypeptide, for example, that functions inside or outside of the non-nuclear organelle. The second polypeptide can function in the nucleus and can be a transcription factor, an enhancer or a suppressor of gene activation or transcription, a subunit of a transcription factor, a DNA binding polypeptide or the like. The second polypeptide can act specifically or non-specifically on one or more specific genes or nucleic acid sequences. The second polypeptide expressed in the non-nuclear organelle can be delivered outside of the non-nuclear organelle. The second polypeptide can comprise at least one PTD and optionally a targeting signal to facilitate translocation of the second polypeptide to a desired intracellular or extracellular location, for example the nucleus, cytoplasm, plasma membrane, etc. It will be appreciated that the second polypeptide can encode a secreted polypeptide, an intracellular polypeptide, a transmembrane polypeptide, or a polypeptide that is at least partially displayed on the exterior surface of a cell membrane. Secreted polypeptides include, but are not limited to, growth factors, cytokines, chemokines, neurotransmitters, insulin, or combinations thereof. 
     Alternatively, the polynucleotide encoding the enzyme can be packaged by a polynucleotide-binding polypeptide and combined with a lipid and/or polyamine vector for delivery to the non-nuclear organelle. An exemplary lipid and/or polyamine vector includes, but is not limited to, LIPOFECTAMINE™. The lipid and/or polyamine vector can be modified to display a targeting signal on the exterior to assist in the delivery of the polynucleotide to the non-nuclear organelle. 
     In some embodiments, the polynucleotide encoding the enzymatic polynucleotide also encodes at least a second polypeptide, for example, a polypeptide that compensates for a mutation in the non-nuclear organelle&#39;s genome. The second polypeptide can compensate for a null mutation, deletion, inversion, substitution, or transposition in the non-nuclear organelle&#39;s genome. Alternatively, the second polypeptide encodes a functional polypeptide that can be delivered to a location outside of the non-nuclear organelle, for example, the nucleus. 
     Still another embodiment provides a method form modifying a genome of a non-nuclear organelle comprising transfecting the non-nuclear organelle with a polynucleotide encoding an enzyme that specifically cleaves heterologous nucleic acids but does not cleave the polynucleotide encoding the enzyme or the endogenous nucleic acids of the non-nuclear organelle. 
     Still another embodiment provides a method for modifying a genome of a non-nuclear organelle comprising contacting a cell with enzymatic polypeptide comprising a PTD and a targeting signal operably linked to the enzymatic polypeptide. The enzymatic polypeptide can be a nuclease or restriction enzyme specific for a restriction site found in the genome of the non-nuclear organelle. Alternatively, the enzymatic polypeptide can cleave nucleic acids at a site found in heterologous nucleic acids and not in nucleic acids endogenous to the non-nuclear organelle. The enzymatic polypeptide can be delivered alone or in combination with a polynucleotide. 
     Suitable mitochondria localization sequences are known to those skilled in the art (see Table 1) and include the mitochondrial localization signal of subunit VIII of human cytochrome oxidase, the yeast cytochrome c oxidase subunit IV presequence and the amino-terminal leader peptide of the rat ornithine-transcarbamylase. In one embodiment the introduced sequences are expressed on the viral capsid head. 
     Organelle localization signals are known to those skilled in the art, and any of those signals can be used to target the complex to the target organelle. Localization sequences suitable for use in the present disclosure are described in Emanuelson et al., Predicting Subcellular Localization of Proteins Based on Their N-terminal Amino Acid Sequence.  Journal of Molecular Biology.  300(4):1005-16, 2000 Jul. 21, and in Cline and Henry, Import and Routing of Nucleus-encoded Chloroplast Proteins.  Annual Review of Cell  &amp;  Developmental Biology.  12:1-26, 1996, the disclosures of which are incorporated herein by reference in their entirety. More particularly, proteins and genes that have mitochondria localization signals for targeting linked proteins or nucleic acids to the mitochondria are listed in TABLE 1. Proteins and Nucleic acids encoding polypeptides that have chloroplast localization signals for targeting linked proteins or nucleic acids to the chloroplasts are listed in TABLE 2. In one embodiment the mitochondria or chloroplast localization signal is operably linked to a virus surface protein. It will be appreciated that part or all of the sequences listed in Tables 1 and 2 can be used as organelle targeting signals. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Localization Signals for Targeting to the Mitochondria. 
               
               
                 (verified using Mitochondrial Project MITOP Database -- 
               
               
                 http://mips.gsf.de/proj/medgen/mitop/) 
               
            
           
           
               
               
               
               
            
               
                 MITOP 
                   
                   
                   
               
               
                 Designation 
                 SEQ. ID. 
                 Gene 
               
               
                 (Accession No.) 
                 NO. 
                 Name 
                 Gene Name Full 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 106092 
                 18 
                 Etfa 
                 electron transfer flavoprotein alpha 
               
               
                 (NP633590) 
                   
                   
                 chain precursor - mouse 
               
               
                 106098 
                 19 
                 Etfb 
                 electron transfer flavoprotein beta 
               
               
                 (Q9DCW4) 
                   
                   
                 chain - mouse 
               
               
                 107450 
                 20 
                 Dld 
                 dihydrolipoamide dehydrogenase 
               
               
                 (NP000099) 
                   
                   
                 precursor - human 
               
               
                 87979 
                 21 
                 Ak3 
                 nucleoside-triphosphate--adenylate 
               
               
                 (NP067274) 
                   
                   
                 kinase 3 - mouse 
               
               
                 88529 
                 22 
                 Cs 
                 citrate synthase, mitochondrial 
               
               
                 (NP080720) 
               
               
                 891996 
                 23 
                 Cps1 
                 carbamoyl-phosphate synthetase 1 
               
               
                 (AA031763) 
               
               
                 97045 
                 24 
                 Mod2 
                 malic enzyme complex, 
               
               
                 (NP032641) 
                   
                   
                 mitochondrial - mouse 
               
               
                 97499 
                 25 
                 Pcca 
                 propionyl-CoA carboxylase alpha 
               
               
                 (AAH49802) 
                   
                   
                 chain precursor - mouse 
               
               
                 A27883 
                 26 
                 PCCA 
                 propionyl-CoA carboxylase alpha 
               
               
                 (NP000273) 
                   
                   
                 chain precursor 
               
               
                 A28053 
                 27 
                 Cbr2 
                 carbonyl reductase (NADPH) - 
               
               
                 NP031647) 
                   
                   
                 mouse 
               
               
                 A29881 
                 28 
                 mpp-2 
                 Mitochondrial processing peptidase 
               
               
                 (XP331748) 
                   
                   
                 beta subunit precursor (beta-mpp) 
               
               
                   
                   
                   
                 (ubiquinol-cytochrome c reductase 
               
               
                   
                   
                   
                 complexcore protein I) 
               
               
                 A30605 
                 29 
                 ACADS 
                 acyl-CoA dehydrogenase 
               
               
                 (NP000008) 
                   
                   
                 precursor, short-chain-specific 
               
               
                 A31998 
                 30 
                 ETFA 
                 electron transfer flavoprotein alpha 
               
               
                 (WP000117) 
                   
                   
                 chain precursor 
               
               
                 A32422 
                 31 
                 DBT 
                 dihydrolipoamide S-(2- 
               
               
                   
                   
                   
                 methylpropanoyl)transferase precursor 
               
               
                 A32800 
                 32 
                 HSPD1 
                 heat shock protein 60 precursor 
               
               
                 (NP002147) 
               
               
                 A36442 
                 33 
                 mpp-1 
                 Mitochondrial processing peptidase 
               
               
                 (XP326125) 
                   
                   
                 alpha chain precursor 
               
               
                 A37033 
                 34 
                 IVD 
                 isovaleryl-CoA dehydrogenase 
               
               
                 (NP002216) 
                   
                   
                 precursor 
               
               
                 A37157 
                 35 
                 BCKD 
                 3-methyl-2-oxobutanoate 
               
               
                 (NP898871) 
                   
                   
                 dehydrogenase (lipoamide) E1-beta 
               
               
                   
                   
                   
                 chain precursor 
               
               
                 A38234 
                 36 
                 OGDH 
                 oxoglutarate dehydrogenase 
               
               
                   
                   
                   
                 (lipoamide) precursor 
               
               
                 A39503 
                 37 
                 ME2 
                 malate dehydrogenase (NAD+) 
               
               
                 (NP002387) 
                   
                   
                 precursor, mitochondrial 
               
               
                 A40487 
                 38 
                 FDXR 
                 ferredoxin--NADP+ reductase, long 
               
               
                 (NP004101) 
                   
                   
                 form, precursor 
               
               
                 A40559 
                 39 
                 ACADL 
                 long-chain-acyl-CoA 
               
               
                 (NP001599) 
                   
                   
                 dehydrogenase (LCAD) 
               
               
                 A40872 
                 40 
                 ALDH5 
                 aldehyde dehydrogenase (NAD+) 5 
               
               
                   
                   
                   
                 precursor, mitochondrial 
               
               
                 A41581 
                 41 
                 CYP3 
                 peptidylprolyl isomerase 3 
               
               
                 (NP005720) 
                   
                   
                 precursor 
               
               
                 A42224 
                 42 
                 arg-2 
                 Carbamoyl-phosphate synthase, 
               
               
                 (P22572) 
                   
                   
                 arginine-specific, small chain 
               
               
                   
                   
                   
                 precursor (arginine-specific 
               
               
                   
                   
                   
                 carbamoyl-phosphate synthetase, 
               
               
                   
                   
                   
                 glutamine chain) (cps-a) 
               
               
                 A42845 
                 43 
                 BDH 
                 D-beta-hydroxybutyrate 
               
               
                   
                   
                   
                 dehydrogenase precursor (3- 
               
               
                   
                   
                   
                 hydroxybutyrate dehydrogenase) 
               
               
                   
                   
                   
                 (fragment) 
               
               
                 A45470 
                 44 
                 HMGC 
                 hydroxymethylglutaryl-CoA lyase 
               
               
                 (AAP88794) 
               
               
                 A47255 
                 45 
                 Pcx 
                 pyruvate carboxylase 
               
               
                 (AAH55030) 
               
               
                 A53020 
                 46 
                 PCCB 
                 propionyl-CoA carboxylase beta 
               
               
                 (AAH53661) 
                   
                   
                 chain precursor 
               
               
                 A53719 
                 47 
                 GLUDP 
                 glutamate dehydrogenase 
               
               
                 (NP036216) 
                   
                   
                 (NAD(P)+) 2 precursor 
               
               
                 A55075 
                 48 
                 HspE1 
                 chaperonin-10 
               
               
                 (NP032329) 
               
               
                 A55680 
                 49 
                 ACADS 
                 short/branched chain acyl-CoA 
               
               
                 (NP001600) 
                   
                   
                 dehydrogenase precursor 
               
               
                 A55723 
                 50 
                 DCI 
                 dodecenoyl-CoA Delta-isomerase 
               
               
                 (P42126) 
                   
                   
                 precursor, mitochondrial 
               
               
                 A55724 
                 51 
                 Acadm 
                 Acyl-CoA dehydrogenase, medium- 
               
               
                 (NP031408) 
                   
                   
                 chain specific precursor (MCAD) 
               
               
                 AA227572 
                 52 
                 WARS2 
                 tryptophanyl-tRNA synthetase 2 
               
               
                 (NM201263) 
                   
                   
                 (mitochondrial) - human 
               
               
                 AB029948 
                 53 
                 SerRS 
                 mitochondrial seryl-tRNA 
               
               
                 (NP060297) 
                   
                   
                 synthetase (cDNA FLJ20450 FIS, 
               
               
                   
                   
                   
                 CLONE KAT05607) - human 
               
               
                 ACDL_MOUSE 
                 54 
                 Acadl 
                 Acyl-CoA dehydrogenase, long- 
               
               
                 (AAH27412) 
                   
                   
                 chain specific precursor (LCAD) 
               
               
                 AF047042 
                 55 
                 CS 
                 citrate synthase, mitochondrial 
               
               
                 (AAC25560) 
               
               
                 AF097441 
                 56 
                 FARS1 
                 phenylalanine-tRNA synthetase 
               
               
                 (NP006558) 
                   
                   
                 (FARS1) mRNA, nuclear gene 
               
               
                   
                   
                   
                 encoding mitochondrial protein - 
               
               
                   
                   
                   
                 human 
               
               
                 ATPO_HUMAN 
                 57 
                 ATP5O 
                 ATP synthase oligomycin sensitivity 
               
               
                 (NP001688) 
                   
                   
                 conferral protein precursor, 
               
               
                   
                   
                   
                 mitochondrial 
               
               
                 AXHU 
                 58 
                 FDX1 
                 adrenodoxin precursor 
               
               
                 (AAP35327) 
               
               
                 CCHU 
                 59 
                 HCS 
                 cytochrome c 
               
               
                 (NP061820) 
               
               
                 CCNC 
                 60 
                 cyc-1 
                 Cytochrome c 
               
               
                 (CAA29050) 
               
               
                 CE06620 
                 61 
                 — 
                 Probable leucyl-tRNA synthetase, 
               
               
                 (NP056155) 
                   
                   
                 mitochondrial 
               
               
                 CE09597 
                 62 
                 — 
                 Pyruvate dehydrogenase (E2) 
               
               
                 (AAG31658) 
                   
                   
                 dihydrolipoamide acetyltransferase 
               
               
                 CH10_MOUSE 
                 63 
                 Hspe1 
                 10 KD heat shock protein, 
               
               
                 (NP032329) 
                   
                   
                 mitochondrial (hsp10) (10K 
               
               
                   
                   
                   
                 chaperonin) mouse 
               
               
                 CH60_CAEEL 
                 64 
                 hsp60 
                 Chaperonin homolog hsp60 
               
               
                 (NP497429) 
                   
                   
                 precursor (heat shock protein 60) 
               
               
                   
                   
                   
                 (hsp-60) 
               
               
                 DEHUE2 
                 65 
                 ALDH2 
                 aldehyde dehydrogenase (NAD+) 2 
               
               
                 (NP000681) 
                   
                   
                 precursor, mitochondrial 
               
               
                 DEHUE 
                 66 
                 GLUD1 
                 glutamate dehydrogenase 
               
               
                 (NP005262) 
                   
                   
                 (NAD(P)+) precursor 
               
               
                 DEHULP 
                 67 
                 DLD 
                 dihydrolipoamide dehydrogenase 
               
               
                 (NP000099) 
                   
                   
                 precursor 
               
               
                 DEHUPA 
                 68 
                 PDHA1 
                 pyruvate dehydrogenase 
               
               
                 (NP000275) 
                   
                   
                 (lipoamide) alpha chain precursor 
               
               
                 DEHUPB 
                 69 
                 PDHB 
                 pyruvate dehydrogenase 
               
               
                 (AAH00439) 
                   
                   
                 (lipoamide) beta chain precursor 
               
               
                 DEHUPT 
                 70 
                 PDHA2 
                 pyruvate dehydrogenase 
               
               
                 (NP005381) 
                   
                   
                 (lipoamide) alpha chain precursor, 
               
               
                   
                   
                   
                 testis-specific (E1) 
               
               
                 DEHUXA 
                 71 
                 BCKDH 
                 3-methyl-2-oxobutanoate 
               
               
                 (NP000700) 
                   
                   
                 dehydrogenase (lipoamide) alpha 
               
               
                   
                   
                   
                 chain precursor 
               
               
                 DEMSMM 
                 72 
                 Mor1 
                 malate dehydrogenase precursor, 
               
               
                 (P08249) 
                   
                   
                 mitochondrial 
               
               
                 DSHUN 
                 73 
                 SOD2 
                 superoxide dismutase (Mn) 
               
               
                   
                   
                   
                 precursor 
               
               
                 ECHM_HUMAN 
                 74 
                 ECHS1 
                 enoyl-CoA hydratase, mitochondrial 
               
               
                 (NP004083) 
                   
                   
                 (short chain enoyl-CoA hydratase 
               
               
                   
                   
                   
                 (SCEH)) 
               
               
                 GABT_HUMAN 
                 75 
                 ABAT 
                 4-aminobutyrate aminotransferase, 
               
               
                 (JC4022) 
                   
                   
                 mitochondrial precursor (gamma- 
               
               
                   
                   
                   
                 amino-N-butyrate-transaminase) 
               
               
                   
                   
                   
                 (GABA transaminase) 
               
               
                 GCDH_HUMAN 
                 76 
                 GCDH 
                 glutaryl-CoA dehydrogenase 
               
               
                 (AAP35352) 
                   
                   
                 precursor (GCD) - human 
               
               
                 GCDH_MOUSE 
                 77 
                 Gcdh 
                 Glutaryl-CoA dehydrogenase 
               
               
                 (NP032123) 
                   
                   
                 precursor (GCD) - mouse 
               
               
                 HCD1_CAEEL 
                 78 
                 — 
                 Probable 3-hydroxyacyl-CoA 
               
               
                 (NP499075) 
                   
                   
                 dehydrogenase F54C8.1 
               
               
                 HCD2_CAEEL 
                 79 
                 — 
                 Probable 3-hydroxyacyl-CoA 
               
               
                 (NP509584) 
                   
                   
                 dehydrogenase B0272.3 
               
               
                 HHMS60 
                 80 
                 Hsp60 
                 heat shock protein 60 precursor 
               
               
                 (NP034607) 
               
               
                 HMGL_MOUSE 
                 81 
                 Hmgcl 
                 hydroxymethylglutaryl-CoA lyase 
               
               
                 (AAB27965) 
                   
                   
                 precursor (HG-CoA lyase) (HL) (3- 
               
               
                   
                   
                   
                 hydroxy-3-methylglutarate-CoA 
               
               
                   
                   
                   
                 lyase) 
               
               
                 I48884 
                 82 
                 — 
                 2-oxoglutarate dehydrogenase E1 
               
               
                 (AAC52130) 
                   
                   
                 component (fragment) 
               
               
                 I48966 
                 83 
                 Aldh2 
                 aldehyde dehydrogenase (NAD+) 2 
               
               
                 (AAH05476) 
                   
                   
                 precursor, mitochondrial 
               
               
                 I49605 
                 84 
                 Acads 
                 Acyl-CoA dehydrogenase, short- 
               
               
                   
                   
                   
                 chain specific precursor (SCAD) 
               
               
                   
                   
                   
                 (butyryl-CoA dehydrogenase) 
               
               
                 I52240 
                 85 
                 ACAD 
                 acyl-CoA dehydrogenase 
               
               
                 (NP000007) 
                   
                   
                 precurser, medium-chain-specific 
               
               
                 I55465 
                 86 
                 PDK1 
                 pyruvate dehydrogenase kinase 
               
               
                 (AAH39158) 
                   
                   
                 isoform 1 - human 
               
               
                 I57023 
                 87 
                 Sod2 
                 superoxide dismutase (Mn) 
               
               
                 (DSHUN) 
                   
                   
                 precursor 
               
               
                 I70159 
                 88 
                 PDK2 
                 pyruvate dehydrogenase kinase 
               
               
                 (AAC42010) 
                   
                   
                 isoform 2 - human 
               
               
                 I70160 
                 89 
                 PDK3 
                 pyruvate dehydrogenase kinase 
               
               
                 (NP005382) 
                   
                   
                 isoform 3 - human 
               
               
                 JC2108 
                 90 
                 HADH 
                 long-chain-fatty-acid beta-oxidation 
               
               
                 (AAA56664) 
                   
                   
                 multienzyme complex alpha chain 
               
               
                   
                   
                   
                 precursor, mitochondrial 
               
               
                 JC2109 
                 91 
                 HADH 
                 long-chain-fatty-acid beta-oxidation 
               
               
                 (NP000174) 
                   
                   
                 multienzyme complex beta chain 
               
               
                   
                   
                   
                 precursor, mitochondrial 
               
               
                 JC2460 
                 92 
                 PC 
                 pyruvate carboxylase precursor 
               
               
                 (AAH11617) 
               
               
                 JC4879 
                 93 
                 SCHAD 
                 3-hydroxyacyl-CoA dehydrogenase, 
               
               
                 (NP005318) 
                   
                   
                 short chain-specific, precursor 
               
               
                 KIHUA3 
                 94 
                 AK3 
                 nucleoside-triphosphate--adenylate 
               
               
                 (AAH16180) 
                   
                   
                 kinase 3 
               
               
                 M2GD_HUMAN 
                 95 
                 DMGD 
                 dimethylglycine dehydrogenase, 
               
               
                 (AAF21941) 
                   
                   
                 mitochondrial precursor 
               
               
                   
                   
                   
                 (ME2GLYDH) - human 
               
               
                 MDHM_HUMA 
                 96 
                 MDH2 
                 malate dehydrogenase 
               
               
                 (AAH01917) 
                   
                   
                 mitochondrial precursor (fragment) 
               
               
                 O75439 
                 97 
                 PMPC 
                 mitochondrial processing peptidase 
               
               
                   
                   
                   
                 beta subunit precursor (beta-MPP) 
               
               
                   
                   
                   
                 (P-52) 
               
               
                 ODO1_MOUSE 
                 98 
                 Ogdh 
                 2-oxoglutarate dehydrogenase E1 
               
               
                 (AAC52130) 
                   
                   
                 component (alpha-ketoglutarate 
               
               
                   
                   
                   
                 dehydrogenase) (fragment) 
               
               
                 ODPA_CAEEL 
                 99 
                 — 
                 Probable pyruvate dehydrogenase 
               
               
                 (NP495693) 
                   
                   
                 E1 component, alpha subunit 
               
               
                   
                   
                   
                 precursor (PDHE1-a) 
               
               
                 OWHU 
                 100 
                 OTC 
                 ornithine carbamoyltransferase 
               
               
                 (NP000522) 
                   
                   
                 precursor 
               
               
                 OWMS 
                 101 
                 Otc 
                 ornithine carbamoyltransferase 
               
               
                 (CAA30121) 
                   
                   
                 precursor 
               
               
                 P21549 
                 102 
                 AGXT 
                 alanine--glyoxylate 
               
               
                 (NP000021) 
                   
                   
                 aminotransferase 
               
               
                 PUT2_HUMAN 
                 103 
                 ALDH4 
                 Delta-1-pyrroline-5-carboxylate 
               
               
                 (NP733844) 
                   
                   
                 dehydrogenase precursor (P5C 
               
               
                   
                   
                   
                 dehydrogenase) 
               
               
                 Q0140 
                 104 
                 VAR1 
                 VAR1 - mitochondrial ribosomal 
               
               
                 (NP009320) 
                   
                   
                 protein 
               
               
                 Q10713 
                 105 
                 KIAA0123 
                 mitochondrial processing peptidase 
               
               
                 (NP055975) 
                   
                   
                 alpha subunit precursor (alpha- 
               
               
                   
                   
                   
                 MPP) (P-55) (HA1523) 
               
               
                 Q16654 
                 106 
                 PDK4 
                 pyruvate dehydrogenase kinase 
               
               
                 (NP002603) 
                   
                   
                 isoform 4 - human 
               
               
                 ROHU 
                 107 
                 TST 
                 thiosulfate sulfurtransferase 
               
               
                 (CAA42060) 
               
               
                 S01174 
                 108 
                 Got2 
                 aspartate transaminase precursor, 
               
               
                 (NP034455) 
                   
                   
                 mitochondrial 
               
               
                 S08680 
                 109 
                 Mut 
                 methylmalonyl-CoA mutase alpha 
               
               
                 (NP032676) 
                   
                   
                 chain precursor 
               
               
                 S13025 
                 110 
                 nuo-40 
                 NADH dehydrogenase (ubiquinone) 
               
               
                 (CAA39695) 
                   
                   
                 40K chain 
               
               
                 S13048 
                 111 
                 cyt 
                 cytochrome c 
               
               
                 (PI9974) 
               
               
                 S16239 
                 112 
                 Glud 
                 glutamate dehydrogenase 
               
               
                 (AAH57347) 
                   
                   
                 (NAD(P)+) precursor 
               
               
                 S23506 
                 113 
                 Pdha1 
                 pyruvate dehydrogenase 
               
               
                 (NP032836) 
                   
                   
                 (lipoamide) 
               
               
                 S25665 
                 114 
                 DLAT_h 
                 dihydrolipoamide S- 
               
               
                 (CAA32052) 
                   
                   
                 acetyltransferase heart - human 
               
               
                   
                   
                   
                 (fragment) 
               
               
                 S26984 
                 115 
                 — 
                 probable DNA-directed RNA 
               
               
                 (P33540) 
                   
                   
                 polymerase - mitochondrion 
               
               
                   
                   
                   
                 plasmid maranhar (SGC3) 
               
               
                 S32482 
                 116 
                 ETFB 
                 electron transfer flavoprotein beta 
               
               
                 (NP001976) 
                   
                   
                 chain 
               
               
                 S38770 
                 117 
                 Dci 
                 3,2-trans-enoyl-CoA isomerase, 
               
               
                 (P42125) 
                   
                   
                 mitochondrial precursor 
               
               
                   
                   
                   
                 (dodecenoyl-CoA delta-isomerase) 
               
               
                 S39807 
                 118 
                 Bckdhb 
                 3-methyl-2-oxobutanoate 
               
               
                   
                   
                   
                 dehydrogenase (lipoamide) beta 
               
               
                   
                   
                   
                 chain 
               
               
                 S40622 
                 119 
                 MUT 
                 methylmalonyl-CoA mutase 
               
               
                 (NP000246) 
                   
                   
                 precursror (MCM) 
               
               
                 S41006 
                 120 
                 — 
                 hypothetical protein t05g5.6 
               
               
                 (CAE65137) 
               
               
                 S41563 
                 121 
                 cit-1 
                 citrate (si)-synthase, mitochondrial 
               
               
                 S42366 
                 122 
                 PRSS15 
                 Lon proteinase homolog 
               
               
                 S42370 
                 123 
                 — 
                 citrate synthase homolog 
               
               
                 (NP499264) 
               
               
                 S47532 
                 124 
                 HSPE1 
                 heat shock protein 10 
               
               
                 (NP002148) 
               
               
                 S53351 
                 125 
                 ME2.1 
                 malate dehydrogenase 
               
               
                 (NP006671) 
                   
                   
                 (oxaloacetate-decarboxylating) 
               
               
                   
                   
                   
                 (NADP+) precursor, mitochondrial 
               
               
                 S60028 
                 126 
                 Fdxr 
                 ferredoxin--NADP+ reductase 
               
               
                 (NP032023) 
                   
                   
                 precursor 
               
               
                 S65760 
                 127 
                 Dbt 
                 dihydrolipoamide transacylase 
               
               
                 (NP034152) 
                   
                   
                 precursor 
               
               
                 S71881 
                 128 
                 Bckdha 
                 branched chain alpha-ketoacid 
               
               
                 (NP031559) 
                   
                   
                 dehydrogenase chain E1-alpha 
               
               
                   
                   
                   
                 precursor 
               
               
                 SCOT_HUMA 
                 129 
                 OXCT 
                 Succinyl-CoA:3-ketoacid-coenzyme 
               
               
                 (NP000427) 
                   
                   
                 A transferase precursor (succinyl 
               
               
                   
                   
                   
                 CoA:3-oxoacid CoA-transferase) 
               
               
                   
                   
                   
                 (OXCT) 
               
               
                 SODM_CAEEL 
                 130 
                 sod-2 
                 Superoxide dismutase precursor 
               
               
                 (NP492290) 
                   
                   
                 (Mn) 
               
               
                 SODN_CAEEL 
                 131 
                 sod-3 
                 Superoxide dismutase precursor 
               
               
                 (NP510764) 
                   
                   
                 (Mn) 
               
               
                 SYHUAE 
                 132 
                 ALAS2 
                 5-aminolevulinate synthase 2 
               
               
                 SYHUAL 
                 133 
                 ALAS1 
                 5-aminolevulinate synthase 1 
               
               
                 (NP000679) 
                   
                   
                 precursor 
               
               
                 SYLM_HUMAN 
                 134 
                 KIAA0028 
                 Probable leucyl-tRNA synthetase, 
               
               
                 (NP056155) 
                   
                   
                 mitochondrial precursor (Leucine-- 
               
               
                   
                   
                   
                 tRNA ligase) (Leurs) (KIAA0028) 
               
               
                 SYMSAL 
                 135 
                 Alas2 
                 5-aminolevulinate synthase 
               
               
                   
                   
                   
                 mitochondrial precursor (erythroid- 
               
               
                   
                   
                   
                 specific) (ALAS-E) 
               
               
                 SYNCLM 
                 136 
                 leu-5 
                 leucine--tRNA ligase precursor, 
               
               
                 (XP323115) 
                   
                   
                 mitochondrial 
               
               
                 SYNCYT 
                 137 
                 cyt-18 
                 tyrosine--tRNA ligase precursor, 
               
               
                   
                   
                   
                 mitochondrial 
               
               
                 SYWM_CAEEL 
                 138 
                 — 
                 Probable tryptophanyl-tRNA 
               
               
                 (T15761) 
                   
                   
                 synthetase, mitochondrial 
               
               
                   
                   
                   
                 (tryptophan--tRNA ligase) (TRPRS) 
               
               
                 THTR_MOUSE 
                 139 
                 Tst 
                 thiosulfate sulfurtransferase 
               
               
                 (NP033463) 
               
               
                 U80034 
                 140 
                 MIPEP 
                 mitochondrial intermediate 
               
               
                 (NP005923) 
                   
                   
                 peptidase 
               
               
                 U82328 
                 141 
                 PDX1 
                 pyruvate dehydrogenase complex 
               
               
                 (NP003468) 
                   
                   
                 protein X subunit precursor 
               
               
                 XNHUDM 
                 142 
                 GOT2 
                 aspartate transaminase precursor, 
               
               
                 (NP002071) 
                   
                   
                 mitochondrial 
               
               
                 XNHUO 
                 143 
                 OAT 
                 ornithine--oxo-acid transaminase 
               
               
                 (NP000265) 
                   
                   
                 precursor 
               
               
                 XNHUSP 
                 144 
                 AGXT 
                 serine--pyruvate aminotransferase 
               
               
                 (NP000021) 
                   
                   
                 (SPT) (alanine--glyoxylate 
               
               
                   
                   
                   
                 aminotransferase) (AGT) 
               
               
                 XNMSO 
                 145 
                 Oat 
                 ornithine--oxo-acid transaminase 
               
               
                 (AAH08119) 
                   
                   
                 precursor 
               
               
                 XXHU 
                 146 
                 DLAT 
                 dihydrolipoamide S- 
               
               
                   
                   
                   
                 acetyltransferase precursor 
               
               
                   
                   
                   
                 (fragment) 
               
               
                 YAL044c 
                 147 
                 GCV3 
                 GCV3 - glycine decarboxylase, 
               
               
                 (P39726) 
                   
                   
                 subunit H 
               
               
                 YBL022c 
                 148 
                 PIM1 
                 PIM1 - ATP-dependent protease, 
               
               
                 (NP009531) 
                   
                   
                 mitochondrial 
               
               
                 YBL038w 
                 149 
                 MRPL16 
                 MRPL16 - ribosomal protein of the 
               
               
                 (NP009515) 
                   
                   
                 large subunit, mitochondrial 
               
               
                 YBL080c 
                 150 
                 PET112 
                 PET112 - required to maintain rho + 
               
               
                 (NP009473) 
                   
                   
                 mitochondrial DNA 
               
               
                 YBL090w 
                 151 
                 MRP21 
                 MRP21 - Mitochondrial ribosomal 
               
               
                 (NP009463) 
                   
                   
                 protein 
               
               
                 YBR120c 
                 152 
                 CBP6 
                 CBP6 - apo-cytochrome B pre- 
               
               
                 (NP009678) 
                   
                   
                 mRNA processing protein 
               
               
                 YBR122c 
                 153 
                 MRPL36 
                 MRPL36 - ribosomal protein YmL36 
               
               
                 (CAA55624) 
                   
                   
                 precursor, mitochondrial 
               
               
                 YBR146w 
                 154 
                 MRPS9 
                 MRPS9 - ribosomal protein S9 
               
               
                 (NP009704) 
                   
                   
                 precursor, mitochondrial 
               
               
                 YBR221c 
                 155 
                 PDB1 
                 PDB1 - pyruvate dehydrogenase 
               
               
                 (NP009780) 
                   
                   
                 (lipoamide) beta chain precursor 
               
               
                 YBR227c 
                 156 
                 MCX1 
                 MCX1 - ClpX homologue in 
               
               
                 (NP009786) 
                   
                   
                 mitochondria 
               
               
                 YBR251w 
                 157 
                 MRPS5 
                 MRPS5 - ribosomal protein S5, 
               
               
                 (NP009810) 
                   
                   
                 mitochondrial 
               
               
                 YBR268w 
                 158 
                 MRPL37 
                 MRPL37 - ribosomal protein 
               
               
                 (NP009827) 
                   
                   
                 YmL37, mitochondrial 
               
               
                 YBR282w 
                 159 
                 MRPL27 
                 MRPL27 - ribosomal protein YmL27 
               
               
                 (NP009841) 
                   
                   
                 precursor, mitochondrial 
               
               
                 YCR003w 
                 160 
                 MRPL32 
                 MRPL32 - ribosomal protein 
               
               
                 (NP009929) 
                   
                   
                 YmL32, mitochondrial 
               
               
                 YCR024c 
                 161 
                 — 
                 asn-tRNA synthetase, mitochondrial 
               
               
                 (NP009953) 
               
               
                 YCR028c-a 
                 162 
                 RIM1 
                 RIM1 - ssDNA-binding protein, 
               
               
                 (NP009958) 
                   
                   
                 mitochondrial 
               
               
                 YCR046c 
                 163 
                 IMG1 
                 IMG1 - ribosomal protein, 
               
               
                 (NP009975) 
                   
                   
                 mitochondrial 
               
               
                 YDL202w 
                 164 
                 MRPL11 
                 MRPL11 - ribosomal protein of the 
               
               
                 (NP010079) 
                   
                   
                 large subunit, mitochondrial 
               
               
                 YDR148c 
                 165 
                 KGD2 
                 KGD2 - 2-oxoglutarate 
               
               
                 (NP010432) 
                   
                   
                 dehydrogenase complex E2 
               
               
                   
                   
                   
                 component 
               
               
                 YDR194c 
                 166 
                 MSS116 
                 MSS116 - RNA helicase of the 
               
               
                 (NP010480) 
                   
                   
                 DEAD box family, mitochondrial 
               
               
                 YDR462w 
                 167 
                 MRPL28 
                 MRPL28 - ribosomal protein of the 
               
               
                 (NP010750) 
                   
                   
                 large subunit (YmL28), 
               
               
                   
                   
                   
                 mitochondrial 
               
               
                 YFL018c 
                 168 
                 LPD1 
                 LPD1 - dihydrolipoamide 
               
               
                 (NP116635) 
                   
                   
                 dehydrogenase precursor 
               
               
                 YGR244c 
                 169 
                 LSC2 
                 succinate-CoA ligase beta subunit 
               
               
                 (NP011760) 
               
               
                 YHR008c 
                 170 
                 SOD2 
                 SOD2 - superoxide dismutase (Mn) 
               
               
                 (NP011872) 
                   
                   
                 precursor, mitochondrial 
               
               
                 YIL070c 
                 171 
                 MAM33 
                 MAM33 - mitochondrial acidic 
               
               
                 (NP012194) 
                   
                   
                 matrix protein 
               
               
                 YJL096w 
                 172 
                 MRPL49 
                 MRPL49 - ribosomal protein 
               
               
                 (CAA89390) 
                   
                   
                 YmL49, mitochondrial 
               
               
                 YJR113c 
                 173 
                 RSM7 
                 RSM7 - similarity to bacterial, 
               
               
                 (NP012647) 
                   
                   
                 chloroplast and mitochondrial 
               
               
                   
                   
                   
                 ribosomal protein S7 
               
               
                 YKL040c 
                 174 
                 NFU1 
                 NFU1 - iron homeostasis 
               
               
                 (NP012884) 
               
               
                 YLL027w 
                 175 
                 ISA1 
                 ISA1 - mitochondrial protein 
               
               
                 (NP013073) 
                   
                   
                 required for normal iron metabolism 
               
               
                 YLR059c 
                 176 
                 REX2 
                 REX2 - putative 3′-5′ exonuclease 
               
               
                 (NP013160) 
               
               
                 YML110c 
                 177 
                 COQ5 
                 COQ5 - ubiquinone biosynthesis, 
               
               
                 (NP013597) 
                   
                   
                 methyltransferase 
               
               
                 YMR062c 
                 178 
                 ECM40 
                 ECM40 - acetylornithine 
               
               
                 (NP013778) 
                   
                   
                 acetyltransferase 
               
               
                 YMR072w 
                 179 
                 ABF2 
                 ABF2 - high mobility group protein 
               
               
                 (NP013788) 
               
               
                 YOL095c 
                 180 
                 HMI1 
                 HMI1 - mitochondrial DNA helicase 
               
               
                 (NP014546) 
               
               
                 YOR040w 
                 181 
                 GLO4 
                 GLO4 - glyoxalase II 
               
               
                 (NP014683) 
                   
                   
                 (hydroxyacylglutathione hydrolase) 
               
               
                 YOR142w 
                 182 
                 LSC1 
                 LSC1 - succinate-CoA ligase alpha 
               
               
                 (NP014785) 
                   
                   
                 subunit 
               
               
                 YPL118w 
                 183 
                 MRP51 
                 MRP51 - strong similarity to 
               
               
                 (NP015207) 
                   
                   
                 S. kluyveri hypothetical protein 
               
               
                 YPL135w 
                 184 
                 ISU1 
                 ISU1 - protein with similarity to iron- 
               
               
                 (NP015190) 
                   
                   
                 sulfur cluster nitrogen fixation proteins 
               
               
                 YPL252c 
                 185 
                 YAH1 
                 YAH1 - similarity to adrenodoxin 
               
               
                 (NP015071) 
                   
                   
                 and ferrodoxin 
               
               
                 YPL262w 
                 186 
                 FUM1 
                 FUM1 - fumarate hydratase 
               
               
                 (NP015061) 
               
               
                 YPR047w 
                 187 
                 MSF1 
                 MSF1 - phenylalanine--tRNA ligase 
               
               
                 (CAA89167) 
                   
                   
                 alpha chain, mitochondrial 
               
               
                 YPR067w 
                 188 
                 ISA2 
                 ISA2 - mitochondrial protein 
               
               
                 (NP015392) 
                   
                   
                 required for iron metabolism 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Localization Signals for Targeting to the Chloroplast: 
               
            
           
           
               
               
               
            
               
                 Designation 
                   
                   
               
               
                 (Accession 
                 SEQ. 
               
               
                 No.) 
                 ID NO. 
                 Description 
               
               
                   
               
               
                 CA782533 
                 189 
                 Transit peptide domain of the apicoblast 
               
               
                   
                   
                 ribosomal protein S9 
               
               
                 P27456 
                 190 
                 Pea glutathione reductase (GR) signal peptide 
               
               
                 (CAA62482) 
               
               
                 BAB91333 
                 191 
                 NH 2 -terminus of Cr-RSH encoding a putative 
               
               
                   
                   
                 guanosine 3′,5′-bispyrophosphate (ppGpp) 
               
               
                   
                   
                 synthase-degradase 
               
               
                 CAB42546 
                 192 
                 14-3-3 proteins 
               
               
                 AAC64139 
                 193 
                 Chloroplast signal recognition particle including 
               
               
                 AAC64109 
                 194 
                 cpSRP54, cpSRP43 subunits or a fragment 
               
               
                 AAD01509 
                 195 
                 thereof 
               
               
                 PWSPG, 
                 196 
                 Chloroplast transit peptides 
               
               
                 FESP1, 
                 197 
               
               
                 P00221, 
                 198 
               
               
                 P05435, 
                 199 
               
               
                 BAA37170, 
                 200 
               
               
                 BAA37171, 
                 201 
               
               
                 AAA81472 
                 202 
               
               
                 X52428 
                 203 
                 AtOEP7, in particular the transmembrane domain 
               
               
                 (CAA36675) 
                   
                 (TMD) and its C-terminal neighboring seven- 
               
               
                   
                   
                 amino acid region (see Lee YJ, Plant Cell 2001 
               
               
                   
                   
                 Oct; 13(10): 2175-90) 
               
               
                 CA757092, 
                 204 
                 THI1 N-terminal chloroplastic transit peptide, in 
               
               
                 CA755666 
                 205 
                 particular 4 to 27 residues 
               
               
                   
               
            
           
         
       
     
     The identification of the specific sequences necessary for translocation of a linked protein into a chloroplast or mitochondria can be determined using predictive software known to those skilled in the art, including the tools located at http://www.mips.biochem.mpg.de/cgi-bin/proj/medgen/mitofilter. 
     4. Transfection of Plants 
     Another embodiment provides methods and compositions for the transfection of plants, for example the delivery of a polynucleotide to a chloroplast. Techniques for plant transfection are known in the art. For example,  Agrobacterium tumefaciens  and  Agrobacterium rhizogenes  both have the ability to transfer portions of their DNA into the genomes of plants and can be used to transfect plant cells. The mechanism by which they transfer DNA is the same, however the differences in the resulting phenotypes are attributed to the presence of a Ti plasmid in  Agrobacterium tumefaciens  and the Ri plasmid in  Agrobacterium rhizogenes . The Ti plasmid DNA induces host plants to grow tumourous masses whereas the Ri plasmid DNA leads to the abundant proliferation of roots.  Agrobacterium tumefacies  is capable of infecting almost any plant tissue whereas  Agrobacterium rhizogenes  can only infect roots. 
     The Ti plasmid of  Agrobacterium  is a large, circular double stranded DNA molecule (T-DNA) of approximately 200 kb, which exist as an autonomous replicating unit. The plasmids are maintained within the bacteria and only a specific region (T-region) approximately 20 kb can be transferred from the bacteria to the host. To accomplish this transfer the Ti plasmid contains a series of genes that code for its own replication, excision from the plasmid, transfer to the host cell, incorporation into the host genome and the induction of tumor formation 
       Agrobacterium  can detect and migrate towards injured plant cells through the detection of chemical signals leaking from the wounded plant. This detection process is referred to as chemotaxis.  Agrobacterium  can recognize plant compounds such as acetosyrinogone, sinapinic acid, coniferyl alcohol, caffeic acid and methylsyringic acid which induce the bacteria&#39;s virulence. To begin the infection process,  Agrobacterium  must bind itself to the host cell. This binding is achieved by a group of genes located within the bacterial chromosome. The bacteria can anchor at the site of injury, by the production of cellulose fibrils. The fibrils attach to the cell surface of the plant host and facilitate the clustering of other bacteria on the cell surface. It is believed that this clustering many help the successful transfer of T-DNA. Once bound to the host, the bacterium is free to begin the processing and transfer of the T-region. One embodiment of the present disclosure discloses transfecting a plant cell with  Agrobacterium  wherein the  Agrobacterium  has been modified to bind to a plant organgelle, for example a chloroplast.  Agrobacterium  can be further modified to encode a nucleic acid of interest for expression in the organelle. Upon binding to the organelle, the  Agrobacterium  can deliver the target nucleic acid into the chloroplast. 
     To transfer the T-region of the Ti plasmid to the host cell organelle, the T-region must be processed such that it is excised from the plasmid and directed to the organelle. The T-region is excised from the Ti plasmid and directed into the host cell or organelle. Once properly packaged, the T-complex transfer is mediated by several proteins and is thought to be similar to bacterial conjugation. Once inside the plant cell or organelle, the T-complex is taken through the membrane. 
     5. Exemplary Cells and Cell Lines 
     In another embodiment, the transfection complex comprises a recombinant polypeptide having a protein transduction domain and an organelle localization signal in combination with a polynucleotide. The complex can be introduced into organelles of cells from a cell line. The cell line can be a transformed cell line that can be maintained indefinitely in cell culture, or the cell line can be a primary cell culture. Exemplary cell lines are those available from American Type Culture Collection including plant cell lines which are incorporated herein by reference. The nucleic acid can be replicated and transcribed within the nucleus of a cell of the transfected cell line. The targeting signal can be enzymatically cleaved if necessary such that the complex is free to remain in the target organelle. 
     Any eukaryotic cell can be transfected to produce organelles that express a specific nucleic acid, for example a metabolic gene, including primary cells as well as established cell lines. Suitable types of cells include but are not limited to undifferentiated or partially differentiated cells including stem cells, totipotent cells, pluripotent cells, embryonic stem cells, inner mass cells, adult stem cells, bone marrow cells, cells from umbilical cord blood, and cells derived from ectoderm, mesoderm, or endoderm. Suitable differentiated cells include somatic cells, neuronal cells, skeletal muscle, smooth muscle, pancreatic cells, liver cells, and cardiac cells. Suitable plant cells can be selected from monocots and dicots, and include corn, soybeans, legumes, grasses, and grains such as rice and wheat. 
     If the organelle to be targeted is a chloroplast, then the host cell can be selected from known eukaryotic photosynthetic cells. If the organelle to be transfected is the mitochondrion, than any eukaryotic cell can be used, including mammalian cells, for example human cells. The cells are transfected to either transiently or stably express the exogenous nucleic acid. In one embodiment a DNA construct encoding a reporter gene is integrated into the mitochondrial genome of a cell to produce a stable transgenic cell line that comprises organelles that express the desired reporter gene. 
     In another embodiment, siRNA or antisense polynucleotides (including siRNA or antisense polynucleotides directed to mtDNA related proteins) can be transfected into an organelle using the compositions described herein. 
     6. Research Tools 
     In one embodiment, the present disclosure is used as a tool to investigate cellular consequences of gene expression, for example mtDNA expression, the mechanisms of heteroplasmy, mtDNA replication and inheritance, as well as threshold effects. Mutant mice can be generated using this approach, allowing investigators to study mutations in nuclear and mtDNA not found in nature. More particularly, the methods and compositions disclosed herein can be used to generate cells that contain mitochondria that have identical genotypes or varying degrees of heteroplasmy. To prepare homoplastic cells, Rho 0  cells (devoid of mtDNA) are first prepared using RNA interference (RNAi). For example Rho 0  cells can be generated using RNAi to the human mitochondrial DNA polymerase. Exemplary Rho 0  cell lines are generated with RNAi to mitochondrial proteins involved in mtDNA maintenance. These Rho 0  cells are maintained and propagated on pyruvate containing supportive media and then transfected with a functional mitochondria genome. After metabolic selection, by removing pyruvate from supportive media, only those cells that contain successfully transfected mitochondria will survive, thus generating a population of cells that all have identical mitochondria genomes. 
     Cell lines having varying degrees of heteroplasmy can then be generated in a controlled manner by fusing two or more homoplasmy cell lines to generate cybrids. Cybrids can be generated using any of the known technique for introducing organelles into a recipient cell, including but not limited to polyethylene glycol (PEG) mediated cell membrane fusion, cell membrane permeabilization, cell-cytoplast fusion, virus mediated membrane fusion, liposome mediated fusion, microinjection or other methods known in the art. 
     7. Transgenic Non-Human Animals 
     The techniques described in the present disclosure can also be used to generated transgenic non-human animals. In particular, zygote microinjection, nuclear transfer, blastomere electrofusion and blastocyst injection of embryonic stem (ES) cell cybrids have each provided feasible strategies for creating hetero- and homoplasmic mice containing mtDNA from transfected cell lines (i.e. cells that containing transfected mitochondria). In one embodiment an embryonic stem (ES) cell is transfected and injected into the blastocyst of a mammalian embryo as a means of generating chimeric mice. In another embodiment, embryonic stem (ES) cell cybrids (from transfected cells and ES cell rhos, or from two separately transfected cells) are first prepared, followed by blastocyst injection into embryos. The use of cells carrying specific mtDNA of interest allows the creation of transmitochondrial mice that are heteroplasmic or even homoplasmic for the transfected DNA. In theory, this technique offers the prospect of transferring any mutant mtDNA that can be obtained from cultured transfected cells into a whole organism model. For example, this disclosed methods and compositions could be used to create mouse models of human mtDNA disease. 
     Using the disclosed compositions and methods for mtDNA transfection will allow investigations into questions such as the effect of varying proportions of the 5000 bp “common deletion”, which accumulates with aging, polymorphisms found in diabetes and neurodegenerative diseases, and dynamics of mtDNA complementation. There are also potential therapeutic uses of this approach. Targeted introduction of the normal mitochondrial genome offers treatment for both classic mtDNA-based diseases and diseases of aging such as neurodegenerative brain conditions and adult-onset diabetes, which have been associated with mtDNA-based mitochondrial dysfunction. 
     Another embodiment of the disclosure provides transfected non-human organisms and methods making and using them. Single or multicellular non-human organisms, preferably non-human mammals, more preferably mice, can be transfected with the compositions described herein by administering the compositions of the present disclosure to the non-human organism. In one embodiment, the polynucleotide remains episomal and does not stably integrate into the genome of the host organism. In another embodiment, the polynucleotide prevents the expression of a gene of interest. Thus, the expression of the polynucleotide in the host can be controlled by the amount of polynucleotide administered to the host. 
     The disclosed transfected non-human organisms have several advantages over traditional transgenic organisms. For example, the transfected organism disclosed herein can be produced in less time that traditional transgenic organisms without sexual reproduction. Moreover, the expression of the polynucleotide of interest in the host can be directly regulated by the amount of polynucleotide of interest administered to the host. Dosage controlled expression of a polynucleotide of interest can be correlated to observed phenotypes and changes in the transfected animal. Additionally, inducible expression and/or replication control elements can be included in the polynucleotide of interest to provide inducible and dosage dependent expression and/or replication. Suitable inducible expression and/or replication control elements are known in the art. 
     8. Kits 
     The present disclosure is also directed to a kit or pack that supplies the elements necessary to conduct transfection of eukaryotic or prokaryotic organisms, in particular the transfection of organelles. In accordance with one embodiment a kit is provided comprising a DNA binding protein construct that includes a protein transduction domain and optionally, a targeting signal and domain. The protein construct can be combined with a polynucleotide to form a complex which can be used to transfect a host or a host cell. In another embodiment the protein construct provided with the kit comprises a nuclear, mitochondrial or chloroplast localization signal selected from those known to target to the organelle, partially listed in Tables I and II. In another embodiment the protein construct comprises a sequence encoding an 11 Arginine residue stretch or HIV- Tat amino acid sequence followed by a mitochondrial localization signal, for example the localization signal of TFAM. optionally operably linked to a targeting signal. 
     In accordance with one embodiment a kit is provided comprising cells that express the protein construct. The cells can be cultured to produce the protein construct in large quantities which can be harvested, purified, and concentrated. The individual components of the kits can be packaged in a variety of containers, e.g., vials, tubes, microtiter well plates, bottles, and the like. Other reagents can be included in separate containers and provided with the kit; e.g., positive control samples, negative control samples, buffers, cell culture media, etc. Preferably, the kits will also include instructions for use. 
     In some embodiment, the kit includes a construct having a polynucleotide binding domain that hybridizes or binds to a predetermined nucleic acid sequence. In another embodiment, kit includes a polynucleotide-binding polypeptide that non-specifically hybridizes or binds to polynucleotides of interest. 
     9. Genetic Diseases or Syndromes 
     The group is comprised of Kearns-Sayre syndrome, Leigh&#39;s syndrome, mitochondrial DNA depletion syndrome (MDS), mitochondrial encephalomyopathy, lactic acidosis and strokelike episodes (MELAS), myoclonus epilepsy with ragged red fibers (MERRF), mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), neuropathy, ataxia and retinitis pigmentosa (NARP), progressive external ophthalmoplegia (PEO). 
     Embodiments of the present disclosure provide compositions and methods applicable for gene therapy protocols and the treatment of gene related diseases or disorders. Suitable genetic based diseases that can be treated with the compositions disclosed herein include but are not limited to: cancer; Canavan disease; Alzheimer&#39;s; Parkinson&#39;s; hypercholesteremia; cystic fibrosis; anemia; artherosclerosis; muscular dystrophy; AIDS; asthma; diabetes; arthritis; adenosine deaminase deficiency; Gaucher&#39;s disease; haemoglobinopathies such as sickle cell anaemia and the thalassemias; and autoimmune diseases. 
     Organelle dysfunction can cause disease in a host, for example a human host or a plant host. In particular, problems with mitochondria or chloroplasts can result in disease. Mitochondrial diseases result from failures of the mitochondria, specialized compartments present in every cell of the body except red blood cells. Cell injury and even cell death are result from mitochondrial failure. If this process is repeated throughout the body, whole systems begin to fail, and the life of the person in whom this is happening is severely compromised. The disease can be in children, for example individuals less that 18 years of age, typically less than 12 years of age, or adults, for example individuals 18 years of age or more. Thus, embodiments of the present disclosure are directed to treating a host diagnosed with an organelle related disease, in particular a mitochondrial disease, by introducing a vector into the host cell wherein the vector specifically binds to the organelle and wherein the vector comprises a nucleic acid encoding mitochondrial protein or peptide. The present disclosure encompasses manipulating, augmenting or replacing portions of the mammalian cell mitochondrial genome to treat diseases caused by mitochondrial genetic defects or abnormalities. 
     Exemplary mitochondrial diseases include but are not limited to: Alpers Disease; Barth syndrome; β-oxidation defects; carnitine-acyl-carnitine deficiency; carnitine deficiency; co-enzyme Q10 deficiency; Complex I deficiency; Complex II deficiency; Complex III deficiency; Complex IV deficiency; Complex V deficiency; cytochrome c oxidase (COX) deficiency; Chronic Progressive External Ophthalmoplegia Syndrome (CPEO); CPT I Deficiency; CPT II deficiency; Glutaric Aciduria Type II; lactic acidosis; Long-Chain Acyl-CoA Dehydrongenase Deficiency (LCAD); LCHAD; mitochondrial cytopathy; mitochondrial DNA depletion; mitochondrial encephalopathy; mitochondrial myopathy; Mitochondrial Encephalomyopathy with Lactic Acidosis and Strokelike episodes (MELAS); Myoclonus Epilepsy with Ragged Red Fibers (MERRF); Maternally Inherited Leigh&#39;s Syndrome (MILS); Myogastrointestinal encephalomyopathy (MNGIE); Neuropathy, ataxia and retinitis pigmentosa (NARP); Leber&#39;s Hereditary Optic Neuropathy (LHON); Progressive external ophthalmoplegia (PEO); Pearson syndrome; Kearns-Sayre syndrome (KSS); Leigh&#39;s syndrome; intermittent dysautonomia; pyruvate carboxylase deficiency; pyruvate dehydrogenase deficiency; respiratory chain mutations and deletions; Short-Chain Acyl-CoA Dehydrogenase Deficiency (SCAD); SCHAD; and Very Long-Chain Acyl-CoA Dehydrongenase Deficiency (VLCAD). 
     Some mitochondrial diseases are a result of problems in the respiratory chain in the mitochondira. The respiratory chain consists of four large protein complexes: I, II, III and IV (cytochrome c oxidase, or COX), ATP synthase, and two small molecules that ferry around electrons, coenzyme Q10 and cytochrome c. The respiratory chain is the final step in the energy-making process in the mitochondrion where most of the ATP is generated. Mitochondrial encephalomyopathies that can be caused by deficiencies in one or more of the specific respiratory chain complexes include MELAS, MERFF, Leigh&#39;s syndrome, KSS, Pearson, PEO, NARP, MILS and MNGIE. 
     The mitochondrial respiratory chain is made up of proteins that come from both nuclear and mtDNA. Although only 13 of roughly 100 respiratory chain proteins come from the mtDNA, these 13 proteins contribute to every part of the respiratory chain except complex II, and 24 other mitochondrial genes are required just to manufacture those 13 proteins. Thus, a defect in either a nuclear gene or one of the 37 mitochondrial genes can cause the respiratory chain to break down. It will be appreciated that the scope of the present disclosure includes transfecting mitochondria with at least one or part of one gene involved in mitochondrial function, in particular at least one or part of the 37 mitochondrial genes to restore or increase the function of the respiratory chain. Any or part of a mitochondrial genome, for example human mitochondrial genome SEQ ID NO: 218, may be introduced into a host mitochondrion using the methods described herein. 
     Diseases of the mitochondria appear to cause the most damage to cells of the brain, heart, liver, skeletal muscles, kidney and the endocrine and respiratory systems. Thus, transfection of mitochondria in these cells and tissues with specific nucleic acids is within the scope of the present disclosure, in particular transfection of mitochondria with nucleic acids encoding mitochondrial-encoded proteins rather than nuclear-encoded proteins. It will be appreciated that the mitochondria can be transfected to express any protein whether naturally present in the mitochondrion or not or naturally encoded by mtDNA or nuclear DNA. Depending on which cells are affected, symptoms may include loss of motor control, muscle weakness and pain, gastro-intestinal disorders and swallowing difficulties, poor growth, cardiac disease, liver disease, diabetes, respiratory complications, seizures, visual/hearing problems, lactic acidosis, developmental delays and susceptibility to infection. 
     Exemplary mtDNA mutations that can be addressed by the present disclosure include but are not limited to: tRNA leu -A3243G, A3251G, A3303G, T3250C T3271C and T3394C; tRNA Lys -A8344G, G11778A, G8363A, T8356C; ND1-G3460A; ND4-A10750G, G14459A; ND6-T14484A; 12S rRNA-A1555G; MTTS2-C12258A; ATPase 6-T8993G, T8993C; tRNA Ser (UCN)-T75110; 11778 and 14484, LHON mutations as well as mutations or deletions in ND2, ND3, ND5, cytochrome b, cytochrome oxidase I-III, and ATPase 8. 
     One embodiment of the present disclosure provides a method for restoring or increasing respiratory chain function in host cell including introducing a polynucleotide-binding polypeptide-polynucleotide complex into the host cell, wherein the complex specifically binds to the mitochondrion and comprises a nucleic acid that encodes a respiratory chain protein or peptide. The nucleic acid of the complex can be injected or otherwise delivered into the interior of the mitochondria when the complex targets the mitochondria. 
     Another embodiment of the present disclosure provides a method for restoring or increasing cytochrome oxidase activity in a host including transfecting mitochondria in a cell, for example a skeletal muscle cell, wherein the complex comprises a nucleic acid that encodes cytochrome oxidase or a functional component thereof. A functional component means a part or fragment of the protein or protein complex or subunit that performs a biological function independently or in combination with another protein, fragment, or subunit. 
     Still another embodiment of the present disclosure provides a method of increasing or restoring β-oxidation in a host including obtaining cells from the host, transfecting an organelle in the cells from the host, introducing a complex comprising a nucleic acid encoding proteins involved in β-oxidation spiral and carnitine transport, wherein the vector specifically binds to the organelle; and introducing the transfected cells of the host back into the host. 
     Other embodiments of the disclosure are directed to methods of restoring mitochondrial function lost or decreased as a result of point mutations or deletions. For example, KSS, PEO and Pearson, are three diseases that result from a type of mtDNA mutation called a deletion (specific portions of the DNA are missing) or mtDNA depletion (a general shortage of mtDNA). Thus, cells from hosts diagnosed with KSS, PEO, Pearson or similar disease can have their mitochondria transfected with the recombinant viral vector. A complex comprising a nucleic acid that corresponds to the deletion in the mtDNA causing the diseased state can be introduced into the cells. The complex will bind the organelle and deliver the nucleic acid into the interior of the mitochondria where the nucleic acid is expressed. The expression product can then incorporate into the mitochondria and increase or restore mitochondrial function. The transfected cells can be reintroduced in the host. It will be appreciated that the host&#39;s cells or other cells can be transfected as described herein and introduced into a host having a dysfunctional organelles, in particular mitochondria. 
     It will be appreciated by those skilled in the art that the present disclosure encompasses delivering either separately or in combination nucleic acids to the mitochondria that are naturally encoded by mtDNA or nuclear DNA. 
     The present disclosure also contemplates alleviating the symptoms of mitochondrial diseases by creating cells having transfected and non-transfected mitochondria. Alternatively, all of the mitochondria in a cell can be transfected or replaced. 
     One embodiment provides a method for compensating for a mtDNA mutation in a host, the method including identifying a host having a mtDNA mutation, obtaining a cell comprising said mtDNA mutation from said host, transfecting a mitochondrion of the host cell, introducing a complex that specifically binds to the organelle into the host cell, wherein the complex comprises a nucleic acid that encodes a functional product corresponding to the mtDNA mutation, introducing said transfected cell into the host. A nucleic acid that encodes a functional product corresponding to the mtDNA mutation means a sequence that produces a protein without the corresponding mutation. For example, if a host cell has an ND4-A10750G mutation, the transfected nucleic acid would encode a wildtype product for the ND4 gene. The non-viral complex can be introduced into the host, for example, intravenously. 
     10. Administration 
     The compositions provided herein may be administered in a physiologically acceptable carrier to a host. Preferred methods of administration include systemic or direct administration to a cell. The compositions can be administered to a cell or patient, as is generally known in the art for gene therapy applications. In gene therapy applications, the compositions are introduced into cells in order to transfect an organelle. “Gene therapy” includes both conventional gene therapy where a lasting effect is achieved by a single treatment, and the administration of gene therapeutic agents, which involves the one time or repeated administration of a therapeutically effective DNA or RNA. 
     The modified complex compositions can be combined in admixture with a pharmaceutically acceptable carrier vehicle. Therapeutic formulations are prepared for storage by mixing the active ingredient having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington&#39;s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween, Pluronics or PEG. 
     The compositions of the present disclosure can be administered parenterally. As used herein, “parenteral administration” is characterized by administering a pharmaceutical composition through a physical breach of a subject&#39;s tissue. Parenteral administration includes administering by injection, through a surgical incision, or through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration includes subcutaneous, intraperitoneal, intravenous, intraarterial, intramuscular, intrasternal injection, and kidney dialytic infusion techniques. 
     Parenteral formulations can include the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Parenteral administration formulations include suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, reconsitutable dry (i.e. powder or granular) formulations, and implantable sustained-release or biodegradable formulations. Such formulations may also include one or more additional ingredients including suspending, stabilizing, or dispersing agents. Parenteral formulations may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. Parenteral formulations may also include dispersing agents, wetting agents, or suspending agents described herein. Methods for preparing these types of formulations are known. Sterile injectable formulations may be prepared using non-toxic parenterally-acceptable diluents or solvents, such as water, 1,3-butane diol, Ringer&#39;s solution, isotonic sodium chloride solution, and fixed oils such as synthetic monoglycerides or diglycerides. Other parentally-administrable formulations include microcrystalline forms, liposomal preparations, and biodegradable polymer systems. Compositions for sustained release or implantation may include pharmaceutically acceptable polymeric or hydrophobic materials such as emulsions, ion exchange resins, sparingly soluble polymers, and sparingly soluble salts. 
     Pharmaceutical compositions may be prepared, packaged, or sold in a buccal formulation. Such formulations may be in the form of tablets, powders, aerosols, atomized solutions, suspensions, or lozenges made using known methods, and may contain from about 0.1% to about 20% (w/w) active ingredient with the balance of the formulation containing an orally dissolvable or degradable composition and/or one or more additional ingredients as described herein. Preferably, powdered or aerosolized formulations have an average particle or droplet size ranging from about 0.1 nanometers to about 200 nanometers when dispersed. 
     As used herein, “additional ingredients” include one or more of the following: excipients, surface active agents, dispersing agents, inert diluents, granulating agents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents, preservatives, physiologically degradable compositions (e.g., gelatin), aqueous vehicles, aqueous solvents, oily vehicles and oily solvents, suspending agents, dispersing agents, wetting agents, emulsifying agents, demulcents, buffers, salts, thickening agents, fillers, emulsifying agents, antioxidants, antibiotics, antifungal agents, stabilizing agents, and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional ingredients” which may be included in the pharmaceutical compositions are known. Suitable additional ingredients are described in Remington&#39;s Pharmaceutical Sciences, Mack Publishing Co., Genaro, ed., Easton, Pa. (1985). 
     Dosages and desired concentrations modified vectors disclosed herein in pharmaceutical compositions of the present disclosure may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary physician. Animal experiments provide reliable guidance for the determination of effective doses for human therapy. Interspecies scaling of effective doses can be performed following the principles laid down by Mordenti, J. and Chappell, W. “The use of interspecies scaling in toxicokinetics” In Toxicokinetics and New Drug Development, Yacobi et al., Eds., Pergamon Press, New York 1989, pp. 42-96. 
     EXAMPLES 
     Example 1 
     Recombinant Constructs 
     The 11 amino acid protein transduction domain (PTD) of the HIV Tat protein was cloned upstream and in frame of the mitochondrial localization signal (MLS) of the human mitochondrial transcription factor A (TFAM). For nuclear transfection, the MLS was replaced with the 9 amino acid nuclear localization signal (NLS) of the SV40 virus T Antigen. The recombinant protein was expressed in bacteria and isolated on a nickel chelating column utilizing a 6× Histidine tag expressed on the C-terminus immediately downstream of an Enterokinase site. The purified protein was incubated with Enterokinase and further purified on a nickel column where the flow-through was collected. The protein was dialyzed overnight at 4° C. against saline containing 33% phospho-buffered saline. Purified protein was concentrated and protein concentration was assessed with the Bradford Assay (Biorad). Purified protein was analyzed with SDS-Page to verify purity. 
     Briefly, full-length human mitochondrial genome was isolated from a mitochondrial preparation from the human neuroblastoma cell line, SH-SY5Y. 1 μg of the DNA was labeled with the Alexa 488 Fluorophore (Molecular Probes). The Alexa-labeled mitochondrial genome was incubated with the PTD-MLS-Tfam recombinant protein for 30 minutes at room temperature. The complexed Alexa-labeled DNA and recombinant protein was introduced into SY5Y cell culture containing MitoTracker Red dye (Molecular Probes) and confocal images were taken at the 30 minute time period post introduction of the complexed DNA-protein mixture. Red indicates mitochondria and green is the Alexa-labeled DNA. Superimposition of the images produces yellow indicating colocalization ( FIG. 1A ). 
     Example 2 
     Mitochondrial Expression of GFP 
     GFP (Green Fluorescent Protein) cDNA was mutated at the 270 nucleotide position to generate the following codon change (UGG→UGA). UGA is read by the mitochondrial translation machinery as a Tryptophan whereas the nuclear translational machinery reads it as a stop codon—producing a non-fluorescing, curtailed GFP when translated by the nuclear machinery but a full-length fluorescent product when translated in the mitochondria. The resultant mitochondrially encoded GFP cDNA was complexed with the PTD-MLS-Tfam as described above and introduced into SY5Y cell culture prestained with MitoTracker Red. The cells were imaged with confocal 24 hours after transfection. GFP signal (green) was observed colocalized with Mitotracker Red (red) producing yellow fluorescence when superimposed ( FIGS. 1B and 1C ). 
     To further verify that the recombinant protein was capable of transfecting cells, Rho cells generated from wild-type mitochondrial genome containing SY5Ys were transfected with mutant mitochondrial genome from LHON (Leber&#39;s Hereditary Optic Neuropathy) cybrids. The LHON mutation is a G-&gt;A transition at nucleotide position 11778 of the mitochondrial genome. This mutation removes a SfaNI Restriction site—thus digestion of a 500 by PCR product generated with primers flanking the restriction site with SfaNI produces in the wild-type condition two smaller products (300 and 200 bp). The PCR product generated from LHON mutant genome cannot be digested and remains as a 500 by product. In  FIG. 2 , the leftmost lane (Lane 1) is the PCR product generated from LHON cells and thus fails restriction digestion. Lane 2 is from Rho cells receiving only naked LHON mitochondrial DNA. Lane 3 is from Rho cells receiving only the recombinant PTD-MLS-Tfam. Lane 4 is from Rho cells receiving LHON mtDNA complexed with the recombinant PTD-MLS-Tfam. Lane 5 is control Rho cells alone. Lane 4 shows that the recombinant protein complexed with mutant DNA is capable of introducing a mutant mitochondrial genome into Rho cells. 
     Example 3 
     Construct Sequence Data 
     
       
         
           
               
            
               
                 TFAM Sequence (Mitochondrial Localization Signal 
               
               
                 (underlined) (Locus Link ID: 7019) 
               
               
                 (SEQ. ID. NO.: 206) 
               
               
                 
                   ATGGCGTTTCTCCGAAGCATGTGGGGCGTGCTGAGTGCCCTGGGAAGG 
                 
               
               
                 
                   TCTGGAGCAGAGCTGTGCACCGGCTGTGGAAGTCGACTGCGCTCCCCC 
                 
               
               
                   TTCAGTTTTGTGTATTTACCGAGGTGGTTT TCATCTGTCTTGGCAAGT 
               
               
                 TGTCCAAAGAAACCTGTAAGTTCTTACCTTCGATTTTCTAAAGAACAA 
               
               
                 CTACCCATATTTAAAGCTCAGAACCCAGATGCAAAAACTACAGAACTA 
               
               
                 ATTAGAAGAATTGCCCAGCGTTGGAGGGAACTTCCTGATTCAAAGAAA 
               
               
                 AAAATATATCAAGATGCTTATAGGGCGGAGTGGCAGGTATATAAAGAA 
               
               
                 GAGATAAGCAGATTTAAAGAACAGCTAACTCCAAGTCAGATTATGTCT 
               
               
                 TTGGAAAAAGAAATCATGGACAAACATTTAAAAAGGAAAGCTATGACA 
               
               
                 AAAAAAAAAGAGTTAACACTGCTTGGAAAACCAAAAAGACCTCGTTCA 
               
               
                 GCTTATAACGTTTATGTAGCTGAAAGATTCCAAGAAGCTAAGGGTGAT 
               
               
                 TCACCGCAGGAAAAGCTGAAGACTGTAAAGGAAAACTGGAAAAATCTG 
               
               
                 TCTGACTCTGAAAAGGAATTATATATTCAGCATGCTAAAGAGGACGAA 
               
               
                 ACTCGTTATCATAATGAAATGAAGTCTTGGGAAGAACAAATGATTGAA 
               
               
                 GTTGGACGAAAGGATCTTCTACGTCGCACAATAAAGAAACAACGAAAA 
               
               
                 TATGGTGCTGAGGAGTGTTAA 
               
               
                   
               
               
                 PTD-MLS-TFAM (PTD underlined; MLS double 
               
               
                 underline) Sequence 
               
               
                 (SEQ. ID. NO.: 208): 
               
               
                 ATGGCG CGTCGTCGTCGTCGTCGTCGTCGTCGTCGTCGT   ATGGCGTTT   
               
               
                 
                   CTCCGAAGCATGTGGGGCGTGCTGAGTGCCCTGGGAAGGTCTGGAGCA 
                 
               
               
                 
                   GAGCTGTGCACCGGCTGTGGAAGTCGACTGCGCTCCCCCTTCAGTTTT 
                 
               
               
                   GTGTATTTACCGAGGTGGTTT TCATCTGTCTTGGCAAGTTGTCCAAAG 
               
               
                 AAACCTGTAAGTTCTTACCTTCGATTTTCTAAAGAACAACTACCCATA 
               
               
                 TTTAAAGCTCAGAACCCAGATGCAAAAACTACAGAACTAATTAGAAGA 
               
               
                 ATTGCCCAGCGTTGGAGGGAACTTCCTGATTCAAAGAAAAAAATATAT 
               
               
                 CAAGATGCTTATAGGGCGGAGTGGCAGGTATATAAAGAAGAGATAAGC 
               
               
                 AGATTTAAAGAACAGCTAACTCCAAGTCAGATTATGTCTTTGGAAAAA 
               
               
                 GAAATCATGGACAAACATTTAAAAAGGAAAGCTATGACAAAAAAAAAA 
               
               
                 GAGTTAACACTGCTTGGAAAACCAAAAAGACCTCGTTCAGCTTATAAC 
               
               
                 GTTTATGTAGCTGAAAGATTCCAAGAAGCTAAGGGTGATTCACCGCAG 
               
               
                 GAAAAGCTGAAGACTGTAAAGGAAAACTGGAAAAATCTGTCTGACTCT 
               
               
                 GAAAAGGAATTATATATTCAGCATGCTAAAGAGGACGAAACTCGTTAT 
               
               
                 CATAATGAAATGAAGTCTTGGGAAGAACAAATGATTGAAGTTGGACGA 
               
               
                 AAGGATCTTCTACGTCGCACAATAAAGAAACAACGAAAATATGGTGCT 
               
               
                 GAGGAGTGTTAA 
               
               
                   
               
               
                 PTD-NLS-TFAM (Nuclear Localization Signal 
               
               
                 (underline) Replaces Mitochondrial; PTD double 
               
               
                 underline) 
               
               
                 (SEQ. ID. NO.: 209): 
               
               
                 ATGGCG CGTCGTCGTCGTCGTCGTCGTCGTCGTCGTCGT   CCGAAAAAA   
               
               
                   AAACGTAAAGTTGAAGACCCG TCATCTGTCTTGGCAAGTTGTCCAAAG 
               
               
                 AAACCTGTAAGTTCTTACCTTCGATTTTCTAAAGAACAACTACCCATA 
               
               
                 TTTAAAGCTCAGAACCCAGATGCAAAAACTACAGAACTAATTAGAAGA 
               
               
                 ATTGCCCAGCGTTGGAGGGAACTTCCTGATTCAAAGAAAAAAATATAT 
               
               
                 CAAGATGCTTATAGGGCGGAGTGGCAGGTATATAAAGAAGAGATAAGC 
               
               
                 AGATTTAAAGAACAGCTAACTCCAAGTCAGATTATGTCTTTGGAAAAA 
               
               
                 GAAATCATGGACAAACATTTAAAAAGGAAAGCTATGACAAAAAAAAAA 
               
               
                 GAGTTAACACTGCTTGGAAAACCAAAAAGACCTCGTTCAGCTTATAAC 
               
               
                 GTTTATGTAGCTGAAAGATTCCAAGAAGCTAAGGGTGATTCACCGCAG 
               
               
                 GAAAAGCTGAAGACTGTAAAGGAAAACTGGAAAAATCTGTCTGACTCT 
               
               
                 GAAAAGGAATTATATATTCAGCATGCTAAAGAGGACGAAACTCGTTAT 
               
               
                 CATAATGAAATGAAGTCTTGGGAAGAACAAATGATTGAAGTTGGACGA 
               
               
                 AAGGATCTTCTACGTCGCACAATAAAGAAACAACGAAAATATGGTGCT 
               
               
                 GAGGAGTGTTAA 
               
               
                   
               
               
                 PTD-MLS-TFAM peptide Length: 259 
               
               
                 (SEQ. ID. NO.: 210) 
               
               
                 MARRRRRRRR RRRMAFLRSM WGVLSALGRS GAELCTGCGS 
               
               
                 RLRSPFSFVY LPRWFSSVLA SCPKKPVSSY LRFSKEQLPI 
               
               
                 FKAQNPDAKT TELIRRIAQR WRELPDSKKK IYQDAYRAEW 
               
               
                 QVYKEEISRF KEQLTPSQIM SLEKEIMDKHLKRKAMTKKK 
               
               
                 ELTLLGKPKR PRSAYNVYVA ERFQEAKGDS 
               
               
                 PQEKLKTVKENWKNLSDSEK ELYIQHAKED ETRYHNEMKS 
               
               
                 WEEQMIEVGR KDLLRRTIKKQRKYGAEEC* 
               
               
                   
               
               
                 PTD-NLS-TFAM peptide Length: 227 
               
               
                 (SEQ. ID. NO.: 211) 
               
               
                 MARRRRRRRR RRRPKKKRKV EDPSSVLASC PKKPVSSYLR 
               
               
                 FSKEQLPIFKAQNPDAKTTE LIRRIAQRWR ELPDSKKKIY 
               
               
                 QDAYRAEWQV YKEEISRFKEQLTPSQIMSL EKEIMDKHLK 
               
               
                 RKAMTKKKEL TLLGKPKRPR SAYNVYVAERFQEAKGDSPQ 
               
               
                 EKLKTVKENW KNLSDSEKEL YIQHAKEDET 
               
               
                 RYHNEMKSWEEQMIEVGRKD LLRRTIKKQR KYGAEEC* 
               
            
           
         
       
     
     Selected Model Organism Protein Similarities That Can Be Used In The Compositions And Methods Disclosed Herein: 
     
       
         
           
               
             
               
                   
               
               
                 Organism, Protein And Percent Identity And Length Of Aligned Region 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 
                   H. sapiens 
                 
                 sp: Q00059 - MTT1_HUMAN Transcription 
                 100%/246 aa 
               
               
                 (SEQ. ID. NO.: 212): 
                 factor 1, mitochondrial precursor (MTTF1) 
                 (see ProtEST) 
               
               
                 
                   M. musculus 
                 
                 ref: NP_033386.1 - transcription factor A, mitochondrial 
                 63%/237 aa 
               
               
                 (SEQ. ID. NO.: 213): 
                 [ Mus musculus ] 
                 (see ProtEST) 
               
               
                 
                   R. norvegicus: 
                 
                 ref: NP_112616.1 - transcription factor A, mitochondrial 
                 64%/237 aa 
               
               
                 (SEQ. ID. NO.: 214): 
                 [ Rattus norvegicus ] 
                 (see ProtEST) 
               
               
                   A. thaliana  (SEQ. ID. 
                 ref: NP_192846.1 - 98b like protein [ Arabidopsis   
                 27%/189 aa 
               
               
                 NO.: 215): 
                   thaliana ] 
                 (see ProtEST) 
               
               
                 
                   C. elegans 
                 
                 ref: NP_501245.1 - F45E4.9.p [ Caenorhabditis elegans ] 
                 27%/189 aa 
               
               
                 (SEQ. ID. NO.: 216): 
                   
                 (see ProtEST) 
               
               
                 
                   D. melanogaster: 
                 
                 ref: NP_524415.1 - mitochondrial transcription factor A 
                 34%/183 aa 
               
               
                 (SEQ. ID. NO.: 217): 
                 [ Drosophila melanogaster ] 
                 (see ProtEST) 
               
               
                   
               
            
           
         
       
     
     Sequence data for the sequences referenced herein are known in the art, for example in GenBank, and are incorporated by reference herein, in their entirety. 
     Example 4 
     Nuclear Transfection Image Data 
     Recombinant PTD-NLS-TFAM was labeled with Alexa 488 dye (Molecular Probes) and visualized using confocal on Sy5y cells pre-incubated with MitoTracker Red dye (Molecular Probes). Green fluorescence indicates the Alexa 488 labeled recombinant PTD-NLS-TFAM and red fluorescence indicates mitochondria, providing a peri-nuclear distribution for analysis of nuclear boundaries. Several images at 5, 10 and 20 minutes post-introduction of the labeled recombinant protein into the cellular media were captured with confocal microscopy. Note that the recombinant construct achieves a more intense localization when within the nucleus (20 minute image capture) implying condensation in the presence of DNA. 
     Example 5 
     Gene Delivery Vector 
     The gene delivery vector molecule depicted in  FIG. 1  was used to deliver DNA. This vector, which combines the three elements of gene delivery described above, allows for the first time the introduction of full-length mitochondrial genomes and exogenous genes cloned into mtDNA into the mitochondrial matrix compartment in both dividing and non-dividing cells. 
     To allow prompt cellular uptake of TFAM-bound DNA, a PTD sequence, specifically, a poly-arginine stretch was added to the N-terminal of the vector molecule. The (+) charge of the PTD aids in the apposition to the (−) charged surface of living cells, while its amphipathic nature leads to invading the cellular membrane. The specific targeting of DNA to the mitochondrial compartment was achieved by adding the amino-terminal mitochondrial targeting signal (MLS) of malate dehydrogenase to the vector between the PTD and TFAM. The combination of PTD and MLS will be further referred to as a Mitochondrial Targeting Domain (MTD). 
       FIG. 2  shows that w.t. mtDNA that has been labeled with Alexa 488 dye and complexed with PTD-MLS-TFAM rapidly enters mitochondria of SY5Y cells within 15 minutes. 
       FIG. 3  shows the rapid restoration of mtDNA replication and bioenergetic function following introduction of w.t. mtDNA by the disclosed methods. The top image (A) is of rho0 cells stained with MitoTracker Red (MTRed), to localize mitochondria as a function of their ΔψM, and following incubation for 12 hours with BrdU and immunostained for BrdU with FITC. Low levels of MTRed accumulation were detected, reflecting low ΔψM, and absence of BrdU staining. Part (B) shows a normal SY5Y cell and part (C) shows a rho0 cell 16 hours after transfection with PTD-MLS-TFAM complexed with w.t. mtDNA. There is a marked increase in MTRed uptake and BrdU staining. 
     Example 6 
     Functionality of Transfected DNA 
     The functionality of transfected DNA was verified by the rescue of the rho0 phenotype. Rho0 cells are cultured cells depleted of endogenous mtDNA by prolonged incubation with ethidium bromide. Such cells survive only in media supplemented with uridine and pyruvate, and do not have measurable activities of the electron transport chain, whose subunits are partially encoded by the mitochondrial genome. MTD-TFAM complexed with LHON mtDNA was added to the media of rho0 cells created from Sy5y cells.  FIG. 4  shows successful introduction into and replication of LHON mtDNA in rho0 cells. The LHON 11778A mutation causes loss of the SfaNI site present in w.t. mtDNA. In rho0 cells a similar w.t.-like pseudogene is amplified and cut by SfaN1. Following transfection of LHON mtDNA into rho0 and passage through metabolic selection, mainly the introduced LHON mtDNA free of the SfaNI site is found. 
     Example 7 
     Transfection of EGFP 
     Additional verification of mitochondrial delivery of DNA was achieved by the introduction of the gene for the green fluorescent protein, EGFP. Since the mitochondrial translation table differs from the nuclear code, the cDNA for EGFP was engineered such that mitochondria will be able to translate the full-length, fluorescing protein (Codon change at nucleotide position 173 of mtEGFP from UGG→UGA), whereas nuclear translation will result in a truncated protein lacking fluorescent activity. The mutagenized, mitochondrial EGFP (mtEGFP) was cloned into the mitochondrial genome at one of two sites (the BamHI site producing ND6 fused to mtEGFP and at the junction of the genes COX3 and ATP6). This ensures that replication and transcription of mitochondrial gene products is undisturbed and that only the mitochondrial replication and transcriptional machinery is capable of maintaining the reporter gene-mtDNA construct. In both cases, due to the transcription of the mitochondrial genome as a polycistronic transcript from a common promoter, the introduced mtEGFP sequence was transcribed alongside the endogenous mitochondrial genes of the mitosome, and translated into a functional, fluorescing product whereas the nuclear transfected mtEGFP failed to produce any fluorescence ( FIG. 5 ). To further verify mtEGFP expression by mitochondria, RNA interference was used to the mtDNA polymerase, polymerase gamma (PoIG). Using a combination of three siRNAs, PoIG levels became undetectable within three days causing the rapid loss of mtDNA. Similarly, mtEGFP was found to be undetectable ( FIG. 6 ) with RNAi to PoIG but not with control siRNA to EGFP. 
     Example 8 
     Translocation of Proteins Encoded By Exogenous DNA 
     The general usefulness of the disclosed compositions and methods to direct gene delivery to mitochondria and express mtDNA and cloned genes was further expanded to test the possibility of having mitochondrially expressed genes directed to other subcellular compartments. To test this possibility mtEGFP was fused with sequences coding for a combination of PTD and the nuclear localization signal (NLS) from Sv40 Large T antigen and cloned into the COX3-ATP6 site. The resulting protein is a fusion of EGFP and NLS+PTD (the Mitochondrial Escape Domain, MED). The MED allows the fusion protein to leave the mitochondria and be targeted to the nucleus. As shown in  FIG. 7 , the MED-NLS-EGFP concentrated in the nuclei to a significant extent and was detectable in mitochondria. The process is rather efficient, with more than 10% of cells showing robust nuclear accumulation of mitochondrially-translated protein. 
     Example 9 
     Digestion of Endogenous DNA 
     Several groups have reported a selective advantage of mutant mtDNA over that of wild-type. To enable the introduced mtDNA to overwhelm any endogenous mtDNA, mutant or otherwise, the BamHI site in ND6 of the cloned mtDNA were abolished. The mutagenesis does not cause an amino acid change. The BamHI restriction endonuclease cDNA was cloned in the COX3-ATP6 site. By abolishing the BamHI site and including the BamHI restriction endonuclease as part of the cloned mtDNA, the BamHI enzyme would restriction digest all endogenous mtDNA and be unable to digest the introduced mtDNA. 24 hours after transfection into Sy5y cells, endogenous mtDNA was undetectable using PCR ( FIG. 8A ). Furthermore, mitochondrial lysates showed significant BamHI activity ( FIG. 8B ). 
     Once delivered, DNA may become permanently integrated into the genome despite losing its therapeutic potential. To overcome this limitation, the BgIII restriction site was introduced into the cloned mtDNA expressing mtEGFP. A recombinant BgIII restriction enzyme was created that contained a MTD on the amino terminus. After several passages in culture, mtEGFP expression was intact. The recombinant MTD-BgIII was added to the mtEGFP expressing Sy5y cells and noted a precipitous drop in EGFP expression and detectable levels of the cloned mtDNA, enabling the possibility of non-permanent genetic modifications ( FIG. 9 ). 
     It should be emphasized that the above-described embodiments of the present disclosure, particularly, any “preferred” embodiments, are merely possible examples of implementations, merely set forth for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the above-described embodiment(s) of the disclosure without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure and the present disclosure and protected by the following claims.