Gamma peptide nucleic acids linked to cell-penetrating motifs

Abstract

A compound, or a stereoisomer, tautomer, or pharmaceutically acceptable salt, comprising: a gamma peptide nucleic acid linked to a cell-penetrating motif.

Description

[0001] 8123-112925-02 03 / 13 / 26 06891

[0002] GAMMA PEPTIDE NUCLEIC ACIDS LINKED TO CELL-PENETRATING MOTIFS

[0003] CROSS REFERENCE TO RELATED APPLICATION

[0004] This application claims the benefit of and priority to the earlier filing date of U. S. Provisional Application No. 63 / 772,087, filed March 14, 2025, which is herein incorporated by reference in its entirety.

[0005] ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

[0006] This invention was made with government support under HD099666 awarded by the National Institutes of Health and under W81XWH-22- 1-0062 awarded by the Defense Health Agency, Medical Research and Development Branch. The government has certain rights in the invention.

[0007] BACKGROUND

[0008] Mitochondria exist in almost every cell of the body and are responsible for creating 90% of the energy needed to sustain life. The mitochondria have their own genome called mitochondrial DNA (mtDNA) that contains 37 genes that encode 13 proteins, and 22 tRNAs and two rRNAs. Mitochondrial disease is a lifelong disorder caused by specific variations in mtDNA that cause mitochondrial dysfunction. Primary mitochondrial disease (PMD) affects roughly one in five thousand individuals, presenting significant challenges due to the lack of effective therapies. These disorders diminish patients' quality of life and burden healthcare systems.

[0009] Some prominent mtDNA mutations include the maternally inherited diabetes and deafness (MIDD) mutation; the myoclonic encephalopathy, lactic acidosis, with or without seizures (MELAS) mutation; and deletion mutations. The most common pathogenic mitochondrial DNA (mtDNA) variant, m.3243A> G, located in the tRNA Leu(UUR) gene (MT-TL1), causes mitochondrial encephalomyopathy, lactic acidosis, and stroke -like episodes (MELAS), and maternally inherited diabetes and deafness (MIDD). Clinical manifestations typically emerge when heteroplasmy exceeds approximately 60%. This tRNA variant disrupts oxidative phosphorylation in energy -demanding tissues, such as brain, pancreas, and heart. Cardiomyopathy is a major cause of morbidity and mortality in affected individuals, making the heart a therapeutic target. Currently, there is no safe and selective8123-112925-02 03 / 13 / 26 06891

[0010] cure or treatment for mitochondrial disease. Thus, there is a need for new mechanisms of targeting mtDNA.

[0011] SUMMARY

[0012] Disclosed herein is a compound, or a stereoisomer, tautomer, or pharmaceutically acceptable salt, comprising: a gamma peptide nucleic acid linked to a cell-penetrating motif.

[0013] Also disclosed herein is a pharmaceutical composition comprising at least one compound disclosed herein and at least one pharmaceutically acceptable excipient.

[0014] Additionally, disclosed herein is a method comprising of treating or preventing a mitochondrial condition, the method comprising administering to a subject in need thereof an effective amount of at least one compound, mixture, or pharmaceutical composition disclosed herein.

[0015] Further disclosed herein is a method for inhibiting replication of a mitochondrial mutation, the method comprising contacting mitochondrial DNA containing the mitochondrial mutation with an effective amount of at least one compound, mixture, or pharmaceutical composition disclosed herein.

[0016] Also disclosed herein are aspects of a novel synthetic route to obtain at least one compound, mixture, or pharmaceutical composition disclosed herein.

[0017] The foregoing will become more apparent from the following detailed description.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows examples of live-cell fluorescence images of CPM3-TAMRA, mitochondria, and nuclear chromatin in iPSC-derived cardiomyocytes according to aspects of the present disclosure.

[0019] FIG. 2 shows a non-competitive strand invasion gel shift assay of mutant and wildtype mtDNA.

[0020] FIG. 3 shows a PCR inhibition assay, illustrating the addition of CPM3 to yPNA, increasing polymerase inhibition characteristics of yPNA without altering selectivity. The molar ratio of yPNA to the template is shown at the bottom.

[0021] FIG. 4 shows a plot of qPCR results (duplex TAQMAN for ND1 and B2M) for 143B carrying either wildtype (0% m.3243A> G) or m.3243A> G (100%) treated with CPM3-yPNA, with each line normalized to the control of the same genotype.

[0022] FIG. 5 shows a plot of qPCR results for 60% m.3243A> G.8123-112925-02 03 / 13 / 26 06891

[0023] FIG. 6 shows high-performance liquid chromatography (HPLC) of a CPM3 coupled to a yPNA according to aspects of the present disclosure.

[0024] FIG. 7 shows mass spectrometry (MS) analysis of a CPM3 coupled to a yPNA according to aspects of the present disclosure.

[0025] FIG. 8 is an image of a gel showing strand invasion and selectivity of m.3243A> G-targeted yPNAs and PNAs according to aspects of the present disclosure.

[0026] FIG. 9 shows an example set of live-cell fluorescence images of CPM3-TAMRA, mitochondria (MITOTRACKER), and nuclear chromatin (Hoechst) in 143B WT and 90% m.3243A> G cells according to aspects of the present disclosure.

[0027] FIG. 10 is a plot of respiration in 143B m.3243A> G (90%) treated with CPM3-yPNA according to aspects of the present disclosure.

[0028] FIG. 11 is a schematic illustration of yPNA engagement with mitochondrial nucleoids and replication dynamics according to aspects of the present disclosure.

[0029] FIG. 12 shows atomic force microscopy images of TF AM-mediated DNA compaction according to aspects of the present disclosure.

[0030] FIG. 13 shows TFAM binding on m.3243A> G DNA and displacement by yPNA according to aspects of the present disclosure.

[0031] FIG. 14 shows Sanger sequencing traces of low and high heteroplasmy (het) iPSC-CMs, and quantitation of duplicate PCR / Sanger reaction of high-heteroplasmy iPSCs and iPSC-CMs, according to aspects of the present disclosure.

[0032] FIG. 15 shows MYOCYTER analysis of isogenic m.3243A> G iPSC-CMs, according to aspects of the present disclosure.

[0033] FIG. 16 shows examples of live-cell fluorescence images of PNA–TAMRA and lysosomes.

[0034] FIG. 17 shows examples of live-cell fluorescence images of γPNA–TAMRA and lysosomes.

[0035] FIG. 18 shows examples of live-cell fluorescence images of a 4:1 (DNA:yPNA) complex formed and delivered with LIPOFECTAMINE 3000.

[0036] FIG. 19 shows examples of live-cell fluorescence images of rFrF–TAMRA and mitochondria in mouse embryonic fibroblasts (MEFs).8123-112925-02 03 / 13 / 26 06891

[0037] DETAILED DESCRIPTION

[0038] Introduction

[0039] The mitochondria contain a small, circular -16,569 base pair (bp) genome that comprises about 1% of the total cellular DNA in humans. Mitochondrial DNA (mtDNA) is present at 100-10,000 copies per human cell, depending on the cell type: a singular mitochondrion contains between 2 and 10 copies of mtDNA. Variants in mtDNA, including single-nucleotide substitutions and large deletions, can impair oxidative phosphorylation (OXPHOS). mtDNA variants are believed to originate from the low fidelity of mtDNA maintenance. The mitochondrion lacks the same repair mechanisms as nuclear DNA. The mitochondrial genome primarily relies on base excision repair (BER), which allows the mtDNA to repair DNA bases affected by oxidation, alkylation, deamination, and singlestrand DNA breaks via DNA glycosylases. Additionally, mtDNA lacks protective histones and is susceptible to oxidative damage from reactive oxygen species generated in the inner mitochondrial membrane. To repair double-strand breaks (DSBs) of nuclear DNA, the nuclear genome relies on homologous recombination (HR) and non-homologous end joining (NHEJ); in contrast, there is little evidence of HR and NHEJ repair mechanisms in mtDNA. Consequently, mtDNA has a mutation rate 10-20 times higher than nuclear DNA.

[0040] Pathogenic mtDNA variants cause incurable primary mitochondrial diseases (PMDs) with high morbidity and mortality. There is a need to develop robust, sequence-specific mtDNA targeting methods, not only as a therapeutic approach but also as a tool to investigate the fundamental principles of mitochondrial DNA replication, packaging, and repair.

[0041] PMDs are genetic disorders caused by known or presumed pathogenic variants in mtDNA or nuclear genes that encode the mitochondrial respiratory chain and mitochondrial proteins. PMDs can be devastating and result in a lifelong care burden with high morbidity and mortality. PMDs often manifest as multisystemic disorders and can present at any age. Currently, there is no effective cure or precision treatment for PMD. mtDNA variation causes PMD in at least 1 in 5,000 people globally, and it is estimated that 1 in 200 people is an asymptomatic carrier of a pathogenic mtDNA variant. PMDs caused by variants in the mitochondrial genome are inherited through direct maternal mtDNA inheritance but can also occur de novo (de novo cases affect -1 / 10,000 live births per year). In individuals with a pathogenic mtDNA variant, the high mtDNA copy number per cell creates an environment for wild-type (WT) and variant mtDNA to coexist, a condition termed heteroplasmy.

[0042] Mitochondrial disease symptoms typically present when variant mtDNA levels exceed a biochemical threshold, which can vary depending on the mtDNA variant but is typically8123-112925-02 03 / 13 / 26 06891

[0043] >60%. Because disease can manifest when mutant load is above this -60%, a successful intervention may only need to lower heteroplasmy below this threshold to restore significant respiratory function.

[0044] The heteroplasmic nature of many mtDNA-bome diseases creates an opportunity for mtDNA-targeted therapies that focus on shifting heteroplasmy levels by depleting variant mtDNA and allowing WT mtDNA to repopulate the genome. Due to the biochemical threshold for mitochondrial disease symptoms (>60% variant mtDNA), a small shift in heteroplasmy levels (e.g., 10% - 15%) could result in functional mitochondria.

[0045] Consequently, there has been significant effort in applying CRISPR / Cas9, zinc finger nucleases (ZFN), transcription-activator-like effector nucleases (TALEN), base editing, and MITOARCUS editing approaches to variant mtDNA. Targeting mtDNA is a two-step challenge; the gene editing moiety must traverse the cell membrane and then get imported into the mitochondria through the mitochondrial double membrane. Mitochondrial import has proved challenging in delivering CRISPR / Cas9 guide RNA to the mtDNA because there is no defined RNA import mechanism for the mitochondria, with some limited success. For these reasons, the status of CRISPR / Cas9 editing of variant mtDNA remains ambiguous. ZFN and TALEN-based editing, in contrast, may be more successful in mtDNA targeting. Unfortunately, there are disadvantages to both approaches. ZFNs are difficult and timeconsuming to design for non-specialists, and using proteins to induce double-stranded breaks raises concerns about off-target effects. TALENs are slightly less cumbersome to create and are shown to produce fewer off-target effects compared to ZFNs; however, their large size (>3kb) is a barrier to mitochondrial delivery. ZFs and TALEs (lacking nuclease activity) can be engineered into base editing technologies by incorporating dsDNA deaminases. Base editing technology allows for correcting single-nucleotide mtDNA variants, limited to CxG to TxA or AxT to GxC conversions, without inducing a DSB. A prominent concern with this approach is bystander editing, which occurs from deaminase processivity, i.e., if multiple Cs or As surround the intended target, those bases could be unintentionally edited.

[0046] MITOARCUS editing technology by Precision BioSciences uses a meganuclease to detect palindromic 22bp DNA sequences and create a DSB in mtDNA. Although MITOARCUS is promising, recent work has shown 226 potential nuclear off-target effects. The above mtDNA editing approaches have demonstrated varying degrees of heteroplasmy shift and restoration of WT mitochondrial function. However, most rely on adeno-associated virus (AAV) delivery, which poses challenges including toxicity, immunogenicity, and limited tropism and accessibility. Thus, there is a need for new mechanisms of targeting mtDNA.8123-112925-02 03 / 13 / 26 06891

[0047] Disclosed herein are aspects of a compound for treating or preventing a mitochondrial condition. In particular disclosed aspects, the compound is capable of inhibiting replication of a mitochondrial mutation, such as the maternally inherited diabetes and deafness (MIDD) mutation; mitochondrial myopathy, encephalopathy, and stroke-like episodes (MELAS) mutation; or any other mutation. Such compounds represent an innovative approach to targeting mtDNA and provide a path for treatment of diseases based on mitochondrial mutation(s).

[0048] Certain aspects relate to a repeatable, sequence-specific therapeutic approach using y-modified peptide nucleic acids (yPNAs) delivered to mitochondria via a cell-penetrating motif (CPM). Peptide nucleic acids (PNAs) and y-modified PNAs offer high binding affinity and mismatch discrimination, with yPNAs capable of strand invasion of double-stranded DNA; however, effective mitochondrial delivery of these relatively large, highly functionalized cargos is challenging. In some instances, small molecule mitochondrial tags (e.g., triphenylphosphonium, TPP) can be appended to PNAs, but this approach fails to deliver the PNAs to the mitochondria. TPP-modified PNAs became trapped at membranes rather than reaching the matrix, illustrating that techniques suitable for small molecules do not translate predictably to nucleic-acid–like cargos.

[0049] Without wishing to be bound by theory, in some aspects, the cell-penetrating motif may exhibit a heretofore undisclosed mechanism of action through endosomal fusion within a mitochondrial network, where timing may differ between different cell types. The yPNA differs from oligonucleotides, which have a phosphodiester backbone and are subject to nuclease destruction. PNAs, including yPNAs, have a protein amide backbone linked to nucleic acids, which may interact more strongly with DNA than oligonucleotides and are resistant to nucleases. PNAs are also less error-tolerant oligonucleotides, making PNAs potentially more sequence-selective than their oligonucleotide equivalents.

[0050] For oligonucleotides or PNAs, their DNA target must be single-stranded, which occurs during replication. yPNAs are derivatives of PNAs that substitute the gamma position in the backbone (e.g., with a serine substituent), increase affinity, and are strand invasive. The yPNA therefore may engage double-stranded DNA, outside of replication, with suitably high affinity to block replication. Blocked replication may result in formation of linear DNA, which is quickly degraded in mitochondria. When directed to the mitochondrial matrix, yPNA can therefore potentially negatively select against specific mtDNA sequences. By defining the molecular determinants of delivery, nucleoid engagement, and functional outcomes across patient-derived models, CPM- yPNAs can enable precision manipulation of8123-112925-02 03 / 13 / 26 06891

[0051] mtDNA heteroplasmy. In some aspects. CPM- yPNAs selectively target variant mtDNA, thereby promoting the replication of wild-type genomes. This approach can provide a safer and more effective means of improving mitochondrial function in heteroplasmic models. The resulting framework can be adapted to other mtDNA variants and provide the foundation for translational development of CPM3-yPNA-based mitochondrial therapeutics.

[0052] Terminology

[0053] The following explanations of terms and methods are provided to better describe the present compounds, compositions and methods, and to guide those of ordinary skill in the art in the practice of the present disclosure. It is also to be understood that the terminology used in the disclosure is for the purpose of describing particular embodiments and examples only and is not intended to be limiting.

[0054] “Administration” as used herein is inclusive of administration by another person to the subject or self-administration by the subject.

[0055] The term "aliphatic" is defined as including alkyl, alkenyl, alkynyl, halogenated alkyl and cycloalkyl groups. A "lower aliphatic" group is a branched or unbranched aliphatic group having from 1 to 10 carbon atoms.

[0056] “Alkanediyl,” “cycloalkanediyl,” “aiyldiyl,” “alkanearyldiyl” refers to a divalent radical derived from aliphatic, cycloaliphatic, aryl, and alkanearyl hydrocarbons.

[0057] “Alkenyl” refers to a cyclic, branched or straight chain group containing only carbon and hydrogen, and contains one or more double bonds that may or may not be conjugated. Alkenyl groups may be unsubstituted or substituted. “Lower alkenyl” groups contain one to six carbon atoms.

[0058] The term “alkoxy” refers to a straight, branched or cyclic hydrocarbon configuration and combinations thereof, including from 1 to 20 carbon atoms, or from 1 to 8 carbon atoms (referred to as a “lower alkoxy”). An example of an “alkoxy group” is represented by the formula -OR, where R can be an alkyl group, optionally substituted with an alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, alkoxy or heterocycloalkyl group. Suitable alkoxy groups include methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy, secbutoxy, tert-butoxy cyclopropoxy, cyclohexyloxy, and the like.

[0059] “Alkoxycarbonyl” refers to an alkoxy substituted carbonyl radical, -C(O)OR, wherein R represents an optionally substituted alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl or similar moiety.8123-112925-02 03 / 13 / 26 06891

[0060] The term “alkyl” refers to a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, / / -propyl, isopropyl, / / -butyl, isobutyl, / -butyl, pentyl, hexyl, heptyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like. A “lower alkyl” group is a saturated branched or unbranched hydrocarbon having from 1 to 6 carbon atoms. Alkyl groups may be “substituted alkyls” wherein one or more hydrogen atoms are substituted with a substituent such as halogen, cycloalkyl, alkoxy, amino, hydroxyl, aryl, alkenyl, or carboxyl. For example, a lower alkyl or (Ci-Celalkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec -butyl, pentyl, 3-pentyl, or hexyl; (C3-Ce)cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; (C3-C6)cycloalkyl(Ci-C6)alkyl can be cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, 2-cyclopropylethyl, 2-cyclobutylethyl, 2-cyclopentylethyl, or 2-cyclohexylethyl; (Ci-Ce)alkoxy can be methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy; (C2-Ce)alkenyl can be vinyl, allyl, 1 -propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1, -pentenyl, 2-pentenyl, 3 -pentenyl, 4-pentenyl, 1- hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, or 5-hexenyl; (C2-Ce)alkynyl can be ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1 -pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1- hexynyl, 2 -hexynyl, 3-hexynyl, 4-hexynyl, or 5-hexynyl; (Ci-Ce)alkanoyl can be acetyl, propanoyl or butanoyl; halo(Ci-Ce)alkyl can be iodomethyl, bromomethyl, chloromethyl, fluoromethyl, trifluoromethyl, 2-chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, or pentafluoroethyl; hydroxy(Ci-C6)alkyl can be hydroxymethyl, 1 -hydroxy ethyl, 2-hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl, 3-hydroxypropyl, 1 -hydroxybutyl, 4-hydroxybutyl, 1-hydroxypentyl, 5-hydroxypentyl, 1 -hydroxyhexyl, or 6-hydroxyhexyl; (Ci-C6)alkoxycarbonyl can be methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, or hexyloxy carbonyl; (Ci-Ce) alkylthio can be methylthio, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, pentylthio, or hexylthio; (C2-Ce)alkanoyloxy can be acetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy, or hexanoyloxy.

[0061] “Alkynyl” refers to a cyclic, branched or straight chain group containing only carbon and hydrogen, and unless otherwise mentioned typically contains one to twelve carbon atoms, and contains one or more triple bonds. Alkynyl groups may be unsubstituted or substituted. “Lower alkynyl” groups are those that contain one to six carbon atoms.

[0062] The term “amine” or “amino” refers to a group of the formula -NRR', where R and R' can be, independently, hydrogen or an alkyl, alkenyl, alkynyl, acyl, aryl, arylalkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl group. For example, an “alkylamino” or8123-112925-02 03 / 13 / 26 06891

[0063] “alkylated amino” refers to -NRR', wherein at least one of R or R' is an alkyl. A suitable amine or amino group is acetamido.

[0064] The term "aminoalkyl" refers to alkyl groups as defined above where at least one hydrogen atom is replaced with an amino group (e.g, -CH2-NH2).

[0065] “Aminocarbonyl” alone or in combination, means an amino substituted carbonyl (carbamoyl) radical, wherein the amino radical may optionally be mono- or di-substituted, such as, for example, with alkyl, aryl, acyl, aralkyl, cycloalkyl, cycloalkylalkyl, alkanoyl, alkoxycarbonyl, aralkoxycarbonyl and the like. For example, an aminocarbonyl may be represented by the formula -C(O)NRR', where R and R' independently can be, for example, a hydrogen, alkyl, alkenyl, alkynyl, acyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl group.

[0066] An “animal” refers to living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term mammal includes both human and nonhuman mammals.

[0067] The term "arylalkyl" refers to an alkyl group wherein an aryl group is substituted for a hydrogen of the alkyl group. An example of an arylalkyl group is a benzyl group.

[0068] “Aryl” refers to a monovalent unsaturated or aromatic (including pseudoaromatic) carbocyclic group having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl), which can optionally be unsubstituted or substituted. The term "pseudoaromatic" refers to a ring system which is not strictly aromatic, but which is stabilized by means of delocalization of electrons and behaves in a similar manner to aromatic rings. A “heteroaryl group,” is defined as an unsaturated or aromatic (including pseudoaromatic) group that has at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorous. Heteroaryl includes, but is not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl, and the like. The aryl or heteroaryl group can be substituted with one or more groups including, but not limited to, alkyl, alkynyl, alkenyl, aryl, halide, nitro, amino, ester, ketone, aldehyde, hydroxy, carboxylic acid, or alkoxy, or the aryl or heteroaryl group can be unsubstituted. The term heteroaryl includes hydroxy-substituted heteroaryls that may exist in tautomeric keto forms, such as 2-hydroxypyridine and pyridine-2-one, and their N-substituted derivatives that necessarily exist in the keto form, such as N-methylpyridin-2-one.8123-112925-02 03 / 13 / 26 06891

[0069] “Aryloxy” or “heteroaryloxy” refers to a group of the formula -OAr, wherein Ar is an aryl group or a heteroaryl group, respectively.

[0070] A “carbonylamino” group may be -N(R)-C(O)-R (wherein each R is independently a substitution group such as, for example, alkyl, alkenyl, alkynyl, acyl, aryl, arylalkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl group, or H). A suitable carbonylamino group is acetamido.

[0071] The term “carboxylate” or “carboxyl” refers to the group -COO" or -COOH. The carboxyl group can form a carboxylic acid. “Substituted carboxyl” refers to -COOR where R is alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl group. For example, a substituted carboxyl group could be a carboxylic acid ester or a salt thereof (e.g., a carboxylate).

[0072] The term “cell-penetrating motif’ or “CPM” generally refers to a chemical structure that is capable of entering a cell. In some aspects disclosed herein, when the cell-penetrating motif is linked to another moiety (such as a gamma peptide nucleic acid), for example by a covalent bond, the cell-penetrating motif enables this moiety to enter the cell or be taken up into the cell at a greater rate than without the cell-penetrating motif.

[0073] The term “cycloalkyl” refers to a non-aromatic carbon-based ring composed of at least three carbon atoms. A cycloalkyl may be a mono or bicyclic ring or ring system.

[0074] Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. The term “heterocycloalkyl group” is a cycloalkyl group as defined above where at least one of the carbon atoms of the ring is substituted with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorous. A heterocycloalkyl may be a mono- or bicyclic ring or ring system.

[0075] The term “ester” refers to a carboxyl group-containing moiety having the hydrogen replaced with, for example, a Ci-ealkyl group (“carboxy IC1-6 alkyl” or “alkylester”), an aryl or aralkyl group (“arylester” or “aralkylester”) and so on. CChCi. / alkyl groups are preferred, such as for example, methylester (CO 2Me), ethylester (CO2EO and propylester (CO2Pr) and includes reverse esters thereof (e.g. -OCOMe, -OCOEt and -OCOPr).

[0076] The terms "halogenated alkyl" or "haloalkyl group" refer to an alkyl group with one or more hydrogen atoms present on these groups substituted with a halogen (F, Cl, Br, I).

[0077] The term “hydroxyl” is represented by the formula -OH.

[0078] The term "hydroxyalkyl" refers to an alkyl group that has at least one hydrogen atom substituted with a hydroxyl group. The term "alkoxyalkyl group" is defined as an alkyl group that has at least one hydrogen atom substituted with an alkoxy group described above.8123-112925-02 03 / 13 / 26 06891

[0079] “Inhibiting” refers to inhibiting the full development of a disease or condition.

[0080] “Inhibiting” also refers to any quantitative or qualitative reduction in biological activity or binding, relative to a control.

[0081] The term “multivalent” generally refers to a moiety that has several sites at which attachment can occur. As used herein, “multivalent moiety” refers to a moiety comprising two or more sites at which A, X, Y, L, M, guanidine or guanidinium groups, and / or cargo can be attached. The multivalent moiety can have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 sites of attachment. At each site of attachment, a lone pair, hydrogen atom, leaving group (e.g., halogen, hydroxy, — OTS, — OMs, — ONs, — OTf, alkoxy, — OSO2R, etc.) or other suitable moiety can be substituted by any of the aforementioned options. In some aspects, the multivalent moiety is divalent, trivalent, or tetravalent. Multivalent and multifunctional may be used interchangeably. Similarly, trivalent and trifunctional can be used interchangeably, as can tetrafunctional and tetrafunctional.

[0082] “N-heterocyclic” refers to mono or bicyclic rings or ring systems that include at least one nitrogen heteroatom. The rings or ring systems generally include 1 to 9 carbon atoms in addition to the heteroatom(s) and may be saturated, unsaturated or aromatic (including pseudoaromatic). The term "pseudoaromatic" refers to a ring system which is not strictly aromatic, but which is stabilized by means of delocalization of electrons and behaves in a similar manner to aromatic rings. Aromatic includes pseudoaromatic ring systems, such as pyrrolyl rings.

[0083] Examples of 5-membered monocyclic N-heterocycles include pyrrolyl, H-pyrrolyl, pyrrolinyl, pyrrolidinyl, oxazolyl, oxadiazolyl, (including 1,2,3 and 1,2,4 oxadiazolyls) isoxazolyl, furazanyl, thiazolyl, isothiazolyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, imidazolyl, imidazolinyl, triazolyl (including 1,2,3 and 1,3,4 triazolyls), tetrazolyl, thiadiazolyl (including 1,2,3 and 1,3,4 thiadiazolyls), and dithiazolyl. Examples of 6-membered monocyclic N-heterocycles include pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, and triazinyl. The heterocycles may be optionally substituted with a broad range of substituents, and preferably with Ci-6 alkyl, Ci-6 alkoxy, C2-6 alkenyl, C2-6 alkynyl, halo, hydroxy, mercapto, trifluoromethyl, amino, cyano or mono or di(Ci-6alkyl)amino. The N-heterocyclic group may be fused to a carbocyclic ring such as phenyl, naphthyl, indenyl, azulenyl, fluorenyl, and anthracenyl.

[0084] Examples of 8, 9 and 10-membered bicyclic heterocycles include 1H thieno[2,3-c]pyrazolyl, indolyl, isoindolyl, benzoxazolyl, benzothiazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolyl, indazolyl, isoquinolinyl, quinolinyl, quinoxalinyl, purinyl,8123-112925-02 03 / 13 / 26 06891

[0085] cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, benzotriazinyl, and the like. These heterocycles may be optionally substituted, for example with Ci-6 alkyl, Ci-6 alkoxy, C2-6 alkenyl, C2-6 alkynyl, halo, hydroxy, mercapto, trifluoromethyl, amino, cyano or mono or di(Ci-6alkyl)amino. Unless otherwise defined optionally substituted N-heterocyclics includes pyridinium salts and the N-oxide form of suitable ring nitrogens.

[0086] The term “peptide nucleic acid” or “PNA" generally refers to a polymer having a protein amide backbone linked to nucleic acids. The term “gamma peptide nucleic acid” or “yPNA” generally refers to a derivative of a peptide nucleic acid that is substituted at its gamma position.

[0087] The term “subject” includes both human and non-human subjects, including birds and non-human mammals, such as non-human primates, companion animals (such as dogs and cats), livestock (such as pigs, sheep, cows), as well as non-domesticated animals, such as the big cats. The term subject applies regardless of the stage in the organism’s life-cycle. Thus, the tern subject applies to an organism in utero or in ovo, depending on the organism (that is, whether the organism is a mammal or a bird, such as a domesticated or wild fowl).

[0088] “Substituted” or “substitution” refers to replacement of a hydrogen atom of a molecule or an R-group with one or more additional R-groups. Unless otherwise defined, the term “optionally-substituted” or “optional substituent” as used herein refers to a group which may or may not be further substituted with 1, 2, 3, 4 or more groups, preferably 1, 2 or 3, more preferably 1 or 2 groups. The substituents may be selected, for example, from C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C3-8cycloalkyl, hydroxyl, oxo, Ci-ealkoxy, aryloxy, C1-6alkoxyaryl, halo, C1-6alkylhalo (such as CF3 and CHF2), C1-6alkoxyhalo (such as OCF3 and OCHF2), carboxyl, esters, cyano, nitro, amino, substituted amino, disubstituted amino, acyl, ketones, amides, aminoacyl, substituted amides, disubstituted amides, thiol, alkylthio, thioxo, sulfates, sulfonates, sulfinyl, substituted sulfinyl, sulfonyl, substituted sulfonyl, sulfonylamides, substituted sulfonamides, disubstituted sulfonamides, aryl, arylC1-6alkyl, heterocyclyl and heteroaryl wherein each alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heterocyclyl and groups containing them may be further optionally substituted. Optional substituents in the case N-heterocycles may also include but are not limited to C1-6alkyl i.e. N-C1-3alkyl, more preferably methyl particularly N-methyl.

[0089] “Sulfinyl” refers to the group -S(=O)H.

[0090] The term “substituted sulfinyl” or “sulfoxide” refers to a sulfinyl group having the hydrogen replaced with, for example a Ci-ealkyl group (“Ci-ealkylsulfinyl” or “Ci-ealkylsulfoxide”), an aryl (“arylsulfinyl”), an arylalkyl (“arylalkyl sulfinyl”) and so on. C1-3alkylsulfinyl groups are preferred, such as for example, -SOmethyl, -SOethyl and -SOpropyl.

[0091] The term “sulfonyl” refers to the group -SO2H. The sulfonyl group can be further substituted with a variety of groups to form, for example, sulfonic acids, sulfonamides, sulfonate esters and sulfones.

[0092] The term “substituted sulfonyl” refers to a sulfonyl group having the hydrogen replaced with, for example a Ci-ealkyl group (“sulfonylCi-ealkyl”), an aryl (“arylsulfonyl”), an arylalkyl (“arylalkylsulfonyl”) and so on. SulfonylC1-3alkyl groups are preferred, such as for example, -SO2Me, -SO2Et and -SO2Pr.

[0093] The term “sulfonylamido” or “sulfonamide” refers to the group -SO2NH2.

[0094] The term “tautomer” refers to constitutional isomers of organic compounds that readily interconvert by migration of a proton, for example, the three tautomers of 3-hydroxypyrazole:

[0095]

[0096] A "therapeutically effective amount" refers to a quantity of a specified agent sufficient to achieve a desired effect in a subject being treated with that agent. Ideally, a therapeutically effective amount of an agent is an amount sufficient to inhibit or treat the disease or condition without causing a substantial cytotoxic effect in the subject. The therapeutically effective amount of an agent will be dependent on the subject being treated, the severity of the affliction, and the manner of administration of the therapeutic composition.

[0097] “Treatment” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop, or administering a compound or composition to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing a pathology or condition, or diminishing the severity of a pathology or condition. As used herein, the term “ameliorating,” with reference to a disease or pathological condition, refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, an improvement in the overall health or well-being of the subject, or by other parameters well known in the art that are specific to the particular disease. The phrase “treating a disease”8123-112925-02 03 / 13 / 26 06891

[0098] refers to inhibiting the full development of a disease, for example, in a subject who is at risk for a disease. “Preventing” a disease or condition refers to prophylactic administration of a composition to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing a pathology or condition, or diminishing the severity of a pathology or condition.

[0099] “Pharmaceutical compositions” are compositions that include an amount (for example, a unit dosage) of one or more of the disclosed compounds together with one or more non-toxic pharmaceutically acceptable additives, including carriers, diluents, and / or adjuvants, and optionally other biologically active ingredients. Such pharmaceutical compositions can be prepared by standard pharmaceutical formulation techniques such as those disclosed in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA (19th Edition).

[0100] The terms “pharmaceutically acceptable salt or ester” refers to salts or esters prepared by conventional means that include salts, e.g., of inorganic and organic acids, including but not limited to hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, malic acid, acetic acid, oxalic acid, tartaric acid, citric acid, lactic acid, fumaric acid, succinic acid, maleic acid, salicylic acid, benzoic acid, phenylacetic acid, mandelic acid and the like. “Pharmaceutically acceptable salts” of the presently disclosed compounds also include those formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc, and from bases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine, ornithine, choline, N, N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)aminomethane, and tetramethylammonium hydroxide. These salts may be prepared by standard procedures, for example by reacting the free acid with a suitable organic or inorganic base. Any chemical compound recited in this specification may alternatively be administered as a pharmaceutically acceptable salt thereof. “Pharmaceutically acceptable salts” are also inclusive of the free acid, base, and zwitterionic forms. Descriptions of suitable pharmaceutically acceptable salts can be found in Handbook of Pharmaceutical Salts, Properties, Selection and Use, Wiley VCH (2002). When compounds disclosed herein include an acidic function such as a carboxy group, then suitable pharmaceutically acceptable cation pairs for the carboxy group are well known to those skilled in the art and include alkaline, alkaline earth, ammonium, quaternary ammonium cations and the like. Such salts8123-112925-02 03 / 13 / 26 06891

[0101] are known to those of skill in the art. For additional examples of “pharmacologically acceptable salts,’’ see Berge et al., J. Pharm. Sci. 66:1 (1977).

[0102] “Pharmaceutically acceptable esters” includes those derived from compounds described herein that are modified to include a carboxyl group. An in vivo hydrolysable ester is an ester, which is hydrolysed in the human or animal body to produce the parent acid or alcohol. Representative esters thus include carboxylic acid esters in which the non-carbonyl moiety of the carboxylic acid portion of the ester grouping is selected from straight or branched chain alkyl (for example, methyl, n-propyl, t-butyl, or n-butyl), cycloalkyl, alkoxyalkyl (for example, methoxymethyl), aralkyl (for example benzyl), aryloxyalkyl (for example, phenoxymethyl), aryl (for example, phenyl, optionally substituted by, for example, halogen, C. sub.1-4 alkyl, or C.sub.1-4 alkoxy) or amino); sulphonate esters, such as alkyl- or aralkylsulphonyl (for example, methanesulphonyl); or amino acid esters (for example, L-valyl or L-isoleucyl). A “pharmaceutically acceptable ester” also includes inorganic esters such as mono-, di-, or tri-phosphate esters. Any aryl moiety present in such esters may comprise a phenyl group, optionally substituted as shown in the definition of carbocycylyl above. Pharmaceutically acceptable esters thus include C1-C22 fatty acid esters, such as acetyl, t-butyl or long chain straight or branched unsaturated or omega-6 monounsaturated fatty acids such as palmoyl, stearoyl and the like. Alternative aryl or heteroaryl esters include benzoyl, pyridylmethyloyl and the like any of which may be substituted, as defined in carbocyclyl above. Additional pharmaceutically acceptable esters include aliphatic L-amino acid esters such as leucyl, isoleucyl and especially valyl.

[0103] For therapeutic use, salts of the compounds are those wherein the counter-ion is pharmaceutically acceptable. However, salts of acids and bases which are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.

[0104] The pharmaceutically acceptable acid and base addition salts as mentioned hereinabove are meant to comprise the therapeutically active non-toxic acid and base addition salt forms which the compounds are able to form. The pharmaceutically acceptable acid addition salts can conveniently be obtained by treating the base form with such appropriate acid. Appropriate acids comprise, for example, inorganic acids such as hydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric, nitric, phosphoric and the like acids; or organic acids such as, for example, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic (i.e. ethanedioic), malonic, succinic (i.e. butanedioic acid), maleic, fumaric, malic (i.e. hydroxybutanedioic acid), tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic,8123-112925-02 03 / 13 / 26 06891

[0105] p-toluenesulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic and the like acids.

[0106] Conversely said salt forms can be converted by treatment with an appropriate base into the free base form.

[0107] The compounds containing an acidic proton may also be converted into their nontoxic metal or amine addition salt forms by treatment with appropriate organic and inorganic bases. Appropriate base salt forms comprise, for example, the ammonium salts, the alkali and earth alkaline metal salts, e.g. the lithium, sodium, potassium, magnesium, calcium salts and the like, salts with organic bases, e.g. the benzathine, N-methyl-D-glucamine, hydrabamine salts, and salts with amino acids such as, for example, arginine, lysine and the like.

[0108] Prodrugs of the disclosed compounds also are contemplated herein. A prodrug is an active or inactive compound that is modified chemically through in vivo physiological action, such as hydrolysis, metabolism and the like, into an active compound following administration of the prodrug to a subject. The term “prodrug” as used throughout this text means the pharmacologically acceptable derivatives such as esters, amides and phosphates, such that the resulting in vivo biotransformation product of the derivative is the active drug as defined in the compounds described herein. Prodrugs preferably have excellent aqueous solubility, increased bioavailability and are readily metabolized into the active inhibitors in vivo. Prodrugs of a compounds described herein may be prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either by routine manipulation or in vivo, to the parent compound. The suitability and techniques involved in making and using prodrugs are well known by those skilled in the art. For a general discussion of prodrugs involving esters see Svensson and Tunek, Drug Metabolism Reviews 165 (1988) and Bundgaard, Design of Prodrugs, Elsevier (1985).

[0109] The term “prodrug” also is intended to include any covalently bonded carriers that release an active parent drug of the present invention in vivo when the prodrug is administered to a subject. Since prodrugs often have enhanced properties relative to the active agent pharmaceutical, such as, solubility and bioavailability, the compounds disclosed herein can be delivered in prodrug form. Thus, also contemplated are prodrugs of the presently disclosed compounds, methods of delivering prodrugs and compositions containing such prodrugs. Prodrugs of the disclosed compounds typically are prepared by modifying one or more functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to yield the parent compound. Prodrugs include compounds having a phosphonate and / or amino group functionalized with any group8123-112925-02 03 / 13 / 26 06891

[0110] that is cleaved in vivo to yield the corresponding amino and / or phosphonate group, respectively. Examples of prodrugs include, without limitation, compounds having an acylated amino group and / or a phosphonate ester or phosphonate amide group. In particular examples, a prodrug is a lower alkyl phosphonate ester, such as an isopropyl phosphonate ester.

[0111] Protected derivatives of the disclosed compounds also are contemplated. A variety of suitable protecting groups for use with the disclosed compounds are disclosed in Greene and Wuts, Protective Groups in Organic Synthesis; 3rd Ed.; John Wiley & Sons, New York, 1999.

[0112] In general, protecting groups are removed under conditions that will not affect the remaining portion of the molecule. These methods are well known in the art and include acid hydrolysis, hydrogenolysis and the like. One preferred method involves the removal of an ester, such as cleavage of a phosphonate ester using Lewis acidic conditions, such as in trimethylsilyl (TMS)-Br mediated ester cleavage to yield the free phosphonate. A second preferred method involves removal of a protecting group, such as removal of a benzyl group by hydrogenolysis utilizing palladium on carbon in a suitable solvent system such as an alcohol, acetic acid, and the like or mixtures thereof. A t-butoxy-based group, including t-butoxy carbonyl protecting groups can be removed utilizing an inorganic or organic acid, such as HC1 or trifluoroacetic acid, in a suitable solvent system, such as water, dioxane and / or methylene chloride. Another exemplary protecting group, suitable for protecting amino and hydroxy functions amino is trityl. Other conventional protecting groups are known and suitable protecting groups can be selected by those of skill in the art in consultation with Greene and Wuts, Protective Groups in Organic Synthesis; 3rd Ed.; John Wiley & Sons, New York, 1999. When an amine is deprotected, the resulting salt can readily be neutralized to yield the free amine. Similarly, when an acid moiety, such as a phosphonic acid moiety is unveiled, the compound may be isolated as the acid compound or as a salt thereof.

[0113] Compounds

[0114] A compound, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, comprising a gamma peptide nucleic acid (yPNA) linked to a cell-penetrating motif (CPM).

[0115] Peptide nucleic acid (PNA) technology is an alternative to nuclease-based and base editing approaches for targeting mitochondrial variants. PNAs are synthetic analogs of DNA or RNA that hybridize single-stranded DNA or RNA with high affinity and sequence specificity. One of the defining features of PNAs is the backbone made of N-2-8123-112925-02 03 / 13 / 26 06891

[0116] aminoethylglycine repeating units, which replace the sugar-phosphate backbone of DNA. The synthetic backbone is linked to natural nucleobases via a carboxymethyl

[0117] linker, allowing PNAs to bind complementary DNA or RNA via Watson-Crick base pairing. Importantly, the PNA backbone does not contain any charged groups. Due to the lack of electrostatic repulsion, PNA / DNA binding is stronger and more stable than DNA / DNA binding. PNA / DNA duplexes have higher thermal stability as reflected by a higher melting temperature than DNA / DNA or DNA / RNA duplexes. Additionally, the unnatural backbone is not recognized by nucleases, proteases, or polymerases. These properties make PNAs an attractive candidate for anti-gene and antisense applications. Subsequently, PNAs can be used to bind specific nuclear DNA targets and can suppress DNA replication. yPNAs are DNA mimics with strong binding affinity and base-pair specificity that can invade doublestranded DNA and can be used for nuclear gene editing in mice, both in vivo and in utero, with no detectable toxicity.

[0118] Although PNAs conjugated with a mitochondrial-targeting moiety (e.g., a lipophilic triphenylphosphonium (TPP) cation) can inhibit variant mtDNA replication in vitro, such systems fail in cells because the mitochondrial targeting moiety (e.g., TPP) can become entrapped in the mitochondrial membrane. Had the PNAs reached the mitochondrial matrix, mixed sequence (i.e., non-homopyrimidine) PNAs can only bind single-stranded DNA, and it is well known that mtDNA is >90% double-stranded in tissues. Therefore, PNAs may have limited access to mtDNA targets.

[0119] Gamma PNAs (yPNAs) are a modified version of PNAs that feature a substituent on the gamma position of the peptide backbone (Formula I), forming a right- handed helical conformation that closely matches the structure of a complementary nucleic acid after hybridization. The gamma substituent creates a chiral center that drives its high DNA affinity and sequence specificity, allowing the yPNA to bind double-stranded DNA (dsDNA) through a strand invasion mechanism, y-modification also changes the hydrophobic character of the PNA backbone, which can shift the size -hydrophobicity character of the compound and affect delivery characteristics.8123-112925-02 03 / 13 / 26 06891

[0120]

[0121] Formula I

[0122] As described in more detail below, in some examples, CPM3-TAMRA colocalizes with mitochondria in 143B cells and iPSC-derived cardiomyocytes (iPSC-CMs). A 15-mer yPNA complementary to the m.3243A> G sequence exhibits selective strand invasion, and CPM3-yPNA treatment reduces heteroplasmy, with a progressive shift observed during compound washout.

[0123] In some aspects, the yPNA is selected to target a mtDNA or mtRNA sequence. These sequences may be related to mtDNA structures, mutations, deletions, or nuclear encoded RNAs imported into the mitochondria. In some aspects, the yPNA has a sequence length selected based on its strand invasiveness and specificity. Longer sequences can increase invasiveness but are less selective or specific than shorter sequences. Shorter sequences can be more specific or selective but can be less strand invasive.

[0124] Several aspects are being considered in the design of yPNAs. In some aspects, yPNAs that are 15-20-mer are strand invasive. In certain aspects, the yPNAs are 15-mer or greater in length. In certain aspects, the yPNAs are 16-mer or greater in length. In certain aspects, the yPNAs are 17-mer or greater in length. In certain aspects, the yPNAs are 18-mer or greater in length. In certain aspects, the yPNAs are 19-mer or greater in length. In certain aspects, the yPNAs are 20-mer or greater in length.

[0125] In some aspects, strand invasion is lost in yPNAs between 10-15 mer in length. In some aspects, yPNAs are selective to a single nucleotide mismatch. In some aspects, certain stretches of DNA could increase the chances of yPNA / yPNA self-hybridization. Lastly, targeting an antisense gene-coding sequence avoids interaction with mitochondrial RNA.

[0126] In certain disclosed aspects, the yPNA comprises the following sequence: C-CGGGACGGTAGAATT-N, which is coded to target mutant sequence m. 3243A> C or m.

[0127] 3243A> G. There are over 250 identified pathogenic mtDNA variants. The most prevalent pathogenic mtDNA variant is a single DNA base pair substitution at m.3243A> G in mitochondrial tRNA leucine 1 (MT-TL1) gene. The m.3243A> G variant impacts the D arm of the tRNA and disrupts post-transcriptional processing, such as methylation and8123-112925-02 03 / 13 / 26 06891

[0128] aminoacylation. These perturbations affect protein synthesis of the electron transport chain, which leads to downstream impairments in OXPHOS. There are several patient phenotypes of m.3243A> G; two of the most prominent are mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), and maternally inherited diabetes and deafness (MIDD). m.3243A> G is observed in 80% of patients with MELAS and causes 85% of cases of MIDD. A clinical study with 55 symptomatic MELAS patients revealed that the median survival time is 16.9 years from the onset of neurological disease. The average age of death was 34.5+19 years (10.2-81.8 years), and 22% of the deaths occurred in patients younger than 18 years of age. Currently, there is no cure or treatment to stop MELAS progression, and treatment consists of vitamins, supplements, and palliative care. MIDD affects 0.5%-2.8% of patients with type 2 diabetes mellitus. MIDD causes insulin-dependent diabetes and progressive sensorineural hearing loss and typically presents at around age 37. The current treatment is the management of symptoms (e.g., controlling diabetes via diet, glucose-lowering agents, or insulin, and hearing augmentations). Although the m.3243A> G mtDNA variation can lead to different phenotypes, patients with cardiovascular manifestations of MELAS or MIDD, such as cardiomyopathy, have increased mortality. Cardiomyopathy is common in patients with mitochondrial disease; 30-38% of MELAS patients and 15-30% of MIDD patients are affected by cardiomyopathy. One study reported that mortality in children with a cardiac phenotype of PMD was higher (71%) than those without the cardiac phenotype (26%). In PMD patients, it is thought that the degree of heteroplasmy in affected tissues determines the manifestation of the phenotype. In the case of cardiac phenotypes, the heterogeneity can be influenced by factors such as the heteroplasmy level of the cardiomyocytes, oxidative stress, and maladaptive remodeling of cardiomyocytes.

[0129] Despite the guidelines described above regarding the design of the yPNA sequence, targeting the m.3243A> G mtDNA variant presents some unique design challenges. The main challenge with targeting the m.3243A> G variant is that the variant is located in a stretch of G-C bases, [GGGCCC], which creates a risk that the yPNAs will self-hybridize (Table 1). Hence, the 15-mer m.3243A> G targeted yPNA only extends two bases past the m.3243A> G variant site on one side. This asymmetry leaves 12 bases of the yPNA that bind the WT DNA sequence before the m.3243A> G variant site.

[0130] Table 1: al5-mer m.3243A> G yPNA is shown with a respective dsDNA target, illustrating the 15-mer yPNA position relative to the m.3243A> G and WT dsDNA sequences. The complementary bases are emphasized for each dsDNA sequence. The GGGCCC region is also emphasized. _

[0131] m.3243A>G mtDNA | 5'-AGGGTTTGTTAAGATGGCAGGGCCCGGTAATCGC-3' |8123-112925-02 03 / 13 / 26 06891

[0132] m.3243A> G mtDNA 3 ' -TCCCAAACAATTCTACCGTCCCGGGCCATTAGCG-5 '

[0133] 15-mer yPNA N-TTAAGATGGCAGGGC-C

[0134] WT mtDNA 5 ' -AGGGTTTGTTAAGATGGCAGAGCCCGGTAATCGC-3 '

[0135] WT mtDNA 3 ' -TCCCAAACAATTCTACCGTCTCGGGCCATTAGCG-5 '

[0136]

[0137] 15-mer yPNA N-TTAAGATGGCAGGGC-C

[0138] In some disclosed aspects, the yPNA comprises the following sequence: N- TAAGAGAACCAACAC-C, which is coded to target mutant sequence m. 8344A> G.

[0139] In some disclosed aspects, the yPNA comprises the following sequence: N- ATAGCCCGGGCCGTA-C, which is coded to target mutant sequence m. 8993T> G.

[0140] In some disclosed aspects, the yPNA comprises the following sequence: N- ATAGCCCCGGCCGTA-C, which is coded to target mutant sequence m. 8993T> C.

[0141] In some disclosed aspects, the yPNA comprises the following sequence: N- ACACTTCCAGTAAGC-C, which is coded to target mutant sequence m. 9176T> C.

[0142] In some disclosed aspects, the yPNA comprises the following sequence: N- TTCGCTGACACCATA-C, which is coded to target mutant sequence m. 3460G> A.

[0143] In some disclosed aspects, the yPNA comprises the following sequence: N- CACAGTCACATCATA-C, which is coded to target mutant sequence m. 11778G> A.

[0144] In some disclosed aspects, the yPNA comprises the following sequence: N- GGAATGGTGGTTGTC-C, which is coded to target mutant sequence m. 14484T> C.

[0145] Referring next to the CPM, the CPM comprises a structure configured to enable the yPNA to enter a cell. The structure may remain within the cytosol or may further localize in subcellular structures such as mitochondria. Cargo size, hydrophobicity, and the site at which the CPM is attached to the yPNA (e.g., N- vs. C-terminus) can materially affect delivery performance.

[0146] Without wishing to be bound by theory, the mass and positive charge of the CPM near a bound yPNA may sterically and electrostatically hinder polymerase progression, increasing functional inhibition even if gel-shift strand-invasion signals are modestly reduced. As described in more detail below in the Examples, coupling CPM3 to yPNA can decrease strand-invasion readouts yet enhance PCR polymerase blockade while maintaining selectivity.

[0147] In some aspects, the CPM comprises guanidine or guanidinium groups of arginines and hydrophobic groups, such as the aromatic rings of naphthylalanine. In some aspects, the CPM has two or more guanidine and / or guanidinium groups (e.g., 2, 3, 5, 6, 7, 8, 9, 10 or more), and one or more hydrophobic groups (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10 or more). Further,8123-112925-02 03 / 13 / 26 06891

[0148] any hydrogen on the CPM can be replaced by a bond to a cargo moiety (e.g., the yPNA) to provide for intracellular (e.g., mitochondrial) delivery of the cargo moiety.

[0149] As introduced above, the attachment site can affect delivery and hybridization geometry. In some aspects, the CPM is bound to a N-terminus of the yPNA. Solubility and delivery can differ with the site of attachment.

[0150] Without wishing to be bound by theory, a CPM positioned near the approach face of the polymerase or proximal to the duplex junction may increase polymerase interference but could dampen apparent strand -invasion mobility shifts, which could have a neutral effect or offset the effects of the polymerase interference. In certain aspects, yPNA-CPM3 demonstrates reduced gel-shift invasion yet stronger PCR inhibition and selectivity.

[0151] In some aspects, the CPM comprises a plurality of guanidine and / or guanidinium groups (two or more, e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) anchored to a scaffold (e.g., an aromatic ring, such as a benzene ring). In some aspects, a hydrophobic group (e.g., an aromatic hydrophobic amino acid including, but not limited to, naphthylalanine (Nal)) can be appended to the scaffold to render the CPM amphipathic, a feature that can facilitate cytosolic entry. Advantageously, cargo can be attached at any suitable position and delivered to the intracellular space of the cell, including the mitochondria. In some aspects, to enable cargo attachment and minimize any potential mutual interference between CPM and the cargo, a flexible linker, e.g., 8-amino-3,6-dioxaoctanoyl-lysine, is added to a carboxyl group of a hydrophobic residue.

[0152] In some aspects, one or more lysine residues can be provided to increase solubility relative to a CPM without the lysine.

[0153] In some aspects, the CPM comprises a structure according to formula II, or an isomer, a tautomer, or a pharmaceutically acceptable salt thereof:

[0154]

[0155] Formula II

[0156] wherein:

[0157] A is a non-peptidic multivalent moiety;

[0158] each X is independently a first bonding group that links R1to A;8123-112925-02 03 / 13 / 26 06891

[0159] each Y is independently a bond or a second bonding group that directly or indirectly links A to a guanidine or guanidinium group; wherein guanidine or guanidinium group refers to the structure[structural formula image showing H₂N-C(=NH)-NH with Z]

[0160] each Z is independently a lone pair, H, halogen, CN, NO2, NH2, or alkyl (when Z is a lone pair, said group is referred to as a guanidine group; when Z is halogen, CN, NO2, NH2, or alkyl, said group is referred to as a guanidinium group);

[0161] each R1is independently a moiety comprising a hydrophobic residue; each R2is independently absent, a moiety comprising a hydrophobic residue, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, — C(O)alkyl, — C(O)alkenyl, — C(O)alkynyl, — C(O)-carbocyclyl, — C(O)-heterocyclyl, each of which is optionally substituted, or NRaRb, wherein Raand Rbare independently selected from — H, alkyl, alkenyl, alkynyl, carbocyclyl, and heterocyclyl each of which are optionally substituted;

[0162] each L is independently a bond, or an alkylene, alkenylene, alkynylene, carbocyclyl, heterocyclyl, each of which are optionally substituted, or — Rc— X1— Rd— wherein each of Rcand Rdare independently selected from alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, each of which are optionally substituted, and X1is O, N, or S;

[0163] each M is independently a bond, or an alkylene, alkenylene, alkynylene, carbocyclyl, heterocyclyl, each of which are optionally substituted, or — Re— X2— Rf— wherein each of Reand Rfare independently selected from alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, each of which are optionally substituted, and X2is O, N, or S;

[0164] when L or Y, or both, is not a bond (i.e., when L or Y, or both, are any of the groups described herein other than a bond), p is an integer from 1 to 3, provided that when Y is an atom, the valence of Y is not violated;

[0165] q is an integer from 1 to 10;

[0166] when L and Y are each a bond, p is an integer from 1 to 10, and q is absent;8123-112925-02 03 / 13 / 26 06891

[0167] s is an integer from 1 to 3; and

[0168] optionally one or more cargo moieties, wherein at least one atom of Formula II is replaced by a cargo moiety or at least one lone pair in Formula II forms a bond to a cargo moiety.

[0169] In some aspects, the compound of Formula II comprises one or more cargo moieties, wherein a hydrogen atom or lone pair is replaced by a cargo moiety. In some aspects, the cargo moiety is the yPNA as disclosed herein. In some aspects, at least one cargo moiety is bonded to A. In some aspects, at least one cargo moiety is bonded to R1. In some aspects, at least one cargo moiety is bonded to R2.

[0170] In some aspects, the cargo moiety is bonded to A, R1, or R2through a linker. In some aspects, the linker comprises an amino acid, alkylene, alkenylene, alkynylene, carbocyclyl, heterocyclyl, or — Rk— X3— R1— wherein Rkand R1are independently selected from alkylene, alkenylene, alkynylene, carbocyclyl, or heterocarbocyclyl, each of which are optionally substituted, and X3is O, N, or S. In some aspects, linkers (e.g., polyethylene glycol (PEG) segments or 8-amino-3,6-dioxaoctanoyl-lysine) may space hydrophobic CPM surfaces from the yPNA, mitigating aggregation while preserving membrane interaction.

[0171] In some aspects, A is a multivalent moiety. In some aspects, A is a trivalent or tetravalent moiety. In some aspects, A is an alkyl, a carbocyclyl, a heterocyclyl, an atom, or an amino acid. In some aspects, A is an aryl or heteroaryl. In some aspects, A is nitrogen. In some aspects, A is a non-peptidic moiety comprising one or more residues of aspartic acid, glutamic acid, lysine or combinations thereof.

[0172] The connectivity of the multivalent moiety A within a compound of Formula II can vary depending on the nature of the L, Y, and M groups specified. Thus, in various aspects of the present disclosure, the multivalent moiety A can be directly bonded to either L, Y, M or a guanidine / guanidinium group. In some aspects, when L is present as a group other than a bond, the multivalent moiety A is directly bonded to L in a compound of Formula II. In other aspects, when L is a bond and Y is a second bonding group, A is directly bonded to Y in a compound of Formula II. Under conditions where L or Y, or both is not a bond, the valence of A is determined by the sum of q and s. In still other aspects, a compound of Formula II is provided where Y and L, but not M, are each a bond, such that A is necessarily directly bonded to one or more M moieties. In yet other aspects, each of Y, L, and M is defined as a bond, such that A is necessarily directly bonded to one or more guanidine and / or guanidinium moieties. Under conditions where at least Y and L are each a bond, the valence of A is determined by the sum of p and s.8123-112925-02 03 / 13 / 26 06891

[0173] In some aspects, the compounds disclosed herein can be prepared by the appropriate substitution of one or more — OH groups on a precursor (e.g., A(OH)n) to form one or more corresponding moieties bonded to A. Some examples of suitable compounds of formula A(OH)ninclude 2-hydroxy-1,3-propanediol, glycerol, thioglycerol, ethylene glycol, polyethylene glycol, polyvinylalcohol, other pharmaceutically acceptable polymers, and the like. Other examples of A(OH)ninclude carbohydrates, sugar alcohols, polymeric alcohols, and the like. Still other examples of A(OH)ninclude polysaccharides such as cellulose or starch, or modified forms thereof (e.g., esters and / or ethers thereof). In one aspect, when A is derived from glycerol, the hydrogen on two of the three OH groups on glycerol may be substituted with a bis(guanidinoalkyl)amine, via formation of carbamate bonding groups, and the hydrogen on the third OH group may be substituted with an amino acid of a short peptide, via formation of a carbamate bonding group.

[0174] Some examples of sugar alcohols include mannitol, sorbitol, xylitol, maltitol, arabitol, ribitol, dulcitol, iditol, isomalt, lactitol, erythritol, and the like. Non-limiting examples of carbohydrates include monosaccharides, disaccharides, oligosaccharides, polysaccharides, celluloses, modified cellulosics, starches, and the like. Further examples of carbohydrates include 5- and 6-membered ring monosaccharides such as ribose, furanose, and mannose, disaccharides such lactose, sucrose, maltose, agrose, polysaccharides and oligosaccharides such as dextrins and maltodextrins, and modified cellulosics such as microcrystalline cellulose, silicified microcrystalline cellulose, mannitol-microcrystalline cellulose, hydroxypropylcellulose, L-hydroxypropylcellulose (low substituted), low molecular weight hydroxypropyl methylcellulose (HPMC) (e.g. Methocel E, F and K from Dow Chemical, Metolose SH from Shin-Etsu, Ltd), hydroxyethylcellulose, sodium carboxymethylcellulose, carboxymethyl hydroxyethylcellulose and other cellulose derivatives.

[0175] As noted above, multivalent moiety A has multiple sites of attachment. In some aspects, the percentage of sites occupied relative to the total available cites of attachment is in the range of from about 10% to about 100%, e.g., about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, inclusive of all values and ranges therebetween.

[0176] In some aspects of Formula II, A is a non-polymeric multivalent moiety. In some aspects, A is a pharmaceutically acceptable polymer moiety. In some aspects, A is selected from carbohydrates, sugar alcohols, and polymeric alcohols. In some aspects, A is a non-polymeric multivalent moiety. In certain aspects, A is a non-polymeric alcohol. In certain8123-112925-02 03 / 13 / 26 06891

[0177] aspects, the non-polymeric alcohol is 2-hydroxy-1,3-propanediol, glycerol, thioglycerol, ethylene glycol.

[0178] In some aspects, each X is independently a first bonding group that links R1to A. In various aspects, X comprises a bonding group selected from:

[0179]

[0180] wherein each a is independently number from 0 to 10, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, inclusive of all values and subranges therebetween. In specific aspects, X is

[0181]

[0182] wherein a is 0. In other specific aspects, X is

[0183]

[0184] wherein a is 0.

[0185] In some aspects, each Y is independently absent or a second bonding group that directly or indirectly links A to a guanidine or guanidinium group. In some aspects, Y is selected from:8123-112925-02 03 / 13 / 26 06891

[0186]

[0187] wherein each b is independently a number from 0 to 10, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, inclusive of all values and subranges therebetween. In certain aspects, Y is absent. In other aspects, Y is N.

[0188] In some aspects, each R1independently comprises a hydrophobic residue selected from alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, or heteroaryl. In some aspects, R1is a residue of an amino acid which has a hydrophobic side chain. In various aspects of R1, the residue comprises a non-aromatic hydrophobic amino acid. In certain aspects, each non-aromatic hydrophobic amino acid is independently selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, cyclohexylalanine, piperidine-2-carboxylic acid, or norleucine, each of which is optionally substituted with one or more substituents. In other aspects, each R1is independently a hydrophobic residue comprising an aromatic ring. In various aspects, each R1is independently a moiety comprising a hydrophobic aromatic residue. In some aspects, each R1is a moiety comprising a hydrophobic aromatic ring.

[0189] R1can include one or more aromatic rings, including fused rings. In some aspects, the hydrophobic residue in R2has a SAS A of at least about 200 A2, at least about 210 A2, at least about 220 A2, at least about 240 A2, at least about 250 A2, at least about 260 A2, at least about 270 A2, at least about 280 A2, at least about 290 A2, at least about 300 A2, at least about 310 A2, at least about 320 A2, or at least about 330 A2. In some aspects, the hydrophobic residue in R2has a SASA of at least about 200 A2, at least about 210 A2, at least about 220 A2, at least about 240 A2, at least about 250 A2, at least about 260 A2, at least about 270 A2, at least about 280 A2, at least about 290 A2, at least about 300 A2, at least about 310 A2, at least about 320 A2, or at least about 330 A2, at least about 350 A2, at least about 360 A2, at least about 370 A2, at least about 380 A2, at least about 390 A2, at least about 400 A2, at least about 410 A2, at least about 420 A2, at least about 430 A2, at least about 440 A2, at least about 450 A2, at least about 460 A2, at least about 470 A2, at least about 480 A2, at least about 490 A2, greater than about 500 A2, at least about 510 A2, at least about 520 A2, at least about 530 A2, at least about 540 A2, at least about 550 A2, at least about 560 A2, at least about 570 A2, at8123-112925-02 03 / 13 / 26 06891

[0190] least about 580 A2, at least about 590 A2, at least about 600 A2, at least about 610 A2, at least about 620 A2, at least about 630 A2, at least about 640 A2, greater than about 650 A2, at least about 660 A2, at least about 670 A2, at least about 680 A2, at least about 690 A2, or at least about 700 A2. In some aspects, R1is an amino acid residue, or analog thereof, having an aromatic side chain. In certain aspects of R1, the aromatic hydrophobic amino acid is naphthylalanine, phenylglycine, homophenylalanine, phenylalanine, tryptophan, or tyrosine, each of which is optionally substituted with one or more substituents. In certain aspects, R1is an amino acid selected from the group consisting of phenylalanine, tryptophan, naphthylalanine, phenylglycine, homophenylalanine, tyrosine, cyclohexylalanine, piperidine-2-carboxylic acid, cyclohexylglycine, 3-(3-benzothienyl)-alanine, 3-(2-quinolyl)-alanine, O-benzylserine, 3-(4-(benzyloxy)phenyl)-alanine, S-(4-methylbenzyl)cysteine, N-(naphthalen-2-yl)glutamine, and 3-(l,l'-biphenyl-4-yl)-alanine, each of which is optionally substituted with one or more substituents. In specific aspects, R1is naphthylalanine, which is optionally substituted. In other specific aspects, R1is phenylalanine, which is optionally substituted. When R1is an amino acid (or analog thereof), the cargo may be conjugated to the termini of R1(either the N- or C-termini of R1depending on how R1is coupled to A) or the termini may be protected with any suitable protecting groups, some of which are listed in R2.

[0191] In various aspects, each R2is independently absent, a moiety comprising a hydrophobic residue, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, — C(O)alkyl, — C(O)alkenyl, — C(O)alkynyl, — C(O)-carbocyclyl, — C(O)-heterocyclyl, or NRaRb, wherein Raand Rbare independently selected from — H, alkyl, alkenyl, alkynyl, carbocyclyl, and heterocyclyl each of which are optionally substituted. When R2is absent, R1is terminated with an appropriate atom. For example, R2is absent and R1is an amino acid residue, the amino acid reside may be terminated with H, or it may have a protecting group, such as an alkyl. Suitable amino acid-protecting groups are described in Isidro-Albert et al. “Amino Acid-Protecting Groups” Chem. Rev. 2009, 6, 2455-2504, which is herein incorporated by reference in its entirety. In various other aspects, each R2independently comprises a hydrophobic residue selected from alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, or heteroaryl. In some aspects, R2is a residue of an amino acid which has a hydrophobic side chain. In various aspects of R2, the residue comprises a non-aromatic hydrophobic amino acid. In certain aspects, each hydrophobic amino acid is independently selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, cyclohexylalanine, piperidine-2-carboxylic acid, or norleucine, each of which is optionally substituted with one or more substituents. When R2is an amino acid (or analog thereof), the termini not covalently bonded8123-112925-02 03 / 13 / 26 06891

[0192] to R1may be protected with any suitable protecting groups, some of which are listed in R3, or the cargo may be conjugated to said termini.

[0193] In other aspects, each R2is independently a hydrophobic residue comprising an aromatic ring. In some aspects, the hydrophobic residue comprising an aromatic ring is an amino acid having a hydrophobic side chain. Non-limiting examples of aromatic hydrophobic amino acids include naphthylalanine, phenylglycine, homophenylalanine, phenylalanine, tryptophan, or tyrosine, each of which is optionally substituted with one or more substituents. In some aspects, each R2is a moiety comprising a hydrophobic aromatic ring. R2can include one or more aromatic rings, including fused rings. In some aspects, the hydrophobic residue in R2has a SAS A of at least about 200 A2, at least about 210 A2, at least about 220 A2, at least about 240 A2, at least about 250 A2, at least about 260 A2, at least about 270 A2, at least about 280 A2, at least about 290 A2, at least about 300 A2, at least about 310 A2, at least about 320 A2, or at least about 330 A2. In some aspects, the hydrophobic residue in R2has a SASA of at least about 200 A2, at least about 210 A2, at least about 220 A2, at least about 240 A2, at least about 250 A2, at least about 260 A2, at least about 270 A2, at least about 280 A2, at least about 290 A2, at least about 300 A2, at least about 310 A2, at least about 320 A2, or at least about 330 A2, at least about 350 A2, at least about 360 A2, at least about 370 A2, at least about 380 A2, at least about 390 A2, at least about 400 A2, at least about 410 A2, at least about 420 A2, at least about 430 A2, at least about 440 A2, at least about 450 A2, at least about 460 A2, at least about 470 A2, at least about 480 A2, at least about 490 A2, greater than about 500 A2, at least about 510 A2, at least about 520 A2, at least about 530 A2, at least about 540 A2, at least about 550 A2, at least about 560 A2, at least about 570 A2, at least about 580 A2, at least about 590 A2, at least about 600 A2, at least about 610 A2, at least about 620 A2, at least about 630 A2, at least about 640 A2, greater than about 650 A2, at least about 660 A2, at least about 670 A2, at least about 680 A2, at least about 690 A2, or at least about 700 A2.

[0194] In some aspects, R2is a residue of an amino acid which has a hydrophobic side chain. In certain aspects, R2is an amino acid selected from the group consisting of phenylalanine, tryptophan, naphthylalanine, phenylglycine, homophenylalanine, tyrosine, cyclohexylalanine, piperidine-2-carboxylic acid, cyclohexylglycine, 3-(3-benzothienyl)-alanine, 3-(2-quinolyl)-alanine, O-benzylserine, 3-(4-(benzyloxy)phenyl)-alanine, S-(4-methylbenzyl)cysteine, N-(naphthalen-2-yl)glutamine, and 3-(l,l'-biphenyl-4-yl)-alanine, each of which is optionally substituted with one or more substituents. In specific aspects, R2is naphthylalanine, which is8123-112925-02 03 / 13 / 26 06891

[0195] optionally substituted. In other specific aspects. R2is phenylalanine, which is optionally substituted.

[0196] In various aspects, each L is independently a bond, or an alkylene, alkenylene, alkynylene, carbocyclyl, heterocyclyl, or — Rc— X1— Rd— wherein each of Rcand Rdare independently selected from alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, each of which are optionally substituted, and X1is O, N, or S. In some aspects, L is an alkylene.

[0197] In various aspects, each M is independently a bond, or an alkylene, alkenylene, alkynylene, carbocyclyl, heterocyclyl, or — Re—X2—Rf— wherein each of Reand Rfare independently selected from alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, each of which are optionally substituted, and X2is O, N, or S. In some aspects, M is an alkylene.

[0198] In various aspects, p is an integer from 1 to 3, provided that when Y is an atom, the valence of Y is not violated. In some aspects, when L or Y, or both, is not a bond, p is an integer from 1 to 3, provided that when Y is an atom, the valence of Y is not violated. In some aspects, p is 2 or 3. In certain aspects, p is 2. In certain other aspects, p is 3.

[0199] In some aspects, q is an integer from 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, inclusive of all values and subranges therebetween. In certain aspects, q is 2 or 3. In specific aspects, q is 2. In other aspects, when L and Y are each a bond, p is an integer from 1 to 10, and q is absent.

[0200] In some aspects, s is an integer from 1 to 3. In specific aspects, s is 1.

[0201] In some aspects, of Formula II, when Y and L are each a bond, q is absent and the sum of p and s is an integer in the range of from 2 to 13, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13, inclusive of all values and subranges therebetween. In some aspects, the sum of p and s is an integer of from 3 to 6. In other aspects, the sum of p and s is 2. In yet other aspects, the sum of p and s is 3. In still other aspects, the sum of p and s is 4. In certain aspects, the sum of p and s is 5. In other certain aspects, the sum of p and s is 6. In still other certain aspects, the sum of p and s is 7. In yet other certain aspects, the sum of p and s is 8. In various aspects, the sum of p and s is 9. In other various aspects, the sum of p and s is 10. In still other various aspects, the sum of p and s is 11. In yet other various aspects, the sum of p and s is 12. In another aspect, the sum of p and s is 13. In specific aspects, the sum of p and s is an integer in the range of from 2 to 13, wherein s is 1. In other specific aspects, the sum of p and s is an integer in the range of from 3 to 6, wherein s is 1.8123-112925-02 03 / 13 / 26 06891

[0202] In some aspects, of Formula II, when Y, L, or both is not a bond, the sum of q and s is an integer in the range of from 2 to 13. In some aspects, the sum of q and s is an integer of from 3 to 6. In other aspects, the sum of q and s is 2. In yet other aspects, the sum of q and s is 3. In still other aspects, the sum of q and s is 4. In certain aspects, the sum of q and s is 5. In other certain aspects, the sum of q and s is 6. In still other certain aspects, the sum of q and s is 7. In yet other certain aspects, the sum of q and s is 8. In various aspects, the sum of q and s is 9. In other various aspects, the sum of q and s is 10. In still other various aspects, the sum of q and s is 11. In yet other various aspects, the sum of q and s is 12. In another aspect, the sum of q and s is 13. In specific aspects, the sum of q and s is an integer in the range of from 2 to 13, wherein s is 1. In other specific aspects, the sum of q and s is an integer in the range of from 3 to 6, wherein s is 1.

[0203] In some aspects of Formula II, the sum of p and s is an integer in the range of from 3 to 6. In other aspects, the sum of p and s is 3 or 4.

[0204] In some aspects of Formula II, the sum of q and s is an integer in the range of from 3 to 6. In other aspects, the sum of q and s is 3 or 4.

[0205] In some aspects of Formula II, Z is H, halogen, CN, NO2, NH2, or alkyl. As noted above, when Z is H, halogen, CN, NO2, NH2, or alkyl, the compounds of Formula II comprise a guanidinium group defined by

[0206]

[0207] which has a positive charge. Therefore, in some aspects, the charged compounds of Formula II further comprise a counterion (i.e., an anion) that includes, but is not limited to halide (e.g., chloride, bromide, or iodide), phosphate, hydrogen phosphate, dihydrogen phosphate, sulfate, hydrogen sulfate, acetate, formate, trifluoroacetate, mesylate, besylate, oxalate, fumarate, lactate, maleate, malonate, citrate, and hexafluorophosphate.

[0208] In some aspects, the present disclosure provides for compounds according to Formula III, or pharmaceutically acceptable salts or tautomers thereof:

[0209]

[0210] wherein:

[0211] A is a non-peptidic multivalent moiety;8123-112925-02 03 / 13 / 26 06891

[0212] each X is independently a first bonding group that links R1to A;

[0213] each Y is independently a bond or a second bonding group that directly or indirectly links A to a guanidine or guanidinium group; wherein

[0214] guanidine or guanidinium group refers to the structure

[0215]

[0216] z

[0217] each Z is independently a lone pair, H, halogen, CN, NO2, NH2, or alkyl;

[0218] each R1is independently a moiety comprising a hydrophobic residue;

[0219] each R2is independently absent, a moiety comprising a hydrophobic residue, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, — C(O)alkyl, — C(O)alkenyl, — C(O)alkynyl, — C(O)- carbocyclyl, — C(O)-heterocyclyl, each of which is optionally substituted, or NRaRb, wherein Raand Rbare independently selected from — H, alkyl, alkenyl, alkynyl, carbocyclyl, and heterocyclyl each of which are optionally substituted;

[0220] each R3is independently a cargo moiety, — H, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, — C(O)alkyl, — C(O)alkenyl, — C(O)alkynyl, — C(O)-carbocyclyl, — C(O)- heterocyclyl, each of which are optionally substituted, or NR'R1, wherein R1and RJare independently selected from — H, alkyl, alkenyl, alkynyl, carbocyclyl, and heterocyclyl each of which are optionally substituted;

[0221] each L is independently a bond, or an alkylene, alkenylene, alkynylene, carbocyclyl, heterocyclyl, each of which are optionally substituted, or — Rc— X1— Rd— wherein each of Rcand Rdare independently selected from alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, each of which are optionally substituted, and X1is O, N, or S;

[0222] each M is independently a bond, or an alkylene, alkenylene, alkynylene, carbocyclyl, heterocyclyl, each of which are optionally substituted, or — Re— X2— Rf— wherein each of Reand R f are independently selected from alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, each of which are optionally substituted, and X2is O, N, or S;

[0223] when L or Y, or both, is not a bond, p is an integer from 1 to 3, provided that when Y is an atom, the valence of Y is not violated;

[0224] q is an integer from 1 to 10;

[0225] when L and Y are each a bond, p is an integer from 1 to 10, and q is absent; and

[0226] s is an integer from 1 to 3.

[0227] In various aspects, A is a multivalent moiety. In some aspects, A is a trivalent or tetravalent moiety. In other aspects, A is an alkyl, a carbocyclyl, a heterocyclyl, an atom, or8123-112925-02 03 / 13 / 26 06891

[0228] an amino acid. In still other aspects, A is an aryl, heteroaryl, or nitrogen. In yet other aspects, A is an aryl or heteroaryl. In some aspects, the aryl or heteroaryl is selected from phenyl, naphthyl, phenanthryl, pyridyl, pyrimidyl, triazinyl, quinolinyl, isoquinolinyl, indolinyl, benzoxazolyl, and benzothiazolyl. In further aspects, A is nitrogen. In various other aspects, A is a non-peptidic moiety comprising one or more residues of aspartic acid, glutamic acid, lysine or combinations thereof.

[0229] The connectivity of the multivalent moiety A within a compound of Formula 111 can vary depending on the nature of the L, Y, and M groups specified. Thus, in various aspects of the present disclosure, the multivalent moiety A can be bonded to either L, Y, M or a guanidine / guanidinium group. In some aspects, when L is present as a group other than a bond, the multivalent moiety A is directly bonded to L in a compound of Formula III. In other aspects, when L is a bond and Y is a second bonding group, A is directly bonded to Y in a compound of Formula III. Under conditions where L or Y, or both is not a bond, the valence of A is determined by the sum of q and s. In still other aspects, a compound of Formula III is provided where Y and L, but not M, are each a bond, such that A is necessarily directly bonded to one or more M moieties. In yet other aspects, each of Y, L, and M is defined as a bond, such that A is necessarily directly bonded to one or more guanidine and / or guanidinium moieties. Under conditions where at least Y and L are each a bond, the valence of A is determined by the sum of p and s.

[0230] In some aspects of Formula III, A is a non-polymeric multivalent moiety. In some aspects, A is a pharmaceutically acceptable polymer moiety. In some aspects, A is selected from carbohydrates, sugar alcohols, and polymeric alcohols. In some aspects, A is a non-polymeric multivalent moiety. In certain aspects, A is a non-polymeric alcohol. In certain aspects, the non-polymeric alcohol is 2 -hydroxy- 1,3 -propanediol, glycerol, thioglycerol, ethylene glycol.

[0231] In some aspects, each X is independently a first bonding group that links R1to A. In various aspects, X comprises a bonding group selected from:8123-112925-02 03 / 13 / 26 06891

[0232]

[0233] wherein each a is independently number from 0 to 10, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, inclusive of all values and subranges therebetween. In specific aspects, X is

[0234]

[0235] wherein a is 0. In other specific aspects, X is

[0236]

[0237] wherein a is 0.

[0238] In some aspects, each Y is independently a bond or a second bonding group that directly or indirectly links A to a guanidine or guanidinium group. In some aspects, Y is selected from:

[0239]

[0240] 8123-112925-02 03 / 13 / 26 06891

[0241] wherein each b is independently a number from 0 to 10, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, inclusive of all values and subranges therebetween. In certain aspects, Y is absent. In other aspects, Y is N.

[0242] In some aspects, each R1independently comprises a hydrophobic residue selected from alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, or heteroaryl. In some aspects, R1is a residue of an amino acid which has a hydrophobic side chain. In various aspects of R1, the residue comprises a non-aromatic hydrophobic amino acid. In certain aspects, each non-aromatic hydrophobic amino acid is independently selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, cyclohexylalanine, piperidine-2-carboxylic acid, or norleucine, each of which is optionally substituted with one or more substituents. In other aspects, each R1is independently a hydrophobic residue comprising an aromatic ring. In various aspects, each R1is independently a moiety comprising a hydrophobic aromatic residue. In some aspects, R1is an amino acid residue, or analog thereof, having an aromatic side chain. In certain aspects of R1, the aromatic hydrophobic amino acid is naphthylalanine, phenylglycine, homophenylalanine, phenylalanine, tryptophan, or tyrosine, each of which is optionally substituted with one or more substituents. In certain aspects, R1is an amino acid selected from the group consisting of phenylalanine, tryptophan, naphthylalanine, phenylglycine, homophenylalanine, tyrosine, cyclohexylalanine, piperidine-2-carboxylic acid, cyclohexylglycine, 3-(3-benzothienyl)-alanine, 3-(2-quinolyl)-alanine, O-benzylserine, 3-(4-(benzyloxy)phenyl)-alanine, S-(4-methylbenzyl)cysteine, N-(naphthalen-2-yl) glutamine, and 3-(l,l'-biphenyl-4-yl)-alanine, each of which is optionally substituted with one or more substituents. In specific aspects, R1is naphthylalanine, which is optionally substituted. In other specific aspects, R1is phenylalanine, which is optionally substituted. When R1is an amino acid (or analog thereof), the cargo may be conjugated to the termini of R1(either the N- or C-termini of R1depending on how R1is coupled to A) or the termini may be protected with any suitable protecting groups, some of which are listed in R2.

[0243] In some aspects, each R1is a moiety comprising a hydrophobic aromatic ring. R1can include one or more aromatic rings, including fused rings. In some aspects, the hydrophobic residue in R1has a SASA of at least about 200 A2, at least about 210 A2, at least about 220 A2, at least about 240 A2, at least about 250 A2, at least about 260 A2, at least about 270 A2, at least about 280 A2, at least about 290 A2, at least about 300 A2, at least about 310 A2, at least about 320 A2, or at least about 330 A2. In some aspects, the hydrophobic residue in R2has a SASA of at least about 200 A2, at least about 210 A2, at least about 220 A2, at least about 240 A2, at least about 250 A2, at least about 260 A2, at least about 270 A2, at least about 280 A2, at8123-112925-02 03 / 13 / 26 06891

[0244] least about 290 A2, at least about 300 A2, at least about 310 A2, at least about 320 A2, or at least about 330 A2, at least about 350 A2, at least about 360 A2, at least about 370 A2, at least about 380 A2, at least about 390 A2, at least about 400 A2, at least about 410 A2, at least about 420 A2, at least about 430 A2, at least about 440 A2, at least about 450 A2, at least about 460 A2, at least about 470 A2, at least about 480 A2, at least about 490 A2, greater than about 500 A2, at least about 510 A2, at least about 520 A2, at least about 530 A2, at least about 540 A2, at least about 550 A2, at least about 560 A2, at least about 570 A2, at least about 580 A2, at least about 590 A2, at least about 600 A2, at least about 610 A2, at least about 620 A2, at least about 630 A2, at least about 640 A2, greater than about 650 A2, at least about 660 A2, at least about 670 A2, at least about 680 A2, at least about 690 A2, or at least about 700 A2.

[0245] In various aspects, each R2is independently absent, a moiety comprising a hydrophobic residue, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, — C(O)alkyl, — C(O)alkenyl, — C(O)alkynyl, — C(O)-carbocyclyl, — C(O) -heterocyclyl, or NRaRb, wherein Raand Rbare independently selected from — H, alkyl, alkenyl, alkynyl, carbocyclyl, and heterocyclyl each of which are optionally substituted. In various other aspects, each

[0246] R2independently comprises a hydrophobic residue selected from alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, or heteroaryl. In some aspects, R2is a residue of an amino acid which has a hydrophobic side chain. In various aspects of R2, the residue comprises a non-aromatic hydrophobic amino acid. In certain aspects, each hydrophobic amino acid is independently selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, cyclohexylalanine, piperidine-2-carboxylic acid, or norleucine, each of which is optionally substituted with one or more substituents. In other aspects, each R2is independently a hydrophobic residue which comprises an aromatic ring. In some aspects, the hydrophobic residue comprising an aromatic ring is an amino acid having an aromatic side chain. Nonlimiting examples of such amino acids include an amino acid selected from the group consisting of phenylalanine, tryptophan, naphthylalanine, phenylglycine, homophenylalanine, tyrosine, cyclohexylalanine, piperidine-2-carboxylic acid, cyclohexylglycine, 3-(3-benzothienyl)-alanine, 3-(2-quinolyl)-alanine, O-benzylserine, 3-(4-(benzyloxy)phenyl)-alanine, S-(4-methylbenzyl)cysteine, N-(naphthalen-2-yl)glutamine, and 3-(l,l'-biphenyl-4-yl)-alanine, each of which is optionally substituted with one or more substituents. In specific aspects, R2is naphthylalanine, which is optionally substituted. In other specific aspects, R2is phenylalanine, which is optionally substituted. When R2is an amino acid (or analog thereof), the termini not covalently bonded to R1may be protected with any suitable protecting groups, some of which are listed in R3, or the cargo may be conjugated to said termini.8123-112925-02 03 / 13 / 26 06891

[0247] In some aspects, each R2is a moiety comprising a hydrophobic aromatic ring. R2can include one or more aromatic rings, including fused rings. In some aspects, the hydrophobic residue in R2has a SASA of at least about 200 A2, at least about 210 A2, at least about 220 A2, at least about 240 A2, at least about 250 A2, at least about 260 A2, at least about 270 A2, at least about 280 A2, at least about 290 A2, at least about 300 A2, at least about 310 A2, at least about 320 A2, or at least about 330 A2. In some aspects, the hydrophobic residue in R2has a SASA of at least about 200 A2, at least about 210 A2, at least about 220 A2, at least about 240 A2, at least about 250 A2, at least about 260 A2, at least about 270 A2, at least about 280 A2, at least about 290 A2, at least about 300 A2, at least about 310 A2, at least about 320 A2, or at least about 330 A2, at least about 350 A2, at least about 360 A2, at least about 370 A2, at least about 380 A2, at least about 390 A2, at least about 400 A2, at least about 410 A2, at least about 420 A2, at least about 430 A2, at least about 440 A2, at least about 450 A2, at least about 460 A2, at least about 470 A2, at least about 480 A2, at least about 490 A2, greater than about 500 A2, at least about 510 A2, at least about 520 A2, at least about 530 A2, at least about 540 A2, at least about 550 A2, at least about 560 A2, at least about 570 A2, at least about 580 A2, at least about 590 A2, at least about 600 A2, at least about 610 A2, at least about 620 A2, at least about 630 A2, at least about 640 A2, greater than about 650 A2, at least about 660 A2, at least about 670 A2, at least about 680 A2, at least about 690 A2, or at least about 700 A2.

[0248] In various aspects, each R3is independently a cargo moiety, carbocyclyl, heterocyclyl, — H, alkyl, alkenyl, alkynyl, — C(O)alkyl, — C(O)alkenyl, — C(O)alkynyl, — C(O)-carbocyclyl, — C(O)-heterocyclyl, or NR'R1, wherein R1and RJare independently selected from — H, alkyl, alkenyl, alkynyl, carbocyclyl, and heterocyclyl each of which are optionally substituted.

[0249] In various aspects, each L is independently a bond, or an alkylene, alkenylene, alkynylene, carbocyclyl, heterocyclyl, or — Rc— X1— Rd— wherein each of Rcand Rdare independently selected from alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, each of which are optionally substituted, and X1is O, N, or S. In some aspects, L is an alkylene.

[0250] In various aspects, each M is independently a bond, or an alkylene, alkenylene, alkynylene, carbocyclyl, heterocyclyl, or — Re— X2— Rf— wherein each of Reand Rfare independently selected from alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, each of which are optionally substituted, and X2is O, N, or S. In some aspects, M is an alkylene.8123-112925-02 03 / 13 / 26 06891

[0251] In various aspects, p is an integer from 1 to 3, provided that when Y is an atom, the valence of Y is not violated. In other aspects, when L or Y, or both, is not a bond, p is an integer from 1 to 3, provided that when Y is an atom, the valence of Y is not violated. In some aspects p is 2 or 3. In certain aspects, p is 2. In certain other aspects, p is 3.

[0252] In some aspects, q is an integer from 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, inclusive of all values and subranges therebetween. In certain aspects, q is 2 or 3. In specific aspects, q is 2. In other aspects, when L and Y are each a bond, p is an integer from 1 to 10, and q is absent.

[0253] In some aspects, s is an integer from 1 to 3. In specific aspects, s is 1.

[0254] In some aspects, of Formula III, when Y and L are each a bond, q is absent and the sum of p and s is an integer in the range of from 2 to 13, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13, inclusive of all values and subranges therebetween. In some aspects, the sum of p and s is an integer in the range of from 3 to 6. In other aspects, the sum of p and s is 2. In yet other aspects, the sum of p and s is 3. In still other aspects, the sum of p and s is 4. In certain aspects, the sum of p and s is 5. In other certain aspects, the sum of p and s is 6. In still other certain aspects, the sum of p and s is 7. In yet other certain aspects, the sum of p and s is 8. In various aspects, the sum of p and s is 9. In other various aspects, the sum of p and s is 10. In still other various aspects, the sum of p and s is 11. In yet other various aspects, the sum of p and s is 12. In another aspect, the sum of p and s is 13. In specific aspects, the sum of p and s is an integer in the range of from 2 to 13, wherein s is 1. In other specific aspects, the sum of p and s is an integer in the range of from 3 to 6, wherein s is 1.

[0255] In some aspects, of Formula III, when Y, L, or both is not a bond, the sum of q and s is an integer in the range of from 2 to 13, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13, inclusive of all values and subranges therebetween. In some aspects, the sum of q and s is an integer in the range of from 3 to 6. In other aspects, the sum of q and s is 2. In yet other aspects, the sum of q and s is 3. In still other aspects, the sum of q and s is 4. In certain aspects, the sum of q and s is 5. In other certain aspects, the sum of q and s is 6. In still other certain aspects, the sum of q and s is 7. In yet other certain aspects, the sum of q and s is 8. In various aspects, the sum of q and s is 9. In other various aspects, the sum of q and s is 10. In still other various aspects, the sum of q and s is 11. In yet other various aspects, the sum of q and s is 12. In another aspects, the sum of q and s is 13. In specific aspects, the sum of q and s is an integer in the range of from 2 to 13, wherein s is 1. In other specific aspects, the sum of q and s is an integer in the range of from 3 to 6, wherein s is 1.8123-112925-02 03 / 13 / 26 06891

[0256] In some aspects of Formula III, the sum of p and s is an integer in the range of from 3 to 6. In other aspects, the sum of p and s is 3 or 4.

[0257] In some aspects of Formula III, the sum of q and s is an integer in the range of from 3 to 6. In other aspects, the sum of q and s is 3 or 4.

[0258] In various aspects, the present disclosure provides for compounds having a structure according to Formula IV or pharmaceutically acceptable salts or tautomers thereof:

[0259] (in;

[0260] ^NH

[0261]

[0262] wherein A, X, Y, Z, R1, R2, R3, p, q, and s are as defined above.

[0263] In some aspects, each m is independently an integer from 0 to 10, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, inclusive of all values and subranges therebetween. In other aspects, each m is independently 4, 5, 6, 7, or 8. In other aspects, m is 3, 4, 5, 6, or 7. In specific aspects, m is 6. In other specific aspects, m is 7. In certain other aspects, m is 5.

[0264] In some aspects, each n is independently an integer from 0 to 6, e.g., 0, 1, 2, 3, 4, 5, 6, inclusive of all values and subranges therebetween. In other aspects, each n is independently 1, 2, or 3. In specific aspects, n is 1. In other specific aspects, n is 2.

[0265] In certain aspects, Y is N, m is 6, and n is 1. In certain aspects, Y is N, m is 5, and n is 1. In certain aspects, Y is N, m is 7, and n is 1.

[0266] In certain aspects, Y is N and p is 2. In certain other aspects, Y is N, p is 2, and s is 1. In certain aspects, Y is N, p is 2, and q is 2. In certain other aspects, Y is N, p is 2, q is 2, and s is 1.

[0267] In various aspects, the present disclosure provides a structure according to Formula V or pharmaceutically acceptable salts or tautomers thereof:8123-112925-02 03 / 13 / 26 06891

[0268]

[0269] where A, X, Z, R1, R2, R3, m, and n are as defined above.

[0270] In various aspects of Formula V, A is a trivalent or tetravalent moiety. In some aspects, A is a carbocyclyl, heterocyclyl, an atom, or an amino acid.

[0271] In various aspects, the compound of Formula V is a compound of Formula V-A to V- C or pharmaceutically acceptable salt or tautomer thereof:8123-112925-02 03 / 13 / 26 06891

[0272]

[0273] wherein X, Z, R1, R2, R3, m, and n are as defined above; T is CH or N; and r is each independently selected from 0, 1, 2, or 3.8123-112925-02 03 / 13 / 26 06891

[0274] In various aspects of Formulas I to IV, the present disclosure provides cell-penetrating motifs having the following structures:

[0275]

[0276] 8123-112925-02 03 / 13 / 26 06891

[0277]

[0278] NH28123-112925-02 03 / 13 / 26 06891

[0279]

[0280] NH2

[0281] or pharmaceutically acceptable salts or tautomers thereof, wherein Z is defined above; each T is independently CH or N; R2is a bond or a moiety comprising an aromatic ring; and R3is a cargo moiety.

[0282] In various aspects of Formulas I to IV, the present disclosure provides cell-penetrating motifs having the following structures:8123-112925-02 03 / 13 / 26 06891

[0283]

[0284] 8123-112925-02 03 / 13 / 26 06891

[0285]

[0286] or pharmaceutically acceptable salts or tautomers thereof, wherein Z is defined above; each T is independently CH or N; R2is a bond or a moiety comprising an aromatic ring; and R3is a cargo moiety.

[0287] In some aspects, the present disclosure provides a cell-penetrating motif having the following structure:

[0288] z

[0289] HN

[0290]

[0291] H2N

[0292] or pharmaceutically acceptable salts or tautomers thereof, wherein Z is defined as above; R2is a bond or a moiety comprising an aromatic ring; and R3is absent or a cargo moiety.

[0293] In some aspects, the present disclosure provides a cell-penetrating motif having the following structure:8123-112925-02 03 / 13 / 26 06891

[0294]

[0295] or pharmaceutically acceptable salts or tautomers thereof, wherein Z is defined as above; R2is a bond or a moiety comprising an aromatic ring; and R3is absent or a cargo moiety.

[0296] In some aspects, the present disclosure provides a cell-penetrating motif having the following structure:

[0297] z

[0298] /

[0299] HN

[0300] )-NH2

[0301] HN

[0302] HN

[0303]

[0304] or pharmaceutically acceptable salts or tautomers thereof, wherein Z is defined as above; R2is a bond or a moiety comprising an aromatic ring; and R3is absent or a cargo moiety.

[0305] In some aspects, the present disclosure provides a cell-penetrating motif having the following structure:8123-112925-02 03 / 13 / 26 06891

[0306] z

[0307] /

[0308] HN

[0309] HN

[0310]

[0311] or pharmaceutically acceptable salts or tautomers thereof, wherein Z is defined as above; R2is a bond or a moiety comprising an aromatic ring; and R3is absent or a cargo moiety.

[0312] In some aspects, the present disclosure provides a cell-penetrating motif having the structure:

[0313] z

[0314] NH

[0315]

[0316] or pharmaceutically acceptable salts or tautomers thereof, wherein Z is defined above; T is CH or N; W is O, S, or NH; R2is a bond or a moiety comprising an aromatic ring; and R3is absent or a cargo moiety.

[0317] In some aspects, the present disclosure provides a cell-penetrating motif having the following structure:8123-112925-02 03 / 13 / 26 06891

[0318] z

[0319] \

[0320] NH

[0321]

[0322] H

[0323] or pharmaceutically acceptable salts or tautomers thereof, wherein Z is defined above; T is CH or N; W is O, S, or NH; R2is a bond or a moiety comprising an aromatic ring; and R3is absent or a cargo moiety.

[0324] In some aspects, the present disclosure provides cell-penetrating motifs having any of the following structures:8123-112925-02 03 / 13 / 26 06891

[0325]

[0326] 8123-112925-02 03 / 13 / 26 06891

[0327]

[0328] 8123-112925-02 03 / 13 / 26 06891

[0329]

[0330] or pharmaceutically acceptable salts or tautomers thereof, wherein Z is defined above; T is CH or N; W is O, S, or NH; R2is a bond or a moiety comprising an aromatic ring; and R3is absent or a cargo moiety.

[0331] In some aspects, the present disclosure provides cell-penetrating motifs having any of the following structures:8123-112925-02 03 / 13 / 26 06891

[0332]

[0333] 8123-112925-02 03 / 13 / 26 06891

[0334]

[0335] 8123-112925-02 03 / 13 / 26 06891

[0336]

[0337] or pharmaceutically acceptable salts or tautomers thereof, wherein Z is defined above; T is CH or N; r is independently selected from 0, 1, 2, or 3; R2is a bond or a moiety comprising an aromatic ring; and R3is absent or a cargo moiety.

[0338] In some aspects, r is 0 or 1. In certain aspects, r is 0. In specific aspects, r is 1. In other specific aspects, r is 2. In further aspects, r is 3.

[0339] In some aspects, the present disclosure provides cell-penetrating motifs having any of the following structures:8123-112925-02 03 / 13 / 26 06891

[0340]

[0341] or pharmaceutically acceptable salts or tautomers thereof, wherein r and Z are defined above; R2is a bond or a moiety comprising an aromatic ring; and R3is absent or a cargo moiety.

[0342] In some aspects, the present disclosure provides cell-penetrating motifs having any of the following structures:8123-112925-02 03 / 13 / 26 06891

[0343]

[0344] or pharmaceutically acceptable salts or tautomers thereof, wherein r and Z are defined above; R2is a bond or a moiety comprising an aromatic ring; and R3is absent or a cargo moiety.

[0345] In various aspects of Formulas I to IV, the present disclosure provides cell-penetrating motifs having the following structures:8123-112925-02 03 / 13 / 26 06891

[0346]

[0347] 8123-112925-02 03 / 13 / 26 06891

[0348]

[0349] or pharmaceutically acceptable salts or tautomers thereof, wherein Z is defined above; R2is a bond or a moiety comprising an aromatic ring; and R3is absent or a cargo moiety.

[0350] In various aspects of Formulas I to IV, the present disclosure provides cell-penetrating motifs having the following structures:8123-112925-02 03 / 13 / 26 06891

[0351]

[0352] 8123-112925-02 03 / 13 / 26 06891

[0353]

[0354] or pharmaceutically acceptable salts or tautomers thereof, wherein Z is defined above; R2is a bond or a moiety comprising an aromatic ring; and R3is absent or a cargo moiety.

[0355] As noted above, in some aspects, the cell-penetrating motifs of the present disclosure possess one or more guanidine moieties, which can each independently be present in a protonated guanidinium form. In some aspects, the percentage of guanidinium moieties to the total combined number of guanidine and guanidinium moieties in each CPM is in the range of from about 10% to about 100%, e.g., about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, inclusive of all values and ranges therebetween.

[0356] In various aspects of the present disclosure, compounds are provided having a cargo moiety comprising a therapeutic agent, such as a gamma peptide nucleic acid.

[0357] In various aspects, a pharmaceutical composition comprising a compound of the present disclosure is provided.8123-112925-02 03 / 13 / 26 06891

[0358] Particular aspects of the presently disclosed compounds may include one or more asymmetric centers; thus these compounds can exist in different stereoisomeric forms.

[0359] Accordingly, compounds and compositions may be provided as individual pure enantiomers or as stereoisomeric mixtures, including racemic mixtures. In certain aspects, the compounds disclosed herein are synthesized in or are purified to be in substantially enantiopure form, such as in a 90% enantiomeric excess, a 95% enantiomeric excess, a 97% enantiomeric excess or even in greater than a 99% enantiomeric excess, such as in enantiopure form.

[0360] The presently disclosed compounds can have at least one asymmetric center or geometric center, cis-trans center(C=C, C=N). All chiral, diasteromeric, racemic, meso, rotational and geometric isomers of the structures are intended unless otherwise specified. The compounds can be isolated as a single isomer or as mixture of isomers. All tautomers of the compounds are also considered part of the disclosure. The presently disclosed compounds also includes all isotopes of atoms present in the compounds, which can include, but are not limited to, deuterium, tritium,18F, etc.

[0361] An illustrative compound is listed below:

[0362]

[0363] General Description of Synthesis

[0364] Also disclosed herein are aspects of a novel synthetic route to obtain at least one compound, mixture, or pharmaceutical composition disclosed herein.

[0365] A compound of formula VI, wherein X is D-napthlalenealanine-R and wherein R is a yPNA, is prepared by reacting trimethyl benzene- 1,3, 5 -tricarboxylate with n- bromosuccinimide (NBS) in the presence of benzoyl peroxide to produce methyl 3,5-bis (hydroxymethyl)benzoate. The methyl 3,5-bis (hydroxymethyl)benzoate is brominated using phosphorus tribromide (PBr3) to produce methyl 3,5-bis(bromomethyl) benzoate.8123-112925-02 03 / 13 / 26 06891

[0366] HN

[0367] CPM1 H

[0368] CPM2 D-Phe

[0369] CPM3 D-Nal

[0370]

[0371] Formula VI

[0372] The methyl 3,5-dibromomethyl-benzoate (2) is reacted with a protected triamine (7) to yield a protected guanidiniumtriamine (8).

[0373] e) DMF / K2CO3 / rt 24 hrs or DMF / DIEA / 2 hrs 40°C

[0374]

[0375] Formula VII

[0376] The ester was removed from the central benzene ring as shown in formula VIII:s r(■;r Y r O G

[0377] X.- -X

[0378]

[0379] Formula VIII

[0380] A compound according to formula IX was prepared by coupling two naphthyl amino acids to a yPNA sequence (formed using Fmoc peptide chemistry). The yPNA was coupled to compound (9) to form the compound of formula IX:

[0381]

[0382] Formula IX

[0383] Pharmaceutical Compositions and Methods of Use

[0384] Also disclosed herein is a pharmaceutical formulation comprising a compound as disclosed herein, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, excipient, or combination thereof. The pharmaceutical formulation may further comprise a pharmacologically active agent other than the compound.

[0385] Also disclosed herein is a method for inhibiting replication of a mitochondrial mutation, comprising contacting mitochondrial DNA containing the mitochondrial mutation with an effective amount of a compound disclosed herein.

[0386] Also disclosed is a method of treating or preventing a mitochondrial condition, comprising administering to a subject an effective amount of a compound disclosed herein. In some aspects, the mitochondrial condition is selected from MIDD, mitochondrial myopathy, MELAS; or a deletion mutation.8123-112925-02 03 / 13 / 26 06891

[0387] The compound may also be administered as a formulation. The formulation may comprise the compound and a pharmaceutically acceptable earner. The formulation also may further comprise at least one additional therapeutic agent, as disclosed herein. The subject may be an animal or human, and any one of the disclosed aspects of the method may be performed in vitro or in vivo.

[0388] Aspects of the disclosed method may be used when the subject is suffering from the mitochondrial condition, or the method may be practiced prophylactically. The mitochondrial condition may be selected from MIDD, mitochondrial myopathy, MELAS, a deletion mutation, or any combination thereof.

[0389] The effective amount used in the disclosed method may be that which is best suited for treating the subject. The effective amount may range from greater than zero to about 1000 mg / kg / day. In particular disclosed aspects, the effective amount ranges from 1 mg / kg / day to about 100 mg / kg / day. The subject of the disclosed method may be human or an animal and the method may be performed in vitro or in vivo.

[0390] The compound disclosed herein may be used in therapy for a mitochondrial disorder. As disclosed herein, the compound may be used to treat and / or inhibit a biological pathway that involves a mitochondrial mutation.

[0391] Particular disclosed aspects of the compound disclosed herein are potent and selective inhibitors of replication or transcription of mitochondrial mutations and therefore may be used in in vitro, in vivo, and ex vivo contexts to regulate or inhibit this activity, prevent any replication, and downregulate mutation expression, as well as the biological responses that result from such activity.

[0392] The compounds may be administered orally, parenterally (including subcutaneous injections (SC or depo-SC), intravenous (IV), intramuscular (IM or depo-IM), intrastemal injection or infusion techniques), sublingually, intranasally (inhalation), intrathecally, topically, ophthalmically, or rectally. The pharmaceutical composition may be administered in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and / or vehicles. The compounds are preferably formulated into suitable pharmaceutical preparations such as tablets, capsules, or elixirs for oral administration or in sterile solutions or suspensions for parenteral administration. Typically, the compounds described above are formulated into pharmaceutical compositions using techniques and procedures well known in the art.

[0393] In some aspects, one or more of the disclosed compounds (including compounds linked to a detectable label or cargo moiety) are mixed or combined with a suitable8123-112925-02 03 / 13 / 26 06891

[0394] pharmaceutically acceptable earner to prepare a pharmaceutical composition.

[0395] Pharmaceutical carriers or vehicles suitable for administration of the compounds provided herein include any such carriers known to be suitable for the particular mode of administration. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21stEdition (2005), describes exemplary compositions and formulations suitable for pharmaceutical delivery of the compounds disclosed herein. In addition, the compounds may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients.

[0396] Upon mixing or addition of the compound(s) to a pharmaceutically acceptable carrier, the resulting mixture may be a solution, suspension, emulsion, or the like. Liposomal suspensions may also be suitable as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. Where the compounds exhibit insufficient solubility, methods for solubilizing may be used. Such methods are known and include, but are not limited to, using cosolvents such as dimethylsulfoxide (DMSO), using surfactants such as Tween®, and dissolution in aqueous sodium bicarbonate. Derivatives of the compounds, such as salts or prodrugs may also be used in formulating effective pharmaceutical compositions. The disclosed compounds may also be prepared with carriers that protect them against rapid elimination from the body, such as time-release formulations or coatings. Such carriers include controlled release formulations, such as, but not limited to, microencapsulated delivery systems.

[0397] The disclosed compounds and / or compositions can be enclosed in multiple or single dose containers. The compounds and / or compositions can also be provided in kits, for example, including component parts that can be assembled for use. For example, one or more of the disclosed compounds may be provided in a lyophilized form and a suitable diluent may be provided as separated components for combination prior to use. In some examples, a kit may include a disclosed compound and a second therapeutic agent for coadministration. The compound and second therapeutic agent may be provided as separate component parts. A kit may include a plurality of containers, each container holding one or more unit dose of the compound. The containers are preferably adapted for the desired mode of administration, including, but not limited to tablets, gel capsules, sustained-release capsules, and the like for oral administration; depot products, pre-filled syringes, ampoules,8123-112925-02 03 / 13 / 26 06891

[0398] vials, and the like for parenteral administration; and patches, medipads, creams, and the like for topical administration.

[0399] The active compound is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the subject treated. A therapeutically effective concentration may be determined empirically by testing the compounds in known in vitro and in vivo model systems for the treated disorder. In some examples, a therapeutically effective amount of the compound is an amount that lessens or ameliorates at least one symptom of the disorder for which the compound is administered. Typically, the compositions are formulated for single dosage administration. The concentration of active compound in the drug composition will depend on absorption, inactivation, and excretion rates of the active compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.

[0400] In some examples, about 0.1 mg to 1000 mg of a disclosed compound, a mixture of such compounds, or a physiologically acceptable salt or ester thereof, is compounded with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, flavor, etc., in a unit dosage form. The amount of active substance in those compositions or preparations is such that a suitable dosage in the range indicated is obtained. The term “unit dosage form” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. In some examples, the compositions are formulated in a unit dosage form, each dosage containing from about 1 mg to about 1000 mg (for example, about 2 mg to about 500 mg, about 5 mg to 50 mg, about 10 mg to 100 mg, or about 25 mg to 75 mg) of the one or more compounds. In other examples, the unit dosage form includes about 0.1 mg, about 1 mg, about 5 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, or more of the disclosed compound(s).

[0401] The disclosed compounds or compositions may be administered as a single dose, or may be divided into a number of smaller doses to be administered at intervals of time. The therapeutic compositions can be administered in a single dose delivery, by continuous delivery over an extended time period, in a repeated administration protocol (for example, by a multi-daily, daily, weekly, or monthly repeated administration protocol). It is understood8123-112925-02 03 / 13 / 26 06891

[0402] that the precise dosage, timing, and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data (such as testing in an animal model). It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. In addition, it is understood that for a specific subject, dosage regimens may be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only.

[0403] When administered orally as a suspension, these compositions are prepared according to techniques well known in the art of pharmaceutical formulation and may contain microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners / flavoring agents. As immediate release tablets, these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and / or other excipients, binders, extenders, disintegrants, diluents and lubricants. If oral administration is desired, the compound is typically provided in a composition that protects it from the acidic environment of the stomach. For example, the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. The composition may also be formulated in combination with an antacid or other such ingredient.

[0404] Oral compositions will generally include an inert diluent or an edible carrier and may be compressed into tablets or enclosed in gelatin capsules. For the purpose of oral therapeutic administration, the active compound or compounds can be incorporated with excipients and used in the form of tablets, capsules, or troches. Pharmaceutically compatible binding agents and adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches, and the like can contain any of the following ingredients or compounds of a similar nature: a binder such as, but not limited to, gum tragacanth, acacia, com starch, or gelatin; an excipient such as microcrystalline cellulose, starch, or lactose; a disintegrating agent such as, but not limited to, alginic acid and com starch; a lubricant such as, but not limited to, magnesium stearate; a gildant, such as, but not limited to, colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; and a flavoring agent such as peppermint, methyl salicylate, or fruit flavoring.

[0405] When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials, which modify the physical form of the dosage unit, for example,8123-112925-02 03 / 13 / 26 06891

[0406] coatings of sugar and other enteric agents. The compounds can also be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings, and flavors.

[0407] When administered orally, the compounds can be administered in usual dosage forms for oral administration. These dosage forms include the usual solid unit dosage forms of tablets and capsules as well as liquid dosage forms such as solutions, suspensions, and elixirs. When the solid dosage forms are used, it is preferred that they be of the sustained release type so that the compounds need to be administered only once or twice daily. In some examples, an oral dosage form is administered to the subject 1, 2, 3, 4, or more times daily. In additional examples, the compounds can be administered orally to humans in a dosage range of 1 to 1000 mg / kg body weight in single or divided doses. One illustrative dosage range is 0.1 to 200 mg / kg body weight orally (such as 0.5 to 100 mg / kg body weight orally) in single or divided doses. For oral administration, the compositions may be provided in the form of tablets containing about 1 to 1000 milligrams of the active ingredient, particularly 1, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900, or 1000 milligrams of the active ingredient. It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.

[0408] Injectable solutions or suspensions may also be formulated, using suitable non-toxic, parenterally-acceptable diluents or solvents, such as mannitol, 1,3 -butanediol, water, Ringer’s solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid. Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include any of the following components: a sterile diluent such as water for injection, saline solution, fixed oil, a naturally occurring vegetable oil such as sesame oil, coconut oil, peanut oil, cottonseed oil, and the like, or a synthetic fatty vehicle such as ethyl oleate, and the like, polyethylene glycol, glycerine, propylene glycol, or other synthetic solvent; antimicrobial agents such as benzyl alcohol and methyl parabens; antioxidants such as ascorbic acid and sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates,8123-112925-02 03 / 13 / 26 06891

[0409] citrates, and phosphates; and agents for the adjustment of tonicity such as sodium chloride and dextrose. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass, plastic, or other suitable material. Buffers, preservatives, antioxidants, and the like can be incorporated as required.

[0410] Where administered intravenously, suitable carriers include physiological saline, phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol, polypropyleneglycol, and mixtures thereof. Liposomal suspensions including tissue -targeted liposomes may also be suitable as pharmaceutically acceptable carriers.

[0411] The compounds can be administered parenterally, for example, by IV, IM, depo-IM, SC, or depo-SC. When administered parenterally, a therapeutically effective amount of about 0.1 to about 500 mg / day (such as about 1 mg / day to about 100 mg / day, or about 5 mg / day to about 50 mg / day) may be delivered. When a depot formulation is used for injection once a month or once every two weeks, the dose may be about 0.1 mg / day to about 100 mg / day, or a monthly dose of from about 3 mg to about 3000 mg.

[0412] The compounds can also be administered sublingually. When given sublingually, the compounds should be given one to four times daily in the amounts described above for IM administration.

[0413] The compounds can also be administered intranasally. When given by this route, the appropriate dosage forms are a nasal spray or dry powder. The dosage of the compounds for intranasal administration is the amount described above for IM administration. When administered by nasal aerosol or inhalation, these compositions may be prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and / or other solubilizing or dispersing agents.

[0414] The compounds can be administered intrathecally. When given by this route, the appropriate dosage form can be a parenteral dosage form. The dosage of the compounds for intrathecal administration is the amount described above for IM administration.

[0415] The compounds can be administered topically. When given by this route, the appropriate dosage form is a cream, ointment, or patch. When administered topically, an illustrative dosage is from about 0.5 mg / day to about 200 mg / day. Because the amount that can be delivered by a patch is limited, two or more patches may be used.8123-112925-02 03 / 13 / 26 06891

[0416] The compounds can be administered rectally by suppository. When administered by suppository, an illustrative therapeutically effective amount may range from about 0.5 mg to about 500 mg. When rectally administered in the form of suppositories, these compositions may be prepared by mixing the drug with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters of polyethylene glycols, which are solid at ordinary temperatures, but liquefy and / or dissolve in the rectal cavity to release the drug.

[0417] It should be apparent to one skilled in the art that the exact dosage and frequency of administration will depend on the particular compounds administered, the particular condition being treated, the severity of the condition being treated, the age, weight, general physical condition of the particular subject, and other medication the individual may be taking as is well known to administering physicians or other clinicians who are skilled in therapy of mitochondrial mutations, diseases, and associated disorders.

[0418] Examples

[0419] General methodology

[0420] Colocalization studies: Mitochondrial uptake of CPM3-yPNAs-TAMRA may be visualized in culture by live-cell confocal microscopy, and sub-mitochondrial localization may be visualized by super-resolution microscopy, structured illumination microscopy (SIM).

[0421] Hoechst stain may be used as a nuclear marker, and

[0422] MITOTRACKER (green) may be used as a mitochondrial marker. Colocalization analysis may be done using NIS ELEMENTS GENERAL ANALYSIS 3 (GA3). Quantification metrics may include yPNA mitochondrial colocalization interpreted by Pearson’s correlation coefficient (PCC) and Mander’ s overlap coefficient (MOC).

[0423] iPSC reprogramming and clonal selection ofiPSCs: Fibroblasts may be reprogrammed by electroporation with four plasmid vectors encoding OCT3 / 4 and p53 shRNA, SOX2 and KLF4, L-MYC, LIN28, and enhanced GFP. Pluripotency may be assessed by immunofluorescence. PCR may be used to evaluate the expression of Yamanaka factors, and multiple clones may be passaged to ensure the isolation of lines without vector-born reprogramming factors and with sufficient differentiation potential for follow-up studies. Once the fibroblasts are reprogrammed into iPSCs, clonal selection may be used to choose iPSC lines to achieve a range of heteroplasmy (e.g., >75%, >50%, <30%, and 0%). iPSC-CM differentiation: iPSC-CMs may be derived using a differentiation protocol.

[0424] Briefly, differentiation may begin once the cells reach -85% confluence. The E8 media may8123-112925-02 03 / 13 / 26 06891

[0425] be replaced, followed by a wash with 1 mL of RPMI 1640 medium, and then the cells may be treated with 2 mL of RPMI 1640+B 27 (no insulin) with 6 pM CHIR99021 (day 0) for two days. On day 2, the media may be changed to RPMI1640 +B27 (no insulin). For 2 days (days 3-4), 2mL RPMI1640 +B27 (no insulin) may be added with 5pM IWR-1. Then, the medium may be changed back to RPMI1640 +B27 (no insulin) for 2 days (day 5 and day 6). For the following days (day 7 forward), the medium (RPMI1640+B27 +insulin) may be changed every 48 hours. Beating cells can be observed on days 9-10. Cells may be kept in RPMI1640+B27+insulin at 37°C 5% CO2 and 21% O2 for further characterization and experiments.

[0426] iPSC-CM characterization: On day 10 of iPSC-CM differentiation, cells may be collected for immunofluorescence and flow cytometry. For immunofluorescence, cells may be fixed, permeabilized, and stained for Troponin T, a-actinin, and NKX2.5 to confirm cardiomyocyte identity by confocal microscopy and flow cytometry.. All samples may be stained with DAPI and imaged using confocal microscopy. The cells may be stained for the same cardiac markers and used for flow cytometry analysis and fluorescence-activated cell sorting (FACS) to select the differentiated iPSC-CMs.

[0427] CPM3- PNA-treatment & imaging: 143B cybrid cells may be plated in 35mm MATTEK dishes at 5 x 105 cells and incubated for 24 hours. After 24 hours, the cells may be treated with fluorescently labeled / PNA-CPM3 (TAMRA or CY5) and stained with Hoechst and MITOTRACKER (green) according to the manufacturer’s protocol. Live-cell microscopy may be performed on a NIKON Al spectral confocal microscope and a NIKON SIM.

[0428] Heteroplasmy quantification: Heteroplasmy may be quantified by ARMS qPCR. ARMS qPCR primers introduce a mismatch 5’ to the variant site, increasing the primers' amplification specificity to the single nucleotide variant, with the following equation:

[0429] Heteroplasmy Level (%) = 1 / [1+(1 / 2)ΔΔCT]x100%. Sanger and deep sequencing and dPCR will verify qPCR results at later timepoints.

[0430] Mitochondrial function: Mitochondrial function may be analyzed using the SEAHORSE XFe96 analyzer mitochondrial stress test, quantifying basal, ATP-linked, and maximal respiration, spare capacity, and non-mitochondrial oxygen consumption. OCR values may be normalized to protein per well, and ECAR to cell number or total protein. In addition, intracellular ATP may be measured by the bioluminescent kit. Lactate may be measured using a colorimetric L-Lactate Assay Kit (ABCAM). Glucose uptake levels may be measured using a 2-NBDG Glucose Uptake Assay Kit (ABCAM) and analyzed using flow cytometry. Western blotting may be performed to quantify the representative OXPHOS8123-112925-02 03 / 13 / 26 06891

[0431] proteins (THERMO or ABCAM). The LUCID RESIPHER may be used for characterizing respiration during differentiation. Cardiomyocyte function: Calcium imaging may be used to evaluate the calcium transients of the iPSC-CMs that regulate their contractile function. Calcium imaging may be performed. Briefly, iPSC-CMs may be plated on MATRIGEL-coated plates and allowed to recover for ~4 days to restore regular beating. Then, the cells may be treated with FLU0-4AM, a calcium indicator dye, and the signals may be recorded using live cell microscopy. Videos may be analyzed using NIS-ELEMENTS and IMAGEJ.

[0432] 15-second movies of beating iPSC-CMs may be captured using a 10x objective on a NIKON ECLIPSE TI-E microscope. The movies may be processed using the “MYOCYTER107” IMAGEJ plug-in.

[0433] Statistical Analysis: Statistical tests may be matched to the design and data structure. Most datasets can be analyzed by t-test or one- or two-way ANOVA with appropriate post -hoc correction; non-parametric or variance-adjusted tests may be used if assumptions are unmet. Batch effects may be assessed and modeled before final analysis. Replicate numbers may follow observed variance and increase if early variability is higher. Significance may be set at p < 0.05 (multiple-testing corrected). Analyses may be performed in PRISM and standard statistical software; all datasets may be archived for reproducibility.

[0434] Example 1

[0435] Preparation of CPM3-m3243-yPNA

[0436]

[0437] a) NSS / benzoyf sroxide / CC^ / reflux 2hrs b> LiAHj THFW’C c) PBiyDCMTC

[0438] Formula X

[0439] Methyl 3,5-bis(bromomethyl) benzoate (2) was prepared as described in Staab and Kirrstefter, Liebigs Ann. Chem. 1979, 886 - 898 (1979), the entirety of which is herein incorporated by reference for all purposes. 16.4g (0.1 mol) Methyl-3,5-dimethylbenzoate (1), 39.2 g (0.22 mol) of N-bromosuccinimide and 0.3 g of dibenzoylperoxide was heated in 200 mL carbon tetrachloride to reflux for 2 hrs. The succinimide was filtered off after cooling8123-112925-02 03 / 13 / 26 06891

[0440] and the solvent was removed on the rotavapor. The residue was crystallized from petroleum ether and recrystallized from cyclohexane to yield 10 g (30 %) with minor byproducts.

[0441] In another preparation method, trimethyl benzene-l,3,5-tricarboxylate (3) (20 g, 79.3 mol) was placed in a 500 mL three-neck flask equipped with a stirrer bar, inner thermometer and dropping funnel. The starting material was dissolved under argon in anhydrous THF (200 mL) and cooled to 0°C. A IM tetrahydrofuran solution of lithium aluminum hydride (80 mL, 80 mmol) was added dropwise at a rate to keep the reaction mixture below 10°C. The reaction mixture turns bright orange during the addition. The reaction mixture was allowed to warm up to room temperature after completion of the addition and stirrer for 1 hr. The reaction was quenched with 20 mL of water, followed by 6N hydrochloric acid until the reaction mixture turned light yellow (pH 3-4). The precipitate was filtered off and the residue was concentrated on the rotary evaporator. The oily residue was treated with 50 mL anhydrous ethanol and concentrated twice. The oily residue was purified by chromatography on silica gel (chloroform / 10% methanol). The first fractions, rf 0.8 contain the mono-reduced product. Methyl 3,5-bis (hydroxymethyl)benzoate (4) elutes with rf 0.2. The product fractions were collected and concentrated to yield 6.5 g, 33 mol (42 %) of product. 1H NMR (500 MHz, Acetone-d6) 87.91 (dt, J = 1.6, 0.7 Hz, 2H), 7.61 (tq, J = 1.6, 0.8 Hz, 1H), 4.71 (d, J = 5.3 Hz, 4H), 4.41 (t, J = 5.6 Hz, 2H), 3.89 (s, 3H).13C NMR (126 MHz, Acetone) 5 143.04, 130.14,

[0442] 129.11, 125.82, 63.29.51.35.

[0443] The methyl 3,5-bis (hydroxymethyl)benzoate (4) (3 g, 15.3 mmol) was dissolved in 60 mL anhydrous DCM under argon and cooled to 0°C. Phosphorus tribromide (1.5 mL, 15.79 mol) was drop wise added keeping the temperature below 6°C. After the addition the addition the reaction mixture as allowed to warm up to room temperature and stirred for 1 hr. The reaction was quenched by the careful addition of water. The reaction mixture was then neutralized by the addition of saturated sodium bicarbonate solution and solid sodium bicarbonate. The organic phase was separated and passed through a short silica gel plug. The filtrate was concentrated to yield 2.4 g, 7.45 mol (48%) of pure, crystalline methyl 3,5-bis(bromomethyl) benzoate (2). 1H NMR (500 MHz, Acetone-d6) 58.05 (d, J = 1.8 Hz, 2H), 7.83 (t, J = 1.8 Hz, 1H), 4.76 (s, 4H), 3.93 (s, 3H).13C NMR (126 MHz, Acetone) 8 165.44, 139.73, 134.14, 131.38, 129.78, 51.75, 32.03.8123-112925-02 03 / 13 / 26 06891

[0444]

[0445] d) CHCl₃ / NEt₃ / 24 hrs rt or MTBE / NEt₃ / 24 hrs rt

[0446] Formula XI

[0447] Bis(hexamethylene)triamine (5) (MW 215.39) 2.15 g (10 mmol) was dissolved in 60 mL methyl-tert-butyl ether. Triethylamine (2.8 mL, 20 mol) was added. Solid N, N'-Bis-Boc-l-guanylpyrazole (6) (6.2 g, 20 mol) was added in portions (MW 310.35 g / mol) under stirring at room temperature. The reaction temperature slightly increased during the addition. The mixture was stirred for 4 hrs. After reducing the reaction volume to 15 mL, the reaction mixture was purified by chromatography on silica gel using methyl-tert-butyl ether / 1% triethylamine as eluent (Rf of product 0.2). After separation of the front spots, 10% methanol was added to the eluent. The viscous oily product (MW 700 g / mol), yield: 5g, was used as such in the next step. The product, bis(N, N'-bis-boc-6-guanyl-hexamethylene)triamine (7), is heat sensitive and thus should not be heated above 40°C. ESI 700.50.1H NMR (500 MHz, chloroform-d) δ 11.52 (s, 2H), 8.32 (t, J = 5.3 Hz, 2H), 3.42 (td, J = 7.3, 5.1 Hz, 4H), 2.62 (t, J = 7.3 Hz, 4H), 1.72 – 1.52 (m, 8H), 1.52 (s, 36H), 1.38 (q, J = 5.0, 4.0 Hz, 8H).13C NMR (126 MHz, CDCl₃) δ 163.65, 156.14, 153.34, 105.07, 83.03, 49.80, 40.90, 29.72, 28.92, 28.32 (Boc), 28.13(Boc), 28.09, 27.02, 26.99, 26.77.

[0448] e) DMF / K2CO3 / rt 24 hrs or DMF / DIEA / 2 hrs 40°C

[0449]

[0450] Formula XII8123-112925-02 03 / 13 / 26 06891

[0451] 1.28 g / 4 mmol of methyl 3,5-dibromomethyl-benzoate (2) and 6g of the protected triamine (7) were dissolved in 30 mL anhydrous acetonitrile. 1.12 g (8 mmol) of potassium carbonate was added. The reaction mixture was vigorously stirred at room temperature for 24 hrs. The solid was filtered off and the filtrate concentrated. The remaining oil was purified on silica gel with methyl-terbutyl ether:hexane: 7N NH3 in methanol =50 / 50 / 1, with a yield of 3.12 g (50 %).

[0452] The structure of the methyl benzoate product (8) was confirmed using NMR.

[0453]

[0454] Formula XIII

[0455] ’H NMR (500 MHz, Chloroform-; / ) 5 11.52 (s, 4H, NH 12,30,62,86), 8.40 (t, J = 5.1 Hz, 4H, NH 14,28,64,88), 7.87 (d, 7= 1.6 Hz, 2H, 1,3), 7.50 - 7.40 (m, 1H, 5), 3.92 (d, J = 1.1 Hz, 3H, 110), 3.57 (s, 4H, 7,9), 3.40 (td, 7= 7.3, 5.1 Hz, 8H, 15,27,65,89), 2.40 (t, 8H, 20,22,70,94), 1.56 (m, 811,16,26,66,90), 1.52 (36 H,s, (43,52,53);(47,59,60);(79,82,83,)(103,106,107);), 1.51 (36H,s,

[0456] (39, 54, 55), (56, 57, 58), (75, 84, 85), (99,108,109)), 1.47 (m, 8H, 19,23,69,93), 1.32

[0457] (m,8H, 17, 25, 67, 91).13C NMR (126 MHz, CDCl₃) δ 167.46 (10), 163.67 (13,29,63,87), 156.09 (13,29,63,87), 153.32 (33,36,72,96), 140.49 (4,6), 133.76 (5), 129.91 (2), 128.40 (1,3), 82.96 (35,38,74,98), 79.17 (42,46,78,102), 58.33 ( 7,9), 53.79 (20,22,70,94), 52.01 (110), 40.98 (15,27,65,89), 29.05 (16,26,66,90), 28,33 (42,46,78,102), 28.33 (18,24,68,92), 28.09 (35,38,74,98), 27.21 (9,18,25,67), 26.90 (19,23,69, 93),. ESI: 1546.58 M+W \ s '] ‘ if Y;v -•%

[0458]

[0459] Formula XIV

[0460] The methyl benzoate product of formula XII (8) was dissolved in 30 mL of tertbutanol. 15 mL of 2N LiOH solution was added. The reaction mixture was vigorously stirred at room temperature for 24 hrs. Stirring was stopped to obtain a two-phase system. The organic layer was spotted on thin-layer chromatography (TLC) for reaction control. When the ester cleavage was complete, the aqueous layer was removed and the organic phase was concentrated down on the rotary evaporator to dryness. The residue still containing some lithium salts was dissolved in chloroform (6 mL) and purified on a silica gel column (150 g silica gel), eluent: chloroform / methanol 7N NH3 in methanol = 90 / 9 / 1.XH NMR (500 MHz, Acetonitrile -A) 5 11.57 (s, 4H), 8.21 (t, J = 5.6 Hz, 4H), 8.03 (s, 2H), 7.45 (s, 1H), 3.69 (s, 4H), 3.29 (p,. / = 7.3, 6.6 Hz, 8H), 2.58 - 2.45 (m, 8H), 1.55 (m,8H), 1.52 (m, 8H), 1.49 (s, 36 H), 1.44 (s, 36 H) 1.30 (16 H,m). *Some signals were determined by HSQC.13C NMR (126 MHz, CD3CN) 5 163.69, 156.06, 152.97, 138.09, 132.93, 129.46, 128.04, 82.96, 78.35, 57.67, 52.83, 40.50, 28.81, 27.57, 27.26, 26.72, 26.41.

[0461] CPM3-γD-γD-PEG2-TTAAGATGGCAGGGC-K-NH2(MW = 6027.30) was synthesized manually using Fmoc peptide chemistry. Solid-phase synthesis was completed on Rink amide-MBHA resin (0.73 mmol / g - Chem-Impex, IL). Coupling reactions generally used 5 equivalents of Fmoc-amino acid (Chem-Impex, IL) or Fmoc-yPNA monomer (Dr NS Labs, Hyderabad, India), 6 equivalents of O-(7-azabenzotriazol-l-yl)-N, N, N’, N’-tetramethyluronium hexafluorophosphate (HATU - Chem-Impex, IL), and 6 equivalents of N, N-Diisopropylethylamine (DIEA - Sigma- Aldrich, MO) in anhydrous N-methylpyrrolidinone (NMP) for 1 hr. Following coupling of the second naphthyl amino acid, the N-terminal Fmoc group was removed using a solution of 20% piperidine in dry DMF and8123-112925-02 03 / 13 / 26 06891

[0462] washed thoroughly with dimethylformamide and dichloromethane. Five equivalents of (9) was activated in a solution containing 8 equivalents of HATU and 8 equivalents of DIEA dissolved in anhydrous NMP, and allowed to react for 12 hrs. The full-length CPM3-yPNA conjugate was cleaved from the resin in a solution containing trifluoroacetic acid (TFA) / triisopropylsilane (95:5, v / v) for 2 hrs. The cleavage cocktail was concentrated to dryness and the residue was resuspended in nanopure water prior to purification. The crude product was purified by reversed-phase HPLC equipped with a Cis column using a linear gradient of 0-40% acetonitrile in water containing 0.1% TFA (FIGS. 5-6).

[0463] Example 2

[0464] Localization of CPM3

[0465] CPM3-TAMRA causes endosomal fusion with the mitochondria network, with the timing differing in different cell types. This result is in contrast to other mechanisms, in which CPM3 undergoes endosomal escape and enters the mitochondria from the cytoplasm.

[0466] The uptake of CPM3- TAMRA (i.e., no yPNA) was tested in iPSC-derived cardiomyocytes (iPSC-CMs). iPSC-CMs were treated with 2pM CPM3-TAMRA for 24 hours. Cells were then washed and treated with MITOTR ACKER Green and Hoechst stain. The cells were imaged live via confocal microscopy on a 100x objective (FIG. 1). These results raise confidence that CPM3 will successfully deliver yPNA to mitochondria in terminally differentiated cardiomyocytes.

[0467] Example 3

[0468] Biochemical assays

[0469] CPM3 was prepared according to Example 1. Click-chemistry was used to couple the CPM3 to m.3243 A> G yPNA. The m.3243A> G-targeted CPM3-modified yPNA was tested for strand invasion and selectivity to m.3243A> G (mut) and wild-type (WT) double-stranded DNA (dsDNA) in a gel-shift assay (FIG. 2). Both dsDNAs were 151 bp in length. CPM3-yPNA was tested in biochemical assays without a fluorophore. As illustrated in FIG. 2, CPM3 decreases yPNA strand invasiveness for m.3243A> G (mut DNA) and m.3243A (wt DNA) without altering the relative selective preference for mut DNA. The square is unbound DNA, and the arrowhead is the strand invasion complex. The yPNA or CPM3-yPNA to DNA ratio is 10:1 in all samples.

[0470] CPM3-yPNA shows preferential binding to the mutant sequence (FIG. 2, lanes 1 and 3) in a non-competitive strand invasion gel shift assay. There is a slight decrease in CPM3-8123-112925-02 03 / 13 / 26 06891

[0471] yPNA binding relative to yPNA but slightly more selectivity based on the lack of a visible shifted band in lane 3. CPM3-yPNA was observed to be strand invasive with a -50% selectivity for m.3243A> C over the wild-type sequence.

[0472] While the large CPM3 moiety reduces strand invasion, its size may be an advantage for inhibiting DNA synthesis. To that end, a PCR inhibition assay (FIG. 3), which removes strand invasion limitations due to the denaturing and annealing step, was used to characterize the impact of the CPM3 addition on the yPNA’s ability to inhibit DNA amplification.

[0473] CPM3-yPNA or yPNA was added to PCR reactions containing m.3243A> G or wild-type plasmid DNA at molar ratios indicated in FIG. 3. The absence of PCR product amplicons indicates the CPM3-yPNA interfered with PCR polymerase elongation; a PCR product amplicon indicates that the CPM3-yPNA did not interfere with PCR polymerase elongation.

[0474] Compared to yPNA alone, CPM3-yPNA is more potent at PCR inhibition, as shown by the absence of an amplicon at a ratio of 8:1 (CPM3-yPNA:plasmid DNA). Additionally, CPM3-yPNA is selective to m.3243A> G DNA at the 8:1 ratio. CPM3 enhances the function of yPNA in selectivity and polymerase interference while directing the yPNA to the mitochondrial compartment.

[0475] While a POLG-primer extension may be used, the impact of CPM3 on DNA copy number and sequence selectivity should be examined in a cellular environment. To that end, a simple cellular model used the 143B cybrid cell line carrying either WT (0% m.3242A> G) or a variant (100% m.3243A> G) as a pilot experiment (FIG. 4). Each line was exposed to 2 pM CPM3-yPNA or a vehicle (water, 0 pM) for two days before cells were collected and total DNA was isolated. Using multiplex qPCR for ND1 (mtDNA) and B2M (nuclear DNA), mtDNA depletion was observed that was specific to the mutant genome.

[0476] A two-day exposure of 2 pM CPM3-yPNA was then tested on the 143B 60% m.3243A> G line as shown in FIG. 5 (n=3 biological replicates). After the treatment, the cells were washed and collected at different time points: 1 hour (1 hr), 1 day, 2 days, 4 days, and 6 days washout time before preparation of DNA. Cells were passaged without further treatment for longer time points.

[0477] An established ND1 / B2M duplex TAQMAN assay was used for qPCR. Here, mtDNA abundance cannot simply be examined directly, as that would reflect an overall score of both genomes. Nor can heteroplasmy be measured directly after exposure because yPNA would block amplification in the qPCR. This interference appears as a significant increase in CT values, usually seen in both the WT and variant primer sets. The interference generally disappeared after four days of growth in medium without yPNA. Interestingly, as the8123-112925-02 03 / 13 / 26 06891

[0478] washout time extended to day 6, a progressive beneficial heteroplasmy shift was observed, suggesting that the yPNA persists for some time after washout, and is consistent with the notion that yPNA-mediated heteroplasmy shift requires mtDNA replication for the elimination of the variant genome. mtDNA depletion by the ND1 / B2M multiplex assay is not observed, as the assay is not sensitive to yPNA carryover into the DNA preparation. The shift in heteroplasmy was also confirmed by amplification-refractory mutation system (ARMS) qPCR analysis on RNA, and by next-generation sequencing of the amplified region.

[0479] ARMS qPCR analysis was performed on no treatment control (NTC) and CPM3-yPNA-treated samples with indicated washout times. Ct values indicated that yPNA was not interfering with treatment samples at these times but did interfere with heteroplasmy assessment of 1- and 2-day samples (not shown), indicates a P-value <0.05 from 2-way ANOVA with Sidak multiple comparison testing.

[0480] The ARMS qPCR did not amplify m.3243A and m.3243A> G, indicating PCR interference. Based on the qPCR results, 1 hour may be insufficient for extensive accumulation of CPM3-yPNA in the mtDNA, and 4 and 6 days showed sufficiently low levels of CPM3-yPNA in the DNA preparations to allow valid assessment of heteroplasmy. The lack of significant shifts in CT values for qPCR at 1 hr, 4 days, and 6 days supports this conclusion.

[0481] A decrease to 40% m.3243A> C mtDNA levels was observed without adverse effects on the normal mtDNA sequence. With additional dose optimization, larger decreases than 40% may be possible. The observed decrease of 40% may be suitable to treat or prevent a mitochondrial condition. Mutations in mtDNA may have fewer effects on mitochondrial function until they cross a critical threshold around 60-70% mutation load. Thus, a heteroplasmy decrease to below that threshold may be sufficient to alleviate symptoms due simply to the presence of the mutation.

[0482] While this is a rigorous treatment of the heteroplasmy shift, there remains a potential for residual yPNA impacting the readouts. Additional days post-exposure are expected to further reduce this potential interference.

[0483] Without wishing to be bound by theory, the heteroplasmy shift may be due to replication fork collapse, leading to double-strand break formation and exonuclease-mediated mtDNA degradation.8123-112925-02 03 / 13 / 26 06891

[0484] Example 4

[0485] yPNA m.3243A> G selectivity

[0486] A 15-mer m.3243A> G-targeted yPNA equipped with a serine-derived hydroxymethyl group at the gamma position and a corresponding unmodified m.3243A> G-targeted PNA were synthesized (Table 1). The 15-mer γPNA is strand-invasive to dsDNA containing the m.3243A> G pathogenic variant. The gel shift seen in lane 6 of FIG. 8 shows the strand invasion and selectivity of the γPNAs and lack of invasion by homologous unmodified PNA (lane 7; no shifted band). The gel shift of FIG. 8 shows γPNA binding to dsDNA at a 20: 1 ratio. Although the γPNA demonstrates strand invasion, the selectivity to m.3243A> G is -30%.

[0487] The selectivity can be visualized in lane 3 of the gel pictured in FIG. 8 as the γPNA appears to also be partially strand-invasive in WT dsDNA (the upper band shows yPNA bound to WT dsDNA). The m.3243A> G yPNA binds the m.3243A> G dsDNA but binds less to the WT DNA. The unmodified PNA is not strand invasive and does not bind dsDNA. More complex in vitro methods can be used to examine selectivity and binding of yPNA m.3243A> G.

[0488] Example 5

[0489] Advancement in mitochondrial targeting

[0490] In Example 2, the non-peptidic cell-penetrating motif CPM3 was shown to deliver small molecules and peptides to the mitochondria in mammalian cells. CPM3, which contains two D-naphthylalanine (Nal) groups, was the most effective for cellular and mitochondrial entry, which was suggested to have enhanced endosomal escape compared to other CPMs. Using a CPM3 coupled at the R group to a TAMRA fluorophore (CPM3-TAMRA), mitochondrial colocalization was demonstrated in WT and 90% m.3243A> G 143B osteosarcoma cells (FIG. 9), demonstrating the ability of CPM3 to direct cargo to mitochondria even when the mitochondria are impaired. 143B cybrids are engineered transmitochondrial cells depleted of endogenous mtDNA and replaced with donor mitochondria containing PMD-causing variants. At early time points, CPM3-TAMRA localized to intracellular bodies consistent with endosomes that, after a few hours, fuse with mitochondria labeled with MITOTRACKER Green. Shown in FIG. 9 is this later time point. CPM3-TAMRA was used to investigate the key parameters for CPM3-mediated cargo delivery.8123-112925-02 03 / 13 / 26 06891

[0491] Example 6

[0492] Effect of CPM3-γPNA on respiration

[0493] A two-day exposure of 2 pM CPM3-yPNA on the 143B 60% m.3243A> G line (FIG.

[0494] 10) tested whether CPM3-γPNA ultimately improves respiration. Cells were trypsinized, counted, and replated onto a SEAHORSE plate without yPNA. The next day, cells underwent a mitochondrial stress test provided by Agilient Technologies, Inc., followed by cell number detection by CYQUANT (Thermo Fisher Scientific Inc.). These experiments had three treatment groups: no treatment (NT), CPM3-γPNA (15-mer), and a shorter version of CPM3-γPNA (13-mer). The 13-mer oxygen consumption rate (OCR) trace was identical to that of NT. The 15-mer showed a trend toward improved basal (before oligomycin application) and maximal respiration (after FCCP and before antimycin A application), but it was not yet statistically significant. These clean traces were encouraging.

[0495] Example 7

[0496] γPNA engagement with mitochondrial nucleoids and replication dynamicsγPNAs bind DNA to form stable heteroduplexes that can displace one strand and are believed to block DNA polymerase progression. Because mtDNA lacks canonical double-strand break (DSB) repair, replication fork collapse leads to linearization, and linear molecules are degraded. yPNA engagement of the m.3243A> G locus may perturb nucleoid architecture, impede replication fork progression, triggering selective removal of mutant genomes through either mitochondrial or lysosomal degradation. Defining how γPNA binding remodels TFAM-compacted DNA, determining whether γPNA binding induces replication stalling or directly triggers degradation, and establishing whether genome elimination occurs within mitochondria or through mitophagy-dependent turnover may reveal the mechanisms of γPNA-DNA interactions alter replication and nucleoid architecture to regulate mitochondrial genome stability (FIG. 11).

[0497] Example 8

[0498] Effects of γPNA on TFAM-compacted DNA structures The mitochondrial genome is compacted by transcription factor A, mitochondrial (TFAM), which organizes the genome into protein-DNA complexes called nucleoids. TFAM loops DNA, loads cooperatively, and is sufficient to compact DNA into nucleoid-like structures. FIG. 12 shows TFAM bound to linearized pUC19, which binds cooperatively to assemble nucleoid-like structures. 0, 7, 15, and 25 pg / ml TFAM was bound for 2 min to8123-112925-02 03 / 13 / 26 06891

[0499] DNA before being spotted onto a fresh mica surface for atomic force imaging. Structural analyses show that TFAM binds the minor groove and sharply bends DNA at each binding site, and super resolution imaging reveals discrete, uniformly sized nucleoids each containing one or a few mtDNA molecules. Whether γPNA molecules can invade these TFAM-coated structures and remodel nucleoid topology is unknown.

[0500] Preliminary data show that γPNA treatment of TFAM-coated DNA leads to displacement of TFAM (FIG. 13). TFAM was loaded onto m.3243A> G DNA for 5 min and then incubated with γPNA for 15 min. DNA has four calculated TFAM dimer binding sites. γPNA does not shift DNA in this timeframe, but does decrease the TFAM bound near TFAM saturation. In this and other timeframes, γPNA concentration and temperature can be altered to tailor invasion behavior and kinetics.

[0501] Example 9

[0502] Effects of γPNA on heavy-strand replication and mtDNA replication intermediates γPNA binding to the light strand DNA at the MT-TL1 locus (antisense to tRNA-Leu(UUR)) may block heavy-strand synthesis as the replication fork encounters the target. Two-dimensional neutral agarose gel electrophoresis (2D-NAGE) may be performed on WT and m.3243A> G homoplasmic cybrids treated with variant-specific or scrambled yPNAs. mtDNA may be digested with restriction enzymes, generating 2-6 kb fragments encompassing nt 3243 but excluding origins OH and OL, following Holt and Goffart protocols. Y-arc analysis can detect replication fork stalling as enhanced intensity or arc distortion.

[0503] Complementary 2D-IMAGE analysis (intact mtDNA agarose gel electrophoresis) will visualize replication and degradation profiles across the genome. RNase H and S 1 nuclease treatments may distinguish RNA-DNA hybrid (RITOLS) intermediates from true stalled forks. Together, these data may determine whether γPNA binding directly obstructs replication or triggers secondary replication-coupled decay.

[0504] Replication intermediates near m.3243A> G may be difficult to resolve by 2D-NAGE. If signals are weak or overlapping, 2D- IMAGE may be used for greater sensitivity. When stalling is modest, RNase H and S1-nuclease treatments can differentiate paused forks from RNA-DNA hybrids, and nascent-strand qPCR will quantify local replication changes. If 2D-NAGE is equivocal, nascent-strand assays, where short-pulse BrdU is immunoprecipitated with anti-BrdU may recover nascent mtDNA, and qPCR across m.3243 may test local8123-112925-02 03 / 13 / 26 06891

[0505] replication slowdown. As an in-situ orthogonal assay, MIRA / mitoSIRF with brief EdU pulses may quantify nascent-mtDNA and fork-proximal proteins in mitochondria.

[0506] γPNA binding can impede heavy-strand synthesis, leading to fork stalling visible as Y-arc distortions or accumulation of linear intermediates. 2D-IMAGE may reveal genomewide replication and degradation intermediates, especially linearization and fragmentation.

[0507] Example 10

[0508] Elimination of m.3243A> G mtDNA

[0509] γPNA-induced heteroplasmy shift may originate from selective degradation of stalled mtDNA either (i) within mitochondria via replication-fork collapse and exonuclease activity or (ii) through mitophagy-mediated nucleoid turnover. 143B m.3243A> G cells may be treated with CPM3-yPNA-TAMRA, and the fate of replicating nucleoids may be tracked using a POLG2-GFP reporter.

[0510] Live-cell confocal imaging can assess colocalization of CPM3-γPNA-TAMRA with POLG2-GFP and lysosomal markers (LAMP1-GFP or LysoTracker Deep Red). If degradation occurs within mitochondria, CPM3-γPNA-positive nucleoids may persist but lose GFP signal due to degradation by MGME1, DNA2, or EXOG. Lysosomal degradation may be indicated by translocation of POLG2-GFP nucleoids to LAMP1-positive vesicles. siRNA knockdown of MGME1, DNA2, and EXOG, along with bafilomycin Al to block lysosomal acidification, may distinguish mitochondrial-intrinsic from lysosomal degradation pathways.

[0511] POLG2-GFP-positive nucleoids may disappear more rapidly after CPM3-γPNA-TAMRA treatment than GFP-negative nucleoids, indicating selective degradation of replicating genomes. Depletion of MGME1 or EXOG may delay this loss, while bafilomycin Al may stabilize lysosomal intermediates. Nucleoids may be degraded too rapidly for tracking or follow mixed mitochondrial and lysosomal routes. Photoactivatable POLG2 reporters and combined nuclease and autophagy perturbations may clarify pathway sequence and timing.

[0512] Example 11

[0513] Exposure-response and persistence of yPNA-mediated heteroplasmy shift Preliminary data show that CPM3 can deliver cargo to iPSC-CMs (FIG. 1), supporting their inclusion in these studies. 143B and iPSC-derived high-heteroplasmy m.3243A> G cells (60%) were treated with CPM3-yPNA, then the compound was withdrawn8123-112925-02 03 / 13 / 26 06891

[0514] (4, 6, 8, and 10 days) and heteroplasmy was monitored, along with mtDNA copy number, and the persistence of effects over time. The target engagement in 143B heteroplasmic cells was measured by total DNA isolation at various times after exposure and PCR interference assay at the locus. Amplification will be compared to zero-heteroplasmy controls. Heteroplasmy was quantified by ARMS qPCR, confirmed by digital PCR and sequencing (watching for γPNA interference), and

[0515] mtDNA copy number by TAQMAN qPCR. To resolve heteroplasmy at the single-cell level, single-cell ATACseq was performed on iPSC-CMs before γPNA treatment and after washout (7 and 14 days) to quantify per-cell chromatin accessibility changes and mtDNA heteroplasmy levels. Analyses compared promoter accessibility within oxidative-metabolism and mitochondrial-biogenesis gene sets between baseline and postwashout samples and correlated accessibility with per-cell heteroplasmy estimates from mtDNA reads.

[0516] Example 12

[0517] Restoration of mitochondrial function

[0518] Respiration, ATP, and mitochondrial protein expression were assessed in heteroplasmic iPSC-cardiomyocytes after γPNA treatment. A well-characterized m.3243A> G iPSC line served as the initial model, with isogenic clones carrying high (50%) and low (< 1%) variant heteroplasmy. These cells successfully differentiated into iPSC-CMs, and cardiomyocyte markers and heteroplasmy were confirmed (FIG. 14).

[0519] Importantly, a beating culture was generated from high and low heteroplasmy isogenic lines. Preliminary MYOCYTER analysis showed increased beat frequency in the high heteroplasmy line, primarily during diastole (FIG. 15). 15-second movies of beating iPSC-CMs were captured at 10x magnification and processed with the IMAGEJ plug-in MYOCYTER. ROIs were selected manually and analyzed using the default MYOCYTER parameters: A) Beat Frequency, B) Beat Times, C) Peak Time, D) Beat Amplitude, E) Systoles, F) Diastoles, and G) MYOCYTER Graph key.

[0520] High-heteroplasmy iPSC-CMs (>75% m.3243A> G) may be expected to exhibit impaired mitochondrial and contractile function, while medium-heteroplasmy lines (~50%) may show partial defects and low-heteroplasmy lines (<30%) may approach normal physiology. Upon γPNA treatment, a significant heteroplasmy shift (>10-15% sustained >6 days without mtDNA depletion) may be expected along with recovery of respiratory and contractile function, with transient mtDNA copy-number reduction followed by repopulation8123-112925-02 03 / 13 / 26 06891

[0521] by wild-type genomes. Isogenic controls (0%) may show no off-target mtDNA loss.

[0522] Minimal cytotoxicity, stable transcriptomes, and reversible mitochondrial depolarization or ROS generation may be expected at higher doses. These outcomes demonstrate that sequence-specific γPNA engagement can produce lasting biochemical correction with acceptable safety margins. Furthermore, successful correlation between the degree of heteroplasmy shift and restored respiratory function may validate γPNA efficacy parameters and replication-blocking mechanisms. Single-cell ATAC-seq after washout may show normalization of accessibility at oxidative and metabolic gene promoters in cells with lower heteroplasmy, establishing a direct molecular link between mtDNA correction and chromatin-state recovery, supported by concordant recovery in metabolic-stress models. Success may show an active dose with <10% A m drop and no γH2AX increase.

[0523] Collectively, these studies may define CPM3-γPNA potential in mitochondrial disease models and clarify the impact of mtDNA replication inhibition

[0524] Comparative Example 1

[0525] PNA alone shows lysosomal, not mitochondrial, localization A PNA bearing the sequence AACTACCACCTACCTC-K-K(TAMRA) was applied to cells and imaged live by confocal microscopy (FIG. 16). Lysosomes were labeled with LYSOTRACKER (FITC / green). Imaging parameters included TRITC (TAMRA / red) channel, laser: 5, gain: 100.

[0526] The PNA-TAMRA signal co-localized with the LYSOTRACKER signal across fields, indicating lysosomal localization rather than mitochondrial distribution. No mitochondrial marker co-localization was observed under these conditions. This suggests that the PNA alone accumulates in lysosomes and fails to deliver cargo to mitochondria.

[0527] Comparative Example 2

[0528] yPNA alone remains lysosomal

[0529] A yPNA equipped with a short cationic-hydrophobic motif ([Cha–Arg]₃) and labeled with TAMRA (([Cha-Arg]3-AACTACCACCTACCTC-K-K(TAMRA)) was applied to cells and imaged live. Lysosomes were labeled with LYSOTRACKER (FITC / green). Confocal settings: TRITC (TAMRA / red): Laser 5, Gain 100; FITC (green): Laser 2, Gain 100.

[0530] The yPNA-TAMRA signal overlapped with LYSOTRACKER (FIG. 17), demonstrating lysosomal localization (both left and right fields). No evidence of mitochondrial distribution was observed under these conditions. This indicates that the8123-112925-02 03 / 13 / 26 06891

[0531] γPNA (with a cationic-hydrophobic motif instead of the disclosed CPM) does not achieve mitochondrial localization.

[0532] Comparative Example 3

[0533] Lipofectamine 3000 Transfection of 4:1 DNA / γPNA A DNA:γPNA complex (4:1) was formed to impart negative charge for delivery via LIPOFECTAMINE 3000. Cells were counterstained with LYSOTRACKER (FITC / green) and imaged by live confocal microscopy using TRITC (TAMRA / red) for the yPNA label (laser 5, gain 130), as shown in FIG. 18.

[0534] The yPNA signal was lysosomally localized in both fields and appeared brighter than 2:1. Mitochondrial co-localization was not observed. This suggests that, lipid-mediated transfection of DNA / γPNA complexes fails to deliver γPNA to mitochondria and instead traps the cargo in lysosomal compartments.

[0535] Comparative Example 4

[0536] rFrF targeting does not localize to mitochondria Mouse embryonic fibroblasts (MEFs) were treated with 10 μM rFrF-TAMRA and stained with MITOTRACKER (green). Confocal images were collected in red (TAMRA) and green (MITOTRACKER) channels with a merged view to assess co-localization (FIG.

[0537] 19). The rFrF-TAMRA signal did not co-localize with MitoTracker; instead, the distribution was consistent with lysosomal localization. This suggests that the rFrF targeting motif fails to deliver cargo to mitochondria in MEFs.

[0538] In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention.

Claims

8123-112925-02 03 / 13 / 26 06891What is claimed is:

1. A compound, or a stereoisomer, tautomer, or pharmaceutically acceptable salt, comprising:a gamma peptide nucleic acid linked to a cell-penetrating motif.

2. The compound of claim 1, wherein the gamma peptide nucleic acid is linked to the cell-penetrating motif by a peptide bond.

3. The compound of claim 1, wherein the gamma peptide nucleic acid comprises a sequence selected to target a mitochondrial DNA mutation.

4. The compound of claim 3, wherein the gamma peptide nucleic acid comprises the sequence C-CGGGACGGTAGAATT-N or the sequence N-CGGGACGGTAGAATT-C.

5. The compound of claim 1, wherein the cell-penetrating motif comprises a nonpeptide scaffold, one or more amidino groups, and one or more hydrophobic groups.

6. The compound of claim 5, wherein the one or more amidino groups include one or more guanidino groups.

7. The compound of claim 5, wherein the one or more hydrophobic groups comprise one or more hydrophobic amino acids8. The compound of claim 7, wherein the one or more hydrophobic amino acids comprise one or more amino acids having aromatic side chains.

9. The compound of claim 7, wherein the gamma peptide nucleic acid is linked to the cell-penetrating motif by a peptide bond to the one or more hydrophobic amino acids.

10. The compound of claim 1, further comprising an ether linkage between the gamma peptide nucleic acid and the cell-penetrating motif.8123-112925-02 03 / 13 / 26 0689111. The compound of claim 1, wherein the gamma peptide nucleic acid is coupled to the cell-penetrating motif at an N-terminus of the gamma peptide nucleic acid.

12. The compound of claim 1, wherein the cell-penetrating motif comprises a structure according to formula I:H2N^NHHNvHN fNHH2NHN)=NHH2NFormula Iwherein R comprises the gamma peptide nucleic acid or a linker coupled to the gamma peptide nucleic acid.

13. The compound of claim 12, wherein the linker comprises one or more hydrophobic amino acids.

14. The compound of claim 13, wherein the one or more hydrophobic amino acids comprise one or more amino acids having aromatic side chains.

15. The compound of claim 12, wherein the linker comprises D-Phe, D-Nal, or a combination thereof.8123-112925-02 03 / 13 / 26 0689116. The compound of claim 1, wherein the compound comprises a structure according to formula II:Formula II.

17. A pharmaceutical composition comprising the compound of claim 1, and at least one pharmaceutically acceptable excipient.

18. A method of treating or preventing a mitochondrial condition, the method comprising administering to a subject in need thereof an effective amount of at least one compound selected from any one of claims 1-3, 5, 10-12, or 16, or the pharmaceutical composition of claim 17.

19. The method of claim 18, wherein the mitochondrial condition is selected from maternally inherited diabetes and deafness (MIDD), mitochondrial myopathy, encephalopathy, and stroke-like episodes (MELAS); or a deletion mutation.

20. A method for inhibiting replication of a mitochondrial mutation, the method comprising contacting mitochondrial DNA containing the mitochondrial mutation with an effective amount of at least one compound of any one of claims 1-3, 5, 10-12, or 16, or the pharmaceutical composition of claim 17.

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