WO2017221271A1

WO2017221271A1 - Organelle-targeting nanocarrier

Info

Publication number: WO2017221271A1
Application number: PCT/IN2017/050254
Authority: WO
Inventors: Archana Chugh; Aastha Jain
Original assignee: Indian Institute Of Technology Delhi
Priority date: 2016-06-22
Filing date: 2017-06-22
Publication date: 2017-12-28

Abstract

Provided herein are peptide based organelle-targeting nanocarriers for macromolecular delivery to targeted sub-cellular locations and methods for transduction and/or transfection of mitochondria.

Description

ORGA NE L L E -TARG ETING NANOCAR RIE R

FIE L D OF T H E INV E NTION

The present invention relates to organelle-targeting nanocarrier and more particularly, to a peptide based organelle-targeting nanocarrier for macromolecular delivery to targeted sub- cellular locations and a method for transduction and transfection of mitochondria for human therapeutics.

BAC K G ROUND OF T H E INV E NT ION Mitochondrion is a highly dynamic and complex organelle actively engaged in various important cellular processes. Commonly known as the powerhouse of the cell, it is important not only for generation of energy but also for thermogenesis, storage of calcium, steriodogenesis and apoptosis. Malfunctioning of mitochondrial processes such as oxidative phosphorylation or apoptosis is responsible for mani f estati on of I i f e-threateni ng di sorders such as cancer.

Mitochondrial malfunction can occur due to mutations in the mitochondrial DNA or nuclear DNA encoding for mitochondrial proteins or even due to reactive oxygen species mediated stress. The treatment of these diseases at the molecular level requires targeting of drugs, genes and functionally active proteins to mitochondria. Several methods such as lipophilic cations, liposome- based carriers ( M IT 0- porter), peptide nucleic acids (PNAs), DQAsomes and cell penetrating peptides (CPPs) have been used for targeting mitochondria for delivery of various macromolecules. However, most of these strategies employ tagging the carrier vehicle with mitochondrial transit targeting peptides (MTP) or signal sequences for targeting the mitochondria. For instance, cell penetrating peptide such as Tat has been conjugated to a M TP and used as a vector for macromolecular delivery in vitro as well as in vivo (Del Gaizo et al, 2003; Horton et al, 2008). Similarly, the mitochondrial transit peptide of human cytochrome c oxidase subunit VIII has been conjugated to PNA for targeting and delivery to isolated mitochondria in vitro (Chinnery et al, 1999). The liposome based carrier, dual function Mito-porter (DF-Mito-porter) also employ tagging of the liposomal vesicle, carrying the macromolecule of interest, to a MTP followed by further coating with another layer of lipid bi layer that is complexed to a CPP on its outer surface (Y amada et al, 2011 ).

Mitochondrial targetingAransit peptides are small peptide sequences that are required by the cell to transport mitochondria localizing proteins. Although widely present in all eukaryotic systems, these peptides do not possess any consensus sequence. However, amphipathicity and presence of Arg residues at position -10 and -2 (with respect to the site of cleavage) has been found to be inevitable in MTPs (von Heijne et al, 1989). Once, translocated inside the cells with the help of a carrier moiety, MT Ps have been reported to carry cargo molecules on their own (Y u et al, 2013). However, to the best of our knowledge no study has reported their cell penetration property. In light of the wide utility of MTPs in targeting the delivery vectors specifically at the site of action i.e mitochondria, it is important to explore their potential as a CPP. MT Ps that could behave as CPPs will possess the ability to act as macromolecule delivery vehicles eliminating the need of a transporter (such as liposomes) for targeting the mitochondria. Apart from targeting and delivering macromolecules, peptide- mediated therapeutics in general, has found a wide application in the treatment of diseases caused due to mitochondrial malfunction. For instance the Szeto-Schiller peptides act as antioxidants and have been shown to be effective in cases of ischemia-reperfusion injury. Moreover, these peptides are cell permeable and do not require any specific carrier moiety to deliver them inside cell cytoplasm They are being currently tested in the phase II of the clinical trials (Szeto, 2006).

Another class of mitochondrial targeting peptide, Mitoparan, derived from the antibacterial peptide mastoparan, has been shown to specifically induce apoptosis in cancer cells by causing mitochondrial membrane swelling resulting in release of cytochrome c (J ones et al, 2008). Although peptides show therapeutic activity themselves, there are still a number of conditions for which delivery of a macromolecule, such as oligonucleotide or drug, is required.

Further, mitochondrial malfunctioning under various circumstances can lead to varied disorders. Effective targeting of macromolecules (drugs) is important for restoration of mitochondrial functioning and treatment of related disorders. Accordingly there is need for an effective organelle targeting nanocarrier that shows improved cell penetration properties and a method thereof that overcomes all the above mentioned drawbacks.

SUM MARY OF T H E INV E NTION Provided herein are nanocarriers for targeted delivery of payload to intracellular organelles. The nanocarriers described herein have dual properties: the ability to penetrate cell membranes/Walls, and specificity, i.e., the ability to target a cell organel I e of i nterest and del i ver the pay I oad sel ecti vely to the targeted organel I e. BRIE F DE SC RIPTION OF T H E DRAWINGS

FIG. 1 shows confocal microscopic images of peptide (CpMTP) uptake in mitochondria of HeLa cells, (a) and (a~) for Mito tracker set, (b) and (b^") for FITC set and (c) and (c~) for merged set as described in more detail in Example 1.

FIG. 2 is a graph showing time-dependent uptake of CpMTP in HeLa cells.

FIG. 3 is a graph showing macromolecular delivery by CpMTP in HeLa cells.

FIG. 4A and 4B are gel electrophoresis images showing determination of DNA binding (FIG. 4A) and protection (FIG. 4B) ability of CpMT P for delivery to mammalian cells using DNA: peptide ratios of 1:1 to 1 :7.

DETAIL E D DE SC RIPTION OF T H E INV E NTION

The invention described herein is explained using specific exemplary details for better understanding. However, the invention disclosed can be worked on by a person skilled in the art without the use of these specific details.

References in the specification to "one embodiment" or "an embodiment" means that a particular feature, structure, characteristic, or function described in connection with the embodiment is included in at least one embodiment of the invention. The appearances of the phrase Ίη one embodiment_, in various places in the specification are not necessari ly al I ref erri ng to the same embodi ment

References in the specification to ^'preferred embodiment_, means that a particular feature, structure, characteristic, or function described in detail thereby omitting known constructions and functions for clear description of the present invention.

In general aspect, the present invention describes an organelle-targeting peptide based nanocarrier for delivery of large macromolecules specifically to the mitochondria and a method thereof for transduction and transfection of mitochondria for human therapeutics. Unexpectedly, it was found that certain transport proteins can themselves serve as cell penetrating peptides, thereby providing the cell penetrating component for the nanocarriers described herein. The cell penetrating acts as a selectivity sequence thereby allowing for delivery of the nanocarrier load to specific cell organelles.

As used herein, a ^'selectivity sequence_, refers to the portion of the nanocarrier which allows for targeted delivery to a cell organelle of interest. The selectivity sequence bears at least some homology to a signaling sequence of a protein within the organelle. As used herein, a ^'signaling sequence_, of a protein within an organelle refers to one or more portion of the protein ^"s sequence which is/are responsible for the appropriate organelle targeting of the protein. In some embodiments, a signaling sequence may be any partial sequence of the protein, or any conserved sequence of vari ous protei ns wi thi n the organel I e or protei ns desti ned to an organel I e.

In a group of embodiments, the nanocarriers described herein comprise peptides identified by a comparison between properties of eel I -penetrating peptides (CPPs) and mitochondrial signal sequences thereby allowing for identification of peptides with dual ability for cellular translocati on/penetration and mitochondrial localization.

The nanocarrier of the present invention establishes a novel pepti de-based vector for efficiently targeting and delivering macromolecules specifically to mitochondria in a simple, one-step mechanism Further, the nanocarrier of the present invention advantageously enables delivery of non-covalently as well as covalently linked macromolecules such as drugs to the mitochondria.

Furthermore, the nanocarrier of the present invention efficiently binds to DNA molecules in vitro, thereby providing a system for gene delivery to the mitochondria for applications in gene therapy. The peptide based nanocarrier of the present invention is cost-effective, since it requires synthesis of only one peptide in comparison to currently available complex lipid-based delivery systems. The present invention is illustrated with reference to the accompanying drawings.

In an embodiment, the present invention discloses an organelle targeting nanocarrier. The organelle targeting nanocarrier is a cell penetrating peptide for delivery of macromolecules to mitochondria. The cell penetrating peptide includes a signal sequence of the protein human mitochondrial, methionine-R-sulfoxide reductase B2 that confers specificity for an intracellular organelle as described herein. Said peptide has a net positive charge of +4 units with the sequence: ARL LWL L RG LTLGTAPRRA (hereinafter, :SEQ IDNO: 1 ^"), sequence L RFLAPRL LSLQGRTA (hereinafter, :SE Q IDNO: 2^"), sequence L PRRPLAWPAWL (hereinafter, :SEQ IDNO: 3^"), sequence L RAAARFG PRLGRRL L (hereinafter, :SEQ IDNO: 4^").

In this one embodiment, the signal sequence is a cell penetrating mitochondrial transit peptide (hereinafter referred to as :CpMTP ~) sequence (also referred to herein as a "nanocarrier"). The macromolecule is linked to CpMTP to form a non-covalent complex. The complex comprises of a CpMTP and a macromolecule, wherein the CpMTP is an agent that transverse a cell membrane and/or cell wall and said complex delivers macromolecules efficiently to the targeted sub-cellular location.

In at least one embodiment, the cell penetrating mitochondrial transit peptide has a sequence which has at least about 20 % to 99% similarity to a sequence selected from:

ARL LWL L RGLTLGTAPRRA; (SEQ ID NO: 1) L RFLAPRL LSLQGRTA; (SEQ ID NO: 2)

L PRRPLAWPAWL; (SE Q ID NO: 3)

L RAAARFG PRLGRRL L; (SEQ ID NO: 4) In another embodiment, a method of preparing an organelle targeting peptide based nanocarrier is disclosed. The method comprises of a macromolecule linked to a CpMTP to form a complex. Said complex comprises of the CpMTP and the macromolecule, wherein the CpMTP is an agent that can transverse a cell membrane and/or cell wall. The complex is formed by various methods like covalent linkage, el ectrostati c I i nkage, hydrophobi c i nteracti on.

The cell penetrating peptides are selected from the group consisting of peptides such as human mitochondrial transit peptides/mitochondrial signal sequences. The macromol ecul es are sel ected from the group consi sti ng of therapeuti c protei ns such as antibodies (IgG, IgM), antibody fragments (scFv, Fab^", Fc fragments), insulin, growth factors, growth hormones, coenzymes, p53, Bcl-2, BAK, anti- cancer proteins, transcriptional factors, oligonucleotides such as siRNA, miRNA, plasmid DNA, mRNA, tm RNA, tRNA, rRNA, shRNA, PNA, ssRNA, dsRNA,ssDNA, dsDNA, DNA:RNA hybrids, cDNA, or combinations thereof and drug molecules.

In one embodiment of the present invention the peptide based nano-carrier enables delivery of covalently as well non-covalently linked macromol ecul es such as therapeutic proteins such as antibodies (IgG, IgM), antibody fragments (scFv, Fab^", Fc fragments), insulin, growth factors, growth hormones, recombinant proteins, coenzymes, p53, Bcl-2, BAK, anti-cancer proteins, transcriptional factors, oligonucleotides such as siRNA, miRNA, plasmid DNA, mRNA, tm RNA, tRNA, rRNA, shRNA, PNA, ssRNA, dsRNA, ssDNA, dsDNA, DNA:RNA hybrids, cDNA, or combinations thereof and drug molecules to the mitochondria for application in human therapeutics. In one embodiment of the present invention the CpMTP translocates across HeLa cells, thereby enabling successful delivery of non-covalently linked macromolecules such as therapeutic proteins such as antibodies (IgG, IgM), antibody fragments (scFv, Fab^", Fc fragments), insulin, growth factors, growth hormones, recombinant proteins, coenzymes, p53, Bcl-2, BAK, anti-cancer proteins, transcriptional factors, oligonucleotides such as siRNA, miRNA, plasmid DNA, mRNA, tm RNA, tRNA, rRNA, shRNA, PNA, ssRNA, dsRNA, ssDNA, dsDNA, DNA:RNA hybrids, cDNA, or combinations thereof and drug molecules to the mitochondria.

In another embodiment the present invention provides a method for the delivery of covalently as well non-covalently linked macromolecules to mitochondria. In a first step, the method comprises of exposing the mitochondria to at least one macromolecule and at least one organelle targeting nanocarrier by treating the cells with a cell penetrating agent. In a next step, the nanocarrier-macromolecule complex interacts with cell membrane. The macromolecule translocates across the cell membrane of the cell and enters the target organelle in the presence of the at least one organel I e targeti ng nanocarri er. In yet another embodiment the present invention provides a method for transduction and transfection of mitochondria for human therapeutics. The method comprises the steps of forming a covalent or non-covalent complex between the therapeutic molecule and the organelle-targeting nanocarrier. Further steps include treating the cells with the organelle-targeting nanocarri er-therapeutic molecule complex. The complex organelle-targeting nanocarrier - therapeutic molecule enters the cells, thereby targeting and delivering the organelle-targeting nanocarrier complexed with therapeutic molecule to the mitochondria. The nanocarriers described herein may be suitably formulated with pharmacologically acceptable excipients to provide compositions useful in treatment of mitochondrial disorders, or any other disorders wherein targeted delivery to an organelle of interest is desired. Mitochondrial diseases are a group of disorders caused by dysfunctional mitochondria. Mitochondria are ubiquitous in the body and in order to minimize side effects, targeted delivery of therapeutics to mitochondria is desirable.

Mitochondrial diseases are typically caused by mutations in the mitochondrial DNA leading to mitochondrial dysfunction. Sometimes mutations in nuclear genes / DNA lead to defective mitochondrial proteins which are translocated in to the mitochondria thereby causing mitochondrial dysfunction. The nanocarriers provided herein and/or compositions thereof may be administered via any suitable delivery route including and not limited to oral, buccal, nasal, intravenous, intraperitoneal, intramuscular, i ntrathecal , and/or i ntracrani al routes.

EXAM PL E S

T he f ol I owi ng exampl es i 11 ustrate the i nventi on, but are not I i mi ti ng thereof. Example 1 : Confocal M icroscopy for uptake of C pM T P in H eL a cells

Cells were observed using a confocal microscope to analyze the CpMTP uptake in mitochondria of HeLa cells. Localization of CpMTP in mitochondria of HeLa cells was assessed by labeling the mitochondria with MitoRed tracking dye. The degree of co- localization between the peptide and the dye was established using Pearson ^"s coefficient A value of 0.87363 was obtained suggesting significant co- localization of CpMTP with Mito-Red tracker dye. The uptake of CpMTP in mitochondria of HeLa cells included a study divided into 3 sets, namely Mito tracker set FITC set and a merged set as shown in Fig. 1. Each set had two HeLa cell sample, wherein the first sample was treated without CpMTP having MitoRed tracking dye only, FITC only and merged dyes respectively and the second sample was treated with CpMT P,[referred to as (a) and (a^") for Mito tracker set, (b) and (b~) for FITC set and (c) and (c~) for merged set].The merge between (a~) and (b~) confirms the localization of CpMT P in mitochondria (c^"). The gamma values for FITC filter were reduced to 0.67 units and MitoRed filter to 0.77 units to enhance the colour contrast and overlap. It was observed that the CpMT P showed efficient cellular internalization and mitochondrial localization in mammalian cells such as HeLa at very low concentration (5n /l) within 5 min of incubation as shown in Fig.1 and 2.

Flow cytometric analysis of time dependent uptake of CpMTP in HeLa cells indicated an increase in uptake of peptide as the time of incubation of peptide with cells increased as shown in Fig. 2.

Example 2: Macromolecular delivery by C pMT P in H eL a cells.

The ability of CpMTP to deliver macromolecular cargos in mitochondria of HeLa cells was evaluated by delivering a high molecular weight protein, DNase I (39kDa). Determination of mtDNA levels in HeLa cells post-delivery of CpMTP-DNase I complex was determined by normalizing the level of ND6 gene (representative for mtDNA levels) with f-actin (representative of nuclear DNA levels). The graph as shown in Fig. 3 represents relative mtDNA levels of cells without any treatment, DNase I alone, Mu-CpMTP, Mu-CpMT P- DNase I complex, CpMTP and CpMTP- DNase I complex.

A non-covalently conjugated complex of CpMTP-DNase I and Mu-CpMTP-DNase I was employed as the carrier complex. The nature of conjugation between the peptides and DNase I was electrostatic since the peptides (both CpMTP and Mu-CpMTP) carry a net charge of +4 units while DNase I carries a net charge of -13 units at physiological pH.

It was hypothesized in this study that the delivery of DNase I by CpMTP to mitochondria would degrade mitochondrial DNA (mtDNA) while the levels of nuclear DNA will not be affected. It was observed that CpMTP-DNase I complex indeed degraded mtDNA by 50% (P -value = 0.00097, = 0.05) with respect to the control (no treatment, peptide only) treatment and 20% with respect to DNase I alone treatment while the levels of nuclear DNA remained unchanged as shown in Fig. 3. However, in the cells treated with Mu-CpMTP-Dnase I complex, no significant reduction in mtDNA and nuclear DNA levels was observed with respect to control (P-value = 0.4269, = 0.05) as shown in Fig. 3.

Example 3: Determination of DNA binding ability of C pMT P for delivery to mammalian cells.

Since CpMTP carries a net charge of +4 units, its ability to bind plasmid DNA, pEGFP-N1 was established by gel retardation and DNase I protection assay. DNA binding occurs via simple electrostatic interaction between the peptide and the DNA molecules. DNA binding efficiency of CpMTPwas determined using pEG FP plasmid. Fig. 4A shows a gel retardation assay for determining the binding efficiency of CpMTP to plasmid DNA: L1, 1 kb ladder; L2, pEGFP plasmid DNA; L3, CpMTP; L4, 1:1 (DNA:peptide); L5, 1:2; L6, 1:3; L7, 1 :4; L8, 1 :5; L9, 1:6; L 10, 1:7.

Fig. 4B shows DNase I protection assay carried out to determine the extent of DNA protection by CpMTP. L1, pEGFP plasmid DNA; L2, CpMTP; L3, 1:1 (DNA:peptide); L4, 1 :2; L5, 1 :3; L6, 1:4; L7, 1:5; L8, 1:6; L9, 1 :7; L10, 1 kb ladder. It was observed that CpMT P binds DNA efficiently and hinders its mobility on agarose gel at a w/W ratio of 1:4 to 1 :7 (DNA: peptide). However, slight retardation was also observed at ratios 1 :2 and 1:3. Upon treating the peptide-DNA complex with DNase I, it was found that DNA remained protected from DNase I from ratio 1 :5 to 1:7 but below that degradation of DNA was observed as shown in Fig. 4A and 4B. Based on these observations, it is proposed that the peptide-DNA complex can be used for mitochondrial transfection with the gene of interest such as those related to mitochondria gene- related disorders. The foregoing descriptions of specific embodiments of the present invention have been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the present invention to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teaching.

The embodiments were chosen and described in order to best explain the principles of the present invention and its practical application, to thereby enable others, skilled in the art to best utilize the present invention and various embodiments with various modifications as are suited to the particular use contemplated.

It is understood that various omission and substitutions of equivalents are contemplated as circumstance may suggest or render expedient, but such are intended to cover the application or implementation without departing from the spirit or scope of the present i nvention.

Claims

C laims :

1. A nanocarrier for delivery of macromolecules to cell organelles comprising a cell penetrating peptide sequence and a selectivity sequence having about 20% to about 99% homology with a signal sequence of an organelle protein in said cell organelle.

2. The nanocarrier of claim 1, wherein the cell penetrating peptide sequence is a mitochondrial transport protein sequence.

3. The nanocarrier of claim 1, wherein the cell organelles are mitochondria and the signal sequence of an organelle protein is a signal sequence of a mitochondrial protein.

4. The nanocarrier of claim 3, wherein the mitochondrial protein is human methionine-R -sulfoxide reductase B2 (SEQ ID NO: 5).

5. The nanocarrier of any one of claims 1 - 4, wherein the selectivity sequence has about 20% to about 99% homology with a signal sequence selected from the group consisting of: ARL LWL L RG LTLGTA PRRA (SEQ ID NO: 1), sequence L RFLAPRL LSLQGRTA (SEQ ID NO: 2), L PRRPLAWPAWL (SE Q ID NO: 3), and L RAAARFGPR LG RRL L (SEQ IDNO: 4).

6. The nanocarrier of claim 1, wherein the nanocarrier carries a net positive charge.

7. T he nanocarri er of cl ai m 1 , wherei n the nanocarri er carri es a net positive charge of +4.

8. A complex comprising a nanocarrier of claim 1, and a macromolecule.

9. The complex of claim 8, wherein the macromolecule and the nanocarrier form a non-covalent complex, or the macromolecule is covalently attached to the nanocarrier.

10. The complex of claim 8, wherein the macromolecule is an antibody, antibody fragments, insulin, growth factors, growth hormones, coenzymes, anti cancer proteins selected from the group consisting of p53, Bel -2, BAX, and BAK, transcri pti onal factors, ol igonuci eoti des sel ected from the group consi sti ng of si R N A , miRNA, plasmid DNA, mRNA, tm RNA, tRNA, rRNA, shRNA, PNA, ssRNA, dsRNA,ssDNA, dsDNA, DNA: RNA hybrids, and cDNA, drug molecules, or combi nati ons thereof.

11. A nanocarrier for delivery of macromolecules to mitochondria comprising a mitochondrial transport protein sequence and a selectivity sequence having about 20% to about 99% homology with a signal sequence of methionine-R-sulfoxide reductase B2 (SEQ ID NO: 5)..

12. The nanocarrier of claim 11, wherein the signal sequence is selected from the group consisting of ARL LWL L RG LT LGTAPRRA (SEQ ID NO: 1), sequence

L RFLAPRL LSLQGRTA (SEQ ID NO: 2), L PRRPLAWPAWL (SE Q ID NO: 3), and L RAAARFGPR LG RRL L (SEQ IDNO: 4).

13. The nanocarrier of claim 11, wherein the nanocarrier carries a net positive charge of +4.

14. A complex comprising a nanocarrier of claim 11, and a macromolecule

15. T he compl ex of cl ai m 14, wherei n the macromol ecul e and the nanocarri er form a non-covalent complex, or the macromolecule is covalently attached to the nanocarrier.

16. A composition comprising the nanocarrier of claim 1 or claim 11.

17. A composition comprising the complex of claim 8 or claim 14.

18. A method for delivery of macromolecules to mitochondria in a cell, the method comprising contacting the cell with a complex of claim 8 or claim 14.

19. A method for treatment of a mitochondrial disease in a subject, the method comprising administering a composition of claim 17 to the subject in need thereof.

20. T he method of cl ai m 19, wherei n the composi ti on i s admi ni stered intravenously, intramuscularly, intraperitoneal ly, intracranially, or intrathecal I y.