Delivery peptides and methods of use thereof

Abstract

The name of the invention is delivery peptides and methods of use thereof. The present invention relates to peptides, and methods of use thereof, such as in delivering an agent into a cell, or modifying a cell.

Description

[0001] This application is a divisional application. The filing date of the original application is August 13, 2020, the application number is 2020800716509 (PCT / IL2020 / 050897), and the name of the invention is “Delivery peptides and methods of use thereof”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 887,050, filed on August 15, 2019, entitled “DELIVERY PEPTIDES AND METHODS OF USING THE SAME,” which is incorporated herein by reference in its entirety. Technical Field

[0004] The present invention belongs to the field of delivery peptides. Background Art

[0005] Vitellogenin (Vg) is a major lipoprotein (LP) in oviparous animals and is the precursor of the yolk protein - vitellin. It is essential for providing the metabolic needs of the developing embryo - and is one of the most abundant LPs in the hemolymph of reproductive (vitellogenous) females. In the giant shrimp Macrobrachium rosenbergii, which is widely cultured worldwide (more than 450,000 tons per year), vitellogenin (MrVg) is synthesized in the hepatopancreas, secreted into the hemolymph, and taken up by the ovary via receptor-mediated endocytosis (RME).

[0006] The RME mechanism involves membrane-bound receptors that specifically bind to ligand molecules in the extracellular fluid. The ligand-receptor complex is located in regions of the plasma membrane called "coated pits" and is internalized by clathrin-coated vesicles. Endocytic events are critical in a variety of different physiological processes with various types of ligands and receptors, and the basic mechanism appears to be conserved in all eukaryotic organisms.

[0007] LP and its membrane receptors are conserved throughout evolution, including human ApoB, several low-density lipoproteins, and crustacean Vg. Vg receptors (VgR) belong to the low-density LP receptor (LDLR) superfamily and contain several conserved domains, including the ligand binding domain (LBD), epidermal growth factor (EGF)-like domain, O-glycosylation domain, transmembrane domain, and short cytoplasmic tail. Studies of vertebrate and invertebrate LDLR and VgR have found that the LBD is composed of several repeats (sequences) of approximately 40 amino acids. Each repeat (sequence) contains six cysteine ​​residues that form three disulfide bonds. These six cysteine ​​repeats (sequences) were found to be important for LDLR-LDL binding.

[0008] In previous studies, the crustacean Macrobrachium rosenbergii VgR (MrVgR) was cloned and sequenced, and its putative protein was found to contain all of the above-mentioned conserved domains, including LBD-I and LBD-II, which are characteristic of arthropod VgR. In addition, MrVgR was shown to specifically bind its ligand MrVg. In addition, receptor blot analysis of Xenopus laevis yolk phosvitin, the ovary-processed form of Vg, showed that the lipid yolk phosvitin subunit containing the lipid binding domain interacted with the receptor. The latter finding is consistent with previous studies on vertebrate Vg, which showed that the lipid binding domain is essential for the interaction with VgR.

[0009] The most extensive study on Vg-VgR interactions was conducted in fish, the blue tilapia, Oreochromis aureus, where VgR was shown to bind to the lipid binding domain of the N-terminal region of Vg. Thus, an 84 amino acid long fragment from the N-terminal portion of Oreochromis aureus Vg was found to be sufficient for VgR binding.

[0010] There remains a great need for agents that are able to specifically deliver cargo molecules such as dyes, polynucleotides, proteins, etc. to cells such as oocytes. Summary of the invention

[0011] According to one aspect, there is provided a peptide consisting of 7 to 23 amino acids derived from the following amino acid sequence:

[0012] DKX1X2X2X3PX4X5GX6YKYVEAX7X8X9SX 10 X 11 (SEQ ID NO: 1), wherein: X1 is selected from the group consisting of amino acid residues N and K; X2 is selected from the group consisting of amino acid residues I and V; X3 is selected from the group consisting of amino acid residues K and R; X4 is selected from the group consisting of amino acid residues A and S; X5 is selected from the group consisting of amino acid residues Y and I; X6 is an amino acid residue selected from the group consisting of S, I, A and T; X7 is an amino acid residue selected from the group consisting of H, S, K and E; X8 is selected from the group consisting of amino acid residues Q and M; X9 is an amino acid residue selected from the group consisting of E, D and M; X 10 is selected from the group consisting of amino acid residues V and T; and X 11 is selected from the group consisting of amino acid residues L and M.

[0013] According to another aspect, a chimera is provided, comprising: a first peptide, wherein the first peptide comprises a peptide disclosed herein; directly or indirectly bound to an agent selected from the group consisting of: a polynucleotide, a second peptide, a small molecule, or any combination thereof.

[0014] In some embodiments, the peptide comprises the amino acid sequence: DKX1X2X2X3PX4X5GX6YKYVEA (SEQ ID NO: 2), wherein: Xi is selected from amino acid residues N and K; X2 is selected from amino acid residues I and V; X3 is selected from amino acid residues K and R; X4 is selected from amino acid residues A and S; X5 is selected from amino acid residues Y and I; X6 is an amino acid residue selected from the following: S, I, A and T.

[0015] In some embodiments, the peptide comprises the amino acid sequence: DKX1X2X2X3PX4 (SEQ ID NO: 3), wherein: X1 is selected from amino acid residues N and K; X2 is selected from amino acid residues I and V; X3 is selected from amino acid residues K and R; and X4 is selected from amino acid residues A and S.

[0016] In some embodiments, the peptide comprises the amino acid sequence: DKX1X2X2X3P (SEQ ID NO: 4), wherein: X1 is selected from amino acid residues N and K; X2 is selected from amino acid residues I and V; and X3 is selected from amino acid residues K and R.

[0017] In some embodiments, the peptide comprises the amino acid sequence: GX6YKYVEA (SEQ ID NO: 5), wherein X6 is an amino acid residue selected from the group consisting of: S, I, A, and T.

[0018] In some embodiments, the agent is selected from the group consisting of a polynucleotide, a second peptide, a small molecule, a dye, an embryo modifying agent, and any combination thereof.

[0019] In some embodiments, polynucleotides encoding the peptides disclosed herein are provided.

[0020] In some embodiments, expression vectors comprising a polynucleotide disclosed herein are provided.

[0021] In some embodiments, a cell is provided that comprises any of the following: (a) a peptide disclosed herein; (b) a chimera comprising the peptide; (c) a polynucleotide encoding the peptide; (d) an expression vector comprising the polynucleotide.

[0022] In some embodiments, a composition is provided, which comprises any of the following: (a) a peptide disclosed herein; (b) a chimera comprising the peptide; (c) a polynucleotide encoding the peptide; (d) an expression vector comprising the polynucleotide; (e) a cell comprising any of (a), (b), (c) and (d); and a carrier.

[0023] In some embodiments, chimeras are used to deliver agents into cells.

[0024] In some embodiments, a method of preparing a chimera disclosed herein is provided, comprising combining a peptide disclosed herein with an agent, wherein the agent is selected from the group consisting of: a polynucleotide, a second peptide, a small molecule, a dye, an embryo modifying agent, and any combination thereof.

[0025] In some embodiments, a method for delivering an agent into a cell is provided, comprising contacting the cell with a chimera disclosed herein, thereby delivering the agent into the cell.

[0026] In some embodiments, a method of modifying a cell is provided, comprising contacting the cell with a chimera disclosed herein, thereby modifying the cell.

[0027] In some embodiments, the cell is a cell of an oviparous animal.

[0028] In some embodiments, the cell is an arthropod cell.

[0029] In some embodiments, the arthropod is a crustacean.

[0030] In some embodiments, the crustacean is a decapod crustacean.

[0031] In some embodiments, the cell is an ovarian cell.

[0032] In some embodiments, the ovarian cell is an oocyte.

[0033] In some embodiments, the contacting comprises any of in vivo contacting, in vitro contacting, and ex vivo contacting.

[0034] In some embodiments, contacting is administering an effective amount of a chimera disclosed herein to an early vitellogenic female.

[0035] In some embodiments, the administration is to the ovary, hemolymph, or both.

[0036] In some embodiments, the administering is at least once a week.

[0037] In some embodiments, the method further comprises the step of crossing the administered female with a male.

[0038] In some embodiments, progeny obtained from a cross performed according to the methods disclosed herein are provided.

[0039] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the invention belongs. Although methods and materials similar or equivalent to those described herein can be used for the practice or testing of embodiments of the present invention, exemplary methods and / or materials are described below. In the event of a conflict, the patent specification (including definitions) shall prevail. In addition, these materials, methods and examples are exemplary only and are not intended to be necessarily restrictive.

[0040] Further embodiments and the full scope of applicability of the present invention will be apparent from the detailed description given below. However, it should be understood that the detailed description and specific examples, although indicating preferred embodiments of the present invention, are given only as examples, because various changes and modifications within the spirit and scope of the present invention will be apparent to those skilled in the art based on the detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Schematic diagram including receptor-mediated vitellogenin endocytosis via clathrin pits and receptor recycling.

[0042] Figure 2 Included are schematic, non-limiting representations of the procedures disclosed herein.

[0043] Figure 3 Included are vertical bar graphs showing the expression of the paired box protein 6 gene (PAX6) during female and male embryonic development in Macrobrachium rosenbergii.

[0044] Figures 4A-4N Included are micrographs showing internalization of vitellogenin (Vg) peptide into early yolk oocytes of Macrobrachium rosenbergii. (4A) TAMRA fluorescence emission image; (4B) overlay of (4A) with a bright field image of an oocyte incubated with Vg peptide labeled with the TAMRA fluorophore. (4C) TAMRA fluorescence emission; (4D) overlay of (4C) with a bright field image of an oocyte incubated with scrambled (sc) Vg peptide labeled with the TAMRA fluorophore. The white arrow in (4B) points to the oocyte membrane, and the black arrow points to Vg accumulation within the oocyte. 4E-4H are images of oocytes from ovarian slices incubated simultaneously with Vg-TAMRA (red) and control-FITC (green) peptides. (4E) and (4F) are confocal images of Vg-TAMRA and control-FITC emissions, respectively. (4G) Nuclear DNA of follicular cells stained with Hoechst (blue). (4H) is Figure 4E-4G Overlay image. Figure 4I-4L Fluorescent images of oocytes and gills taken from early vitellogenic females injected simultaneously with Vg-TAMRA and control-FITC peptides. Figures 4I-4NFluorescence micrographs of oocytes and gills from early vitellogenic females injected with either Vg-TAMRA peptide or control-FITC peptide. Confocal images of Hoechst-stained oocytes and Vg-TAMRA (4I) and control-FITC (4J) peptides at their respective excitation wavelengths, and an overlay image (4K). Both Vg-TAMRA and control-FITC peptides can be detected in the gills (4L and 4M, respectively) and in the overlay image (4N).

[0045] Figures 5A-5P Included are micrographs showing that Vg peptide accumulates in oocytes in a dose-dependent manner. Ovarian slices incubated with decreasing concentrations of Vg or scVg peptide: 12 μM (5A, 5E, 5I and 5M), 6 μM (5B, 5F, 5J and 5N), 1.5 μM (5C, 5G, 5K and 5O) and 0.75 μM (5D, 5H, 5L and 5P). Oocytes incubated with Vg-TAMRA peptide are shown in fluorescent micrographs (5A-5D) and in overlays with bright field (5E-5H). Oocytes incubated with scVg-TAMRA peptide are shown in fluorescent micrographs (5I-5L) and in overlays with bright field (5M-5P).

[0046] Figures 6A-6G Included are micrographs and graphs showing that the Vg-TAMRA peptide (Vg) is capable of piggybacking conjugated dsRNA-FITC into oocytes in vitro. (6A) UV images of agarose gel separated double-stranded (ds) RNAs of: dsPAX6A (198 bp), dsPAX6B (230 bp), and ds epidermal growth factor receptor (dsEGFR) (700 bp). (6B) DLS measurements of particle radius of dsRNA-peptide conjugates with molar ratios ranging from 1:1 to 1:30 (dsRNA was kept at 25 pmol). Fluorescence images of agarose gel loaded with lysine-histidine tag (KH9) Vg conjugated to 210 bp long dsRNA of PAX6 (Vg-dsPAX6) under 550 nm excitation (6C) and UV (6D). A constant amount (25 pmole) of dsPAX6 was combined with increasing amounts of Vg to produce the following molar ratios: 1:1, 1:5, 1:10, 1:20, and 1:50, as shown in lanes (6C-6D). Fluorescence images of ovarian slices incubated with Vg-dsPAX6 (6C), white arrows point to orange spots indicating internalized dsPAX6-FITC and Vg-TAMRA superposition (6E), white arrows indicate scVg-dsPAX6 and dsPAX6 control labeled with FITC located on the outside of the oocyte membrane (6F and 6G, respectively).

[0047] 7A-7C include micrographs and graphs showing that Vg-dsRNA conjugates injected into reproductive females conferred gene silencing and delayed eye development in embryos. Figures 7A-I to 7A-VI Included are images of embryos isolated from mothers injected with Vg-dsPAX6 (7A-I to 7A-III) or from mothers injected with scVg-dsPAX6 (7A-IV to 7A-VI). Embryos with no or residual eye development (7A-II, white arrows), cyclops embryos (7A-II, black arrows), or partially developed eyes (7A-III, gray arrows) were observed. (7B) Included are vertical bar graphs showing the average length / width index of embryonic eyes of 9-day-old embryos sampled from mothers injected with Vg-dsPAX6 (black bars) or from mothers injected with scVg-dsPAX6 (gray bars). (7C) Included are vertical bar graphs showing the expression of epidermal growth factor receptor (EGFR) in larvea of ​​mothers injected with Vg-dsEGFR or control mothers (i.e., injected with Vg-dsGFP or scVg-dsEGFR).

[0048] Figures 8A-8C include photomicrographs and graphs showing that high dose injections into females induced for vitellogenesis result in significant eye development retardation. (8A) Embryos isolated from mothers injected with Vg-dsPAX6 (8A-I to 8A-III) or injected with dsPAX6 (8A-IV to 8VI). (8B) Includes vertical bar graphs showing the average length / width index of embryonic eyes taken from 10-day embryos from mothers injected with Vg-dsPAX6 (dotted bars) or dsPAX6 (ribbond bars). (8C) Includes vertical bar graphs showing the average length / width index of embryonic eyes taken from females currently injected with Vg-dsPAX6 (dotted bar data from Figure 9B) - compared to the average length / width index of embryonic eyes taken from 10-day embryos from mothers injected with Vg-dsPAX6 (dotted bar data from Figure 9B) - compared to the average length / width index of embryonic eyes taken from 10-day embryos from mothers injected with Vg-dsPAX6 (dotted bar data from Figure 9B) Figure 7B Compared with the black column data.

[0049] Figures 9A-9C include micrographs showing that PAX6 silencing in embryos affects the shape of ommatids at late larval stages. (9A-9C) Scanning electron microscopy (SEM) images of normal eyes (9A) and irregular eyes (9B-9C) of larvae of treated females. I, II, III represent different magnifications of ×500, ×1,000, and ×2,000, respectively.

[0050] Figures 10A-10EIncludes examples, micrographs, multiple sequence alignments, and chromatograms showing that Cas9-Vg active hybrid proteins were cloned, expressed, purified, and administered. (10A) Organizational scheme of the Cas9-Vg construct cloned into the pET28B plasmid and 3D structure prediction of the cloned fusion protein. (10B) UV irradiation of sybersafe-stained agarose gels showing PAX6 DNA fragmentation in the presence of commercial Cas9 or recombinant Cas9-Vg, in the presence + or absence - of sgRNA. The assay has been performed using 600ng of recombinant or commercial Cas9. (10C) Coomassie-stained gel of recombinant His-Cas9-Vg Ni-NTA elution fractions. (10D-10E) are multiple sequence alignments and chromatograms showing case analysis and sequencing of DNA extracted from embryos, which are the offspring of mothers administered Cas9-Vg conjugated to PAX6 gRNA. (10D) Sanger chromatogram of the forward template (top) and the relative contribution of each sequence (bottom), depicting the deletion of the 3rd or 4th base upstream of the PAM (CGG, underlined dashed line). (10E) Sanger chromatogram of the reverse template (top) and the relative contribution of each sequence (bottom), depicting the deletion of the 4th base downstream of the PAM.

[0051] Figures 11A-11G Included are graphs and fluorescence micrographs showing that the VgR interaction region of vitellogenin is highly conserved. (11A) Graph showing a multiple sequence alignment of the 26 amino acid VgR interaction region from Macrobrachium rosenbergii and six other crustacean species. The filled areas depict identical amino acids between the sequences. The line above the sequence of each species describes the similarity to the Macrobrachium rosenbergii sequence. The plus sign (+) indicates an amino acid exchange with another amino acid from the same feature group. (11B-11G) Fluorescence micrographs showing in vitro incubation of ovarian slices in the presence of the Vg-derived peptide TAMRA and scVg-FITC (peptide sequences disclosed in Table 4). (11B) Vg235-260, (11C) Vg237-244, (11D) Vg246-253, (11E) Vg237-253, (11F) Lv-Vg238-245, and (11G) Lv-Vg247-254. DETAILED DESCRIPTION

[0052] The present invention relates to peptides that are capable of being internalized or endocytosed into ovarian cells, such as oocytes. In some embodiments, the peptides or fragments thereof are capable of delivering an agent into ovarian cells.

[0053] In some embodiments, the peptide comprises or consists of 7 to 23 amino acids derived from the following amino acid sequence: DKX1X2X2X3PX4X5GX6YKYVEAX7X8X9SX 10 X 11 (SEQ ID NO: 1), wherein: X1 is selected from the group consisting of amino acid residues: N and K; X2 is selected from the group consisting of amino acid residues: I and V; X3 is selected from the group consisting of amino acid residues: K and R; X4 is selected from the group consisting of amino acid residues: A and S; X5 is selected from the group consisting of amino acid residues: Y and I; X6 is selected from the group consisting of amino acid residues: S, I, A and T; X7 is selected from the group consisting of amino acid residues: H, S, K and E; X8 is selected from the group consisting of amino acid residues: Q and M; X9 is selected from the group consisting of amino acid residues: E, D and M; X 10 is selected from the group consisting of amino acid residues: V and T; and X 11 Selected from the amino acid residues: L and M.

[0054] As used herein, the phrase "7 to 23 amino acids" includes peptides of at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 17, at least 19, at least 21, or at least 23 amino acids or any value and range therebetween. Each possibility represents a separate embodiment of the present invention. In some embodiments, 7 to 23 amino acids includes 7 to 22, 8 to 20, 9 to 23, 10 to 17, 8 to 12, 9 to 19, 11 to 15, 10 to 13, 16 to 22, 17 to 19, 11 to 23, 14 to 19, 16 to 20, 9 to 16, or 8 to 11 amino acids. Each possibility represents a separate embodiment of the present invention.

[0055] In some embodiments, the peptide comprises the amino acid sequence: DKX1X2X2X3PX4X5GX6YKYVEA (SEQ ID NO: 2), wherein: X1 is selected from the amino acid residues: N and K; X2 is selected from the amino acid residues: I and V; X3 is selected from the amino acid residues: K and R; X4 is selected from the amino acid residues: A and S; X5 is selected from the amino acid residues: Y and I; and X6 is an amino acid residue selected from the following: S, I, A and T.

[0056] In some embodiments, the peptide comprises the amino acid sequence: DKX1X2X2X3PX4 (SEQ ID NO: 3), wherein: X1 is selected from the amino acid residues: N and K; X2 is selected from the amino acid residues: I and V; X3 is selected from the amino acid residues: K and R; and X4 is selected from the amino acid residues: A and S.

[0057] In some embodiments, the peptide comprises the amino acid sequence: DKX1X2X2X3P (SEQ ID NO: 4), wherein: X1 is selected from the group consisting of amino acid residues: N and K; X2 is selected from the group consisting of amino acid residues: I and V; and X3 is selected from the group consisting of amino acid residues: K and R.

[0058] In some embodiments, the peptide comprises the amino acid sequence: GX6YKYVEA (SEQ ID NO: 5), wherein: X6 is an amino acid residue selected from the group consisting of: S, I, A and T.

[0059] In one embodiment, the peptide comprises the amino acid sequence: DKNIIKPAYGSYKYVEA (SEQ ID NO: 6).

[0060] In one embodiment, the peptide comprises the amino acid sequence: DKNIIKP (SEQ ID NO: 7).

[0061] In one embodiment, the peptide comprises the amino acid sequence: DKNIVRPA (SEQ ID NO: 8).

[0062] In one embodiment, the peptide comprises the amino acid sequence: GSYKYVEA (SEQ ID NO: 9).

[0063] In one embodiment, the peptide comprises the amino acid sequence: GIYKYVEA (SEQ ID NO: 10).

[0064] In some embodiments, the peptide is further functionalized by adding a functional group to the N' terminus, the C' terminus, or both of the peptide. In some embodiments, the functional group is an amino acid. In some embodiments, the functional group is selected from a cysteine ​​residue or a lysine residue.

[0065] The present invention includes derivatives of the peptides of the present invention. The term "derivative" or "chemical derivative" includes any chemical derivative of a peptide having one or more residues chemically derivatized by reaction of a side chain or functional group, as long as the derivatized peptide retains the peptide disclosed herein, i.e., Vg receptor (VgR) binding and subsequent induction of receptor-mediated endocytosis. Such derivatized molecules include, for example, those molecules in which the free amino group has been derivatized to form an amine hydrochloride group, p-toluenesulfonyl group, benzyloxycarbonyl group, tert-butyloxycarbonyl group, chloroacetyl group or formyl group. Free carboxyl groups can be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups can be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine can be derivatized to form N-im-benzylhistidine. Chemical derivatives also include those peptides containing one or more naturally occurring amino acid derivatives of the twenty standard amino acid residues. For example: 4-hydroxyproline can replace proline; 5-hydroxylysine can replace lysine; 3-methylhistidine can replace histidine; homoserine can replace serine; and ornithine (O) can replace lysine.

[0066] In addition, the peptide derivatives can differ from the native sequence of the peptide of the invention in chemical modifications, including but not limited to terminal-NH2 acylation, acetylation, methylation, phosphorylation, PEGylation or thioglycolic acid amidation; and in terminal carboxyl amidation, for example, by ammonia, methylamine, etc. The peptides can be linear, cyclic or branched, etc., with any conformation achievable using methods known in the art, as long as the derivatized peptide is able to bind to VgR and subsequently induce receptor-mediated endocytosis.

[0067] As used herein, the terms "peptide," "polypeptide," and "protein" are interchangeable and refer to a polymer of amino acid residues, such as the amino acid residue polymer disclosed in SEQ ID NO: 1, or a fragment thereof comprising or consisting of 7-23 amino acids derived from SEQ ID NO: 1 (e.g., SEQ ID NOs: 2-10).

[0068] As used herein, the term "amino acid" means an organic compound containing both a basic amino group and an acidic carboxyl group.

[0069] As used herein, the term "amino acid residue" refers to the portion of an amino acid present in a peptide.

[0070] The term "peptide bond" means a covalent amide linkage formed by the loss of a molecule of water between the carboxyl group of one amino acid and the amino group of a second amino acid.

[0071] As used herein, the terms "peptide" and "protein" encompass natural peptides, peptidomimetics (generally including non-peptide bonds or other synthetic modifications), and peptide analogs peptoids and hemi-peptoids, or any combination thereof. In another embodiment, the terms "peptide" and "protein" apply to amino acid polymers in which at least one amino acid residue is an artificial chemical analog of the corresponding naturally occurring amino acid.

[0072] Those skilled in the art will recognize that individual substitutions, deletions or additions to a peptide or protein sequence, which alter, add or delete a single amino acid or a small percentage of amino acids in the encoded sequence, are conservatively modified variants in which the change results in the substitution of an amino acid with similar charge, size and / or hydrophobicity characteristics, such as, for example, substitution of glutamic acid (E) for aspartic acid (D).

[0073] As used herein, the phrase "conservative substitution" also encompasses the use of a chemically derivatized residue in place of a non-derivatized residue, provided that such peptide exhibits the necessary functions as specified herein.

[0074] The peptide derivatives may also include side chain bond modifications, including but not limited to -CH2-NH-, -CH2-S-, -CH2-S=O, OC-NH-, -CH2-O-, -CH2-CH2-, S=C-NH- and -CH=CH-; as well as backbone modifications, such as modified peptide bonds. The peptide bonds (-CO-NH-) within the peptide can be replaced by, for example, N-methylated bonds (-N(CH3)-CO-); ester bonds (-C(R)HCOOC(R)HN); ketomethylene bonds (-CO-CH2-); α-aza bonds (-NH-N(R)-CO-), where R is any alkyl group, such as methyl; carba bonds (-CH2-NH-); hydroxyethylene bonds (-CH(OH)-CH2-); thioamide bonds (-CS-NH); olefin double bonds (-CH=CH-); and peptide derivatives (-N(R)-CH2-CO-), where R is a "normal" side chain, naturally occurring on a carbon atom. These modifications can occur at one or more of the bonds along the peptide chain, or even at several (e.g., 2-3) at the same time.

[0075] Peptide synthesis

[0076] According to one embodiment, the peptides of the present invention can be synthesized or prepared by any method and / or technique known in the art for peptide synthesis.

[0077] According to another embodiment, the peptide can be synthesized by the solid phase peptide synthesis method of Merrifield (see J. Am. Chem. Soc, 85: 2149, 1964). According to another embodiment, the peptide of the present invention can be synthesized using standard solution methods known in the art (see, for example, Bodanszky, M., Principles of Peptide Synthesis, Springer-Verlag, 1984).

[0078] In general, the synthetic method comprises adding one or more amino acids or appropriately protected amino acids sequentially to a growing peptide chain bound to a suitable resin. Typically, the amino or carboxyl group of the first amino acid is protected by an appropriate blocking group. The protected or derivatized amino acid can then be attached to an inert solid support (resin), or used in a solution, adding the next amino acid with a complementary (amino or carboxyl) group that is appropriately protected in the sequence under conditions suitable for forming an amide connection. The blocking group is then removed from the newly added amino acid residue and the next amino acid (appropriately protected) is added, and the rest is deduced. After all desired amino acids are connected in an appropriate order, any remaining blocking group is removed sequentially or simultaneously, and if the peptide chain is synthesized by a solid phase method, the peptide chain is cut from the solid support to obtain final peptide.

[0079] In the solid phase peptide synthesis method, the α-amino group of the amino acid is protected by an acid or base sensitive group. Such a protecting group should have a property that is stable to the peptide connection forming conditions and is easily removed without destroying the growing peptide chain. Suitable protecting groups are tert-butyloxycarbonyl (BOC), benzyloxycarbonyl (Cbz), biphenyl isopropyloxycarbonyl, tert-pentyloxycarbonyl, isobornyloxycarbonyl, (α,α)-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfinyl, 2-cyano-tert-butyloxycarbonyl, 9-fluorenylmethoxycarbonyl (Fmoc), etc. In the solid phase peptide synthesis method, the C-terminal amino acid is attached to a suitable solid support. Suitable solid supports that can be used for the above synthesis are those materials that are inert to the reagents and reaction conditions of the stepwise condensation-deprotection reaction and are insoluble in the solvent medium used. Suitable solid supports are chloromethyl polystyrene-divinylbenzene polymers, hydroxymethyl-polystyrene-divinylbenzene polymers, etc. The coupling reaction is carried out in a solvent such as ethanol, acetonitrile, N,N-dimethylformamide (DMF), etc. The coupling of consecutive protected amino acids can be carried out in an automatic peptide synthesizer known in the art.

[0080] In another embodiment, peptide of the present invention can be synthesized so that one or more of the bonds connecting the amino acid residues of the peptide are non-peptide bonds. In another embodiment, non-peptide bonds include but are not limited to imino, ester, hydrazide, semicarbazide and azo bonds, which can be formed by reactions well known to those skilled in the art.

[0081] The present invention further includes polynucleotide sequences comprising nucleic acids encoding any peptide of the present invention. In another embodiment, the nucleic acid sequence encoding the peptide has at least 70%, or alternatively at least 80%, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 99% or any value and scope homology therebetween with the nucleic acid sequence encoding the peptide of the present invention or its derivative. Each possibility represents a separate embodiment of the present invention.

[0082] In some embodiments, the present invention provides a polynucleotide encoding a peptide of the present invention. In some embodiments, the present invention provides a polynucleotide encoding a chimera of the present invention.

[0083] In some embodiments, the polynucleotide molecule encodes a peptide comprising atypical amino acids.

[0084] In some embodiments, polynucleotides of the present invention are connected to expression vectors, which include transcriptional control of cis-regulatory sequences (e.g., promoter sequences). In some embodiments, cis-regulatory sequences are suitable for guiding the constitutive expression of peptides of the present invention. In some embodiments, cis-regulatory sequences are suitable for guiding the tissue-specific expression of peptides of the present invention. In some embodiments, cis-regulatory sequences are suitable for guiding the inducible expression of peptides of the present invention.

[0085] The term "polynucleotide" refers to a nucleic acid (e.g., DNA or RNA) sequence comprising a coding sequence required for producing a peptide. In one embodiment, a polynucleotide refers to a single-stranded or double-stranded nucleic acid sequence isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence, and / or a composite polynucleotide sequence (e.g., a combination of the above).

[0086] In one embodiment, "complementary polynucleotide sequence" refers to the sequence produced by reverse transcription of messenger RNA using reverse transcriptase or any other RNA-dependent DNA polymerase. In one embodiment, the sequence can be subsequently amplified in vivo or in vitro using a DNA polymerase.

[0087] In one embodiment, a "genomic polynucleotide sequence" refers to a sequence derived or isolated from a chromosome, and thus represents a contiguous portion of a chromosome.

[0088] In one embodiment, a "composite polynucleotide sequence" refers to a sequence that is at least partially complementary and at least partially genomic. In one embodiment, a composite sequence may include some exon sequences required to encode a peptide of the present invention, as well as some intron sequences intervening therebetween. In one embodiment, intron sequences may be from any source, including other genes, and may generally include conserved splicing signal sequences. In one embodiment, intron sequences include cis-acting expression regulatory elements.

[0089] In some embodiments, the polynucleotides of the present invention are prepared using PCR technology or any other method or procedure known to those of ordinary skill in the art.

[0090] In some embodiments, an expression vector comprising a polynucleotide encoding a peptide of the present invention or a chimera comprising the peptide is provided.

[0091] In one embodiment, the polynucleotide of the present invention is inserted into an expression vector (i.e., a nucleic acid construct) to enable the recombinant peptide to be expressed. In one embodiment, the expression vector includes additional sequences that render the vector suitable for replication and integration in prokaryotes. In one embodiment, the expression vector includes additional sequences that render the vector suitable for replication and integration in eukaryotes. In one embodiment, the expression vector includes a shuttle vector that renders the vector suitable for replication and integration in both prokaryotes and eukaryotes. In some embodiments, the cloning vector comprises a transcription and translation initiation sequence (e.g., a promoter, an enhancer) and a transcription and translation terminator (e.g., a polyadenylation signal).

[0092] In some embodiments, a cell is provided that comprises any of: a peptide of the present invention; a chimera comprising the peptide; a polynucleotide encoding the peptide of the present invention; and an expression vector comprising a polynucleotide encoding the peptide of the present invention.

[0093] In one embodiment, a variety of prokaryotic or eukaryotic cells can be used as host expression systems to express the peptides of the present invention. In some embodiments, these include, but are not limited to, microorganisms, such as bacteria transformed with recombinant phage DNA, plasmid DNA or cosmid DNA expression vectors containing peptide coding sequences; yeast transformed with recombinant yeast expression vectors containing peptide coding sequences; plant cell systems infected with recombinant viral expression vectors containing peptide coding sequences (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors containing peptide coding sequences, such as Ti plasmids.

[0094] In some embodiments, a non-bacterial expression system (eg, a mammalian expression system) is used to express the peptides of the invention. In one embodiment, an expression vector is used to express a polynucleotide of the invention in a mammalian cell.

[0095] In some embodiments, in bacterial systems, a variety of expression vectors can be advantageously selected according to the intended use of the expressed peptide. In one embodiment, a large amount of peptide is required. In one embodiment, a vector that is expected to direct the expression of a high-level protein product may be used as a fusion with a hydrophobic signal sequence that directs the expressed product to the periplasm or culture medium of the bacteria, where the protein product is easily purified. In one embodiment, a certain fusion protein is modified with a specific cleavage site to facilitate peptide recovery. In one embodiment, vectors suitable for such manipulation include, but are not limited to, the pET series of Escherichia coli (E. coli) expression vectors [Studier et al., Methods in Enzymol. 185: 60-89 (1990)].

[0096] In one embodiment, a yeast expression system is used. In one embodiment, a plurality of vectors containing constitutive or inducible promoters can be used in yeast, as disclosed in US Pat. No. 5,932,447. In another embodiment, a vector is used that promotes integration of foreign DNA sequences into yeast chromosomes.

[0097] In one embodiment, the expression vector may further include additional polynucleotide sequences that, for example, allow translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES).

[0098] In some embodiments, mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(±), pGL3, pZeoSV2(±), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81, available from Invitrogen, pCI available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV available from Strategene, pTRES available from Clontech, and derivatives thereof.

[0099] In some embodiments, expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses can be used. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papilloma virus include pBV-1MTHA, and vectors derived from Epstein Bar virus include pHEBO and p2O5. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and any other vectors that allow expression of proteins under the guidance of SV-40 early promoter, SV-40 late promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters that have been shown to be effectively expressed in eukaryotic cells.

[0100] In some embodiments, recombinant viral vectors providing advantages such as lateral infection and targeting specificity are used to express peptides of the present invention in vivo. In one embodiment, lateral infection is intrinsic in the life cycle of, for example, retroviruses, and is the process in which a single infected cell produces a plurality of progeny virions that bud and infect adjacent cells. In one embodiment, the result is that a large area is rapidly infected, wherein most of them are not initially infected by the original virus particles. In one embodiment, the viral vector produced can not spread laterally. In one embodiment, this feature can be useful if the desired purpose is to introduce a given gene only into the target cell of a local quantity.

[0101] Various methods can be used to introduce expression vectors into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986], and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. Additionally, positive-negative selection methods are described in US Pat. Nos. 5,464,764 and 5,487,992.

[0102] In one embodiment, a plant expression vector is used. In one embodiment, expression of the peptide coding sequence is driven by multiple promoters. In some embodiments, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al., Nature 310:511-514 (1984)], or the coat protein promoter of TMV [Takamatsu et al., EMBO J. 6:307-311 (1987)] are used. In another embodiment, a plant promoter is used, such as, for example, the small subunit of RUBISCO [Coruzzi et al., EMBO J. 3: 1671-1680 (1984); and Brogli et al., Science 224: 838-843 (1984)] or a heat shock promoter, such as soybean hsp17.5-E or hsp17.3-B [Gurley et al., Mol. Cell. Biol. 6: 559-565 (1986)]. In one embodiment, the construct is introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, and other techniques known to the skilled artisan. See, for example, Weissbach & Weissbach [Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463 (1988)]. Other expression systems known in the art, such as insect and mammalian host cell systems, may also be used with the present invention.

[0103] It will be appreciated that, in addition to the elements required for transcription and translation of the inserted coding sequence (encoding the peptide), the expression construct may also include sequences engineered to optimize the stability, production, purification, yield or activity of the expressed peptide.

[0104] In some embodiments, transformed cells are cultured under effective conditions that allow expression of a large amount of recombinant peptides. In some embodiments, effective culture conditions include, but are not limited to, effective culture medium, bioreactor, temperature, pH and oxygen conditions that allow protein production. In one embodiment, effective culture medium refers to any culture medium for cultivating cells to produce recombinant peptides of the present invention. In some embodiments, culture medium generally includes an aqueous solution with assimilable carbon, nitrogen and phosphate sources and suitable salts, minerals, metals and other nutrients such as vitamins. In some embodiments, cells can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter plates and culture dishes. In some embodiments, culture is carried out under temperature, pH and oxygen content that are suitable for recombinant cells. In some embodiments, culture conditions are within the professional knowledge of those of ordinary skill in the art.

[0105] In some embodiments, depending on the vector and host system used for production, the resulting peptide of the invention is retained within the recombinant cell, secreted into the fermentation medium, secreted into the space between two cell membranes, such as the periplasmic space in E. coli; or retained on the outer surface of the cell or virus membrane. In one embodiment, after a predetermined period of culture, the recovery of the recombinant peptide is affected.

[0106] In one embodiment, the phrase "recovering the recombinant peptide" as used herein refers to collecting the entire fermentation medium containing the peptide and does not necessarily imply additional separation or purification steps.

[0107] In one embodiment, the peptides of the invention are purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing, and differential solubilization.

[0108] In one embodiment, to facilitate recovery, the expressed coding sequence can be modified to encode the peptide of the invention and the fused cleavable portion. In one embodiment, the fusion protein can be designed so that the peptide can be easily separated by affinity chromatography; for example, by being fixed on a column specific for the cleavable portion. In one embodiment, the cleavage site is engineered between the peptide and the cleavable portion, and the peptide can be released from the chromatographic column by treatment with an appropriate enzyme or agent that specifically cleaves the fusion protein at this site [e.g., see Booth et al., Immunol. Lett. 19: 65-70 (1988); and Gardella et al., J. Biol. Chem. 265: 15854-15859 (1990)].

[0109] In one embodiment, the peptides of the invention are recovered in a "substantially pure" form that permits effective use of the protein in the applications described herein.

[0110] As used herein, the term "substantially pure" describes a peptide or other material that has been separated from its natural contaminants. Generally, a monomeric peptide is substantially pure when at least about 60% to 75% of the sample presents a single peptide backbone. Minor variants or chemical modifications generally share the same peptide sequence. Substantially pure peptides can account for more than about 85% to 90% of the peptide sample, and can be more than 95% pure, more than 97% pure, or more than about 99% pure, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention. Purity can be measured on a polyacrylamide gel, with homogeneity determined by staining. Alternatively, for some purposes, high resolution may be necessary, and HPLC or similar purification methods may be utilized. For most purposes, a simple chromatographic column or polyacrylamide gel may be utilized to determine purity.

[0111] The term "purified" does not require that the material be present in an absolutely pure form that does not include the presence of other compounds. Rather, it is a relative definition. A peptide is in a "purified" state after the starting material or natural material has been purified by at least one order of magnitude, 2 or 3 or 4 or 5 orders of magnitude.

[0112] In one embodiment, the peptides of the invention are substantially free of naturally associated host cell components. The term "substantially free of naturally associated host cell components" describes a peptide or other substance that is separated from natural contaminants associated with its natural host cell state. Thus, a peptide that is chemically synthesized or synthesized in a cell system different from its natural source host cell will be free of its naturally associated host cell components.

[0113] In one embodiment, the peptides of the present invention can also be synthesized using an in vitro expression system. In one embodiment, in vitro synthesis methods are well known in the art, and the components of the system are commercially available. Non-limiting examples of in vitro systems include, but are not limited to, in vitro translation, as exemplified below.

[0114] chimera

[0115] As used herein, the term "chimera" includes any conjugate comprising two or more parts, wherein the two or more parts are directly or indirectly bound to each other, and wherein the parts are derived from different sources or are not naturally bound to each other. In some embodiments, the two or more parts have: different functions, are derived from or derived from different genes, peptides, genomic regions or species, different chemical classifications (e.g., peptides and polynucleotides, as exemplified herein).

[0116] In some embodiments, the chimera of the present invention comprises a first peptide, wherein the first peptide comprises a peptide of the present invention directly or indirectly bound to an agent, wherein the agent is selected from: nucleotides, oligonucleotides, polynucleotides, amino acids, peptides, peptides, proteins, small molecules, synthetic molecules, organic molecules, inorganic molecules, polymers, synthetic polymers or any combination thereof.

[0117] In some embodiments, the chimera of the present invention comprises a first peptide comprising an amino acid sequence as shown in SEQ ID NO: 1. In some embodiments, the chimera of the present invention comprises a first peptide comprising an amino acid sequence as shown in SEQ ID NO: 2. In some embodiments, the chimera of the present invention comprises a first peptide comprising an amino acid sequence as shown in SEQ ID NO: 3. In some embodiments, the chimera of the present invention comprises a first peptide comprising an amino acid sequence as shown in SEQ ID NO: 4. In some embodiments, the chimera of the present invention comprises a first peptide comprising an amino acid sequence as shown in SEQ ID NO: 5.

[0118] In some embodiments, the chimera of the present invention comprises a first peptide comprising an amino acid sequence as shown in any one of SEQ ID No.: 1-5, provided that it is a full-length vitellogenin protein. In some embodiments, the chimera of the present invention comprises a first peptide comprising an amino acid sequence as shown in any one of SEQ ID No.: 1-5, wherein the first peptide is not a full-length vitellogenin protein.

[0119] As used herein, the term "full length" refers to the native protein being the translation product of the vitellogenin encoding gene (e.g., from the start codon, encoding methionine to the stop codon). In some embodiments, the full-length protein comprises a native, linear, folded, unfolded, misfolded, denatured form of vitellogenin protein, or any combination thereof.

[0120] As used herein, the term "vitelinogen" refers to a precursor protein of yolk protein (e.g., yolk phosvitin). In some embodiments, the vitellogenin is an arthropod vitellogenin. In some embodiments, the vitellogenin is a crustacean vitellogenin. In some embodiments, the vitellogenin is a vitellogenin of a crustacean of the genus Macrobrachium. In some embodiments, the vitellogenin is of a crustacean of the family Penaeidae.

[0121] In some embodiments, the full-length vitellogenin is full-length vitellogenin from Macrobrachium rosenbergii (Accession No. BAB69831.1).

[0122] As used herein, the term "directly" refers to the situation where the peptide of the present invention is bound to the agent by a covalent bond.

[0123] As used herein, the term "indirectly" refers to a situation in which the peptide and agent of the invention are each bound to a linker or spacer element and are not directly bound to each other. In some embodiments, the peptide is covalently bound to the linker. In some embodiments, the agent is covalently or non-covalently bound to the linker.

[0124] As used herein, the term "covalent bond" refers to any bond that includes or involves the sharing of electrons. Non-limiting examples of covalent bonds include, but are not limited to, peptide bonds, glycosidic bonds, ester bonds, phosphodiester bonds.

[0125] As used herein, the term "non-covalent bond" includes any bond or interaction between two or more moieties that does not contain or involve electron sharing. Non-limiting examples of non-covalent bonds or interactions include, but are not limited to, electrostatics, π effects, van der Waals forces, hydrogen bonds, and hydrophobic effects.

[0126] The term "linker" refers to a molecule or macromolecule used to connect the different parts of the chimera (i.e., the peptide and agent of the present invention). In one embodiment, the linker can also promote other functions, including but not limited to maintaining biological activity, maintaining subunit and domain interactions, etc.

[0127] In another embodiment, the connector can be a monomeric entity, such as a single amino acid. In another embodiment, amino acids with small side chains, or peptide chains, or polymer entities of several amino acids are particularly preferred. In another embodiment, the peptide connector is 2 to 30 amino acids long, 2 to 25 amino acids long, 4 to 23 amino acids long, 4 to 20 amino acids long, 5 to 22 amino acids long, or 2 to 28 amino acids long. Each possibility represents a separate embodiment of the present invention. In another embodiment, the peptide connector is at least 6 amino acids long, at least 8 amino acids long, at least 10 amino acids long, at least 12 amino acids long, at least 15 amino acids long, at least 17 amino acids long, at least 20 amino acids long, at least 22 amino acids long, at least 25 amino acids long, at least 27 amino acids long, or at least 30 amino acids long, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention. In one embodiment, the connector can be a nucleic acid encoding a small peptide chain. In another embodiment, the connector encodes a peptide connector of 6 to 30 amino acids long, 6 to 25 amino acids long, 7 to 23 amino acids long, 8 to 20 amino acids long, 10 to 22 amino acids long, or 12 to 28 amino acids long. Each possibility represents a separate embodiment of the present invention. In another embodiment, the connector encodes a peptide connector of at least 6 amino acids long, at least 8 amino acids long, at least 10 amino acids long, at least 12 amino acids long, at least 15 amino acids long, at least 17 amino acids long, at least 20 amino acids long, at least 22 amino acids long, at least 25 amino acids long, at least 27 amino acids long, or at least 30 amino acids long, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention.

[0128] In some embodiments, the peptides and peptide connectors of the present invention are transcribed from a single polynucleotide sequence. In some embodiments, the peptides and peptide connectors of the present invention are transcribed from a single polynucleotide sequence, thereby providing a chimera of the present invention. In some embodiments, the peptides and peptide connectors of the present invention are located within a single peptide chain. In some embodiments, the peptides and peptide connectors of the present invention are adjacent to each other in a manner where the last amino acid at the C' terminus of the peptide of the present invention is bound to the first amino acid at the N' terminus of the peptide connector by a peptide bond. In some embodiments, the peptides and peptide connectors of the present invention are adjacent to each other in a manner where the first amino acid at the N' terminus of the peptide of the present invention is bound to the last amino acid at the C' terminus of the peptide connector by a peptide bond.

[0129] In some embodiments, the peptides of the present invention can be attached or connected to an agent via a chemical linker. Chemical linkers are well known in the art and include, but are not limited to, dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), N-isobutoxy-carbonyl-2-isobutoxy-1,2-dihydroquinoline (IIDQ).

[0130] Recombinant techniques can be used to express the peptides of the present invention and are well known in the art. In another embodiment, the linker can be a cleavable linker, resulting in the peptide of the present invention being cleaved after being delivered to a selected tissue or cell. In such an embodiment, the cell or tissue will have an endogenous enzyme (a naturally occurring enzyme or recombinantly engineered to express an enzyme) that can cleave the cleavable linker or an exogenous enzyme (e.g., by injection, absorption, etc.) that can cleave the cleavable linker.

[0131] In another embodiment, the linker can be biodegradable, so that the peptide of the present invention is further processed by intracellular hydrolysis and / or enzymatic cleavage. In some embodiments, the cleavable group includes acetyl, trimethylacetyl, butyryl, methylsuccinyl, tert-butylsuccinyl, ethoxycarbonyl, methoxycarbonyl, benzoyl, 3-aminocyclohexylidenyl, etc.

[0132] In some embodiments, the peptide linker has a charge at a pH value in the range of 6.5 to 8.

[0133] In some embodiments, the linker has a positive charge. In some embodiments, the linker has a negative charge.

[0134] In one embodiment, the peptide linker comprises 2-30 amino acid residues selected from the group consisting of lysine, arginine, histidine, aspartic acid, glutamic acid, and any combination thereof.

[0135] In one embodiment, the peptide linker comprises a polynucleotide binding region or domain, wherein the polynucleotide is selected from DNA, RNA, or a hybrid thereof.

[0136] In some embodiments, the chimera comprising a peptide linker and a polynucleotide agent comprises a peptide linker comprising 4 to 8, 4 to 12, 6 to 14, 8 to 16, 8 to 20, 10 to 24, 6 to 28, or 2 to 30 amino acids, or any value and range therebetween, and a polynucleotide agent comprising 200 to 500, 150 to 750, 250 to 650, 500 to 700, 450 to 550, 250 to 475, 350 to 650, or 200 to 700 base pairs, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0137] In some embodiments, the chimera comprises a polynucleotide agent and a peptide linker in a weight / weight ratio ranging from 1:1 (w / w) to 1:50 (w / w). In some embodiments, 1:1 (w / w) to 1:50 (w / w) includes 1:1 (w / w) to 1:2 (w / w), 1:1 (w / w) to 1:5 (w / w), 1:1 (w / w) to 1:15 (w / w), 1:1 (w / w) to 1:20 (w / w), 1:1 (w / w) to 1:25 (w / w), 1:1 (w / w) to 1:30 (w / w), 1:1 (w / w) to 1:35 (w / w), 1:1 (w / w) to 1:40 (w / w), or 1:1 (w / w) to 1:45 (w / w). Each possibility represents a separate embodiment of the invention.

[0138] In some embodiments, chimera has the following size: at most 50nm, at most 75nm, at most 100nm, at most 150nm, at most 200nm, at most 250nm, at most 350nm, at most 450nm or at most 500nm or any value and scope therebetween. Each possibility represents a separate embodiment of the present invention. In some embodiments, chimera has a size of 50 to 550nm, 100 to 500nm, 150 to 550nm, 125 to 375nm, 50 to 300nm, 225 to 435nm, 200 to 400nm, 175 to 385nm, 215 to 305nm, or 250 to 325nm. Each possibility represents a separate embodiment of the present invention.

[0139] In one embodiment, the chimera size is the diameter or radius of the chimera. In one embodiment, the structure or shape of the chimera is particle-related, particle-equivalent, or represented by particles, such as round or spherical particles. In one embodiment, the chimera size represents the average size of a plurality of chimeras. In one embodiment, the chimera size is the average diameter or average radius of a plurality of chimeras. As used herein, the terms "chimera size" and "particle size" are interchangeable.

[0140] In some embodiments, the chimera comprises a peptide connector comprising an amino acid sequence KHKHKHKHKHKHKHKHKHKH (SEQ ID NO: 11). In some embodiments, the chimera comprises a peptide connector comprising an amino acid sequence KHKHKHKHKHKHKHKH (SEQ ID NO: 12). In some embodiments, the chimera comprises a peptide connector comprising an amino acid sequence KHKHKHKHKHKHKH (SEQ ID NO: 13). In some embodiments, the chimera comprises a peptide connector comprising an amino acid sequence KHKHKHKHKHKH (SEQ ID NO: 14). In some embodiments, the chimera comprises a peptide connector comprising an amino acid sequence KHKHKHKHKH (SEQ ID NO: 15). In some embodiments, the chimera comprises a peptide connector comprising an amino acid sequence KHKHKHKH (SEQ ID NO: 16). In some embodiments, the chimera comprises a peptide connector comprising an amino acid sequence KHKHKH (SEQ ID NO: 17). In some embodiments, the chimera comprises a peptide connector comprising the amino acid sequence KHKH (SEQ ID NO: 18). In some embodiments, the chimera comprises a peptide connector comprising the amino acid sequence KH. In some embodiments, the chimera comprises a peptide connector comprising the amino acid sequence KKKKKKKKK (SEQ ID NO: 19).

[0141] In some embodiments, the chimera comprises a peptide linker comprising the amino acid sequence PVSLLQELCMRRGISPKYDLLQIEGAVHEPTFVYRVTVGEFAANGSGQSKKKAKHAAAKAVLDIII (SEQ ID NO: 20).

[0142] In some embodiments, the agent is selected from the group consisting of: a polynucleotide, a peptide, and a small molecule.

[0143] As used herein, the term "agent" or "embryo modifier" encompasses any compound that can change the activity, functionality, survival, fitness, appearance, structure, development, behavior, or any combination thereof of an embryo, a juvenile developed therefrom, or a post-larvae developed therefrom. In some embodiments, the agent is in an endogenous or exogenous molecule or compound. As used herein, the term "endogenous" refers to the fact that the compound is naturally produced in or by the contacted cell. As used herein, the term "exogenous" refers to the fact that the compound is not naturally produced in or by the contacted cell. In some embodiments, the exogenous agent is synthetically produced. In some embodiments, the exogenous agent is derived from or obtained from any source or species other than the source or species from which the contacted cell is derived.

[0144] In some embodiments, the agent is a dye. In one embodiment, the dye is a fluorophore. In one embodiment, the dye is a quantum dot, a semiconductor particle of a few nanometers (generally 5 to 50 nanometers) in size that has optical and electronic properties.

[0145] In some embodiments, modification includes changing the genomic DNA sequence. In some embodiments, changing the genomic DNA sequence includes introducing: point mutation, insertion, deletion, inversion, recombination, gap, double-strand break or any combination. In some embodiments, changing the genomic DNA sequence includes introducing exogenous DNA (i.e., transgenic). In some embodiments, modification includes changing the expression spectrum of the gene. In some embodiments, changing the expression spectrum of the gene is at the mRNA level, protein level or both. In some embodiments, modification is silencing (or knocking down) the expression of the gene. In some embodiments, modification is to overexpress (or upregulate) the expression of the gene. In some embodiments, the gene expression changed at the mRNA level includes: induction of mRNA degradation, increased mRNA instability or reduced mRNA stability, reduced mRNA transcription rate, reduced mRNA level, reduced mRNA-ribosome interaction or recognition or any combination thereof. In some embodiments, the gene expression changed at the protein level includes: reduced protein level, reduced protein translation rate, suppressed or blocked protein translation or any combination thereof.

[0146] In some embodiments, the agent as a polynucleotide is selected from: single-stranded RNA, antisense RNA, siRNA, dsRNA, shRNA, guide RNA, microRNA (miRNA) and DNA. As used herein, DNA refers to any deoxyribonucleic acid polymer, such as complementary DNA (cDNA), digested cDNA or genomic DNA (gDNA), plasmid DNA, etc. In some embodiments, the agent is any RNA interference (RNAi) inducing polynucleotide.

[0147] Antisense sequences as described herein include any of the following: antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, siRNA compounds, single-stranded or double-stranded RNA interference (RNAi) compounds such as siRNA compounds, modified bases / locked nucleic acids (LNA), antagomirs, peptide nucleic acids (PNA) or any other oligomeric compounds or oligonucleotide mimetics that can hybridize with at least a portion of a target nucleic acid such as a gene or its transcript and modulate its function. In some embodiments, antisense sequences include antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides comprising modified linkages, microinterfering RNA (miRNA) and short hairpin RNA (shRNA).

[0148] As used herein, the term "interfering RNA" refers to any double-stranded or single-stranded RNA sequence that is capable of inhibiting or down-regulating gene expression by mediating RNA interference, either directly or indirectly (i.e., after transformation). Interfering RNAs include, but are not limited to, siRNAs and shRNAs. RNAi refers to the selective degradation of sequence-compatible messenger RNA transcripts.

[0149] As used herein, the term "shRNA" refers to an RNA molecule comprising an antisense region, a loop portion, and a sense region, wherein the sense region has complementary nucleotides that base-pair with the antisense region to form a duplex stem. After post-transcriptional processing, the small hairpin RNA is converted into a small interfering RNA by a cleavage event mediated by Dicer, a member of the RNase III family.

[0150] As used herein, the term siRNA refers to any small RNA molecule capable of inhibiting or down-regulating gene expression by mediating RNA interference in a sequence-specific manner.The small RNA can be, for example, about 18 to 21 nucleotides long.

[0151] As used herein, the term dsRNA refers to any double-stranded RNA molecule that can inhibit or down-regulate gene expression by mediating RNA interference in a sequence-specific manner. The dsRNA can be, for example, about 50 to 1,000 nucleotides long, about 50 to 500 nucleotides long, about 150 to 750 nucleotides long, or about 100 to 500 nucleotides long, or any value and scope therebetween. Each possibility represents a separate embodiment of the present invention.

[0152] In some embodiments, the agent is a peptide. In some embodiments, the agent as a peptide is an enzyme. In some embodiments, the agent as a peptide is an apoptosis-inducing agent. The term "apoptosis-inducing agent" includes any molecule or compound that can induce or promote programmed cell death, or any molecule or compound involved in the process of programmed cell death.

[0153] As used herein, the term "enzyme" includes any peptide that is capable of specifically catalyzing a reaction (i.e., an enzymatic process). As used herein, an "enzymatic process" is any process catalyzed or performed by an enzyme, such as nucleic acid molecule (s) ligation, reverse transcription, amplification, digestion, dephosphorylation, etc. The results of an enzymatic process include desired products and by-products.

[0154] In some embodiments, the agent as an enzyme is a DNA binding protein. In some embodiments, the DNA binding protein comprises a clustered regularly spaced short palindromic repeat-associated protein 9 system (CRISPR / Cas9). In some embodiments, the agent according to the present invention comprises a Cas9 protein.

[0155]

[0156] According to some embodiments, an agent as an enzyme, such as Cas9, unwinds the DNA duplex and searches for a sequence that matches the crRNA to cut. Target recognition occurs after detecting the complementarity between the "protospacer" sequence in the target DNA and the spacer sequence left in the crRNA. Importantly, Cas9 will only cut DNA when the correct protospacer adjacent motif (PAM) is also present at the 3' end. According to certain embodiments, different protospacer adjacent motifs can be used. For example, the S.pyogenes system requires an NGG sequence, where N can be any nucleotide. The S.thermophilus type II system requires NGGNG (Horvath and Barrangou, 2010) and NNAGAAW (Deveau, Barrangou et al. 2008). Bioinformatics analysis has generated a wide database of CRISPR loci in various bacteria, which can be used to identify other useful PAMs and expand the collection of CRISPR targetable objects.

[0157] The term "single guide RNA" (sgRNA) is a 20 bp RNA molecule that can form a complex with Cas9 and act as a DNA recognition module. sgRNA is generally designed as a synthetic fusion of CRISPR RNA (crRNA) and a trans-activating crRNA.

[0158] In some embodiments, the chimera comprises a peptide of the invention, a Cas9 protein, a TRACER (ie, a trans-activating crRNA), and an sgRNA.

[0159] In some embodiments, chimera comprises multiple chimeras. As used herein, multiple chimeras comprise at least 2 chimeras, at least 3 chimeras, at least 4 chimeras, or at least 5 chimeras, or any range therebetween. Each possibility represents a separate embodiment of the present invention. In some embodiments, multiple chimeras comprise 2 to 3, 2 to 4, 2 to 5, 3 to 4, 3 to 5, or 4 to 5 chimeras. Each possibility represents a separate embodiment of the present invention.

[0160] In some embodiments, a plurality of chimeras include a first chimera, the first chimera including a fusion peptide, the fusion peptide including a peptide of the invention, a Cas9 protein; and a second chimera, the second chimera including a peptide of the invention, a TRACER, and an sgRNA. In some embodiments, among the plurality of chimeras, at least one chimera includes a TRACER. In some embodiments, among the plurality of chimeras, no more than one chimera includes a TRACER.

[0161] Those skilled in the art will appreciate that any Cas9 known in the art can be used in the chimeras and methods described herein. Cas9 (e.g., SaCas9 as described below) can be used as a platform for DNA transcription regulators to activate or inhibit gene expression by fusing an inactive enzyme to a known regulatory domain. For example, binding of a single dCas9 to a target sequence in genomic DNA can interfere with gene transcription.

[0162] There are multiple publicly available tools that aid in the selection and / or design of target sequences and lists of bioinformatically determined unique gRNAs for different genes in different species, including but not limited to Target Finder (e.g., E-CRISP), RGEN Tools: Cas-OFFinder, CasFinder: A Flexible Algorithm for Identifying Specific Cas9 Targets in the Genome, and CRISPROptimal Target Finder.

[0163] According to some embodiments, the method of the present invention utilizes inactivated (dead) Cas9 (dCas9). As used herein, the term "dCas9" refers to a Cas9 nuclease failure variant that is altered or otherwise modified to inactivate nuclease activity. This change or modification includes changing one or more amino acids to inactivate nuclease activity or nuclease domain. This modification includes removing one or more peptide sequences that present nuclease activity, i.e., nuclease domain, so that one or more peptide sequences (i.e., nuclease domain) that present nuclease activity are not present in the DNA binding protein. Based on the present disclosure, other modifications that inactivate nuclease activity will be apparent to those skilled in the art. Therefore, the nuclease failure DNA binding protein includes a peptide sequence that is modified to inactivate nuclease activity or removes a peptide sequence (one or more) to inactivate nuclease activity. Even if the nuclease activity has been inactivated, the nuclease failure DNA binding protein still retains the ability to bind to DNA. Therefore, the DNA binding protein includes one or more peptide sequences required for DNA binding, but may lack one or more or all of the nuclease sequences that present nuclease activity. Thus, a DNA binding protein includes one or more peptide sequences required for DNA binding, but may have one or more or all of the nuclease sequences that render the nuclease activity inactive.

[0164] In some embodiments, the complementarity of a polynucleotide (such as an antisense polynucleotide disclosed herein, such as an sgRNA or dsRNA) to a target nucleotide (such as a gene or its transcript) is at least 75%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% complementary, or any range and value therebetween. Each possibility represents a separate embodiment of the present invention. In some embodiments, the complementarity of a polynucleotide (such as an antisense polynucleotide disclosed herein, such as an sgRNA or dsRNA) to a target nucleotide (such as a gene or its transcript) is 70-85%, 80-90%, 92-97%, 95-99% or 97-100%. Each possibility represents a separate embodiment of the present invention.

[0165] In some embodiments, the method comprises mixing: (a) a chimera of the present invention comprising a peptide of the present invention and a Cas9 protein, and (b) a guide RNA configured to hybridize with a target gene; and contacting a cell with an effective amount of the resulting mixture.

[0166] In some embodiments, the method comprises mixing: (a) a chimera of the present invention comprising a peptide of the present invention and a Cas9 protein, and (b) a guide RNA configured to hybridize with a target gene; and administering an effective amount of the resulting mixture to an early vitellogenic female.

[0167] In some embodiments, the method comprises co-administering to early vitellogenic females an effective amount of any of the following: (a) a chimera of the present invention comprising a peptide of the present invention and a Cas9 protein, and (b) a guide RNA configured to hybridize with a target gene.

[0168] In some embodiments, co-administration includes simultaneous administration. In some embodiments, co-administration includes, not at the same time or injection event, administering each of the following: (a) a chimera of the present invention comprising a peptide of the present invention and a Cas9 protein, and (b) a guide RNA configured to hybridize with a target gene. In some embodiments, co-administration includes, at least 5 seconds, at least 1 minute, at least 3 minutes, at least 5 minutes, at least 7 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 1 hour, at least 6 hours, at least 12 hours or at least 1 day or any value and range therebetween, administering a chimera of the present invention comprising a peptide of the present invention and a Cas9 protein and a guide RNA configured to hybridize with a target gene. Each possibility represents a separate embodiment of the present invention.

[0169] In some embodiments, co-administration includes administering a chimera of the invention comprising a peptide of the invention and a Cas9 protein at least 5 seconds, at least 1 minute, at least 3 minutes, at least 5 minutes, at least 7 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 1 hour, at least 6 hours, at least 12 hours, or at least 1 day after administering a guide RNA configured to hybridize to a target gene, or vice versa, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0170] In some embodiments, the term "target gene" refers to any gene that is desired to be edited, such as a developmental gene, a gene that affects survival, or other gene.

[0171] In some embodiments, "configured to hybridize" refers to the guide RNA comprising a nucleic acid sequence that is at least partially complementary to the nucleic acid sequence of the target gene and is thereby capable of at least partially hybridizing thereto.

[0172] Composition

[0173] In some embodiments, a composition is provided, which comprises any of the following: a peptide of the present invention; a chimera comprising the peptide of the present invention; a polynucleotide encoding the peptide of the present invention; an expression vector comprising the polynucleotide; a host cell comprising any of the above; and a carrier.

[0174] In some embodiments, a composition is provided comprising two or more peptides derived from SEQ ID NO: 1, wherein the two or more peptides have up to 99% sequence identity. In some embodiments, the amino acid sequences of the at least two peptides partially overlap or do not overlap.

[0175] In some embodiments, the compositions disclosed herein are used to deliver an agent to a cell. In some embodiments, the compositions disclosed herein are used to modify a cell.

[0176] method

[0177] In some embodiments, a method for preparing a chimera disclosed herein is provided, comprising the step of combining a peptide of the present invention with an agent selected from the group consisting of a polynucleotide, a second peptide, a small molecule, or any combination thereof. In some embodiments, the method for preparing a chimera comprises generating a peptide of the present invention. In some embodiments, generating a peptide of the present invention comprises expressing a peptide of the present invention from a polynucleotide encoding the peptide or from an expression vector comprising the polynucleotide, wherein the expression is in a living cell, an artificial cell, or a cell-free system. In some embodiments, the protein is produced synthetically, such as by a solid-state method. In some embodiments, the peptide of the present invention is combined with an agent to provide a chimera, including combining by polymerization. The term "combining by polymerization" refers to the case where the agent is a peptide, and the peptide of the present invention and the agent are transcribed from a single encoding polynucleotide as a single peptide. In some embodiments, the peptide of the present invention is combined with an agent to provide a chimera, including combining by connection. The term "combining by connection" refers to the case where each of the peptide of the present invention and the agent is first generated (i.e., provided) separately, and then connected or combined to each other to provide a chimera. In some embodiments, the connection can be performed enzymatically or chemically. In some embodiments, the linkage of a peptide of the invention to an agent is direct or indirect, as disclosed above.

[0178] In some embodiments, methods are provided for delivering an agent into a cell comprising contacting the cell with a chimera disclosed herein.

[0179] In some embodiments, methods are provided for modifying a cell comprising contacting the cell with a chimera disclosed herein.

[0180] In some embodiments, the cell is an oviparous cell. As used herein, the term "oviparous" refers to any organism that lays eggs, in which most embryonic development occurs, rather than in the mother. Non-limiting examples of oviparous species include arthropods, molluscs, fish, amphibians, reptiles, birds, and monotremes. Non-limiting examples of arthropods include, for example, crustaceans, insects, arachnids, chelicerae, etc.

[0181] In some embodiments, the cell is an arthropod cell.

[0182] In some embodiments, the arthropod is a crustacean. In some embodiments, the crustacean is a decapod crustacean. Non-limiting examples of decapod crustaceans include, but are not limited to, prawns, shrimps, lobsters, crabs, and crayfishes.

[0183] In some embodiments, the cell is an ovarian cell. In some embodiments, the ovarian cell is an oocyte. In some embodiments, the oocyte is selected from: a primary oocyte, a secondary oocyte, a mature oocyte, a pre-vitellogenous oocyte, a partially vitellogenous oocyte, and a vitellogenous oocyte. In some embodiments, the ovarian cell is a follicular cell. In some embodiments, the ovarian cell is an ovarian epithelial cell and a germinal epithelial cell. In some embodiments, the ovarian cell is an oocyte trophoblast.

[0184] According to the method of the present invention, in some embodiments thereof, contacting the cell with the chimera of the present invention comprises any one of in vivo contact, in vitro contact or ex vivo contact.

[0185] In some embodiments, the method comprises administering an effective amount of a chimera of the invention to a previtellogenic female.

[0186] In some embodiments, the method further comprises the step of determining that the female is a previtellogenic female and thus suitable for administration as disclosed herein.

[0187] Methods of determining the state or stage of vitellogenesis are common and apparent to one of ordinary skill in the art. Non-limiting examples include, but are not limited to, quantification of circulating vitellogenin levels using an immunoassay (eg, enzyme-linked immunosorbent assay).

[0188] In some embodiments, administration is an injection. In some embodiments, administration is administration to the ovaries. In some embodiments, administration is an injection. In some embodiments, administration is administration to the hemolymph. In some embodiments, administration is administration to the ovaries and hemolymph. In some embodiments, administration is at least once a week, at least twice a week, or at least three times a week, or any range therebetween. Each possibility represents a separate embodiment of the present invention. In some embodiments, administration is once or twice a week, once to three times a week, or twice or three times a week. Each possibility represents a separate embodiment of the present invention.

[0189] As used herein, the term "hemolymph" refers to the circulatory system.

[0190] In some embodiments, the method further comprises the step of hybridizing the administered female with a male. In some embodiments, hybridization comprises mating with a male. In some embodiments, hybridization comprises in vitro fertilization or IVF. Methods of in vitro fertilization or IVF are common and obvious to those of ordinary skill in the art. Non-limiting examples include, but are not limited to, collecting sperm or sperm cells and cultivating or placing them on a recipient female respectively with the ovum produced.

[0191] In some embodiments, progeny obtained from a cross performed according to the methods of the present invention are provided.

[0192] As used herein, the term "subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject for whom treatment is desired, particularly a mammalian subject, such as a human.

[0193] In the discussion, unless otherwise stated, adjectives such as "substantially" and "about" that modify conditions or relational features of one or more features of embodiments of the present invention are understood to mean that the condition or feature is defined to be within an acceptable tolerance range for the operation of the embodiment for the intended application. Unless otherwise stated, the word "or" in the specification and claims is considered to be an inclusive "or", not an exclusive or, and means at least one or any combination of the items it connects.

[0194] It should be understood that the terms "a" and "an" as used above and elsewhere herein refer to "one or more" of the listed components. It will be clear to one of ordinary skill in the art that the use of the singular includes the plural unless otherwise expressly stated. Therefore, the terms "a", "an" and "at least one" are used interchangeably in this application.

[0195] In order to better understand the teachings herein and without limiting the scope of the teachings in any way, unless otherwise specified, all numbers indicating quantities, percentages or ratios and other numerical values ​​used in the specification and claims will be understood to be modified by the term "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the following specification and the attached claims are approximate values, which may vary depending on the desired properties to be obtained. At a minimum, each numerical parameter should at least be interpreted in light of the number of reported significant figures and by applying conventional rounding techniques.

[0196] In the specification and claims of this application, each of the verbs "comprise", "include" and "have" and their cognates is used to indicate that one or more objects of the verb are not necessarily a complete list of components, elements or parts of the one or more subjects of the verb.

[0197] Other terms as used herein are intended to be defined by their commonly understood meanings in the art.

[0198] Unless otherwise stated or obvious from the context, as used herein, the term "or" is to be understood as being inclusive.

[0199] Throughout the specification and claims, the word "comprise" or variations such as "comprising" or "including" means the inclusion of any stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0200] As used herein, the term "consisting essentially of," or variations such as "consisting essentially of," used throughout the specification and claims, means the inclusion of any recited integer or group of integers, and optionally the inclusion of any recited integer or group of integers that does not materially change the basic or novel property of a specified method, structure, or composition.

[0201] As used herein, the terms "comprising", "including", "containing", "having", etc. may mean "including", "comprising", etc.; "consisting mainly of" or "consisting mainly of" have the meanings specified in the US Patent Law and the term is open-ended, allowing for the existence of contents beyond the narrative, as long as the basic or novel features of the narrative are not changed by the existence of contents beyond the narrative, but excluding prior art implementations. In one embodiment, the terms "comprising", "including", "having" are interchangeable with "consisting of".

[0202] Other objects, advantages and novel features of the present invention will become apparent to those skilled in the art after examining the following embodiments, which are not intended to be limiting. In addition, each of the various embodiments and aspects of the present invention as described above and as claimed in the appended claims section can find experimental support in the following embodiments.

[0203] Example

[0204] In general, the nomenclature used herein and the laboratory procedures used in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are explained in detail in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM, ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds.) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); U.S. Patent Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, JE, ed. (1994); "Culture of Animal Cells-A Manual of Basic Technique", Freshney, Wiley-Liss, NY (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan JE, ed. (1994); Stites et al.(eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization-A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this article. .

[0205] Materials and methods

[0206] Custom Peptides

[0207] and

[0208] KHKHKHKHKHKHKHKHKHCQAPVKLIAYDKNKYEHEYRISVSGK-FITC (KHscVg; SEQ ID NO: 25) was purchased from Peptron (Yuseong-gu, Daejeon, South Korea). For in vitro tissue culture experiments, Schneider's Drosophila medium, fetal bovine serum (FBS) and antibiotic cocktail - penicillin, streptomycin and amphotericin B (PSA) at concentrations: penicillin G sodium salt: 10,000 units / mL, streptomycin sulfate: 10 mg / mL, amphotericin B: 25 μg / mL were purchased from Biological Industries (Beit HaEmek, Israel). Insulin and UTP-FITC were purchased from Sigma-Aldrich (St. Louis, Missouri).

[0209] animal

[0210] Female Macrobrachium rosenbergii were collected from the Aquaculture Station of the Ministry of Agriculture in Dor, Israel and were kept at the Ben-Gurion University facility (27°C, 12 hours of daylight, ad libitum access to food). Females used for in vivo experiments were kept in tanks (5 females per tank), each female being kept in an individual cage. Fertile males were released in the tanks.

[0211] Oocyte imaging

[0212] Ovarian sheets from in vitro or in vivo experiments were washed with Schneider's Drosophila medium without peptides for 5 minutes. The sheet was then torn apart in a drop of culture medium on a slide to better observe individual oocytes. The slides were examined and images were taken at × 60 magnification using a confocal microscope FV1000 (Olympus). The excitation laser wavelength for the TAMRA-labeled peptide was 561 nm, the excitation laser wavelength for the FITC-labeled control peptide was 488 nm, and emission (light) was obtained at 490-530 nm and 540-640 nm, respectively.

[0213] Synthesis of double-stranded RNA (dsRNA)

[0214] In vitro synthesis of dsRNA of green fluorescent protein (GFP), epidermal growth factor receptor (EGFR) and paired box protein 6 (PAX6). The pGEM-T Easy plasmid containing the gene ORE sequence serves as a template for dsRNA synthesis. The template is amplified by PCR, initiated by two gene-specific primers, and has a T7 promoter site at the 5' of one primer (T7P) (see the primers and T7 promoter sequences for dsRNA synthesis in Table 1). The primer pairs are as follows: the sense strand is synthesized using primer T7P forward vs reverse primer, and the antisense strand is synthesized by T7P reverse vs forward primer. The PCR amplicon is electrophoresed on a 1.3% agarose gel, visualized with SYBR Safe DNA Gel Stain (Invitrogen) and ultraviolet light, and purified with a PCR purification kit (NucleoSpin Gel and PCR Clean-up, Machery-Nagel). Single-stranded RNA was generated using the Transcript Aid T7 High Yield Transcription Kit (ThermoScientific, Lithuania) according to the manufacturer's instructions. RNA molecules were purified by phenol-chloroform (1:1) and sodium acetate and precipitated with ethanol. The sense and antisense strands were hybridized by incubation at 70°C for 15 minutes, at 65°C for 15 minutes, and at room temperature for 30 minutes. The dsRNA quality was assessed on an agarose gel and diluted to 5 mg / ml. The dsRNA was kept at -80°C until use. FITC-labeled GFP dsRNA (dsGFP) was synthesized by incorporating UTP-FITC (the ratio between UTP and UTP-FITS was 1:1) in the synthesis reaction as described above. RNA molecules were purified by the RNeasy Minelute clean-up kit (QIAGEN) and then hybridized and quality assessed as described above.

[0215] Table 1. Primers used for dsRNA synthesis and real-time RT-PCR

[0216]

[0217] Peptide-dsRNA conjugation

[0218] A constant amount of dsRNA (25 pmol) solution was mixed with increasing amounts of peptide solution in an Eppendorf vial (~1.5 mL). The total conjugation reaction volume was made up to 15 μl with DEPC-treated DDW. The solution was incubated at room temperature for 20 minutes. To evaluate peptide-dsRNA conjugation, a 1.3% agarose gel was prepared in TAE buffer. The conjugate was mixed with an equal volume of loading buffer (50% glycerol and 0.5 M EDTA). The peptide-dsRNA conjugate was separated at 120 V for 30 minutes. The gel was separated by using a Typhoon FLA 9500 gel system. The gel was scanned for fluorescence emission of the conjugate.

[0219] In vitro incubation of ovarian slices with peptides and peptide-dsRNA conjugates

[0220] Ovarian pieces of Macrobrachium rosenbergii (early vitellogenic ovaries) with oocyte diameters of 1-3 mm and ~150 mm were placed in a sterile 24-well plate (2 cm per well) in a biological hood. 2 Growth area ) were cultivated in 250 μl Schneider's Drosophila medium, in which the osmotic pressure was fixed to 420 mOsm / L and supplemented with FBS (10%), PSA (final concentrations: penicillin-100 units / mL, streptomycin-0.1 mg / mL, amphotericin B-0.25 μg / mL and insulin 10 mg / ml in 25 mM HEPES). The ovarian slices were cultivated with Vg-TAMRA or scVg-TAMRA or with both Vg-TAMRA and scVg-FITC at the same time (6 μM each). The plates were incubated at room temperature for 24 and 48 hours. To test dsRNA piggybacking, 55 pmol of dsRNA was conjugated to 275 pmol of Vg-TAMRA or scVg-FITC peptides. The ovarian slices were incubated and cultured as described above with dsGFP-Vg-TAMRA or dsGFP-scVg-FITC conjugates.

[0221] In vivo assay - injection of peptide or peptide-dsRNA into vitellogenic females

[0222] A solution containing culture medium, Vg-TAMRA and scVg-FITC peptide (5 μg / gr body weight) was injected into the hemolymph sinus at the base of the fifth walking leg of reproductive females of Macrobrachium rosenbergii (14.3 ± 0.4 gr, opaque white to yellowish ovaries, Figure 2 , left). For injection, a manual microdispenser syringe was used. After injection, animals were kept at 28°C for 24 h in the shrimp culture facility of Ben-Gurion University. Vg-dsEGFR or Vg-dsPAX6 conjugates were injected twice a week for the first two weeks (conjugation molar ratio of 5:1, 5 μg dsRNA / g body weight). From the third week onwards, injections were given once a week. Females were monitored daily and, after the occurrence of reproductive molts, were released into tanks with males for fertilization. Embryonic development on the ventral feet of their mothers was monitored under a stereoscope ( Figure 2 , middle), and fractions were collected for RNA extraction and subsequent quantitative PCR analysis.

[0223] Effects on embryonic eye development

[0224] To evaluate the ability to deliver functional dsRNA into developing embryos in vivo ( Figure 2 ), and injected the Vg-dsPAX6 conjugate into reproductive females of Macrobrachium rosenbergii. PAX6 of Macrobrachium rosenbergii is homologous to insect eye development genes, and we found that it is expressed during mid- and late-stage embryonic development in males and females in embryonic transcriptome libraries ( Figure 3 ). To record the effect of PAX6 silencing on embryonic eye development, egg samples containing embryos were collected from ovipositor females on day 9 and monitored under a stereoscope. Embryos were counted and classified into embryos with normal eyes, embryos with delayed eyes, and embryos with residual eye development or no eye development. In addition, the size of the eye pigment (length and width) was measured and the eye length / width index was calculated. The length / width index of ten colored eyes was measured under a microscope in both eyes of the experimental and control groups.

[0225] Quantitative PCR analysis

[0226] RNA was extracted from 9-day-old embryos. Total RNA was isolated using TRI RNA Isolation Reagent (Sigma-Aldrich, Israel) according to the manufacturer's instructions. First-strand cDNA was synthesized in a reverse transcriptase reaction using 1 μg of total RNA using the qScript cDNA Synthesis Kit (QuantaBiosciences, Gaithersburg, MD, USA) according to the manufacturer's instructions.

[0227] SensiFAST Probe Hi-ROX Mix (BIOLINE), specific primers and Universal Probe Library (Roche) (see Table 1) are used to realize the relative quantification of Mr-EGFR transcript levels. Mr-18S rRNA (GenBank accession number GQ131934)--serving as a standard gene--is also quantitatively analyzed by real-time RT-PCR using specific primers (see Table 1) with the above mixture and Universal Probe Library Probe 152 (Roche). The reaction is carried out using ABIPrism 7300 sequence detection system (Applied Biosystems, Foster City, California).

[0228] Example 1

[0229] Vg peptide can be internalized into oocytes

[0230] In order to evaluate the ability of oocytes to internalize the newly designed Vg-derived peptides, in vitro experiments were performed. Vg-derived peptides and control peptides labeled with TAMRA fluorophores were detected by confocal microscopy. Both peptides were detected near the oocyte membrane (Figure 4). Unlike the control peptide, the Vg peptide was mainly distributed on the cytoplasmic side of the oocyte membrane that formed the yolk droplet ( Figure 4A and 4B , black arrows), and mainly accumulated in the peripheral region near the membrane of the oocyte ( Figure 4B , white arrows), and scattered on the outer surface of the newly formed droplets. In contrast, the control peptide was mainly dispersed on the outer surface of the oocyte ( Figure 4C and 4D To obtain the highest certainty regarding the internalization specificity of the Vg-derived peptide, Vg and control peptides were labeled with an additional fluorophore ( Figure 4E-4H ), the Vg peptide was labeled with TAMRA ( Figure 4E and 4H ), while the control peptide was labeled with FITC molecule ( Figure 4F ). Similar results were obtained in this experiment, showing different distributions of the two fluorescently labeled peptides. The Vg-TAMRA peptide was clearly observed to be internalized into the oocyte and spread along the inner side of the membrane. In contrast, the control-FITC peptide could only be observed on the outer surface of the membrane ( Figure 4F ).

[0231] To determine whether the Vg-TAMRA peptide has the ability to be specifically internalized into oocytes in vivo, a mixture of Vg-TAMRA and control-FITC peptide was injected into the shrimp circulatory system. The control-FITC peptide (with an emission wavelength of 515 nm) was absent in oocytes dissected 24 hours after injection. Fig. 4IFor the Vg-TAMRA peptide, clear 564 nm emission was observed in oocytes ( Figure 4J and 4K ), mainly in the yolk droplets. To verify that the two peptides were indeed circulating in the female shrimp, the gills of the shrimp were dissected and examined under a confocal microscope. Clear dual (TAMRA and FITC) emission was observed ( Figure 4L ), indicating that most of the injected peptide was washed away through the osmotic pressure regulating system (i.e., gills).

[0232] Example 2

[0233] Vg peptide is internalized into oocytes in a dose-responsive manner

[0234] Further, a dose-dependent test of the penetration of Vg-derived peptides into oocytes using decreasing concentrations of Vg was performed ( FIG. 5 ). Unlike the control scrambled peptide (scVg), the Vg peptide maintained a similar distribution pattern in the oocyte throughout the decreasing concentrations ( FIG. 5 , upper row). Another difference between Vg and the control peptide is the fluorescence intensity. In the fluorescence and overlay images, the Vg peptide exhibited a higher intensity of red TAMRA emission ( FIG. 5 , upper two rows) compared to the control peptide image ( FIG. 5 , lower two rows). For Vg, clear fluorescence signals were detected at concentrations of 12 μM, 6 μM, and 1.5 μM, while fluorescence signals of the control peptide were detected around the oocyte, only weak fluorescence signals at high concentrations (12 μM and 6 μM), and no obvious signals were detected at 1.5 μM. No signals were detected for both peptides at 0.75 μM ( FIG. 5 ).

[0235] Example 3

[0236] Internalization of peptide-dsRNA conjugates into oocytes

[0237] In order to verify whether Vg-derived peptides can carry macromolecules (such as dsRNA) and internalize them into oocytes, a conjugation method between peptides and dsRNA was developed. Vg-TAMRA and control-FITC peptides were synthesized with cationic polylysine-histidine tails (KH) to allow electrostatic interactions between the peptide and negatively charged dsRNA. The dsRNA-peptide complexes prepared at different molar ratios (peptide / dsRNA ratio: 1, 5, 10, 20 and 30) were characterized by two different assays: dynamic light scattering (DLS) and separation on agarose gel.

[0238] A mixture of 700 bp dsRNA (dsEGFR) and another 200 and 230 bp dsRNA (dsPAX6) was prepared ( Figure 3A). Dynamic light scattering was performed by conjugation of peptides KH4-Vg and KH9-Vg to dsEGFR, while dsPAX6 was conjugated only to KH9-Vg. DLS results showed that conjugation of 700 bp dsRNA to KH4-Vg or KH9-Vg in a 1:1 ratio resulted in the formation of particles of almost similar size (400-500 μm, Figure 6B , squares and dots). However, at higher peptide ratios (1:30), the particles became smaller and the radius decreased to ∼100 nm ( Figure 6B When KH9-Vg was mixed with a short dsRNA form (dsPAX6), the particle radius was less than 100 μm at a dsPAX6:peptide ratio of 1:5 ( Figure 6B ,triangle).

[0239] A constant amount of dsRNA (25 pmol) obtained for the PAX6 gene (dsPAX6) was mixed with increasing amounts of peptide. A gradual hysteresis of the conjugate occurred in the agarose gel according to the increasing amount of peptide ( Figure 6C-6D The migration of individual cationic peptides toward the cathode is clearly visible in the gel fluorescence image ( Figure 6C At a dsPAX6:peptide ratio of 1:50, excess free peptide or large aggregates stagnating on or near the loading well were clearly observed ( Figure 6C-6D ). However, at a ratio of 1:5, almost no fluorescent signal was seen on or near the loading well. Therefore, to avoid any competition between the conjugated peptide and free peptide and to maximize the internalization of the conjugate into the oocyte, a dsPAX6:peptide ratio of 1:5 was selected for subsequent in vitro and in vivo experiments. dsPAX6-peptide conjugates at a 1:5 ratio were incubated with ovarian slices in vitro. Orange fluorescent spots can be seen on confocal images of oocytes incubated with dsPAX6-Vg ( Fig. 6E , yellow arrow), indicating that green fluorescent dsPAX6-FITC conjugated with red fluorescent Vg-TAMR can be internalized into oocytes. The internalization of dsPAX6-FITC is accompanied by the internalization of Vg-TAMRA ( Fig. 6E However, in oocytes of ovarian slices incubated with dsPAX6 conjugated to a control peptide ( Fig. 6F ) or in ovarian slices incubated with dsPAX6 alone ( Figure 6G )The green fluorescent spot cannot be seen.

[0240] Example 4

[0241] Silencing ability of dsRNA-peptide conjugates in embryos

[0242] At the time of spawning and on the 9th day of development, a portion of the incubated eggs ( Figure 2, middle) and embryonic eye development was recorded. Almost one fifth of the embryos removed from Vg-dsPAX6-injected females showed reduced or absent eye development ( Fig. 7A-I , 7A-II and 7A-III). In those with weakened eye embryos, the fine eye pigment line was abundant ( Fig.7A-III , grey arrows). Single-eyed embryos (cyclops) were also observed ( Figure 7A-II , black arrows). A complete lack of eye development or remnants of pigment was also observed ( Figure 7A-II , white arrows). The mean length / width index of eye pigment was found to be significantly higher in embryos from Vg-dsPAX6-treated females compared to embryos from scVg-dsPAX6-treated females ( Figure 7B ).

[0243] Example 5

[0244] Silencing ability of dsRNA-peptide conjugates in larvae

[0245] When Vg was conjugated to EGFR dsRNA (dsEGFR) and injected into yolk-laying females, the hatchlings of these females ( Figure 2 , left) Compared with pups from females injected with dsEGFR-control peptide or dsEGFR alone, pups expressed significantly lower levels of EGFR ( Figure 7C ). Of note, for dsEGFR quantitative PCR, mRNA was collected from juveniles. These juveniles hatched from eggs laid 21 days earlier, and the mothers received the dsEGFR-peptide conjugate a few days before laying eggs. This means that the injected dsEGFR was still present more than three weeks after the last injection and effectively induced gene silencing in the offspring.

[0246] Example 6

[0247] Optimization of dsRNA:peptide particle size

[0248] The inventors investigated the effects of dsRNA size (i.e., length), linker tail length, and dsRNA:peptide ratio on particle size. The linker used included a lysine-histidine repeat sequence (KH). Dynamic light scattering (DLS) was used to determine the resulting particle size (expressed in nanometers of particle radius). The results are shown below (Tables 2-3).

[0249] Table 2. Particle size of dsRNA:(KH)9-Vg peptide measured by DLS

[0250]

[0251] Table 3. Particle size of dsRNA:(KH)4-Vg peptide measured by DLS

[0252]

[0253] Example 7

[0254] Doubling injection events enhances dsRNA silencing effects

[0255] Females induced for vitellogenesis (details in Materials and Methods) were injected with KH9-Vg peptide conjugated to dsPAX6 or dsPAX6 alone at doses higher than in previous experiments (4 μg each dsPAX6A and B / gr body weight, peptide:dsPAX6 5:1 molar ratio). After spawning, embryos on the ventral pedicles of treated mothers were monitored for eye development. 60% of the eyes of embryos removed from females injected with Vg-dsPAX6 showed irregular development ( Figures 8A-I to 8A-III The average eye line length / width index of 10-day-old embryos taken from mothers injected with Vg-dsPAX6 was 5.6±0.37 ( Fig. 8A-III ), which was a significantly larger index ( Figure 8B Comparison of the eyeline length / width indices obtained from two separate experiments showed that the index in the previous experiment (4.03 ± 0.18) was significantly lower than the eye index obtained from the current experiment ( Figure 8C ). The latter showed that in vitellogenesis-induced females, a higher dose provided a greater silencing efficacy, thus significantly retarding eye development in the presence of PAX6 silencing.

[0256] Example 8

[0257] Long-term effects of PAX6 silencing delivered via Vg peptide

[0258] To test whether early stages of silencing of targeted developmental genes such as PAX6 affect later developmental stages, for example, stage 10 and 11 larvae (from higher dose injection experiments) were taken for 3D eye surface analysis by scanning electron microscopy (SEM). SEM analysis showed that the shape of the ommatids in the experimental larvae was deformed. While the control larvae presented well-shaped hexagonal ommatids, the ommatids of the experimental group larvae presented irregular, elongated, continuous or elevated hinges between the ommatids (Figures 9B-9C).

[0259] Example 9

[0260] Developing delivery tools for CRISPR-Cas9

[0261] As mentioned previously, dsRNA silencing with a Vg-dsRNA conjugate affects approximately 60% of embryos. dsRNA is used for short-term silencing, so the effect ceases shortly after dsRNA treatment. For long-term gene editing or knockout, a recombinant Vg peptide fused to Cas9 was designed. The complex contains His×6, a VgR interaction domain derived from an 84 amino acid sequence of vitellogenin, and a CAS9 sequence ( Fig. 10A ). 3D prediction of the hybrid protein showed that the Vg-derived peptides were arranged as random coils and β-sheet structures ( Fig. 10A ). The His-tagged Cas9-Vg construct was cloned into the pET28A plasmid and expressed in E. coli BL21. The expressed recombinant protein Cas9-Vg-His was purified on Ni-NTA, then passed through an ion exchange column, and the eluted fractions were separated on SDS-PAGE ( Fig. 10C A clear thick band of purified Cas9-Vg protein was observed in SDS-PAGE ( Fig. 10C ). Mass spectrometry (MS) analysis confirmed that the Coomassie blue-stained band observed at ~160 kDa was indeed Cas9 fused to Vg. The activity of Cas9 was tested. The Cas9-Vg complex showed DNA cleavage activity only when mixed with a specific gRNA ( Fig. 10B ). Incubation of Cas9-Vg with a complementary specific gRNA to the PAX6 560 bp cDNA resulted in the latter being cleaved into smaller fragments, e.g., 460 bp and 100 bp. Cas9-Vg without gRNA did not cleave DNA. In addition, the cleavage activity was similar to that of commercial Cas9 ( Fig. 10B ). Thereafter, Cas9-Vg was mixed with PAX6 gRNA and the mixture was injected into yolk-producing females. Ten (10) days after egg laying, several embryos were collected and their DNA was extracted. In fact, the inventors have demonstrated that gene editing events have occurred ( Figures 10D-10E ). Figures 10D-10E Represents the positive template of the Sanger chromatogram ( Fig. 10D ) and the reverse template ( Fig.10E ) in addition to the original peaks, there are also new different small peaks in the sequence. The chromatogram shows that the PAX6 gene has a single nucleotide deletion at the 4th position upstream of PAM, indicating that the gene encoding PAX6 is edited in the embryo.

[0262] Example 10

[0263] Internalization of Vg-derived peptides into decapod oocytes

[0264] The inventors further investigated the ability and efficacy of shorter / partial sequences derived from the peptides of the present invention to be internalized into Macrobrachium rosenbergii oocytes. In addition, the inventors investigated whether homologous peptides derived from different decapod crustaceans, such as Litopenaeus vannamei, would also be internalized into Macrobrachium rosenbergii oocytes.

[0265] Table 4. Custom peptides designed to evaluate minimum peptide length and Litopenaeus vannamei peptides that can be internalized into M. rosenbergii oocytes

[0266]

[0267]

[0268] The peptides were incubated with ovarian slices in vitro to evaluate their internalization. The scVg-FITC peptide used as a control was observed only in the outer regions of the oocytes, around and above the follicle cells ( Figures 11B-11G ). In contrast, the Vg-derived peptide was able to enter the oocyte, as it was observed outside the follicular cells within the oocyte (red dots, Figures 11B-11G ). When using Vg235-260-TAMRA, Vg237-244-TAMRA, Vg237-253-TAMRA ( Figures 11B-11D ) compared to the intensity obtained with the Vg246-253-TAMRA peptide ( Fig.11E In addition, both Vg-derived peptides from Penaeus vannamei were able to be internalized into the oocytes of Macrobrachium rosenbergii ( Figures 11F-11G ).

[0269] Although the present invention has been described in detail, those skilled in the art will appreciate that many changes and modifications may be made. Therefore, the present invention should not be interpreted as being limited to the specifically described embodiments, and the scope and mind of the present invention will be more easily understood by referring to the appended claims.

Claims

1. A peptide consisting of 7 to 23 amino acids derived from the following amino acid sequence: DKX1X2X2X3PX4X5GX6YKYVEAX7X8X9SX 10 X 11 (SEQ ID NO:1), wherein: X1 is selected from the group consisting of amino acid residues N and K; X2 is selected from the group consisting of amino acid residues I and V; X3 is selected from the group consisting of amino acid residues K and R; X4 is selected from the group consisting of amino acid residues A and S; X5 is selected from the group consisting of amino acid residues Y and I; X6 is selected from the group consisting of amino acid residues S, I, A and T; X7 is selected from the group consisting of amino acid residues H, S, K and E; X8 is selected from the group consisting of amino acid residues Q and M; X9 is selected from the group consisting of amino acid residues E, D and M; X 10 is selected from the group consisting of amino acid residues V and T; and X 11 is selected from the group consisting of amino acid residues L and M.

2. The peptide according to claim 1, comprising the amino acid sequence: DKX1X2X2X3PX4X5GX6YKYVEA (SEQ ID NO: 2), in: X1 is selected from the group consisting of amino acid residues N and K; X2 is selected from the group consisting of amino acid residues I and V; X3 is selected from the group consisting of amino acid residues K and R; X4 is selected from the group consisting of amino acid residues A and S; X5 is selected from the group consisting of amino acid residues Y and I; and X6 is an amino acid residue selected from the group consisting of: S, I, A and T.

3. The peptide according to claim 1, comprising the amino acid sequence: DKX1X2X2X3PX4 (SEQ ID NO: 3), in: X1 is selected from the group consisting of amino acid residues N and K; X2 is selected from the group consisting of amino acid residues I and V; X3 is selected from the group consisting of amino acid residues K and R; and X4 is selected from the group consisting of amino acid residues A and S.

4. The peptide according to claim 1, comprising the amino acid sequence: DKX1X2X2X3P (SEQ ID NO: 4), in: X1 is selected from the group consisting of amino acid residues N and K; X2 is selected from the group consisting of amino acid residues I and V; and X3 is selected from the group consisting of amino acid residues K and R.

5. The peptide according to claim 1, comprising the amino acid sequence: GX6YKYVEA (SEQ ID NO: 5), wherein X6 is an amino acid residue selected from the group consisting of S, I, A and T.

6. A chimera comprising: a first peptide, wherein the first peptide comprises the peptide according to any one of claims 1 to 5; and is directly or indirectly bound to an agent selected from the group consisting of: a polynucleotide, a second peptide, a small molecule, or any combination thereof.

7. The chimera of claim 6, wherein the agent is selected from the group consisting of a dye and an embryo modifying agent.

8. A polynucleotide encoding a peptide according to any one of claims 1 to 5 or a chimera according to claim 6 or 7.

9. An expression vector comprising the polynucleotide according to claim 8.

10. A cell comprising any of the following: a. A peptide according to any one of claims 1 to 5; b. The chimera according to claim 6 or 7; c. The polynucleotide according to claim 8; and d. The expression vector according to claim 9.

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