Peptide-based DNA vaccine platform

Abstract

The present disclosure relates to methods of generating a peptide-based DNA vaccine platform. In particular, the present disclosure relates to a method of generating the peptide-based DNA vaccine platform against COVID-19. A method of introducing a cargo into the cells of a living body is disclosed. The method of introducing the cargo into the cells of a living body may include administering a nanocomplex to the cells of the living body. The nanocomplex includes at least one chimeric peptide linked to the cargo. The chimeric peptide includes an endosomal escape motif, a nucleus entry motif, and a DNA condensing motif linked together in any order. The cargo is preferably comprising a genetically engineered construct comprising a nucleic acid sequence. The present disclosure further relates to the uses of the present disclosure in the methods of gene delivery, gene therapy, and cancer treatment.

Description

PEPTIDE-BASED DNA VACCINE PLATFORMTECHNICAL FIELD

[0001] The present disclosure relates to methods for generating a peptide -based DNA vaccine platform and more particularly, relates to methods of generating a peptide-based DNA vaccine platform against COVID-19. The present disclosure further relates to methods of gene delivery, gene therapy, and cancer treatment using the present disclosure.BACKGROUND ART

[0002] Vaccination involves stimulating the immune system with an infectious agent or its components, modified in such a manner that no harm or disease is caused. When the host is confronted with the modified infectious agent, the immune system can adequately neutralize it before it causes any ill effect. For example, introduction of specific antigens or live attenuated infectious agents that replicate within the host have affected vaccination.

[0003] New approaches to vaccination involve the direct introduction into appropriate tissues of an mRNA or plasmid containing the DNA sequence encoding the antigen(s) against which an immune response is sought. This approach to vaccination relies on in situ production of a target antigen. DNA vaccine approach has a number of potential advantages over traditional approaches, including stimulation of both B- and T-cell responses, improved vaccine stability, absence of any infectious agent and relative ease of large-scale manufacture. However, current DNA vaccine platforms are unable to elicit sufficient immune response mostly due to lack of ideal delivery platform. An ideal vaccine platform should be simple to deploy, rapid to develop, reproducible, temperature stable, and consistently manufacturable, thus lowering costs and development risks while providing an important new tool. The synthetic DNA (SynDNA) platform addresses many of these important goals. DNA immunogens can be directly designed and optimized from pathogen sequences and synthesized allowing flexibility and speed in preclinical testing with rapid transition to clinical scale up. In vivo expression of the constructed sequences facilitates rapid screening and down selection of potential vaccine candidates. Multiple studies have reported that synDNA allows for the generation of cellular and humoral responses against pathogens with impact in challengemodel systems. Originally intramuscular (IM) inoculation and more recently intradermal (ID) delivery using highly concentrated formulations have induced consistent immunity in clinic.

[0004] Delivery methods such as jet delivery, gene gun delivery, nanoparticle delivery, and others have demonstrated increased DNA uptake in vivo. Adaptive electroporation (EP) which controls the energy delivered during in vivo EP increases transformation efficiency over needle and syringe delivery. Following local injection by needle and syringe plasmid DNA is taken up by a limited number of cells at the site of injection, where the DNA is transcribed into mRNA and translated into antigen intracellularly. Adaptive EP increases the initial uptake of plasmid by local cells approximately 500x. Locally transfected antigen-presenting cells (APCs) can directly traffic to the regional lymph node (LN) which is critical for initiating immune response. Translated antigen can be shed exogenously and picked up by APCs for cross presentation. Shed exogenous soluble antigen can drain locally to the regional LN and extracellular spaces in local environment allowing for engagement of B cell immunity. Local tissue becomes a protein factory for presentation of antigen on major histocompatibility complex- 1 (MHC I) or MHC II molecules for re-expansion of LN primed CD8+ T cells (cytotoxic T lymphocytes, or CTLs) and CD4+ T cells, respectively.

[0005] There are many methods and platforms used for vaccination. For example, Huang Weijin Zhao Aihua Xu Miao et al., presented a patent on “Novel corona DNA vaccine containing adjuvant”, (CN113633764A). Huang Weijin Zhao Aihua Xu Miao invented a DNA vaccine composition for CO VID- 19 wherein the DNA vaccine includes first nucleic acid molecule as an adjuvant component, and a second nucleic acid molecule as an immunogenic component. David Gordon Bermudes et al., presented a patent on “Expression of SARS-CoV-2 spike protein receptor binding domain in attenuated Salmonella as a vaccine” (US11406702B1). David Gordon Bermudes introduced a live genetically engineered bacterium that includes at least one portion of a SARS-CoV-2 antigen and the live genetically engineered bacterium. Nevertheless, DNA vaccine platforms face several challenges. Conventional DNA vaccine carrier platforms each have distinct disadvantages.

[0006] There is, therefore, a need for a vaccine composition and a method of synthesizing thereof which has a cost-efficient development process, safe demonstration, and be size tailorable, function tailorable, and capable of targeting different organs and cell types, simultaneously. There is further a need for carrier platforms that are stable, nontoxic, and capable of eliciting promising humoral and cellular immune response upon administration.SUMMARY OF THE DISCLOSURE

[0007] This summary is intended to provide an overview of the subject matter of this patent, and is not intended to identify essential elements or key elements of the subject matter, nor is it intended to be used to determine the scope of the claimed implementations. The proper scope of this patent may be ascertained from the claims set forth below in view of the detailed description below and the drawings.

[0008] In one general aspect, the present disclosure describes a method of introducing a cargo into cells of a living body. In an exemplary embodiment, an exemplary method may include administering a nanocomplex to exemplary cells of an exemplary living body. In an exemplary embodiment, an exemplary nanocomplex may include at least one chimeric peptide linked to an exemplary cargo, in which an exemplary chimeric peptide may include an endosomal escape motif, a nucleus entry motif, and a DNA condensing motif linked together in any order.

[0009] In an exemplary embodiment, an exemplary chimeric peptide may further include a targeting motif. In an exemplary embodiment, an exemplary chimeric peptide may include MIRGD or CTATMPG2H. In an exemplary embodiment, an exemplary MIRGD may include amino acid sequences identical to amino acid sequences of SEQ ID NO. 2. In an exemplary embodiment, an exemplary CTAMPG2H may include amino acid sequences identical to amino acid sequences of SEQ ID NO. 3.

[0010] In an exemplary embodiment, an exemplary MIRGD may include an endosomal escape motif, including an amino acid sequence identical to an amino acid sequence of SEQ ID NO. 5, a nucleus entry motif, including an amino acid sequence identical to an amino acid sequence of SEQ ID NO. 6, a DNA condensing motif, including an amino acid sequence identical to an amino acid sequence of SEQ ID NO. 7, and a targeting motif, including an amino acid sequence identical to an amino acid sequence of SEQ ID NO. 8.

[0011] In an exemplary embodiment, the CTAMPG2H may include a cell-penetrating motif, including an amino acid sequence identical to an amino acid sequence of SEQ ID NO. 9, an endosomal escape motif, including an amino acid sequence identical to an amino acid sequence of SEQ ID NO. 5, a nucleus entry motif, including an amino acid sequence identical to an amino acid sequence of SEQ ID NO. 6, and a DNA condensing motif, including an amino acid sequence identical to an amino acid sequence of SEQ ID NO. 7.

[0012] In an exemplary embodiment, an exemplary cargo may include at least one of a nucleic acid, a protein, a lipid, a polymer, a carbohydrate, a chemical compound, a nanoparticle, a microsphere containing a diagnostic or therapeutic agent, and combination thereof.

[0013] In an exemplary embodiment, administering an exemplary nanocomplex to exemplary cells of an exemplary living body may include vaccination of a subject organism to elicit adaptive immunity. In an exemplary embodiment, administering an exemplary nanocomplex to exemplary cells of an exemplary living body may include in vivo or in vitro gene delivery as a DNA vaccine platform to elicit adaptive immunity.

[0014] In an exemplary embodiment, an exemplary in vivo gene delivery may inlcude administering an exemplary nanocomplex including a DNA vaccine platform to elicit adaptive immunity against SARS-CoV-2. In an exemplary embodiment, an exemplary in vivo gene delivery may include RBD-encoding gene delivery. In an exemplary embodiment, an exemplary RBD- encoding gene may include a SARS-CoV-2 antigen. In an exemplary embodiment, an exemplary RBD-encoding gene may be sub cloned in PCDNA3.1 for vaccination against COVID- 19.

[0015] In an exemplary embodiment, administering an exemplary nanocomplex to exemplary cells of an exemplary living body may include gene delivery of an exemplary nanocomplex as a DNA vaccine platform to one of elicit adaptive immunity against infectious diseases, elicit adaptive immunity to therapy of cancer, elicit adaptive immunity to therapy of allergies, therapy of autoimmune diseases, and gene therapy.

[0016] In an exemplary embodiment, gene delivery of an exemplary nanocomplex as a DNA vaccine platform to elicit adaptive immunity to therapy of cancer may include targeted gene delivery to tumor tissues. In an exemplary embodiment, administering an exemplary nanocomplex to exemplary cells of an exemplary living body may include targeted gene suppression through at least one of a microRNA, a SiRNA, a DNAi, a peptide nucleic acid, and combination thereof against any target gene.

[0017] In one general aspect, the present disclosure describes a plurality of nanocomplexes for diagnostic / therapeutic purposes. In an exemplary embodiment, each nanocomplex of an exemplary plurality of nanocomplexes may include a cargo and at least one chimeric peptide linked to a cargo. In an exemplary embodiment, an exemplary cargo may include at least one of a nucleic acid, a protein, a lipid, a polymer, a carbohydrate, a chemical compound, a nanoparticle, a microsphere containing a diagnostic or therapeutic agent, and combination thereof, the chimeric peptidecomprising an endosomal escape motif, a nucleus entry motif and a DNA condensing motif linked together in any order.

[0018] In an exemplary embodiment, an exemplary plurality of nanocomplexes may include DNA vaccine nano assembly platform capable for inducing a specific immune response against an antigenic peptide encoded by expression vectors, in which an exemplary cargo may include a nucleic acid including a DNA or an RNA. In an exemplary embodiment, the linkage between an exemplary chimeric peptide and an exemplary nucleic acid may be noncovalent including electrostatic interactions and an exemplary nano assembly size tailorability ranges between 20 nm- 2000nm.

[0019] In an exemplary embodiment, an exemplary chimeric peptide may include MIRGD or CTATMPG2H or their derivatives. In an exemplary embodiment, an exemplary MIRGD may include amino acid sequences identical to amino acid sequences of SEQ ID NO. 2. In an exemplary embodiment, an exemplary CTAMPG2H may include amino acid sequences identical to amino acid sequences of SEQ ID NO. 3.

[0020] In an exemplary embodiment, an exemplary MIRGD may include an endosomal escape motif, including an amino acid sequence identical to an amino acid sequence of SEQ ID NO. 5, a nucleus entry motif, including an amino acid sequence identical to an amino acid sequence of SEQ ID NO. 6, a DNA condensing motif, including an amino acid sequence identical to an amino acid sequence of SEQ ID NO. 7, and a targeting motif, including an amino acid sequence identical to an amino acid sequence of SEQ ID NO. 8.

[0021] In an exemplary embodiment, an exemplary CTAMPG2H may include a cell-penetrating motif, including an amino acid sequence identical to an amino acid sequence of SEQ ID NO. 9, an endosomal escape motif, including an amino acid sequence identical to an amino acid sequence of SEQ ID NO. 5, a nucleus entry motif, including an amino acid sequence identical to an amino acid sequence of SEQ ID NO. 6, and a DNA condensing motif, including an amino acid sequence identical to an amino acid sequence of SEQ ID NO. 7.

[0022] In an exemplary embodiment, each nanocomplex of an exemplary plurality of exemplary nanocomplexes may have a zeta potential in a range of +1 to +20, and each nanocomplex of an exemplary plurality of exemplary nanocomplexes may have an average particle size in a range of 50 nm to 1000 nm.

[0023] In one general aspect, the present disclosure describes a method of administering a nanocomplex for diagnostic / therapeutic purposes. In an exemplary embodiment, an exemplary method may include forming an exemplary nanocomplex and injecting an exemplary nanocomplex into a living body intramuscularly. In an exemplary embodiment, forming an exemplary nanocomplex may include forming a plurality of MIRGD peptides by transferring at least one MIRGD peptide into a bacteria, an exemplary at least one MIRGD peptide may include amino acid sequences identical to amino acid sequences of SEQ ID NO. 2, purifying an exemplary plurality of MIRGD peptides using Ni-NTA agarose affinity chromatography, and forming a linkage between a cargo and at least one MIRGD peptide of an exemplary plurality of MIRGD peptides. In an exemplary embodiment, an exemplary cargo may include at least one of a nucleic acid, a protein, a lipid, a polymer, a carbohydrate, a chemical compound, a nanoparticle, a microsphere containing a diagnostic or therapeutic agent, and combination thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The drawings and data described herein are for illustrative purposes only of selected embodiments and not all possible implementations, and are not intended to limit the scope of the present disclosure. In the figures, like reference numerals refer to the same or similar elements.

[0025] FIG. 1 illustrates order of motifs within exemplary chimeric peptides as one of main counterparts of an exemplary peptide-based DNA vaccine, consistent with one or more exemplary embodiments of the present disclosure;

[0026] FIG. 2 illustrates an image of sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blot results proving expression and purification of MIRGD and CTATMPG2H, consistent with one or more exemplary embodiments of the present disclosure;

[0027] FIG. 3A illustrates a circular dichroism spectroscopy graph depicting an exemplary secondary structure of MIRGD and CTATMPG2H, consistent with one or more exemplary embodiments of the present disclosure;

[0028] FIG. 3B illustrates a circular dichroism spectroscopy reference graph for an exemplary secondary structure of proteins, consistent with one or more exemplary embodiments of the present disclosure;

[0029] FIG. 4 illustrates an image of agarose gel proving formation of nanocomplexes used as a peptide based DNA vaccine, consistent with one or more exemplary embodiments of the present disclosure;

[0030] FIG. 5 illustrates images of agarose gels proving stability of nanocomplexes used as a peptide based DNA vaccine under variety of time and temperature conditions, consistent with one or more exemplary embodiments of the present disclosure;

[0031] FIG. 6 illustrates graphs depicting hydrodynamic size and zeta potential of nanocomplexes used as a peptide based DNA vaccine, consistent with one or more exemplary embodiments of the present disclosure;

[0032] FIG. 7 illustrates an image of agarose gel proving stability of nanocomplexes used as a peptide based DNA vaccine, consistent with one or more exemplary embodiments of the present disclosure;

[0033] FIG. 8 illustrates an image of Transmission electron microscopy (TEM) results proving formation of nanocomplexes besides depicting the morphology and size of nanocomplexes used as a peptide based DNA vaccine, consistent with one or more exemplary embodiments of the present disclosure;

[0034] FIG. 9A illustrates a fluorescent microscope image depicting transfection of HEK293T cells utilizing the nanocomplexes containing CTATMPG2H as a chimeric peptide carrier and pDB2 plasmid as a reported plasmid. Showing results of utilizing an exemplary platform (Peptide based DNA vaccine) for in vitro gene delivery, consistent with one or more exemplary embodiments of the present disclosure;

[0035] FIG. 9B illustrates a fluorescent microscope image depicting transfection of Hek293T cells utilizing exemplary nanocomplexes containing MIRGD as a chimeric peptide carrier and pDB2 plasmid as a reported plasmid, showing results of utilizing an exemplary platform (Peptide based DNA vaccine) for in vitro gene delivery, consistent with one or more exemplary embodiments of the present disclosure;

[0036] FIG. 9C illustrates a fluorescent microscope image depicting transfection of Hek293T cells utilizing poly ethylene imine (PEI) complexes containing PEI as a carrier and pDB2 plasmid as a reported plasmid, used as a positive control, consistent with one or more exemplary embodiments of the present disclosure;

[0037] FIG. 10 illustrates a graph depicting transfection rate of Hek293T cells utilizing exemplary nanocomplexes containing CTATMPG2H and MiRGD as a chimeric peptide carrier and psiCHECK plasmid as a reported plasmid. Showing results of utilizing an exemplary platform (Peptide based DNA vaccine) for in vitro gene delivery, consistent with one or more exemplary embodiments of the present disclosure;

[0038] FIG. 11 illustrates a graph depicting results of assessing humoral immune response of utilization of an exemplary platform (Peptide based DNA vaccine) for in vivo gene delivery, consistent with one or more exemplary embodiments of the present disclosure;

[0039] FIG. 12A illustrates image of H and E stained slides of mouse lung, heart and spleen, consistent with one or more exemplary embodiments of the present disclosure;

[0040] FIG. 12B illustrates image of H and E stained slides of mouse kidney and liver, consistent with one or more exemplary embodiments of the present disclosure;

[0041] FIG. 13 illustrates a table describing optimized biosynthesis condition of CTATMPG2H and MIRGD, consistent with one or more exemplary embodiments of the present disclosure;

[0042] FIG. 14 illustrates a table describing optimized purification protocol for CTATMPG2H and MIRGD, consistent with one or more exemplary embodiments of the present disclosure;

[0043] FIG. 15 illustrates a table showing results of virus neutralization assay, proving the functionality of antibodies detected in serum of animal models that were injected with nanocomplexes, consistent with one or more exemplary embodiments of the present disclosure; and

[0044] FIG. 16 illustrates a diagram illustrating an exemplary peptide based DNA nanoassembly platform, consistent with one or more exemplary embodiments of the present disclosure.DESCRIPTION OF EMBODIMENTS

[0046] In the following detailed description, numerous specific details are set forth by way of examples in order to provide a thorough understanding of the relevant teachings. However, it should be apparent that the present teachings may be practiced without such details. In other instances, well known methods, procedures, components, and / or circuitry have been described at a relatively high-level, without detail, in order to avoid unnecessarily obscuring aspects of the present teachings.

[0047] The novel features which are believed to be characteristic of the present disclosure, as to its structure, organization, use and method of operation, together with further objectives and advantages thereof, will be better understood from the following discussion. In the following detailed description, numerous specific details are set forth by way of examples in order to provide a thorough understanding of the relevant teachings. However, it should be apparent that the present teachings may be practiced without such details. In other instances, well known methods, procedures, components, and / or circuitry have been described at a relatively high-level, without detail, in order to avoid unnecessarily obscuring aspects of the present teachings. The following detailed description is presented to enable a person skilled in the art to make and use the methods and devices disclosed in exemplary embodiments of the present disclosure. For purposes of explanation, specific nomenclature is set forth to provide a thorough understanding of the present disclosure. However, it will be apparent to one skilled in the art that these specific details are not required to practice the disclosed exemplary embodiments. Descriptions of specific exemplary embodiments are provided only as representative examples. Various modifications to the exemplary implementations will be readily apparent to one skilled in the art, and the general principles defined herein may be applied to other implementations and applications without departing from the scope of the present disclosure. The present disclosure is not intended to be limited to the implementations shown, but is to be accorded the widest possible scope consistent with the principles and features disclosed herein.

[0048] The present disclosure is directed to exemplary embodiments of a DNA vaccine for COVID-19. In an exemplary embodiment, an exemplary DNA vaccine may include at least a chimeric peptide, and a cargo. In an exemplary embodiment, an exemplary at least a chimeric peptide may be attached to an exemplary cargo using a linkage. In an exemplary embodiment, an exemplary chimeric peptide may include at least an endosomal escape motif, a nucleus entry motif,a DNA condensing motif, and combinations thereof. In an exemplary embodiment, an exemplary at least an endosomal escape motif, a nucleus entry motif, a DNA condensing motif may be linked together in any order. In an exemplary embodiment, an exemplary chimeric peptide may include MIRGD and CTATMPG2H. In an exemplary embodiment, an exemplary cargo may include at least at least a nucleic acid, a protein, a lipid, a polymer, a carbohydrate, a chemical compound, a nanoparticle, a microsphere containing a diagnostic or therapeutic agent, and combinations thereof.

[0049] In an exemplary embodiment, MIRGD may be developed and employed to deliver cargos(s) to cells in culture and in vivo. In an exemplary embodiment, exemplary cargos may include genes but are not limited to just this type of cargo. In an exemplary embodiment, an exemplary MIRGD may form a nonacomplex when linked to an exemplary cargo. In an exemplary embodiment an exemplary nanocomplex may be capable of transferring an exemplary cargo to cells in culture and in vivo. In an exemplary embodiment an exemplary MIRGD may include four motifs. In an exemplary embodiment, an exemplary each motif of an exemplary MIRGD may be responsible for a function.

[0050] In an exemplary embodiment, exemplary four motifs may include endosomal escape motif, nucleus entry motif, DNA condensing motif, and targeting motif. In an exemplary embodiment, an exemplary endosomal escape motif may include HIV gp41. In an exemplary embodiment, HIV gp41 may have amino acid sequences similar to SEQ ID NO. 5. In an exemplary embodiment, an exemplary endosomal escape motif may equip an exemplary nanocomplex with capability of escaping endosomes to protect an exemplary intended gene. In an exemplary embodiment, an exemplary endosomal escape motif may facilitate further process may be required for expression of an intended gene.

[0051] In an exemplary embodiment, an exemplary nucleus entry motif may include NLS (SV40 large T-antigen). In an exemplary embodiment, an exemplary NLS (SV40 large T-antigen) may have an amino acid sequence similar to SEQ ID NO. 6. In an exemplary embodiment, an exemplary nucleus entry motif may be responsible for enhancing pDNA trafficking in cytosol. In an exemplary embodiment, an exemplary nucleus entry motif may help to enter an exemplary intended gene into a nucleus that may be a necessary step for gene expression and eliciting a promising immune response. In an exemplary embodiment, nucleus entry motif: NLS (SV40 large T-antigen) may have an amino acid sequence similar to SEQ ID NO: 6 responsible for enhancingpDNA trafficking in cytosol and to enter an exemplary intended gene into a nucleus that is a necessary step for gene expression and eliciting a promising immune response.

[0052] In an exemplary embodiment, an exemplary DNA condensing motif may include 2Histonl. In an exemplary embodiment, an exemplary 2Histonl may have an amino acid sequence similar to SEQ ID NO. 7. In an exemplary embodiment, an exemplary DNA condensing motif may be responsible for DNA binding in order to form exemplary nanocomplexes that may protect an exemplary intended gene. In an exemplary embodiment, an exemplary DNA condensing motif may also facilitate gene delivery.

[0053] In an exemplary embodiment, an exemplary targeting motif may include iRGD. In an exemplary embodiment, an exemplary iRGD may have an amino acid sequence similar to SEQ ID NO. 8. In an exemplary embodiment, an exemplary targeting motif may be targeted through intended cells by targeting variety of cell surface markers. In an exemplary embodiment, an exemplary iRGD may target avP3, a5pi and allbp3 integrin. Additional and alternative motifs may be used in exemplary chimeric peptides of the present disclosure.

[0054] In an exemplary embodiment, an exemplary MIRGD may have four motifs. FIG. 1 illustrates an order of exemplary four motifs in MIRGD, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, an exemplary order of exemplary four motifs of an exemplary MIRGD may be endosomal escape motif, nucleus entry motif, DNA condensing motif, and targeting motif, respectively.

[0055] In an exemplary embodiment, an exemplary chimeric peptide may be represented as Gp41- -NLS-2Histonl-iRGD.

[0056] Alternatively, in an exemplary embodiment, an exemplary MIRGD chimeric peptide may have four motifs may be arranged sequentially in a different order form what is demonstrated in FIG. 1. FIG. 1 illustrates order of motifs within exemplary chimeric peptides as one of the main counterparts of an exemplary peptide-based DNA vaccine, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, analogs of MIRGD with exemplary motifs arranged in a different order may be prepared. In an exemplary embodiment, analogs of MIRGD with exemplary motifs arranged in a different order may be evaluated in exemplary nanocomplexes for gene delivery. In an exemplary embodiment, changing order of exemplary motifs of MIRGD may result in a different rate of gene delivery. In an exemplary embodiment, changing order of exemplary motifs of MIRGD may reduce or increase rate of genedelivery. In an exemplary embodiment, all exemplary chimeric peptides and exemplary nanocomplexes formed therefrom are considered to fall within the scope of the present disclosure. In an exemplary embodiment, diagram 102 illustrates SEQ ID NO. 1. In an exemplary embodiment, diagram 102 illustrates an amino acid sequential of MPG2H. In an exemplary embodiment, diagram 104 illustrates SEQ ID NO. 2. In an exemplary embodiment, diagram 104 illustrates an amino acid sequential of MIRGD. In an exemplary embodiment, diagram 106 illustrates an amino acid sequential of CTATMPG2H. In an exemplary embodiment, diagram 106 illustrates SEQ ID NO. 3. In an exemplary embodiment, diagram 106 illustrates an amino acid sequential of CTATMPG2H.

[0057] In an exemplary embodiment, an exemplary MIRGD chimeric peptide may have an amino acid sequence similar amino acid sequence of SEQ ID NO. 2. In an exemplary embodiment, an exemplary MIRGD chimeric peptide may have a molecular weight of 9682.39. In an exemplary embodiment, an exemplary MIRGD chimeric peptide may have an isoelectric point of 10.93. In an exemplary embodiment, an exemplary MIRGD chimeric peptide may have 91 amino acids.

[0058] In an exemplary embodiment, chimeric peptides may be designed with 2 to 8 motifs. In an exemplary embodiment, number of exemplary motifs in exemplary chimeric peptides may result in different gene delivery rates. In an exemplary embodiment, the best gene delivery rate in vitro and in vivo and also in a case of DNA vaccine may be MIRGD structure. In an exemplary embodiment, MIRGD derivatives may be developed when one repeat of each motif is added. In an exemplary embodiment, exemplary derivatives of MIRGD chimeric peptide within the scope of the present disclosure may be similar to amino acid sequence of SEQ ID NO: 2 in which one amino acid or two amino acids may be either deleted, substituted by another amino acid, or one or two amino acid may be added. In an exemplary embodiment, exemplary MIRGD derivatives may be developed using different linker sequences between functional motifs. In an exemplary embodiment, exemplary MIRGD derivatives may be developed by adding tag motifs for a purification process.

[0059] In an exemplary embodiment, exemplary MIRGD derivatives may be developed when an exemplary HIV gp41 motif may be substituted with other endosomal escape motifs. In an exemplary embodiment, MIRGD derivatives may be developed when an exemplary NLS (SV40 large T-antigen) motif is substituted with other nucleus entry motifs. In an exemplary embodiment, MIRGD derivatives may be developed when an exemplary 2Histon 1 motif is substituted with otherDNA condensing motifs. In an exemplary embodiment, MIRGD derivatives may be developed when an exemplary iRGD motif is substituted with other targeting motifs. In an exemplary embodiment, MIRGD derivatives may be developed when other endosomal escape motif is added besides an exemplary HIV gp41 motif. In an exemplary embodiment, MIRGD derivatives may be developed when other nucleus entry motif is added besides NLS (SV40 large T-antigen) motif. In an exemplary embodiment, MIRGD derivatives may be developed when other DNA condensing motif is added besides an exemplary 2Histonl motif. In an exemplary embodiment, MIRGD derivatives may be developed when other targeting motif is added besides an exemplary iRGD motif. In an exemplary embodiment, MIRGD derivatives may be developed when other functional motifs are added. In an exemplary embodiment, MIRGD derivatives may be developed when one of exemplary motifs is deleted. In an exemplary embodiment, MIRGD derivatives may be developed when more than one of exemplary motifs is deleted.

[0060] In another embodiment, useful within exemplary methods of the present disclosure, of a chimeric peptide designated CTATMPG2H may be developed and utilized to deliver cargos(s) including genes to cells in culture and in vivo. CTATMPG2H forms a nanocomplex when linked to cargo that transfer cargo to cells in culture and in vivo. CTATMPG2H may include four motifs each responsible for a function. Each motif may be described in detail in following order. In an exemplary embodiment, exemplary four motifs of CTATMPG2H may include cell-penetrating motif, endosomal escape motif, nucleus entry motif, and DNA condensing motif.

[0061] In an exemplary embodiment, cell-penetrating motif may include TAT. In an exemplary embodiment, TAT may have an amino acid sequence similar to an amino acid sequence of SEQ ID NO: 9. In an exemplary embodiment, in order to equip the platform, a cell entryway may be formed besides exemplary endosomes-based ways. In an exemplary embodiment, reports also may indicate that an exemplary CTAT motif may help endosomal escape.

[0062] In an exemplary embodiment, endosomal escape motif may include HIV gp41. In an exemplary embodiment, HIV gp41 may have an amino acid sequence similar to an amino acid sequence of SEQ ID NO: 5. In an exemplary embodiment, endosomal escape motif may be used to equip an exemplary platform with a capability of escaping endosomes to protect an exemplary intended gene and facilitating further process required for expression of an intended gene.In an exemplary embodiment, nucleus entry motif may include NLS (SV40 large T-antigen). In an exemplary embodiment, NLS may have an amino acid sequence similar to an amino acidsequence of SEQ ID NO: 6. In an exemplary embodiment, NLS may be responsible for enhancing pDNA trafficking in cytosol. In an exemplary embodiment, NLS may also be responsible to enter an exemplary intended gene into an exemplary nucleus that may be a necessary step for gene expression and eliciting a promising immune response.

[0063] In an exemplary embodiment, DNA condensing motif may include 2Histonl. In an exemplary embodiment, 2Histonl may have an amino acid sequence similar to an amino acid sequence of SEQ ID NO: 7. In an exemplary embodiment, 2Histonl may be responsible for DNA binding in order to form nanocomplexes that may protect an exemplary intended gene. In an exemplary embodiment, 2Histonl may facilitate gene delivery.

[0064] In a representative embodiment, CTATMPG2H may have an amino acid sequence similar to an amino acid sequence of SEQ ID NO: 3. In an exemplary embodiment, CTATMPG2H may have four motifs in order with reference to FIG. 1. Additional exemplary chimeric peptides in which order of motifs may be changed are considered to be within the scope of the present disclosure. In other embodiments an exemplary chimeric peptide may include a variation of an exemplary amino acid sequence of CTATMPG2H in which one or two amino acid may either be deleted, substituted by another amino acid, or one or two amino acid may be added.

[0065] In a representative embodiment, an exemplary CTATMPG2H chimeric peptide may have a molecular weight of 11589.67, an isoelectric point of CTATMPG2H 11.85, and may contain 103 amino acids. In an exemplary embodiment, CTATMPG2H demonstrated no toxicity in cell culture. Here is provided a list of other functional motifs to better illustrate the concept.

[0066] In an exemplary embodiment, examples of Alternatives for DNA condensing motif may include any amino acid sequence that can condense nucleotide sequence to some degree. In an exemplary embodiment, capability of an exemplary nanocomplex may be measured by the retardation of nucleotide sequence mobility with electrophoresis. In an exemplary embodiment, DNA condensing motif may also be responsible for protecting nucleotide sequences from serum nucleases and other hazards through reaching an exemplary intended cell. In an exemplary embodiment, Alternatives for DNA condensing motif may include Mu peptide with amino acid sequence of SEQ ID NO. 10, poly-L-lysine, poly-L-Arginine with an amino acid sequence of (RRXRRXHHXHHX)n where X is any amino acid except D and E, TAT with an amino acid sequence of SEQ ID NO. 11, Tyr-TAT with an amino acid sequence of SEQ ID NO. 12, Ploy- TAT, Histones H2B, H1.4F, and Hl.

[0067] In an exemplary embodiment, examples of alternatives for endosomal escape motif may include any amino acid sequence that may disrupt endosomes membrane to some degree. In an exemplary embodiment, disruption in endosome membrane may come from different mechanisms including proton sponge, change in secondary structure, etc. In an exemplary embodiment, alternatives for endosomal escape motif may include GALA peptide (SEQ ID NO. 13), KALA peptide (SEQ ID NO. 14), RALA (SEQ ID NO. 15), Inf-7 (SEQ ID NO. 16), and H5WYG peptide (SEQ ID NO. 17).

[0068] In an exemplary embodiment, examples of alternatives for Nucleus entry motif (NLS) may include any amino acid sequence that may traffic nucleotide sequences into an exemplary nucleus. In an exemplary embodiment, alternatives for Nucleus entry motif may include HIV Rev protein (SEQ ID NO. 18), and M9 peptide derived from hnRNP protein.

[0069] In an exemplary embodiment, examples of alternatives for targeting motifs may include any amino acid sequence that may target inter / intracellular receptor or marker. In an exemplary embodiment, targeting motifs may include FGF growth factor, scFv anti-ERbB2.

[0070] In an exemplary embodiment, other functional motifs may include any amino acid sequence responsible for a function that may be added to an exemplary chimeric peptide sequence. For example, CTAT peptide may be a functional motif that penetrate a cell membrane and provide an additional cell entry pathway besides endosome -based cell entry.

[0071] In an exemplary embodiment, exemplary chimeric peptides may be linked with a cargo or cargo molecule to form a nanocomplex. In an exemplary embodiment, Cargo, when present as a cargo molecule may be selected from a nucleic acid, a protein, a lipid, a polymer, a carbohydrate, a chemical compound, and combinations thereof.

[0072] In embodiments wherein an exemplary cargo may be a nucleic acid, an exemplary nucleic acid may be selected, without limitation, from DNA, RNA, SiRNA, microRNA, antisense RNA, catalytic RNA or catalytic DNA. In an exemplary embodiment, an exemplary cargo may also be a nanoparticle or a microsphere containing a diagnostic or therapeutic agent. In an exemplary embodiment, alternatively, an exemplary cargo may be a detectable compound. In an exemplary embodiment, detectable compounds mean any known label compound known in the art and include, by way of illustration, a fluorophore compound, a fluorophore-conjugated compound, a bioluminescent compound, a bioluminescent-conjugated compound, a chemiluminescentcompound, a chemiluminescent-conjugated compound, or an enzyme substrate that may react with an enzyme to produce a detectable compound.

[0073] In other embodiments, an exemplary cargo may be a nucleic acid. In an exemplary embodiment, one exemplary nucleic acid cargo may be a 5047 bp vector encoding GFP as a reporter known under the name of pDB2. In an exemplary embodiment, an exemplary pDB2 vector may be used to assess gene transfer efficiency in cell culture. In an exemplary embodiment, another exemplary nucleic acid may be a 6273 bp vector encoding Luciferase as a reporter known under the name of psiCHECK(TM)2. In an exemplary embodiment, 6273 bp vector may be used to assess gene transfer efficiency in cell culture. In an exemplary embodiment, another exemplary nucleic acid may be a 6117 bp vector encoding RBD (SEQ ID NO:4) as a SARS-CoV-2 antigen. In certain embodiments, an exemplary RBD encoding vector may be used to assess gene transfer efficiency in vivo besides eliciting immune responses.

[0074] In an exemplary embodiment, Nanocomplexes (platforms) may be developed when chimeric peptides and an exemplary cargo are linked with covalent or non-covalent interactions. In an exemplary embodiment, When Nanocomplexes and an exemplary cargo may be linked with covalent interaction, one or more linker molecules may optionally be used to achieve a linkage as may be generally known in the art. In other embodiments, nanocomplexes may be developed when chimeric peptides and an exemplary intended gene may be linked with non-covalent interactions like electrostatic interaction between a positive charge of chimeric peptides and a negative charge of nucleic acid with reference to FIG. 16. FIG. 16 illustrates a diagram illustrating the peptide based DNA nanoassembly platform. Chimeric peptides and nucleic acids are linked with non- covalent interaction to form nanocomplexes with distinct features, consistent with one or more exemplary embodiments of the present disclosure. As shown in FIG. 16, part 1602 is a chimeric peptide, part 1604 is a nucleic acid (expression vector), and part 1606 is a nanocomplex. In an exemplary embodiment, chimeric peptides and nucleic acids may be linked with non-covalent interaction to form nanocomplexes with distinct features

[0075] In an exemplary embodiment, representative exemplary nanocomplexes according to the present disclosure may include a nanocomplex of MIRGD as a chimeric peptide and pDB2 as a nucleic acid vector, a nanocomplex of MIRGD as a chimeric peptide and psiCHECK(TM)2 as a nucleic acid vector, a nanocomplex of MIRGD as a chimeric peptide and the RBD encoding vector as a nucleic acid vector, a nanocomplex of CTATMPG2H as a chimeric peptide and pDB2 as anucleic acid vector, a nanocomplex of CTATMPG2H as a chimeric peptide and psiCHECK(TM)2 as a nucleic acid vector, and a nanocomplex of CTATMPG2H as a chimeric peptide and an exemplary RBD encoding vector as a nucleic acid vector.

[0076] In an exemplary embodiment, exemplary presently disclosed nanocomplexes may generally exhibit certain physical, structural and reactivity properties. In an exemplary embodiment, for example, nanocomplexes typically may have a zeta potential in a range between +1 and +20. In an exemplary embodiment, nanocomplexes typically may have a size range between 50 nm and 1000 nm. In certain embodiments, exemplary nanocomplexes may have stability against serum nucleases. In certain embodiments, exemplary nanocomplexes may have stability against time range between 15 minutes and 24 hours. In certain embodiments, exemplary nanocomplexes may have stability between 15 °C and 37 °C. In certain embodiments, exemplary nanocomplexes may be stable up to 24 hours within 15 °C to 37 °C.

[0077] In an exemplary embodiment, exemplary nanocomplexes may include MIRGD / pDB2. In an exemplary embodiment, MIRGD / pDB2 may demonstrate high gene transfer efficiency in cell culture, in comparison with PEI as a gold standard gene carrier. In an exemplary embodiment, exemplary nanocomplexes may include MIRGD / psiCHECK(TM)2. In an exemplary embodiment, MIRGD / psiCHECK(TM)2 may demonstrate high gene transfer efficiency in cell culture, in comparison with PEI as a gold standard gene carrier. In an exemplary embodiment, exemplary nanocomplexes may include CTATMPG2H / pDB2. In an exemplary embodiment, CTATMPG2H / pDB2 may demonstrate high gene transfer efficiency in cell culture, in comparison with PEI as a gold standard gene carrier. In an exemplary embodiment, exemplary nanocomplexes may include CTATMPG2H / psiCHECK(TM)2. In an exemplary embodiment, CTATMPG2H / psiCHECK(TM)2 may demonstrate high gene transfer efficiency in cell culture, in comparison with PEI as a gold standard gene carrier. . In an exemplary embodiment, exemplary nanocomplexes may include MIRGD / RBD encoding vector demonstrate high gene transfer efficiency in vivo, by eliciting promising immune response against CO VID- 19. In an exemplary embodiment, exemplary nanocomplexes may include MIRGD / RBD encoding vector demonstrate no cytotoxicity in vivo.

[0078] In an exemplary embodiment, an exemplary present disclosure may provide for a method of introducing a cargo into cells in cell culture and in vivo, including administering, to the cells with a nanocomplex including chimeric peptides linked to an exemplary cargo. The presenttechnology may provide for a method of selectively introducing a cargo into cells in cell culture and in vivo, including administering, to exemplary cells with a nanocomplex including chimeric peptides linked to an exemplary cargo. The present technology may provide for a method of eliciting humoral and cellular immunity in subject in risk from a disease, including administering, to an exemplary subject a nanocomplex including chimeric peptides linked to an exemplary intended gene encoding vector, where sufficient rate of gene transfers in vivo result in expression of intended protein that may elicit immune response. The present technology may provide for a method of eliciting humoral and cellular immunity in subject in risk from an infection disease, including administering, to an exemplary subject a nanocomplex including chimeric peptides linked to an exemplary intended gene encoding vector, where sufficient gene transfers in vivo may result in expression of intended protein that elicit immune response.

[0079] The present technology may provide for a method of eliciting a memory immune in subject in risk from an infection disease, including administering, to an exemplary subject a nanocomplex including chimeric peptides linked to an exemplary intended gene encoding vector, where sufficient rate of gene transfer in vivo result in expression of intended protein that elicit immune response. The present technology may provide for a method of treating a subject suffering from an infection disease, including administering, to the subject a therapeutic amount of nanocomplex including chimeric peptides linked to an exemplary intended gene encoding vector, where the sufficient rate of gene transfer in vivo result in expression of intended protein that elicit immune response. The present technology may provide for a method of eliciting humoral and cellular immunity in subject in risk from COVID-19, including administering, to an exemplary subject nanocomplex including chimeric peptides linked to the RBD encoding vector, where sufficient rate of gene transfer in vivo result in expression of RBD that may elicit immune response.

[0080] In an exemplary embodiment, exemplary chimeric peptides, as generally described above can be used in any of presently described uses and applications. In an exemplary embodiment, without limitation the class of exemplary chimeric peptides may include an exemplary above- mentioned MIRGD, CTATMPG2H and analogs and derivatives of MIRGD and CTATMPG2H.

[0081] In an exemplary embodiment, non-limiting example of gene encoding vector may be RBD encoding vector. In an exemplary embodiment, non-limiting examples of cargo may be nucleic acid vectors. In an exemplary embodiment, nucleic acid vectors may include psiCHECK(TM)2, pDB2 and RBD encoding vector. In an exemplary embodiment, non-limiting example of cells incell culture may be HEK293T. In an exemplary embodiment, non-limiting examples of disease may be cancers and infection disease like COVID-19. In an exemplary embodiment, non-limiting examples of subjects may be a human subject or a non-human subject, such as a primate, a companion animal, a laboratory animal or a farm animal.

[0082] In an exemplary embodiment, the present disclosure may include methods, materials and devices for gene delivery. The present technology may further include methods, materials and devices for in vivo gene delivery. In particular, the present disclosure may concern safe carrierbased in vivo delivery of intended DNA to specific cells in order to elicit promising immune response against intended illness. In an exemplary embodiment, exemplary specific cells may include immune cells.

[0083] In the present disclosure, a designed vector containing the DNA sequence responsible for coding the intended antigen may be delivered in vivo by a designed, bio- synthesized and characterized peptide based platform. In an exemplary embodiment, an exemplary designed vector containing the DNA sequence may elicit promising immune response to be considered as a DNA vaccine platform. In the present disclosure, the peptide based DNA vaccine, may generally be described as a nanocomplex including chimeric peptide and a vector containing an exemplary DNA sequence of intended gene.

[0084] The present disclosure may concern methods, materials and devices for design, biosynthesis and characterization of an exemplary multi-functional chimeric peptide as one of main counterparts of an exemplary peptide based DNA vaccine. In an exemplary embodiment, peptides were designed in order to perform a safe gene delivery. In an exemplary embodiment, exemplary designs may be based on functional motifs, each responsible for a purpose, in an order to keep each motif functional. In an exemplary embodiment, exemplary motifs may be chosen and designed to purpose answers to important challenges that gene delivery and DNA vaccine’s areas of research face. In an exemplary embodiment, each exemplary motif may be responsible for a function. The present disclosure may concern methods, materials and devices for an exemplary design, of plasmid containing an exemplary DNA sequence of intended gene as one of exemplary main counterparts of an exemplary peptide based DNA vaccine. The present disclosure may concern methods, materials and devices for an exemplary design, development and characterization of a gene delivery platform (nanocomplex) including an exemplary chimeric peptide and plasmid containing an exemplary intended DNA sequence.

[0085] In an exemplary embodiment, an exemplary platform may be readily redesigned in order to target different cell lines and organs to transfer genes at different rates to be used as a gene delivery platform for DNA vaccines, cancer vaccines, and gene therapy. In one embodiment of the present disclosure, there may be provided materials and methods to design, biosynthesis and purify a chimeric peptide called MIRGD, used as one of exemplary main counterparts of an exemplary peptide based DNA vaccine. In one embodiment of the present disclosure, there may be provided materials and methods to design, biosynthesis and purify a chimeric peptide called CTATMPG2H, used as one of exemplary main counterparts of an exemplary peptide based DNA vaccine. In another embodiment of the present disclosure, there may be provided materials, methods and exemplary results for characterization of exemplary chimeric peptides called MIRGD and CTATMPG2H, used as one of exemplary main counterparts of an exemplary peptide based DNA vaccine. In another embodiment of the present disclosure, there may be provided exemplary materials, methods and exemplary results for developing intended antigen expressing plasmid, used as one of exemplary main counterparts of an exemplary peptide based DNA vaccine. FIG. 2 illustrates an image of sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blot results proving the expression and purification of MIRGD and CTATMPG2H, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, image 202 shows WESTERN-BLOT including MIRGD, Protein Ladder, and CTATMPG2H. . In an exemplary embodiment, image 204 shows SDS-PAGE including MIRGD, Protein Ladder, and CTATMPG2H.

[0086] In another embodiment of the present disclosure, there may be provided exemplary materials, methods and exemplary results for nanocomplex preparation and characterization, introduced as an exemplary platform of peptide based DNA vaccine. In another embodiment of the present disclosure, there may be provided exemplary materials, methods and exemplary results of utilizing an exemplary platform (Peptide based DNA vaccine) for in vitro gene delivery.

[0087] In another embodiment of the present disclosure, there may be provided exemplary materials and methods of utilizing an exemplary platform (peptide based DNA vaccine) for in vivo gene delivery. In an exemplary embodiment, an exemplary platform may be used for mouse model immunization.

[0088] In another embodiment of the present disclosure, there may be provided exemplary materials and methods and exemplary results of assessing an exemplary immune response ofutilization of an exemplary platform for in vivo gene delivery. In an exemplary embodiment, exemplary materials and methods may be used for assessing an exemplary humoral response of animal models to the utilization of an exemplary platform.

[0089] In another embodiment, there may be provided exemplary materials and methods and exemplary results of assessing safety of utilization an exemplary platform for in vivo gene delivery. In an exemplary embodiment, exemplary materials and methods may be used for assessing cytotoxicity of an exemplary platform in animal models.

[0090] In an exemplary embodiment, a nano assembly, peptideticles may also be designed to identify tumor vasculature zip code, penetrate in tumor tissue and target the cancer microenvironment for gene delivery. In an exemplary embodiment, exemplary essential functional motifs may be simultaneously embedded into two main peptides, MIRGD and HNH, using genetic engineering. In an exemplary embodiment, an exemplary MIRGD as an exemplary first peptide, may constitute an exemplary core of nano assembly. In an exemplary embodiment, an exemplary second peptide and an exemplary HNH may cover an exemplary nano assembly. In an exemplary embodiment, an exemplary MIRGD may include DNA condensing, endosome escaping, nuclear delivery, and tissue penetrating elements. In an exemplary embodiment, an exemplary HNH may include DNA binding and pH-responsive elements. In an exemplary embodiment, an exemplary MIRGD and an exemplary HNH peptides may be formed tightly stable peptideticles in presence of DNA that successfully identified the tumor zip code, penetrated into tumor spheres tuned via environment pH, and initiated an exemplary cellular uptake, followed by high gene expression. In an exemplary embodiment, exemplary peptideticles proficiently may be accumulated in tumor tissues, while sparing exemplary healthy ones. In an exemplary embodiment, exemplary peptideticles may deliver luciferase gene with high efficiency into tumor tissue by overcoming difficulties met under physiological and cellular condition.

[0091] In an exemplary embodiment, there may be many aspects regarding exemplary challenges gene delivery platforms may face. Exemplary conventional platforms of gene delivery each may have distinct disadvantages. Regarding exemplary disadvantages which may be common among exemplary conventional platforms may include lack of safe, size tailorable, function tailorable, organ targetable, cell type targetable, stable and nontoxic carrier capable of having a promising delivery rate in vivo.

[0092] In an exemplary embodiment, the present disclosure may concern methods, materials and devices for an exemplary design, biosynthesis and characterization of multi-functional chimeric peptide as a carrier which may answer each of exemplary common disadvantages of conventional gene delivery platforms mentioned above. In an exemplary embodiment, the present disclosure may concern methods, materials and devices for an exemplary design, development and characterization of a gene delivery platform (nanocomplexes) including an exemplary chimeric peptide and plasmid containing the intended DNA sequence which may answer each of exemplary common disadvantages of conventional gene delivery platforms mentioned above. In an exemplary embodiment, features of the present disclosure may be responsible for purpose answers for each of exemplary mentioned disadvantages fully described in details with reference to drawings.

[0093] In an exemplary embodiment, safety of an exemplary carrier may mostly be result of the fact that an exemplary carrier may be peptide based and an exemplary nature inspired motif design. In an exemplary embodiment, an exemplary nature inspired motif design may refer that each motif already may be utilized in different cells pathway. In an exemplary embodiment, safety of an exemplary chimeric peptide based carrier may be examined with variety of tests that some may be described with reference to FIG. 9A, FIG. 9B, FIG. 12A and FIG. 12B that may indicate high cell viability rate in cell culture as it may be assessed by imaging and no sign of cytotoxicity in vivo as organs may be collected on day 30 and H&E stained and analyzed. FIG. 9A illustrates a fluorescent microscope image depicting the transfection of HEK293T cells utilizing the nanocomplexes containing CTATMPG2H as a chimeric peptide carrier and pDB2 plasmid as a reported plasmid. Showing the results of utilizing the platform (Peptide based DNA vaccine) for in vitro gene delivery, consistent with one or more exemplary embodiments of the present disclosure. FIG. 9B illustrates a fluorescent microscope image depicting the transfection of Hek293T cells utilizing the nanocomplexes containing MIRGD as a chimeric peptide carrier and pDB2 plasmid as a reported plasmid. Showing the results of utilizing the platform (Peptide based DNA vaccine) for in vitro gene delivery, consistent with one or more exemplary embodiments of the present disclosure.

[0094] In an exemplary embodiment, size of the carrier and the nanocomplex may be readily tailored with adding or omitting different motifs besides an exemplary nanocomplex formation condition as regard to N / P ratio, time and temperature. With reference to FIG. 1 and FIG. 6 thatmay indicate order of motifs and exemplary N / P ratios result in different nanocomplex sizes as measured by dynamic light scattering.

[0095] In an exemplary embodiment, function of an exemplary carrier and an exemplary nanocomplex may be readily tailored with adding or omitting different motifs besides an exemplary nanocomplex formation condition as regard to N / P ratio, time and temperature. In an exemplary embodiment, as described for an example in this disclosure, adding an exemplary CTAT motif to an exemplary backbone of previously designed chimeric peptide known as MPG2H, an exemplary carrier and nanocomplex may be equipped to another cell entering pathway known as penetrating with reference to FIG. 1, FIG. 9 A and FIG. 10 that may indicate addition of an exemplary CTAT motif may increase an exemplary gene transfer efficiency in cell culture. In an exemplary embodiment, an exemplary gene transfer efficiency in cell culture may be assessed by imaging (green fluorescent signal shows the cells transfected with GFP reporter) and luciferase assay technique.

[0096] In an exemplary embodiment, chimeric peptide carrier may be readily redesigned by adding or omitting different motifs to target intended organs. In an exemplary embodiment, exemplary nanocomplexes may be developed to achieve intended size needed to target intended organs.

[0097] In an exemplary embodiment, chimeric peptide carrier may be readily redesigned by adding or omitting different motifs besides being tailored to achieve intended size needed to target intended cell types with reference to FIG. 9A and FIG. 9B that may indicate use of iRGD motif may target cells with RGD binding integrin(receptor). In an exemplary embodiment, can exemplary chimeric peptide carrier may be assessed by imaging (green fluorescent signal shows the cells transfected with GFP reporter).

[0098] In an exemplary embodiment, stability of an exemplary chimeric peptide based carrier and nanocomplex may be examined with a variety of tests that some described regarding serum stability, time stability and temperature stability with reference to FIG. 5 and FIG. 7.

[0099] In an exemplary embodiment, nontoxicity of an exemplary chimeric peptide based carrier and an exemplary nanocomplex may be examined with a variety of tests that some described regarding cytotoxicity with reference to FIG. 9A, FIG. 9B, FIG. 12A and FIG. 12B that indicates high cell viability rate in cell culture as it was assessed by imaging and no sign of cytotoxicity in vivo as organs were collected on day 30 and H&E stained and analyzed.

[0100] In an exemplary embodiment, rate of in vivo delivery of an exemplary chimeric peptide carrier and an exemplary nanocomplex may be examined with a variety of tests that some described regarding the delivery rate of plasmid containing RBD DNA sequence in mouse models with reference to FIG. 11 that indicates high titer of antibody that was produced in animal models after 2 dose injection as it was assessed by ELISA assay.

[0101] The foregoing description of the features of the present disclosure may be responsible for purpose answers for each of the mentioned disadvantages has been provided for purposes of illustration and description. It is not intended to be exhaustive or to be limit the feature of the technology.

[0102] The present disclosure may concern methods, materials and devices for design, development and characterization of a DNA vaccine platform (nanocomplexes) including an exemplary chimeric peptide and plasmid containing an exemplary intended DNA sequence which answers each of an exemplary common disadvantages of conventional DNA vaccine platforms mentioned above. Features of the present disclosure responsible for purpose answers for each of exemplary mentioned disadvantages were fully described in details with reference to drawings.

[0103] In an exemplary embodiment, the process of design and biosynthesis of both counterparts of an exemplary platform (chimeric peptide based carrier and plasmid containing intended DNA sequence) and developing an exemplary nanocomplex, may be all from well-established methods which are both time and cost efficient and may readily be utilized toward relative ease of large- scale manufacture with reference to FIG. 13 and FIG. 14 that may indicate exemplary methods and materials of expression and purification of chimeric peptides.

[0104] In an exemplary embodiment, capability of an exemplary platform to elicit promising immune response may be examined with variety of tests that some described regarding humoral immunity against SARS-CoV-2 with reference to FIG. 11 and FIG. 15 that indicates high titer of antibody produced in animal models after 2dose injection as it was assessed by ELISA assay, and the functionality of produced antibodies that was assessed by virus neutralization test respectively.

[0105] In an exemplary embodiment, methods of an exemplary present disclosure may find many areas of applicability by making use of the fact that an exemplary introduced platform may be readily designed to purpose answers to challenges gene delivery areas of research including DNA vaccine, cancer vaccine, therapeutic vaccine, infection disease and immunotherapy face including the lack of ideal carrier platform be able of forming stable, safe nanocomplex at differentnano size range, tailorable to target different cell lines and organs in order to show promising delivery and expression rate.

[0106] In an exemplary embodiment, an exemplary platform may be used as a targeted delivery of intended gene in vitro to cell lines as described in detailed description of an embodiment with reference to FIG. 9B and FIG. 10 that indicates promising rate of gene transfer to cells with RGD binding integrin (receptor) as it was assessed by imaging (green fluorescent signal shows the cells transfected with GFP reporter) and luciferase assay technique. Also depicting the PEI complexes containing PEI as a carrier and psiCHECK plasmid as a reported plasmid, used as a positive control and untreated cells and cells that were treated with naked psiCHECK plasmid without any carrier as a negative control.

[0107] In an exemplary embodiment, an exemplary platform may be used as an un-targeted delivery of intended gene in vitro to cell lines as described in detailed description of an embodiment with reference to FIG. 9A and FIG. 10 that indicates promising rate of gene transfer to cells in culture as it was assessed by imaging (green fluorescent signal shows the cells transfected with GFP reporter) and luciferase assay technique. FIG. 10 illustrates a graph depicting the transfection rate of Hek293T cells utilizing the nanocomplexes containing CTATMPG2H and MiRGD as a chimeric peptide carrier and psiCHECK plasmid as a reported plasmid. Showing the results of utilizing the platform (Peptide based DNA vaccine) for in vitro gene delivery, consistent with one or more exemplary embodiments of the present disclosure. As shown in FIG. 10, image 1020 is a blank sample. Image 1002 and 1004 are for untreated cells and naked plasmid (psiCHECK.2), respectively. Image 1006 is PEI, N / P=5. Image 1022 is for N / P=12. Image 1008 is for CTATMPG2H. Image 1010 is for MIRGO, image 1012 is for CTATMPG2H, image 1014 is for MIRGO, image 1016 is for CTATMPG2H, and image 1018 is for MIRGO. Image 1024 is for N / P=8. Image 1026 is for N / P=4.

[0108] In an exemplary embodiment, an exemplary platform may be used as a targeted delivery of intended gene in vivo to animal models as described in detailed description of an embodiment with reference to FIG. 11 that indicates high titer of antibody that was produced in animal models after 2dose injection as it was assessed by ELISA assay. In an exemplary embodiment, an exemplary platform may be used as an un-targeted delivery of intended gene in vivo to animal models.

[0109] In an exemplary embodiment, an exemplary platform application in targeted delivery of intended gene in vivo to animal models may be considered as a DNA vaccine platform as described in detailed description of an embodiment with reference to FIG. 11 that indicates high titer of antibody that produced in animal models after 2 dose injection as it was assessed by ELISA, and the functionality of produced antibodies that were assessed by virus neutralization test respectively.

[0110] In an exemplary embodiment, an exemplary platform application in targeted delivery of RBD gene in vivo to animal models may be considered as a DNA vaccine platform against COVID-19 as described in detailed description of an embodiment with reference to FIG. 11 and FIG. 15 that indicates high titer of antibody that may be produced in animal models after 2 dose injection as it was assessed by ELISA assay, and the functionality of produced antibodies that were assessed by virus neutralization test respectively. FIG. 11 illustrates a graph depicting the results of assessing the humoral immune response of utilization of the platform (Peptide based DNA vaccine) for in vivo gene delivery, consistent with one or more exemplary embodiments of the present disclosure. As shown in FIG. 11, image 1136 is for sample 1 (peptide control (48.4 pg)- MIRGD), image 1134 is for sample 2 (plasmid control (10 pg)- PCDNA3.1 containing RBD sequence), image 1132 is for sample 3 (Control- non-injected), image 1130 is for sample 4 (: Nanocomplex (48.8 pg MIRGD + 10 pg PCDNA3.1 containing RBD sequence + 500 mM chloroquine + 20 ng GM-CSF)) and image 1128 is for sample 5 (Nanocomplex (48.8 pg MIRGD + 10 pg PCDNA3.1 containing RBD sequence + 500 mM chloroquine + Freund's Complete Adjuvant)), image 1126 is for O.OOlserum dilution, image 1124 is for 0.002 serum dilution, image 1122 is for 0.01 serum dilution, image 1120 is for 0.001 serum dilution, image 1118 is for 0.002 serum dilution, image 1116 is for 0.01 serum dilution, image 1114 is for 0.001 serum dilution, image 1112 is for 0.002 serum dilution, image 1110 is for 0.01 serum dilution, image 1108 is for 0.001 serum dilution, image 1106 is for 0.002 serum dilution, image 1104 is for 0.01 serum dilution, image 1103 is for 0.001 serum dilution, and image 1102 is for 0.002 serum dilution.

[0111] In an exemplary embodiment, an exemplary platform may be used to identify an exemplary tumor vasculature zip code, penetrate in tumor tissue and target an exemplary cancer microenvironment for gene delivery. In an exemplary embodiment, an exemplary carrier counterpart of an exemplary platform introduced as multi-functional chimeric peptide may be readily redesigned (methods fully described) as in adding or omitting variety of motifs in order toperform gene delivery within targeted or untargeted toward variety of cells and organs in vitro or in vivo. In an exemplary embodiment, an exemplary methods mentioned above may find applicability in order to suppress or overexpress of intended gene.

[0112] In an exemplary embodiment, an exemplary gene counterpart of the platform introduced as a plasmid containing intended gene may be readily redesigned (methods fully described) as in adding or omitting variety of gene sequences in order to perform gene delivery within targeted or untargeted toward variety of cells and organs in vitro or in vivo. In an exemplary embodiment, exemplary methods mentioned above may find applicability in order to suppress or overexpress of intended gene.

[0113] In an exemplary embodiment, exemplary nanocomplexes may include additional elements. For instance, chimeric peptides or cargo may be conjugated to one or more molecules to perform a variety of functions like theranostic applications. In general, an exemplary introduced platform may be used tailored or untailored as many as areas of research regarding gene delivery.

[0114] In an exemplary embodiment, example embodiments will now be described more fully with reference to the accompanying drawings. The description, function and order of motifs used in the design of multi-functional chimeric peptides used as a gene delivery carrier more particular as one of the main counterparts of DNA vaccine platform with reference to FIG. 1 that may indicate the non-limiting example of motifs orders. In an exemplary embodiment, exemplary multi-functional chimeric peptides used as a gene delivery carrier may include 5 motifs. In an exemplary embodiment, exemplary motifs may include cell-penetrating motif, endosomal escape motif, nucleus entry motif, DNA condensing motif, and targeting motif.

[0115] In an exemplary embodiment, an exemplary cell -penetrating motif may include TAT: In an exemplary embodiment, an exemplary cell-penetrating motif may be used to equip an exemplary platform, a cell entryway besides exemplary endosomes-based ways.

[0116] In an exemplary embodiment, an exemplary endosomal escape motif may include gp41. In an exemplary embodiment, an exemplary endosomal escape motif may be used to equip an exemplary platform, capability of escaping endosomes to protect an exemplary intended gene and facilitating the further process required for expression of an intended gene. In an exemplary embodiment, reports also indicate that the CT AT motif can help endosomal escape.

[0117] In an exemplary embodiment, an exemplary nucleus entry motif may include NLS. In an exemplary embodiment, an exemplary nucleus entry motif may be responsible to enter theintended gene into an exemplary nucleus that may be a necessary step for gene expression and eliciting a promising immune response.

[0118] In an exemplary embodiment, an exemplary DNA condensing motif may include 2Histon 1. In an exemplary embodiment, an exemplary DNA condensing motif may be responsible for DNA binding in order to form nanocomplexes that may protect an exemplary intended gene besides facilitating gene delivery.

[0119] In an exemplary embodiment, an exemplary targeting motif may include iRGD. In an exemplary embodiment, an exemplary targeting motif may be used as an example of capability of an exemplary platform that may be targeted through intended cells by targeting variety of cell surface markers.

[0120] The order of functional motifs was designed in order to not only keep each motif functional but also add other capabilities as an environmental targeting (Nanocomplexes smaller than 100 nm in size can enter lymphatic system).Example 1: Forming an exemplary DNA vaccine platform

[0121] FIG. 13 and FIG. 14 indicates the methods and materials of expression and purification of chimeric peptides including MIRGD and CTATMPG2H.

[0122] Biosynthesis: MIRGD construct previously designed by Hosseinkhani and colleagues was transformed into E. coli BL21 strain and expressed under an inducible condition. (FIG. 13)

[0123] Purification: Two-step purification was performed by Ni-NTA affinity chromatography and dialysis. After expression, the cells were harvested by centrifugation at 5500 RPM for 6 min. the pallets were suspended in lysis buffer (FIG. 14) and then pulsed by probe sonicator for 10 minutes (10 seconds on and 20 seconds off- 20 pulse). The suspensions were centrifuged at 12000 RPM for 30 minutes. The supernatants were added to Ni-NTA chromatography columns and incubated for 1 hour. The columns were washed by wash buffers (FIG. 14) in order to eliminate unwanted proteins. Later the desired peptides were eluted by elusion buffer (FIG. 14) and stored at -20 °C. For extra purification and desalting, the purified peptides were dialyzed against PBS buffer containing 3 percent glycerol by dialysis tubing for 48 hours at 4 °C. The buffer was replaced every 6 hours. The extra purified peptide is preserved under cold condition to prevent aggregation.

[0124] Primer designing: Two set of primers designed in order to add another functional motif to N-terminal of previous introduced chimeric peptide(MPG2H).

[0125] PCR Amplification: The PCR product was achieved under the optimized terms and later was extracted from agarose gel and cleaned up.

[0126] Cloning: The cleaned up PCR product was digested with restriction enzymes, then was ligated in pet28a vector. To verifying the cloning, colony PCR, double enzymatic digestion and sequencing were conducted.

[0127] Biosynthesis: After approved cloning, the construct was transformed into E. coli Rosetta and expressed under an inducible condition. (FIG. 13). FIG. 13 illustrates a table describing the optimized biosynthesis condition of CTATMPG2H and MIRGD, consistent with one or more exemplary embodiments of the present disclosure.

[0128] Purification: Two step purification was performed by Ni-NTA affinity chromatography and dialysis. After expression, the cells were harvested by centrifugation at 5500 RPM for 6 minutes. The pallets were suspended in lysis buffer (FIG. 14) and then pulsed by probe sonicator for 10 minutes (10 seconds on and 20 seconds off - 20 pulse). The suspensions were centrifuged at 12000 RPM for 30 minutes. The supernatants were added to Ni-NTA chromatography columns and incubated for 1 hour. The columns were washed by wash buffers (FIG. 14) in order to eliminate unwanted proteins. Later the desired peptides were eluted by elusion buffer (FIG. 14) and stored at -20 °C. For extra purification and desalting, the purified peptides were dialyzed against PBS buffer containing 3 percent glycerol by dialysis tubing for 48 hours at 4 °C. The buffer was replaced every 6 hours. The extra purified peptide is preserved under cold condition to prevent aggregation. FIG. 14 illustrates a table describing the optimized purification protocol for CTATMPG2H and MIRGD, consistent with one or more exemplary embodiments of the present disclosure.

[0129] FIG. 2, FIG. 3A and FIG. 3B indicate the purity and the identity of purified chimeric peptides as assessed by SDS-PAGE electrophoresis and western blotting, and the secondary structure of both chimeric peptides as assessed by circular dichroism respectively. FIG. 3A illustrates a circular dichroism spectroscopy graph depicting the secondary structure of MIRGD and CTATMPG2H, consistent with one or more exemplary embodiments of the present disclosure. FIG. 3B illustrates a circular dichroism spectroscopy reference graph for the secondary structure of proteins, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, image 302 shows diagrams of MIRGD. In an exemplary embodiment, image 304 shows diagrams of CTATMPG2H. In an exemplary embodiment, image 306 shows acircular dichroism spectroscopy reference graph for a-helix. In an exemplary embodiment, image 308 shows a circular dichroism spectroscopy reference graph for beta-sheet. In an exemplary embodiment, image 310 shows a circular dichroism spectroscopy reference graph for random coil.Example 2: Expression and purification of an exemplary DNA vaccine platform

[0130] Expression and purification analysis: The expression of intended peptides was determined by western blot using anti His-tag antibody against the His-tag motifs designed at the C-terminal and N-terminal of the peptides. (FIG. 2) The purification rate was analyzed with 12.5 % Tris-Glycine SDS-PAGE and optical density at 214 nm. (FIG. 2)Results: based on bioinformatics calculation the theoretical pI / Mw of MIRGD and CTATMPG2H evaluated about: 10.93 / 9682.39 and 11.85 / 11589.67 respectively. But since the peptides are highly cationic and as a result of these conformations, peptides bonds appear about 15 kD on SDS- PAGE electrophoresis result.Example 3: Secondary structure analysis of an exemplary DNA vaccine platform

[0131] Secondary structure analysis: The secondary structure of the peptide was determined by CD spectropolarimetry at far-UV spectral region (190-250 nm). (FIG. 3A)Results: The results demonstrated that CTATMPG2H construct represents a considerable peak in the 200-210 nm region, and another peak in 220 nm. According to the graphical representation of the model spectra illustrated in Figure 3.B. it seems that, the CTATMPG2H construct is folded into a structure with a major propensity of a a-helix structure and MIRGD had shown the secondary structure with a combination of P-sheet and random coil structures.Example 4: Testing an exemplary DNA vaccine platform

[0132] Description of the embodiment regarding the materials, methods and the results for developing intended antigen expressing plasmid, used as one of the main counterparts of the peptide based DNA vaccine.

[0133] The desire gene expressing RBD (part of the first subunit of spike) considered as proper SARS-CoV-2 antigen, was sub cloned in PCDNA3.1. Sub cloning approved by colony PCR, double digestion and sequencing.

[0134] The designed plasmid was transformed into E. coli DH5a. The plasmid was replicated within 2 days in LB culture media and extracted using midi MN plasmid extraction kit commercially available.

[0135] Also commercially available plasmids carrying GFP (pDB2) and luciferase (psiCHECK2) were used as reporters for optimization steps.Example 5: Gel electrophoresis of an exemplary DNA vaccine platform

[0136] Description of the embodiment regarding the materials, methods and the results for nanocomplex preparation and characterization, introduced as the platform of peptide based DNA vaccine in reference to FIG. 4, FIG. 5, FIG. 6, FIG. 7, and FIG. 8 that indicates the formation of nanocomplexes at different N / P ratios as it assessed by agarose gel retardation assay the shows gene loses its mobility due binding to chimeric peptide and formation of nanocomplex, the stability of formed nanocomplexes at range of temperature and time as it assessed by gel retardation assay the shows different condition cannot disturb the nanocomplex formation, the size and zeta potential of formed nanocomplexes as it assessed by dynamic light scattering, the stability of formed nanocomplexes at presence of serum nucleases as it assessed by gel retardation assay the shows presence of serum nuclease cannot reduce the quality of gene when linked o chimeric peptide and form the nanocomplex, the image of nanocomplexes as it assessed by Transmission electron microscopy (TEM), respectively. FIG. 4 illustrates an image of agarose gel proving the formation of nanocomplexes used as a peptide based DNA vaccine, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embedment, image 402 shows agarose gel containing CTATMPG2H. In an exemplary embedment, image 404 shows agarose gel containing MIRGD. In an exemplary embedment, image 406 shows N / P=l. In an exemplary embedment, image 408 shows N / P=2. In an exemplary embedment, image 410 shows N / P=4. In an exemplary embedment, image 412 shows N / P=8. In an exemplary embedment, image 414 shows plasmid. In an exemplary embedment, image 416 shows N / P=8. In an exemplary embedment, image 418 shows N / P=4. In an exemplary embedment, image 420 shows N / P=2. In an exemplary embedment, image 422 shows N / P=l. FIG. 5 illustrates images of agarose gels proving the stability of nanocomplexes used as a peptide based DNA vaccine under variety of time and temperature conditions, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embedment, image 502 shows agarose gels containing CTATMPG2G. In an exemplary embedment, image 504 shows agarose gels containing MIRGD. In an exemplary embedment, image 506 shows stability of an exemplary nanocomplex at a temperature 15°C of for 30 minutes. In an exemplary embedment, image 508 shows stability of an exemplary nanocomplex at a temperature 28°C of for 24 hours. In an exemplary embedment, image 510 shows stability of anexemplary nanocomplex at a temperature 37°C of for 24 hours. In an exemplary embedment, image 512 shows stability of DNA ladder. In an exemplary embedment, image 514 shows stability of an exemplary nanocomplex at a temperature 37°C of for 24 hours. In an exemplary embedment, image 516 shows stability of an exemplary nanocomplex at a temperature 28°C of for 24 hours. In an exemplary embedment, image 518 shows stability of an exemplary nanocomplex at a temperature 15°C of for 30 minutes.

[0137] FIG. 6 illustrates graphs depicting the hydrodynamic size and zeta potential of nanocomplexes used as a peptide based DNA vaccine, consistent with one or more exemplary embodiments of the present disclosure. FIG. 7 illustrates an image of agarose gel proving the stability of nanocomplexes used as a peptide based DNA vaccine, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, image 702 shows agarose gel containing MIRGD. In an exemplary embodiment, image 704 shows agarose gel containing CTATMPG2G. In an exemplary embodiment, image 706 shows N / P=0.5. In an exemplary embodiment, image 708 shows N / P= 1. In an exemplary embodiment, image 710 shows N / P=4. In an exemplary embodiment, image 712 shows N / P=4+serum. In an exemplary embodiment, image 714 shows N / P=4+SDS. In an exemplary embodiment, image 716 shows plasmid+serum. In an exemplary embodiment, image 718 shows N / P=4+SDS. In an exemplary embodiment, image 720 shows N / P=4+serum. In an exemplary embodiment, image 722 shows N / P=4. In an exemplary embodiment, image 724 shows N / P= 1. In an exemplary embodiment, image 726 shows N / P=0.5. FIG. 8 illustrates an image of Transmission electron microscopy (TEM) results proving the formation of nanocomplexes besides depicting the morphology and size of nanocomplexes used as a peptide based DNA vaccine, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, image 802, image 804, and image 806 show TEM images of nanocomplexes with different sized.

[0138] Predefined amounts of peptides in HBG and PBS buffer were mixed with equal amounts of plasmid (PBDb2 and PCDNA3.1+RBD) by gentle pipetting in several N: P ratios and incubated at 15 °C for 30 minutes. The formation of nanocomplexes was evaluated by Gel retardation assay using 1 % agarose gel and visualizing the electrophoresis mobility of complexes by UV illuminator.(FIG. 4)

[0139] Results: Based on the gel electrophoresis results, both peptides were able to form stable complexes with plasmids and retard the mobility of plasmids on the agarose gel.Example 6: Time and temperature stability analysis of an exemplary DNA vaccine platform

[0140] Time and temperature stability analysis: Retardation assay was performed on each sample in several N: P ratios after incubating for 24 hours at 28 °C and 37 °C. (FIG. 5)

[0141] Results: Based on the results of gel electrophoresis analysis both nanocomplexes (MIRGD + PLASMID and CTATMPG2H + PLASMID) stayed completely intact and stable after 24 hours at 37 °C and 28 °C.Example 7: Hydrodynamic size, zeta potential and distribution analysis of an exemplary DNA vaccine platform

[0142] Hydrodynamic size, zeta potential and distribution analysis: The average particle size, size distribution and zeta potential of each nanocomplex in variety of N: P ratios were determined by A Zetasizer Nano ZS instrument at 25 °C. (FIG. 6)

[0143] Results: Based on the time, temperature, purification procedure and nanocomplex fabrication process, variety of sizes and zeta potential can be obtained and it is manageable based on the goal. The size of nanocomplex can be in range of 50-1000 nanometer and the zeta potential can be in range of -10 till +20 mv. The nanocomplexes used for mouse immunization were managed to be about 100 nm and +10 mV. The positive surface charge and the nanometer range of the nanocomplexes are two major factors that help the platform to attach and enter the cells.Example 8: Serum stability of an exemplary DNA vaccine platform

[0144] Serum stability: The stability of nanocomplexes in presence of serum was examined by gel retardation assay. The nanocomplexes incubated with 10%FBS for 60 minutes at 37 °C. to dissociate DNA from nanocomplexes SDS was added. Samples were electrophoresed on 1% agarose and visualized by UV. (FIG. 7)

[0145] Results: Based on the results of gel electrophoresis analysis both nanocomplexes (MIRGD + PLASMID and CTATMPG2H + PLASMID) shows promising ability of protecting the Plasmid against serum nucleases.

[0146] TEM: The nanocomplexes were prepared at a N / P ratio of 10 and stained with 2% uranyl acetate for 2 minutes at room temperature on a formvar carbon-coated grid (Cu Mesh 300). The grids were imaged using a Transmission electron microscope (TEM, Zeiss - EM10C - 80 kV). (FIG. 8).Example 9: Analyzing rate of gene delivery of an exemplary DNA vaccine platform

[0147] Description of the embodiment regarding the materials, methods and the results of utilizing the platform (peptide based DNA vaccine) for in vitro gene delivery with reference to FIG. 9A, FIG. 9B and FIG. 9C as it was assessed by imaging (green fluorescent signal shows the cells transfected with GFP reporter) and luciferase assay technique that show promising rate of gene transfer in comparison with PEI as a gold standard gene carrier (control+) and untreated cells and cells treated with naked gene without carrier(Control-). FIG. 9C illustrates a fluorescent microscope image depicting the transfection of Hek293T cells utilizing the poly ethylene imine (PEI) complexes containing PEI as a carrier and pDB2 plasmid as a reported plasmid, used as a positive control, consistent with one or more exemplary embodiments of the present disclosure. FIG. 9C also depletes untreated cells and cells that were treated with naked pDB2 plasmid without any carrier as a negative control. As shown in FIG. 9A, image 902 is a fluorescent microscope image of CTATMPG2H-N / P:4, image 904 is a fluorescent microscope image of CTATMPG2H- N / P:8, and image 906 is a fluorescent microscope image of CTATMPG2H-N / P: 12. As shown in FIG. 9B, image 908 is a fluorescent microscope image of MIRGD-N / P:4, image 910 is a fluorescent microscope image of MIRGD-N / P:8, and image 912 is a fluorescent microscope image of MIRGD-N / P: 12. As shown in FIG. 9C, image 914 is a fluorescent microscope image of PEI- N / P:8, image 916 is a fluorescent microscope image of PEI-N / P:5, image 918 is a fluorescent microscope image of untreated samples, and image 920 is a fluorescent microscope image of Naked plasmid.

[0148] HEK293T cells were seeded in 48-well plate at 60000 Density in DMEM supplemented with 10% FBS and PenStrep antibiotic and incubated 24 hours. Cells were incubated with serum free media 1 hour prior to transfection. Cells were transfected with nanocomplexes with several N: P ratios with and without of chloroquine. After 4 hours, cells were re incubated for 48 hours in fresh media. Cellular bio imaging was conducted with fluorescent microscope. The transfection rate was also analyzed with luciferase assay technique. Cells were lysed with cell lysis buffer(hypotonic). Luciferase activity of lysed cells were measured in presence of luciferin, Mg2+and ATP with a luminometer. (FIG. 10)

[0149] Transfections were performed with MIRGD (FIG. 9B) and CTATMPG2H (FIG. 9A) as gene carriers. PEI also used as a positive control. (FIG. 9C) Also un-treated cells and cells that were treated with naked plasmid considered as a negative control. (FIG. 9C)

[0150] Results: Both peptides show remarkable transfection efficiency in comparison with PEI as a gold standard. The rate of gene delivery was assessed at variety of N / P ratios with complementary methods (luciferase assay and imaging).Example 10: Analyzing in vivo gene delivery of an exemplary DNA vaccine platform

[0151] Description of the embodiment regarding the materials and methods and the results of assessing the immune response of utilization of the platform (peptide based DNA vaccine) for in vivo gene delivery. In particular, assessing the humoral response of animal models to the utilization of the platform (peptide based DNA vaccine) with reference to FIG. 11 and FIG. 15 that indicates high titer of antibody that produced in animal models after 2dose injection as it assessed by ELISA assay, and the functionality of produced antibodies that assessed by virus neutralization test respectively.

[0152] The immunogenicity of exemplary vaccine candidates (nanocomplexes) may be carried out in inbred BALB / c mouse, (female 6-8 weeks) mouse models were injected IM with nanocomplexes containing 10 pg of designed plasmid and 500 mM chloroquine on day 0 and 15. Models also injected with plasmid and peptides alone, considered as controls. Also 20 ng of GM- CSF used as adjuvant. Blood samples and organs were collected on day 30.

[0153] Measurement of antibody titres by ELISA assay: ELISA assay was performed to determine antibody titres in animal sera samples. 96-well RBD pre coated plates were incubated with several dilutions of animal’s sera samples for 2 hours at 37 °C. Wells were washed 5 times and incubated with HRP goat anti-mouse IgG (minimal x-reactivity) antibody (BioLegend, San Diego, CA, USA) for 1 hour at 37 °C. Wells were washed 5 times, then were incubated with TMB / H2O2 substrate. Reaction stopped with 1 M HCL, plates were read at 450 nm with microplate spectrophotometer. (FIG. 11)

[0154] Results: Meaningful differences were observed between the antibody titres of main groups and controls. Main groups were those which received nanocomplex (48.8 pg MIRGD + 10 pg PCDNA3.1 containing RBD sequence + 500 mM chloroquine) with + 20 ng GM-CSF or Freund's Complete Adjuvant, as adjuvants. And control groups were those who received 10 pg PCDNA3.1 containing RBD sequence or 48.8 pg MIRGD or nothing.

[0155] Based on the mentioned results the introduced DNA vaccine platform shows promising ability of in vivo gene delivery in order to elicit humoral immunity by showing high titres of anti- RBD antibodies.Example 11: Virus Neutralization Test (VNT) of an exemplary DNA vaccine platform

[0156] Virus neutralization test was performed in Amirabad Virology Laboratory (Tehran, Iran). Briefly, twofold serial dilutions of BALB / c mice sera were prepared in 2% DMEM. The sera were obtained from the mice that received nanocomplex (48.8 pg MIRGD + 10 pg PCDNA3.1 containing RBD sequence + 500 mM chloroquine + 20 ng GM-CSF) Afterwards, 100 TCID50 of SARS-CoV-2 (Wuhan strain isolated from infected patients) was added and a 60-min incubation at 37 C was conducted. The mixtures were added to VeroE6 cells and incubated at 37 °C and 5% CO2 atmosphere. Finally, the cytopathic effect (CPE) was investigated and scored. (FIG. 15).

[0157] Result: serum dilutions were tested and 1 / 2, 1 / 4, 1 / 8 dilutions of serum were able to deactivate viruses and prevent CPE, it shows the effective functionality of antibodies that were made in animal models in response to the DNA vaccine platform. It can be concluded that by the optimization of the sequence of the intended plasmid higher VNT titres could be achieved.

[0158] With the help of this result it can be claimed that not only the introduced DNA vaccine platform can elicit promising humoral immunity by showing high titer of antibodies (ELISA results) against introduced plasmid (PCDNA3.1 + RBD), but also the antibodies are functional. FIG. 15 illustrates a table showing the results of virus neutralization assay, proving the functionality of antibodies detected in serum of animal models that were injected with nanocomplexes, consistent with one or more exemplary embodiments of the present disclosure. The nanocomplex includes 48.8 pg MIRGD + 10 pg PCDNA3.1 containing RBD sequence + 500 mM chloroquine + 20 ng GM-CSF. The results demonstrate the ability of the introduced DNA vaccine platform to elicit humoral immune response.Example 12: Assessing the cytotoxicity of an exemplary DNA vaccine platform

[0159] Description of the embodiment regarding the materials and methods and the results of assessing the safety of utilization the platform (peptide based DNA vaccine) for in vivo gene delivery. In particular, assessing the cytotoxicity of the platform (peptide based DNA vaccine) in animal models with reference to FIG. 12A and FIG. 12B that indicates high cell viability rate in cell culture as it assessed by imaging and no sign of cytotoxicity in vivo as organs were collected on day 30 and H&E stained and analyzed. FIG. 12A illustrates image of H and E stained slides of mouse lung, heart and spleen, consistent with one or more exemplary embodiments of the present disclosure. FIG. 12B illustrates image of H and E stained slides of mouse kidney and liver,consistent with one or more exemplary embodiments of the present disclosure. As shown in FIG. 12A, image 1202 is a control sample, image 1204 is when using MIRGD, image 1206 is when using pcDNA3.1+RBD, and image 1208 is when using the nanocopmlex. As shown in FIG. 12B, image 1210 is for control sample, image 1212 is when using MIRGD, image 1214 is when using pcDNA3.1+RBD, and image 1216 is when using the nanocopmlex.

[0160] In order to assess the biosafety of the candidate platform, organs of all groups were collected, fixed, H and E staining was performed and analyzed, (heart, lung, liver, kidney, spleen and lymph nodes)

[0161] Results: Control group: liver: some aggregates of poly-morphonuclear and mononuclear inflammatory cells causing cholangitis. Kidney: some vacuolated renal tubular epithelial cells, tubular casts and some diffused lymphocytes. Spleen: some neutrophils. Heart: mild mononuclear myocarditis. Lung: alveolar atelectasis, hemorrhage, hemosiderosis, fibrin deposits, one portion of alveolar ectasia, and infiltration of some poly-morphonuclear and mononuclear inflammatory cells causing moderate pneumonia. Lymph node: some neutrophils

[0162] MIRGD: liver: some hepatocytes with losing their cytoplasm due to glycogen-storage and some small foci composed of necrotic hepatocytes, neutrophils and mononuclear inflammatory cells. Kidney: tubular cast, some vacuolated renal tubular epithelial cells and sporadic and diffused mononuclear inflammatory cells. Spleen: hemorrhage (hematoma) and some polymorphonuclear inflammatory cells. Heart: mild lymphocytic myocarditis. Lung: alveolar atelectasis, hyperemia, hemosiderosis, fibrin deposits, infiltration of some mononuclear and polymorphonuclear inflammatory cells causing alveolitis and bronchiolitis.

[0163] Plasmid (PCDNA3.1 containing RBD sequence): liver: some small aggregates of inflammatory cells. Kidney: tubular cast, some vacuolated renal tubular epithelial cells and sporadic mononuclear inflammatory cells. Spleen: some poly-morphonuclear inflammatory cells. Heart: mild mononuclear myocarditis. Lung: alveolar atelectasis, hemorrhage, hemosiderosis, scant fibrin deposits and infiltration of some mononuclear and poly-morphonuclear inflammatory cells. Lymph node: normal

[0164] Nanocomplex (MIRGD + PCDNA3.1 containing RBD sequence + 500 mM chloroquine + 20 ng GM-CSE): liver: some small aggregates of inflammatory cells. Kidney: tubular cast, some vacuolated renal tubular epithelial cells and diffused mononuclear inflammatory cells. Spleen: some neutrophils. Heart: mild lymphocytic myocarditis. Lung: alveolar atelectasis, edema, scantfibrin deposits, perivascular cuffing and moderate infiltration of mononuclear and polymorphonuclear inflammatory cells causing interstitial pneumonia. Lymph node: sporadic polymorphonuclear inflammatory cells

[0165] Note: The majority of histopathological findings represented some lesions due to inflammatory response with an almost analogous severity. Other observed changes were either repetitive in all slides or insignificant.

[0166] There was NO remarkable sign of cytotoxicity.

[0167] Also group that was injected with nanocomplex (48.8 pg MIRGD + 10 pg PCDNA3.1 containing RBD sequence + 500 mM chloroquine + 20ng GM-CSF) were followed over 5 months and 100 percent probability of survival was observed.

[0168] The foregoing description of the embodiments has been provided for purposes of illustration and description. It is not intended to be exhaustive or to be limit the disclosure. Individual elements or features of a particular embodiment are generally not limited to that particular embodiment, but, where applicable are interchangeable and can be used in a selected embodiment, even if no specifically shown or described. The same may also be varied in many ways. Such variations are not to be regarded as a departure from the disclosure, and all such modifications are intended to be included within the scope of the disclosure.

[0169] The foregoing description of the features of the present disclosure responsible for purpose answers for each of the mentioned disadvantages has been provided for purposes of illustration and description. It is not intended to be exhaustive or to be limit the feature of the technology.

[0170] Industrial Applicability

[0171] The present disclosure may use chimeric peptides in constructing exemplary nanocomplexes and methods of using exemplary nanocomplexes as gene carriers. The particular advantages conferred by construction and use of exemplary nanocomplexes address challenges related to DNA vaccines, most specifically incapability of conventional gene delivery methods to be readily engineered in a short and logical period of time, toward different cells and organs with intended functions such as gene transfer rate. Methods of the present disclosure may find many areas of applicability by making use of the fact that an exemplary nanocomplexes may be readily designed to purpose answers to challenges gene delivery areas of research including DNA vaccine, cancer vaccine, therapeutic vaccine, infection disease and immunotherapy may face including lack of ideal carrier platform be able of forming stable, safe nanocomplex at different nano size range,tailorable to target different cell lines and organs in order to showing promising delivery and expression rate.

[0172] While the foregoing has described what are considered to be the best mode and / or other examples, it is understood that various modifications may be made therein and that the subject matter disclosed herein may be implemented in various forms and examples, and that the teachings may be applied in numerous applications, only some of which have been described herein. It is intended by the following claims to claim any and all applications, modifications and variations that fall within the true scope of the present teachings.

[0173] Unless otherwise stated, all measurements, values, ratings, positions, magnitudes, sizes, and other specifications that are set forth in this specification, including in the claims that follow, are approximate, not exact. They are intended to have a reasonable range that is consistent with the functions to which they relate and with what is customary in the art to which they pertain.

[0174] The scope of protection is limited solely by the claims that now follow. That scope is intended and should be interpreted to be as broad as is consistent with the ordinary meaning of the language that is used in the claims when interpreted in light of this specification and the prosecution history that follows and to encompass all structural and functional equivalents. Notwithstanding, none of the claims are intended to embrace subject matter that fails to satisfy the requirement of Sections 101, 102, or 103 of the Patent Act, nor should they be interpreted in such a way. Any unintended embracement of such subject matter is hereby disclaimed.

[0175] Except as stated immediately above, nothing that has been stated or illustrated is intended or should be interpreted to cause a dedication of any component, step, feature, object, benefit, advantage, or equivalent to the public, regardless of whether it is or is not recited in the claims.

[0176] It will be understood that the terms and expressions used herein have the ordinary meaning as is accorded to such terms and expressions with respect to their corresponding respective areas of inquiry and study except where specific meanings have otherwise been set forth herein. Relational terms such as first and second and the like may be used solely to distinguish one entity or action from another without necessarily requiring or implying any actual such relationship or order between such entities or actions. The terms “comprises,” “comprising,” or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, orapparatus. An element proceeded by “a” or “an” does not, without further constraints, preclude the existence of additional identical elements in the process, method, article, or apparatus that comprises the element.

[0177] The Abstract of the Disclosure is provided to allow the reader to quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. In addition, in the foregoing Detailed Description, it can be seen that various features are grouped together in various implementations. This is for purposes of streamlining the disclosure, and is not to be interpreted as reflecting an intention that the claimed implementations require more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive subject matter lies in less than all features of a single disclosed implementation. Thus, the following claims are hereby incorporated into the Detailed Description, with each claim standing on its own as a separately claimed subject matter.

[0178] While various implementations have been described, the description is intended to be exemplary, rather than limiting and it will be apparent to those of ordinary skill in the art that many more implementations and implementations are possible that are within the scope of the implementations. Although many possible combinations of features are shown in the accompanying figures and discussed in this detailed description, many other combinations of the disclosed features are possible. Any feature of any implementation may be used in combination with or substituted for any other feature or element in any other implementation unless specifically restricted. Therefore, it will be understood that any of the features shown and / or discussed in the present disclosure may be implemented together in any suitable combination. Accordingly, the implementations are not to be restricted except in light of the attached claims and their equivalents. Also, various modifications and changes may be made within the scope of the attached claims.

Claims

What is claimed is:

1. A method of introducing a cargo into cells of a living body, the method comprising: administering a nanocomplex to the cells of the living body, the nanocomplex comprising at least one chimeric peptide linked to the cargo, wherein the chimeric peptide comprises an endosomal escape motif, a nucleus entry motif, and a DNA condensing motif linked together in any order.

2. The method of claim 1 wherein the chimeric peptide further comprises a targeting motif.

3. The method of claim 1 wherein the chimeric peptide comprises MIRGD or CTATMPG2H, the MIRGD comprising amino acid sequences identical to amino acid sequences of SEQ ID NO.2, the CTAMPG2H comprising amino acid sequences identical to amino acid sequences of SEQ ID NO. 3.

4. The method of claim 3, wherein the MIRGD comprises: an endosomal escape motif, comprising an amino acid sequence identical to an amino acid sequence of SEQ ID NO. 5; a nucleus entry motif, comprising an amino acid sequence identical to an amino acid sequence of SEQ ID NO. 6; a DNA condensing motif, comprising an amino acid sequence identical to an amino acid sequence of SEQ ID NO. 7; and a targeting motif, comprising an amino acid sequence identical to an amino acid sequence of SEQ ID NO. 8.

5. The method claim 3, wherein the CTAMPG2H comprises: a cell-penetrating motif, comprising an amino acid sequence identical to an amino acid sequence of SEQ ID NO. 9; an endosomal escape motif, comprising an amino acid sequence identical to an amino acid sequence of SEQ ID NO. 5; a nucleus entry motif, comprising an amino acid sequence identical to an amino acid sequence of SEQ ID NO. 6; and a DNA condensing motif, comprising an amino acid sequence identical to an amino acid sequence of SEQ ID NO. 7.

6. The method of claim 1 , wherein the cargo comprises at least one of a nucleic acid, a protein, a lipid, a polymer, a carbohydrate, a chemical compound, a nanoparticle, a microsphere containing a diagnostic or therapeutic agent, and combination thereof.

7. The method of claim 1, wherein administering the nanocomplex to the cells of the living body comprises vaccination of a subject organism to elicit adaptive immunity.

8. The method of claim 1, wherein administering the nanocomplex to the cells of the living body comprises in vivo or in vitro gene delivery as a DNA vaccine platform to elicit adaptive immunity.

9. The method of claim 8, wherein the in vivo gene delivery comprises administering the nanocomplex comprising a DNA vaccine platform to elicit adaptive immunity against SARS-CoV-2.

10. The method of claim 9, wherein the in vivo gene delivery comprises RBD-encoding gene delivery, the RBD-encoding gene comprising a SARS-CoV-2 antigen, the RBD-encoding gene being sub cloned in PCDNA3.1 for vaccination against CO VID- 19.

11. The method of claim 8, wherein administering the nanocomplex to the cells of the living body comprises gene delivery of the nanocomplex as a DNA vaccine platform to one of elicit adaptive immunity against infectious diseases, elicit adaptive immunity to therapy of cancer, elicit adaptive immunity to therapy of allergies, therapy of autoimmune diseases, and gene therapy.

12. The method of claim 11, wherein gene delivery of the nanocomplex as a DNA vaccine platform to elicit adaptive immunity to therapy of cancer comprises targeted gene delivery to tumor tissues.

13. The method of claim 1, wherein administering the nanocomplex to the cells of the living body comprises targeted gene suppression through at least one of a microRNA, a SiRNA, a DNAi, a peptide nucleic acid, and combination thereof against any target gene.

14. A plurality of nanocomplexes for diagnostic / therapeutic purposes, each nanocomplex of the plurality of nanocomplexes comprising:a cargo, comprising at least one of a nucleic acid, a protein, a lipid, a polymer, a carbohydrate, a chemical compound, a nanoparticle, a microsphere containing a diagnostic or therapeutic agent, and combination thereof; and at least one chimeric peptide linked to a cargo, the chimeric peptide comprising an endosomal escape motif, a nucleus entry motif and a DNA condensing motif linked together in any order.

15. The plurality of nanocomplexes of claim 14, wherein the plurality of nanocomplexes comprises DNA vaccine nano assembly platform capable for inducing a specific immune response against an antigenic peptide encoded by expression vectors, wherein the cargo comprises a nucleic acid comprising a DNA or an RNA, wherein the linkage between the chimeric peptide and the nucleic acid is noncovalent including electrostatic interactions and the nano assembly size tailorability ranges between 20 nm- 2000nm.

16. The plurality of nanocomplexes of claim 15, wherein the chimeric peptide comprises MIRGD or CTATMPG2H or their derivatives, the MIRGD comprising amino acid sequences identical to amino acid sequences of SEQ ID NO. 2, the CTAMPG2H comprising amino acid sequences identical to amino acid sequences of SEQ ID NO. 3.

17. The plurality of nanocomplexes of claim 16, wherein the MIRGD comprises: an endosomal escape motif, comprising an amino acid sequence identical to an amino acid sequence of SEQ ID NO. 5;a nucleus entry motif, comprising an amino acid sequence identical to an amino acid sequence of SEQ ID NO. 6; a DNA condensing motif, comprising an amino acid sequence identical to an amino acid sequence of SEQ ID NO. 7; and a targeting motif, comprising an amino acid sequence identical to an amino acid sequence of SEQ ID NO. 8.

18. The plurality of nanocomplexes of claim 16, wherein the CTAMPG2H comprises: a cell-penetrating motif, comprising an amino acid sequence identical to an amino acid sequence of SEQ ID NO. 9; an endosomal escape motif, comprising an amino acid sequence identical to an amino acid sequence of SEQ ID NO. 5; a nucleus entry motif, comprising an amino acid sequence identical to an amino acid sequence of SEQ ID NO. 6; and a DNA condensing motif, comprising an amino acid sequence identical to an amino acid sequence of SEQ ID NO. 7.

19. The nanocomplex of claim 14, wherein: each nanocomplex of the plurality of the nanocomplexes has a zeta potential in a range of +1 to +20; and each nanocomplex of the plurality of the nanocomplexes has an average particle size in a range of 50 nm to 1000 nm.

20. A method of administering a nanocomplex for diagnostic / therapeutic purposes, comprising: forming the nanocomplex, comprising: forming a plurality of MIRGD peptides by transferring at least one MIRGD peptide into a bacteria, the at least one MIRGD peptide comprising amino acid sequences identical to amino acid sequences of SEQ ID NO. 2; purifying the plurality of MIRGD peptides using Ni-NTA agarose affinity chromatography; and forming a linkage between a cargo and at least one MIRGD peptide of the plurality of MIRGD peptides, the cargo comprising at least one of a nucleic acid, a protein, a lipid, a polymer, a carbohydrate, a chemical compound, a nanoparticle, a microsphere containing a diagnostic or therapeutic agent, and combination thereof; and injecting the nanocomplex into a living body intramuscularly.

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