Peptide-based DNA vaccine platform

Abstract

The present disclosure relates to a method for constructing a peptide-based DNA vaccine platform. In particular, the invention provides a construction strategy of a peptide-based DNA vaccine platform oriented to COVID-19. Further disclosed herein is a method of delivering a drug to living cells, wherein the delivery process can be accomplished by applying a nanocomposite to living cells. The nano-composite comprises at least one chimeric peptide connected with the medicine, and the chimeric peptide is formed by connecting an endosome escape motif, a cell nucleus entry motif and a DNA (deoxyribonucleic acid) condensation motif in any sequence. The drug is preferably a genetic engineering construct containing a nucleic acid sequence. The disclosure further relates to potential uses of the technology in application scenarios such as gene delivery, gene therapy and cancer therapy.

Description

Technical Field

[0001] This disclosure relates to methods for constructing peptide-based DNA vaccine platforms, and more specifically, to a method for constructing a peptide-based DNA vaccine platform for COVID-19. This disclosure further relates to methods for gene delivery, gene therapy, and cancer treatment using this platform. Background Technology

[0002] Vaccination stimulates the immune system by introducing a pathogen or a component thereof into the body and modifying it in a way that does not cause harm or disease. When the host is exposed to the modified pathogen again, the immune system is able to effectively neutralize it before it causes adverse effects. For example, introducing specific antigens or using attenuated live pathogens that can replicate in the host have been used to achieve vaccine immunization.

[0003] Novel vaccination strategies involve the direct delivery of mRNA or plasmids containing DNA sequences encoding target antigens into suitable tissues to induce the desired immune response. These vaccine approaches rely on the in situ expression of the target antigen in vivo. Compared to traditional vaccines, DNA vaccines offer several potential advantages, such as simultaneous activation of B and T cell responses, higher vaccine stability, absence of any infectious pathogens, and easier large-scale production. However, most current DNA vaccine platforms fail to induce sufficiently strong immune responses, primarily due to the lack of ideal delivery systems. An ideal vaccine delivery platform should be easy to deploy, rapidly develop, highly reproducible, temperature-stable, and capable of stable large-scale production, thereby reducing costs and R&D risks and providing important new tools for vaccine development. Synthetic DNA (SynDNA) platforms largely meet these key requirements. DNA immunogens can be directly designed and optimized based on pathogen sequences and obtained through chemical synthesis, enabling high flexibility and development speed in preclinical studies and a rapid transition to clinical-scale production. The in vivo expression of the constructed sequences further facilitates the rapid screening and selection of candidate vaccines. Multiple studies have reported that SynDNA can induce cellular and humoral immune responses against pathogens and has shown significant effects in challenge model systems. While early DNA vaccines were mostly administered intramuscularly (IM), in recent years, intradermal (ID) delivery with high-concentration formulations has also demonstrated consistent and reliable immune responses in clinical practice.

[0004] Microfluidic drug delivery, gene gun delivery, and nanoparticle delivery have all been shown to improve DNA uptake efficiency in vivo. Adaptive electroporation (EP) significantly improves transfection efficiency compared to traditional syringe injection by modulating the energy released during in vivo electroporation. After local injection using a syringe, plasmid DNA is only taken up by a small number of cells at the injection site, transcribed into mRNA, and then translated into antigen proteins. Adaptive electroporation can increase the initial uptake of plasmids by local cells by approximately 500-fold. Locally transfected antigen-presenting cells (APCs) can migrate directly to regional lymph nodes (LNs), which is crucial for initiating an immune response. Cell-produced antigens can also be secreted extracellularly, taken up by APCs, and cross-presented. Furthermore, secreted soluble exogenous antigens can drain locally into regional lymph nodes and surrounding tissue spaces, thereby activating B-cell immune responses. Therefore, local tissues become "protein factories" for antigens, capable of presenting antigens on major histocompatibility complex class I (MHC I) or class II (MHC II) molecules, thereby promoting the proliferation of CD8+ T cells (cytotoxic T lymphocytes, CTLs) and CD4+ T cells activated by lymph nodes, respectively.

[0005] Currently, various methods and platforms exist for vaccine administration. For example, Huang Weijin, Zhao Aihua, Xu Miao, and others proposed a COVID-19 DNA vaccine composition in their patent "Novel corona DNA vaccine containing adjuvant" (CN113633764A), in which the DNA vaccine contains a first nucleic acid molecule as an adjuvant component and a second nucleic acid molecule as an immunogen component. David Gordon Bermudes et al., in their patent "Expression of SARS-CoV-2 spike protein receptor binding domain in attenuated Salmonella as a vaccine" (US11406702B1), proposed a live genetically engineered bacterium containing at least one SARS-CoV-2 antigen sequence, and used this live bacterium as a vaccine vector for immunization. However, DNA vaccine platforms still face several challenges. Traditional DNA vaccine delivery vector platforms each have significant limitations.

[0006] Therefore, there is an urgent need for a vaccine composition and its synthesis method that is cost-effective in the development process, has verifiable safety profiles, and is customizable in size and function, capable of simultaneously targeting different organs and cell types. Furthermore, a delivery vector platform that remains stable and non-toxic after administration and can induce significant humoral and cellular immune responses is also required. Summary of the Invention

[0007] This summary is intended to provide a general overview of the subject matter of this patent and is not intended to limit the key or essential elements of that subject matter, nor to define the scope of the claimed embodiments. The appropriate scope of protection for this patent shall be determined in conjunction with the detailed description and drawings below, and by the appended claims.

[0008] In one general aspect, this disclosure describes a method for delivering a drug to living cells. In one exemplary embodiment, the method may include administering a nanocomposite to target cells in living organisms. In this exemplary embodiment, the nanocomposite may comprise at least one chimeric peptide linked to the drug, wherein the chimeric peptide may be composed of endosome escape motifs, nuclear entry motifs, and DNA condensation motifs linked in any order.

[0009] In one exemplary embodiment, the chimeric peptide may further comprise a targeting motif. In another exemplary embodiment, the chimeric peptide may comprise MIRGD or CTATMPG2H. In one exemplary embodiment, MIRGD may comprise an amino acid sequence that is completely identical to the amino acid sequence of SEQ ID NO.2. In another exemplary embodiment, CTATMPG2H may comprise an amino acid sequence that is completely identical to the amino acid sequence of SEQ ID NO.3.

[0010] In one exemplary embodiment, the MIRGD may comprise: an endosome escape motif whose amino acid sequence is identical to that of SEQ ID NO. 5; a nuclear entry motif whose amino acid sequence is identical to that of SEQ ID NO. 6; a DNA condensation motif whose amino acid sequence is identical to that of SEQ ID NO. 7; and a targeting motif whose amino acid sequence is identical to that of SEQ ID NO. 8.

[0011] In one exemplary embodiment, the CTAGMPG2H may comprise: a cell penetration motif whose amino acid sequence is identical to that of SEQ ID NO. 9; an endosome escape motif whose amino acid sequence is identical to that of SEQ ID NO. 5; a nucleus entry motif whose amino acid sequence is identical to that of SEQ ID NO. 6; and a DNA condensation motif whose amino acid sequence is identical to that of SEQ ID NO. 7.

[0012] In one exemplary embodiment, the medicament may include at least one of the following: nucleic acids, proteins, lipids, polymers, carbohydrates, chemical compounds, nanoparticles, microspheres containing diagnostic or therapeutic agents, or any combination thereof.

[0013] In one exemplary embodiment, administering the nanocomposite to target cells in vivo may include vaccinating a test organism to induce an adaptive immune response. In another exemplary embodiment, administering the nanocomposite to target cells in vivo may include serving as a DNA vaccine platform for in vivo or in vitro gene delivery to induce an adaptive immune response.

[0014] In one exemplary embodiment, the in vivo gene delivery may include administering a nanocomposite comprising a DNA vaccine platform to induce an adaptive immune response against SARS-CoV-2. In another exemplary embodiment, the in vivo gene delivery may include the delivery of an RBD-encoded gene. In one exemplary embodiment, the RBD-encoded gene may contain a SARS-CoV-2 antigen. In another exemplary embodiment, the RBD-encoded gene may be subcloned into PCDNA3.1 for use in vaccination against COVID-19.

[0015] In one exemplary embodiment, administering the nanocomposite to target cells in vivo may include using it as a DNA vaccine platform for gene delivery for any of the following purposes: inducing an adaptive immune response against an infectious disease, inducing an adaptive immune response for cancer treatment, inducing an adaptive immune response for allergy treatment, for the treatment of an autoimmune disease, or for gene therapy.

[0016] In one exemplary embodiment, using the nanocomposite as a DNA vaccine platform for gene delivery to induce an adaptive immune response for cancer treatment may include targeted gene delivery to tumor tissue. In another exemplary embodiment, administering the nanocomposite to living target cells may include targeted gene repression of any target gene by at least one of the following: microRNA, siRNA, DNAi, peptide nucleic acid, or any combination thereof.

[0017] In one general aspect, this disclosure describes a series of nanocomposites for diagnostic and / or therapeutic uses. In one exemplary embodiment, each of the plurality of nanocomposites may comprise a drug and at least one chimeric peptide linked to the drug. In one exemplary embodiment, the drug may comprise at least one of the following: nucleic acid, protein, lipid, polymer, carbohydrate, chemical compound, nanoparticle, microsphere containing a diagnostic or therapeutic agent, or any combination thereof; wherein the chimeric peptide comprises an endosome escape motif, a nuclear entry motif, and a DNA condensation motif, and these three may be linked in any order.

[0018] In one exemplary embodiment, the plurality of nanocomposites may include a DNA vaccine nanoassembly platform capable of inducing a specific immune response against an antigenic peptide encoded by an expression vector. In one exemplary embodiment, the drug may include nucleic acids, such as DNA or RNA. In another exemplary embodiment, the connection between the chimeric peptide and the nucleic acid may be non-covalent, including electrostatic interactions; the size of the nanoassembly structure is tunable from 20 nm to 2000 nm.

[0019] In one exemplary embodiment, the chimeric peptide may include MIRGD, CTAMPG2H, or derivatives thereof. In one exemplary embodiment, the MIRGD may contain an amino acid sequence that is completely identical to the amino acid sequence of SEQ ID NO. 2. In another exemplary embodiment, the CTAMPG2H may contain an amino acid sequence that is completely identical to the amino acid sequence of SEQ ID NO. 3.

[0020] In one exemplary embodiment, the MIRGD may comprise: an endosome escape motif whose amino acid sequence is identical to that of SEQ ID NO. 5; a nuclear entry motif whose amino acid sequence is identical to that of SEQ ID NO. 6; a DNA condensation motif whose amino acid sequence is identical to that of SEQ ID NO. 7; and a targeting motif whose amino acid sequence is identical to that of SEQ ID NO. 8.

[0021] In one exemplary embodiment, the CTAGMPG2H may comprise: a cell penetration motif whose amino acid sequence is identical to that of SEQ ID NO. 9; an endosome escape motif whose amino acid sequence is identical to that of SEQ ID NO. 5; a nucleus entry motif whose amino acid sequence is identical to that of SEQ ID NO. 6; and a DNA condensation motif whose amino acid sequence is identical to that of SEQ ID NO. 7.

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

[0023] In one general aspect, this disclosure describes a method for administering a nanocomposite for diagnostic and / or therapeutic purposes. In one exemplary embodiment, the method may include preparing the nanocomposite and delivering it into a living organism via intramuscular injection. In one exemplary embodiment, the preparation of the nanocomposite may include: generating a plurality of MIRGD peptides by transfecting at least one MIRGD peptide into bacteria, wherein the amino acid sequence of said MIRGD peptide is identical to the amino acid sequence of SEQ ID NO. 2; subsequently purifying the plurality of MIRGD peptides using Ni-NTA agarose affinity chromatography; and further linking a drug to at least one of the plurality of MIRGD peptides. In one exemplary embodiment, the drug may include at least one of the following: nucleic acids, proteins, lipids, polymers, carbohydrates, chemical compounds, nanoparticles, microspheres containing diagnostic or therapeutic agents, or any combination thereof. Attached Figure Description

[0024] The accompanying drawings and data described herein are for illustrative purposes only and do not cover all possible implementations, nor are they intended to limit the scope of this disclosure. In the drawings, the same or similar elements are designated with the same reference numerals.

[0025] Figure 1 The arrangement of motifs in an exemplary chimeric peptide is shown, which serves as a key component of an exemplary peptide-based DNA vaccine and is consistent with one or more exemplary embodiments of this disclosure.

[0026] Figure 2Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) images and Western blotting results are shown to demonstrate the expression and purification of MIRGD and CTATMPG2H, consistent with one or more exemplary embodiments of this disclosure.

[0027] Figure 3A A circular dichroism spectroscopy diagram is shown to illustrate exemplary secondary structures of MIRGD and CTATMPG2H, consistent with one or more exemplary embodiments of this disclosure.

[0028] Figure 3B A circular dichroism spectral reference diagram of the protein secondary structure is shown, consistent with one or more exemplary embodiments of this disclosure.

[0029] Figure 4 Agarose gel electrophoresis images are shown to demonstrate the formation of nanocomplexes used as peptide-based DNA vaccines, consistent with one or more exemplary embodiments of this disclosure.

[0030] Figure 5 Agarose gel electrophoresis images are shown to demonstrate the stability of the nanocomposite used as a peptide-based DNA vaccine under different time and temperature conditions, consistent with one or more exemplary embodiments of this disclosure.

[0031] Figure 6 A diagram illustrating the hydrodynamic particle size and zeta potential of a nanocomposite for a peptide-based DNA vaccine is shown, consistent with one or more exemplary embodiments of this disclosure.

[0032] Figure 7 Agarose gel electrophoresis images are shown to demonstrate the stability of the nanocomposite used as a peptide-based DNA vaccine, consistent with one or more exemplary embodiments of this disclosure.

[0033] Figure 8 Transmission electron microscopy (TEM) images are shown to demonstrate the formation of the nanocomposite and to show the morphology and size of the nanocomposite used as a peptide-based DNA vaccine, consistent with one or more exemplary embodiments of this disclosure.

[0034] Figure 9AFluorescence microscopy images are shown to demonstrate the transfection of HEK293T cells using a nanocomposite containing CTATMPG2H as a chimeric peptide carrier and pDB2 plasmid as a reporter plasmid. These results demonstrate the effectiveness of in vitro gene delivery using the exemplary platform (peptide-based DNA vaccine) of this disclosure, consistent with one or more exemplary embodiments of this disclosure.

[0035] Figure 9B Fluorescence microscopy images are shown to demonstrate the transfection of HEK293T cells using an exemplary nanocomposite containing MIRGD as a chimeric peptide carrier and pDB2 plasmid as a reporter plasmid. These results demonstrate the effectiveness of in vitro gene delivery using the exemplary platform (peptide-based DNA vaccine) of this disclosure, consistent with one or more exemplary embodiments of this disclosure.

[0036] Figure 9C Fluorescence microscopy images are shown to demonstrate the transfection of HEK293T cells with a polyethyleneimine (PEI) complex (where PEI is used as a vector and pDB2 plasmid is used as a reporter plasmid), which serves as a positive control and is consistent with one or more exemplary embodiments of this disclosure.

[0037] Figure 10 A graph is shown to illustrate the transfection rate of HEK293T cells using an exemplary nanocomposite containing CTATMPG2H and MIRGD as chimeric peptide carriers and psiCHECK plasmid as a reporter plasmid. This result demonstrates the effectiveness of in vitro gene delivery using the exemplary platform (peptide-based DNA vaccine) of this disclosure, consistent with one or more exemplary embodiments of this disclosure.

[0038] Figure 11 A graph is shown to illustrate the results of humoral immune response assessments during in vivo gene delivery using the exemplary platform (peptide-based DNA vaccine) of this disclosure, consistent with one or more exemplary embodiments of this disclosure.

[0039] Figure 12A Images of hematoxylin-eosin (H&E) stained sections of mouse lungs, heart, and spleen are shown, consistent with one or more exemplary embodiments of this disclosure.

[0040] Figure 12B Images of hematoxylin-eosin (H&E) stained sections of mouse kidneys and livers are shown, consistent with one or more exemplary embodiments of this disclosure.

[0041] Figure 13A table is shown to describe optimized biosynthetic conditions for CTATMPG2H and MIRGD, consistent with one or more exemplary embodiments of this disclosure.

[0042] Figure 14 A table describing an optimized purification process for CTATMPG2H and MIRGD is shown, consistent with one or more exemplary embodiments of this disclosure.

[0043] Figure 15 A table showing the results of virus neutralization experiments demonstrates the functionality of antibodies detected in the serum of animal models injected with the nanocomposite, consistent with one or more exemplary embodiments of this disclosure.

[0044] Figure 16 A schematic diagram of an exemplary peptide-based DNA nanoassembly platform is shown, consistent with one or more exemplary embodiments of this disclosure. Detailed Implementation

[0045] In the following detailed description, numerous specific details are illustrated by way of examples to provide a more comprehensive understanding of the relevant technical content of this disclosure. However, it should be understood that the technical solutions of this disclosure are not necessarily implemented only with all of these specific details. In other instances, to avoid unnecessarily obscuring certain aspects of this disclosure, well-known methods, steps, components, and / or circuits are described at a higher level without elaboration.

[0046] The novel features of this disclosure in terms of its structure, organization, use, and method of operation, as well as its further purposes and advantages, will be more fully understood through the following discussion. In the detailed description below, numerous specific details are set forth by way of example to provide a more comprehensive understanding of the relevant technical content of this disclosure. However, it should be understood that the technical solutions of this disclosure are not necessarily implemented only with all these specific details. In other instances, to avoid unnecessarily obscuring certain aspects of this disclosure, well-known methods, steps, components, and / or circuits are described at a higher level without elaboration. The following detailed description is intended to enable those skilled in the art to manufacture and use the methods and apparatus disclosed in the exemplary embodiments of this disclosure. For ease of explanation, specific terminology is used herein to provide a more comprehensive understanding of this disclosure. However, those skilled in the art will understand that these specific details are not necessary for implementing the exemplary embodiments of this disclosure. The description of specific exemplary embodiments is merely representative examples. Those skilled in the art will readily recognize that various modifications can be made to these exemplary embodiments, and that the general principles defined herein can be applied to other embodiments and applications without departing from the scope of this disclosure. This disclosure is not intended to be limited to the embodiments shown, but should be given the broadest possible scope, as long as it still conforms to the principles and features disclosed herein.

[0047] This disclosure aims to provide exemplary embodiments of DNA vaccines against COVID-19. In one exemplary embodiment, the DNA vaccine may comprise at least one chimeric peptide and one drug. In one exemplary embodiment, the at least one chimeric peptide may bind to the drug via a linkage. In one exemplary embodiment, the chimeric peptide may comprise at least one endosome escape motif, one nuclear entry motif, one DNA condensation motif, or any combination thereof. In one exemplary embodiment, the endosome escape motif, the nuclear entry motif, and the DNA condensation motif may be linked to each other in any order. In one exemplary embodiment, the chimeric peptide may comprise MIRGD and CTATMPG2H. In one exemplary embodiment, the drug may comprise at least nucleic acids, proteins, lipids, polymers, carbohydrates, chemical compounds, nanoparticles, microspheres containing diagnostic or therapeutic agents, or any combination thereof.

[0048] In one exemplary embodiment, MIRGDs can be developed and used to deliver drugs to cultured cells and cells in vivo. In one exemplary embodiment, the drug may include, but is not limited to, genes. In one exemplary embodiment, the MIRGD can form a nanocomposite after being linked with a drug. In one exemplary embodiment, the nanocomposite can be used to deliver drugs to cultured cells and cells in vivo. In one exemplary embodiment, a MIRGD may contain four motifs. In one exemplary embodiment, each motif of the MIRGD performs a specific function.

[0049] In one exemplary embodiment, the four motifs may include: an endosome escape motif, a nuclear entry motif, a DNA condensation motif, and a targeting motif. In one exemplary embodiment, the endosome escape motif may include HIV gp41. In one exemplary embodiment, the amino acid sequence of HIV gp41 may be similar to the amino acid sequence of SEQ ID NO. 5. In one exemplary embodiment, the endosome escape motif may confer the ability of the nanocomplex to escape endosomes, thereby protecting the target gene. In another exemplary embodiment, the endosome escape motif may facilitate subsequent steps required for target gene expression.

[0050] In one exemplary embodiment, the nuclear entry motif may include an NLS (SV40 large T-antigen). In one exemplary embodiment, the NLS (SV40 large T-antigen) may have an amino acid sequence similar to that of SEQ ID NO. 6. In one exemplary embodiment, the nuclear entry motif may be responsible for enhancing the transport of pDNA in the cytoplasm. In another exemplary embodiment, the nuclear entry motif may facilitate the entry of a target gene into the nucleus, a step essential for gene expression and the induction of an effective immune response.

[0051] In one exemplary embodiment, the DNA condensation motif may include 2Histon1. In one exemplary embodiment, 2Histon1 may have an amino acid sequence similar to that of SEQ ID NO. 7. In one exemplary embodiment, the DNA condensation motif may be responsible for binding to DNA to form a nanocomplex capable of protecting a target gene. In another exemplary embodiment, the DNA condensation motif may also facilitate gene delivery.

[0052] In one exemplary embodiment, the targeting motif may include an iRGD. In one exemplary embodiment, the iRGD may have an amino acid sequence similar to that of SEQ ID NO. 8. In one exemplary embodiment, the targeting motif can achieve targeting of target cells by recognizing multiple cell surface markers. In one exemplary embodiment, the iRGD may target αvβ3, α5β1, and αIIbβ3 integrins. Other additional or alternative motifs may also be used in the exemplary chimeric peptides of this disclosure.

[0053] In one exemplary implementation, the MIRGD may comprise four motifs. Figure 1 The arrangement of the four motifs in MIRGD is shown, consistent with one or more exemplary embodiments of this disclosure. In one exemplary embodiment, the order of the four motifs in MIRGD may be: endosome escape motif, nuclear entry motif, DNA condensation motif, and targeting motif.

[0054] In one exemplary embodiment, the chimeric peptide may be represented as Gp41-NLS-2Histon1-iRGD.

[0055] Alternatively, in one exemplary embodiment, the four motifs of the MIRGD chimeric peptide may be arranged differently from those of the other two. Figure 1 Arrange them in the order shown. Figure 1 The diagram illustrates the motif sequence in exemplary chimeric peptides, as a key component of exemplary peptide-based DNA vaccines, consistent with one or more exemplary embodiments of this disclosure. In one exemplary embodiment, MIRGD analogs with motifs arranged in different orders can be prepared. In another exemplary embodiment, the gene delivery performance of these MIRGD analogs with different motif sequences can be evaluated in exemplary nanocomposites. In one exemplary embodiment, changing the motif sequence of MIRGD may result in a change in gene delivery rate. In another exemplary embodiment, changing the motif sequence may decrease or increase the gene delivery rate. In one exemplary embodiment, all such chimeric peptides and the nanocomposites formed therefrom are considered to be within the scope of protection of this disclosure. In one exemplary embodiment, schematic diagram 102 shows SEQ ID NO.1. In one exemplary embodiment, schematic diagram 102 shows the amino acid sequence of MPG2H. In one exemplary embodiment, schematic diagram 104 shows SEQ ID NO.2 and illustrates the amino acid sequence of MIRGD. In one exemplary embodiment, schematic diagram 106 shows the amino acid sequence of CTATMPG2H and corresponds to SEQ ID NO.3.

[0056] In one exemplary embodiment, the MIRGD chimeric peptide may have an amino acid sequence similar to that of SEQ ID NO. 2. In one exemplary embodiment, the molecular weight of the MIRGD chimeric peptide may be 9682.39. In one exemplary embodiment, the isoelectric point of the MIRGD chimeric peptide may be 10.93. In one exemplary embodiment, the MIRGD chimeric peptide may consist of 91 amino acids.

[0057] In one exemplary embodiment, the chimeric peptide may be designed to contain 2 to 8 motifs. In one exemplary embodiment, the number of motifs in the chimeric peptide may result in different gene delivery efficiencies. In one exemplary embodiment, the MIRGD structure exhibits optimal gene delivery efficiency, whether used in vitro, in vivo, or as a DNA vaccine. In one exemplary embodiment, MIRGD derivatives can be developed by adding a repeating unit to each motif. In one exemplary embodiment, MIRGD chimeric peptide derivatives within the scope of this disclosure may have an amino acid sequence similar to SEQ ID NO. 2, wherein one or two amino acids may be deleted, replaced with other amino acids, or one or two amino acids may be added. In one exemplary embodiment, MIRGD derivatives can be developed by introducing different linker peptide sequences between functional motifs. In another exemplary embodiment, MIRGD derivatives can be developed by adding tag motifs for purification procedures.

[0058] In one exemplary embodiment, MIRGD derivatives can be developed by replacing the HIV gp41 motif with other endosome escape motifs. In one exemplary embodiment, MIRGD derivatives can be developed by replacing the NLS (SV40 large T-antigen) motif with other nuclear entry motifs. In one exemplary embodiment, MIRGD derivatives can be developed by replacing the 2Histon1 motif with other DNA condensation motifs. In one exemplary embodiment, MIRGD derivatives can be developed by replacing the iRGD motif with other targeting motifs. In one exemplary embodiment, MIRGD derivatives can be developed by adding other endosome escape motifs in addition to the HIV gp41 motif. In one exemplary embodiment, MIRGD derivatives can be developed by adding other nuclear entry motifs in addition to the NLS (SV40 large T-antigen) motif. In one exemplary embodiment, MIRGD derivatives can be developed by adding other DNA condensation motifs in addition to the 2Histon1 motif. In one exemplary embodiment, MIRGD derivatives can be developed by adding other targeting motifs in addition to the iRGD motif. In one exemplary embodiment, MIRGD derivatives can be developed by adding other functional motifs. In one exemplary embodiment, MIRGD derivatives can be developed by deleting a motif. In another exemplary embodiment, MIRGD derivatives can be developed by deleting multiple motifs.

[0059] In another exemplary embodiment, a chimeric peptide labeled CTATMPG2H can be developed and used in the exemplary methods of this disclosure for delivering drugs (including genes) to cultured cells and in vivo cells. When CTATMPG2H is linked to a drug, a nanocomplex can be formed, thereby enabling drug delivery to cultured cells in vitro and in vivo cells. CTATMPG2H may contain four motifs, each responsible for a specific function. The motifs can be described in detail in the following order. In one exemplary embodiment, the four motifs of CTATMPG2H may include: a cell penetration motif, an endosome escape motif, a nuclear entry motif, and a DNA condensation motif.

[0060] In one exemplary embodiment, the cell penetration motif may include a TAT. In one exemplary embodiment, the TAT may have an amino acid sequence similar to that of SEQ ID NO. 9. In one exemplary embodiment, to enhance platform functionality, an additional cell entry pathway may be formed through this motif in addition to the endosomal pathway. In one exemplary embodiment, it has been reported that the CTAT motif also facilitates endosome escape.

[0061] In one exemplary embodiment, the endosome escape motif may include HIV gp41. In one exemplary embodiment, HIV gp41 may have an amino acid sequence similar to that of SEQ ID NO. 5. In one exemplary embodiment, the endosome escape motif may endow the platform with the ability to escape endosomes, thereby protecting the target gene and facilitating subsequent processes required for target gene expression. In one exemplary embodiment, the nuclear entry motif may include NLS (SV40large T-antigen). In one exemplary embodiment, NLS may have an amino acid sequence similar to that of SEQ ID NO. 6. In one exemplary embodiment, NLS may enhance the transport of pDNA in the cytoplasm. In another exemplary embodiment, NLS may facilitate the entry of the target gene into the nucleus, a step necessary for gene expression and the induction of an effective immune response.

[0062] In one exemplary embodiment, the DNA condensation motif may include 2Histon1. In one exemplary embodiment, 2Histon1 may have an amino acid sequence similar to that of SEQ ID NO. 7. In one exemplary embodiment, 2Histon1 may be responsible for binding to DNA to form a nanocomplex capable of protecting a target gene. In another exemplary embodiment, 2Histon1 may also facilitate gene delivery.

[0063] In one exemplary embodiment, CTATMPG2H may have an amino acid sequence similar to that of SEQ ID NO.3. In one exemplary embodiment, CTATMPG2H may contain four motifs, the order of which can be referenced. Figure 1 Other exemplary chimeric peptides with altered motif sequences are also considered to fall within the scope of this disclosure. In other embodiments, the chimeric peptide may include variants of the CTATMPG2H amino acid sequence, wherein one or two amino acids may be deleted, replaced with other amino acids, or one or two amino acids may be added.

[0064] In one exemplary embodiment, the CTATMPG2H chimeric peptide may have a molecular weight of 11589.67, an isoelectric point of 11.85, and consist of 103 amino acids. In one exemplary embodiment, CTATMPG2H exhibits non-toxicity in cell culture. A list of other functional motifs is provided herein to better illustrate the technical concepts of this disclosure.

[0065] In one exemplary embodiment, alternatives to the DNA condensation motif may include any amino acid sequence capable of causing nucleotide sequences to condense to a certain extent. In one exemplary embodiment, this capability of the nanocomplex can be measured by a slowdown in the migration rate of the nucleotide sequence during electrophoresis. In one exemplary embodiment, the DNA condensation motif may also be responsible for protecting the nucleotide sequence from degradation by serum nucleases and other harmful factors before it reaches the target cell. In one exemplary embodiment, alternatives to the DNA condensation motif may include: Mu peptide having the amino acid sequence of SEQ ID NO. 10, poly-L-lysine, poly-L-arginine, whose amino acid sequence may be represented as (RRXRRXHHXHHX)n (where X is any amino acid other than D and E), TAT peptide having SEQ ID NO. 11, Tyr-TAT having SEQ ID NO. 12, poly-TAT, histone H2B, H1.4F, and H1.

[0066] In one exemplary embodiment, alternatives to the endosome escape motif may include any amino acid sequence capable of disrupting the endosome membrane to some extent. In one exemplary embodiment, endosome membrane disruption may originate from various mechanisms, including the proton sponge effect, alterations in secondary structure, etc. In one exemplary embodiment, alternatives to the endosome escape motif may include the following peptides: •GALA peptide (SEQ ID NO.13). •KALA peptide (SEQ ID NO.14) • RALA peptide (SEQ ID NO.15) •Inf-7 (SEQ ID NO.16) • H5WYG peptide (SEQ ID NO.17).

[0067] In one exemplary embodiment, alternatives to the nuclear entry motif (NLS) may include any amino acid sequence capable of transporting a nucleotide sequence to the cell nucleus. In one exemplary embodiment, alternatives to the nuclear entry motif may include: •HIV Rev protein (SEQ ID NO.18), • The M9 peptide derived from the hnRNP protein.

[0068] In one exemplary embodiment, alternatives to the targeting motif may include any amino acid sequence capable of recognizing intracellular or extracellular receptors or markers. In one exemplary embodiment, the targeting motif may include FGF growth factor or scFv anti-ERbB2 single-chain antibody fragments.

[0069] In one exemplary embodiment, other functional motifs may include any amino acid sequence responsible for a specific function that can be added to a chimeric peptide sequence. For example, the CTAT peptide may serve as a functional motif capable of penetrating the cell membrane and providing an additional cell entry pathway beyond the endosome-mediated cell entry pathway.

[0070] In one exemplary embodiment, the chimeric peptide may be linked to a drug or drug molecule to form a nanocomposite. In one exemplary embodiment, when the drug is present in the form of a drug molecule, it may be selected from the following types: nucleic acid, protein, lipid, polymer, carbohydrate, chemical compound, or any combination thereof.

[0071] In embodiments where the drug is a nucleic acid, the nucleic acid may be selected without limitation from the following types: DNA, RNA, siRNA, microRNA, antisense RNA, catalytic RNA, or catalytic DNA. In one exemplary embodiment, the drug may also be nanoparticles or microspheres containing diagnostic or therapeutic agents. In another exemplary embodiment, the drug may be a detectable compound. In one exemplary embodiment, a detectable compound refers to any labeled compound known in the art, examples of which include: fluorophores, fluorophore-coupled compounds, bioluminescent compounds, bioluminescent conjugates, chemiluminescent compounds, chemiluminescent conjugates, or enzyme substrates capable of reacting with enzymes to generate a detectable signal.

[0072] In other embodiments, the drug may be a nucleic acid. In one exemplary embodiment, an exemplary nucleic acid drug may be a 5047 bp vector encoding GFP as a reporter gene, referred to as pDB2. In one exemplary embodiment, the pDB2 vector can be used to assess gene transfer efficiency in cell culture. In another exemplary embodiment, another exemplary nucleic acid may be a 6273 bp vector encoding Luciferase as a reporter gene, referred to as psiCHECK™2. In one exemplary embodiment, this 6273 bp vector can be used to assess gene transfer efficiency in cell culture. In another 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 some embodiments, this RBD-encoded vector can be used to assess gene transfer efficiency in vivo while inducing an immune response.

[0073] In one exemplary embodiment, a nanocomplex (platform) can be formed when the chimeric peptide is linked to a drug via covalent or non-covalent interactions. In one exemplary embodiment, when the nanocomplex is linked to a drug via covalent interactions, one or more linker molecules can be selectively used to achieve the connection, methods known in the art for this purpose. In other embodiments, a nanocomplex can also be formed when the chimeric peptide is linked to a target gene via non-covalent interactions, for example, through electrostatic interactions between the positive charge of the chimeric peptide and the negative charge of the nucleic acid. Figure 16 As shown. Figure 16 A schematic diagram of a peptide-based DNA nanoassembly platform is shown. Chimeric peptides and nucleic acids bind through non-covalent interactions to form nanocomposites with unique characteristics, consistent with one or more exemplary embodiments of this disclosure. Figure 16 As shown: Component 1602 is a chimeric peptide; Component 1604 is a nucleic acid (expression vector); Component 1606 is a nanocomposite.

[0074] In one exemplary embodiment, chimeric peptides and nucleic acids can be linked through non-covalent interactions to form nanocomposites with unique properties.

[0075] In one exemplary embodiment, representative exemplary nanocomposites according to this disclosure may include: a nanocomposite with MIRGD as a chimeric peptide and pDB2 as a nucleic acid carrier; a nanocomposite with MIRGD as a chimeric peptide and psiCHECK(TM)2 as a nucleic acid carrier; a nanocomposite with MIRGD as a chimeric peptide and RBD-encoded carrier as a nucleic acid carrier; a nanocomposite with CTATMPG2H as a chimeric peptide and pDB2 as a nucleic acid carrier; a nanocomposite with CTATMPG2H as a chimeric peptide and psiCHECK(TM)2 as a nucleic acid carrier; and a nanocomposite with CTATMPG2H as a chimeric peptide and RBD-encoded carrier as a nucleic acid carrier.

[0076] In one exemplary embodiment, the exemplary currently disclosed nanocomposites typically exhibit certain physical, structural, and reactive properties. In one exemplary embodiment, for example, the nanocomposites typically have a zeta potential in the range of +1 to +20. In one exemplary embodiment, the nanocomposites typically have a size range of 50 nm to 1000 nm. In some embodiments, the exemplary nanocomposites are stable against serum nucleases. In some embodiments, the exemplary nanocomposites remain stable over a time range of 15 minutes to 24 hours. In some embodiments, the exemplary nanocomposites remain stable in a range of 15°C to 37°C. In some embodiments, the exemplary nanocomposites are stable for up to 24 hours in a range of 15°C to 37°C.

[0077] In one exemplary embodiment, the exemplary nanocomposite may include MIRGD / pDB2. In one exemplary embodiment, MIRGD / pDB2 may exhibit higher gene transfer efficiency in cell culture, showing advantages over PEI as a gold standard gene carrier. In one exemplary embodiment, the exemplary nanocomposite may include MIRGD / psiCHECK(TM)2. In one exemplary embodiment, MIRGD / psiCHECK(TM)2 may exhibit higher gene transfer efficiency in cell culture, showing advantages over PEI as a gold standard gene carrier. In one exemplary embodiment, the exemplary nanocomposite may include CTATMPG2H / pDB2. In one exemplary embodiment, CTATMPG2H / pDB2 may exhibit higher gene transfer efficiency in cell culture, showing advantages over PEI as a gold standard gene carrier. In one exemplary embodiment, the exemplary nanocomposite may include CTATMPG2H / psiCHECK(TM)2. In one exemplary embodiment, CTATMPG2H / psiCHECK(TM)2 may exhibit higher gene transfer efficiency in cell culture, showing advantages over PEI as a gold standard gene carrier. In one exemplary embodiment, the exemplary nanocomposite may include a MIRGD / RBD-encoded vector that can exhibit high gene transfer efficiency in vivo, achieved by inducing a favorable immune response against COVID-19. In one exemplary embodiment, the exemplary nanocomposite may include a MIRGD / RBD-encoded vector that does not exhibit cytotoxicity in vivo.

[0078] In one exemplary embodiment, the exemplary technology of this disclosure can provide a method for delivering a drug into cells in a cell culture and into cells in vivo, including administering a nanocomplex to the cells, the nanocomplex comprising a chimeric peptide linked to an exemplary drug. The technology can provide a method for selectively delivering a drug into cells in a cell culture and into cells in vivo, including administering a nanocomplex comprising a chimeric peptide linked to an exemplary drug to exemplary cells. The technology can provide a method for inducing humoral and cellular immunity in subjects at risk of disease, including administering a nanocomplex comprising a chimeric peptide linked to an exemplary target gene encoding vector to an exemplary subject, wherein sufficient levels of gene transfer in vivo will result in the expression of a target protein, which can induce an immune response. The technology can provide a method for inducing humoral and cellular immunity in subjects at risk of infectious disease, including administering a nanocomplex comprising a chimeric peptide linked to an exemplary target gene encoding vector to an exemplary subject, wherein sufficient levels of gene transfer in vivo will result in the expression of a target protein, which can induce an immune response.

[0079] This technology can provide a method for inducing memory immunity in subjects at risk of infectious diseases, including administering a nanocomposite containing a chimeric peptide linked to an exemplary target gene-encoding vector to an exemplary subject, wherein sufficient levels of gene transfer in vivo will result in the expression of a target protein, which can induce an immune response. This technology can provide a method for treating patients with infectious diseases, including administering a therapeutic amount of a nanocomposite containing a chimeric peptide linked to an exemplary target gene-encoding vector to a subject, wherein sufficient levels of gene transfer in vivo will result in the expression of a target protein, which can induce an immune response. This technology can provide a method for inducing humoral and cellular immunity in subjects at risk of COVID-19, including administering a nanocomposite containing a chimeric peptide linked to an RBD-encoding vector to an exemplary subject, wherein sufficient levels of gene transfer in vivo will result in the expression of the RBD, which can induce an immune response.

[0080] In one exemplary embodiment, the exemplary chimeric peptides generally described above can be used in any of the purposes and applications currently described. In one exemplary embodiment, without limitation, the class of exemplary chimeric peptides may include MIRGD, CTATMPG2H as described above, as well as analogues and derivatives of MIRGD and CTATMPG2H.

[0081] In one exemplary embodiment, a non-limiting example of a gene-coding vector may be an RBD-coding vector. In one exemplary embodiment, a non-limiting example of a drug may be a nucleic acid vector. In one exemplary embodiment, a nucleic acid vector may include psiCHECK™ 2, pDB2, and an RBD-coding vector. In one exemplary embodiment, a non-limiting example of a cell culture may be HEK293T. In one exemplary embodiment, a non-limiting example of a disease may be cancer and infectious diseases such as COVID-19. In one exemplary embodiment, a non-limiting example of a test subject may be a human test subject or a non-human test subject, such as a primate, companion animal, laboratory animal, or farm animal.

[0082] In one exemplary embodiment, this disclosure may include methods, materials, and apparatus for gene delivery. The technology may also include methods, materials, and apparatus for in vivo gene delivery. In particular, this disclosure may relate to the in vivo delivery of a target DNA to specific cells based on a safe vector to induce a favorable immune response against a target disease. In one exemplary embodiment, the specific cells may include immune cells.

[0083] In this disclosure, a designed vector comprising a DNA sequence encoding a target antigen can be delivered in vivo via a designed, biosynthesized, and characterized peptide-based platform. In one exemplary embodiment, the designed vector containing the DNA sequence can induce a favorable immune response, thereby being considered a DNA vaccine platform. In this disclosure, a peptide-based DNA vaccine can generally be described as a nanocomposite comprising a chimeric peptide and a vector containing an exemplary target gene DNA sequence.

[0084] This disclosure relates to methods, materials, and apparatus for designing, biosynthesizing, and characterizing an exemplary multifunctional chimeric peptide, which is a key component of an exemplary peptide-based DNA vaccine. In one exemplary embodiment, the peptide is designed to achieve safe gene delivery. In one exemplary embodiment, the exemplary design may be based on functional domains, each domain serving a specific purpose and arranged in an order that preserves the function of each domain. In one exemplary embodiment, the exemplary domains may be selected and designed based on key challenges faced in the fields of gene delivery and DNA vaccine research. In one exemplary embodiment, each exemplary domain may serve one function. This disclosure relates to methods, materials, and apparatus for designing plasmids containing an exemplary target gene DNA sequence, which is another key component of an exemplary peptide-based DNA vaccine. This disclosure also relates to methods, materials, and apparatus for designing, developing, and characterizing a gene delivery platform (nanocomplex) comprising an exemplary chimeric peptide and a plasmid containing an exemplary target DNA sequence.

[0085] In one exemplary embodiment, the exemplary platform can be rapidly redesigned to achieve gene transfer at different rates for different cell lines and organs, thereby serving as a gene delivery platform for DNA vaccines, cancer vaccines, and gene therapies. In one embodiment of this disclosure, materials and methods are provided for designing, biosynthesizing, and purifying a chimeric peptide called MIRGD, which is a key component of an exemplary peptide-based DNA vaccine. In one embodiment of this disclosure, materials and methods are provided for designing, biosynthesizing, and purifying a chimeric peptide called CTATMPG2H, which is a key component of an exemplary peptide-based DNA vaccine. In another embodiment of this disclosure, materials, methods, and exemplary results are provided for characterizing exemplary chimeric peptides called MIRGD and CTATMPG2H, which are key components of an exemplary peptide-based DNA vaccine. In another embodiment of this disclosure, exemplary materials, methods, and exemplary results are provided for developing target antigen expression plasmids, which are key components of an exemplary peptide-based DNA vaccine. Figure 2 Images of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot are shown to demonstrate the expression and purification of MIRGD and CTATMPG2H, consistent with one or more exemplary embodiments of this disclosure. In one exemplary embodiment, image 202 shows a Western blot image containing MIRGD, a protein gradient, and CTATMPG2H. In one exemplary embodiment, image 204 shows an SDS-PAGE image containing MIRGD, a protein gradient, and CTATMPG2H.

[0086] In another embodiment of this disclosure, exemplary materials, methods, and results for the preparation and characterization of nanocomposites introduced as an exemplary peptide-based DNA vaccine platform are provided. In another embodiment of this disclosure, exemplary materials, methods, and results for in vitro gene delivery using this exemplary platform (peptide-based DNA vaccine) are provided.

[0087] In another embodiment of this disclosure, exemplary materials and methods for in vivo gene delivery using an exemplary platform (peptide-based DNA vaccine) are provided. In one exemplary embodiment, the exemplary platform can be used for immunization of a mouse model.

[0088] In another embodiment of this disclosure, exemplary materials, methods, and results may be provided for evaluating exemplary immune responses resulting from in vivo gene delivery using an exemplary platform. In one exemplary embodiment, the exemplary materials and methods may be used to evaluate humoral immune responses in animal models to the exemplary platform.

[0089] In another embodiment, exemplary materials, methods, and results may be provided for evaluating the safety of in vivo gene delivery using an exemplary platform. In one exemplary embodiment, the exemplary materials and methods may be used to evaluate the cytotoxicity of the exemplary platform in animal models.

[0090] In one exemplary embodiment, a nanoassembly (peptideticles) can be designed to recognize the "zip code" characteristics of tumor blood vessels, enter tumor tissue, and target the tumor microenvironment for gene delivery. In one exemplary embodiment, exemplary key functional domains can be genetically engineered to simultaneously embed into two main peptides, MIRGD and HNH. In one exemplary embodiment, the exemplary MIRGD, as the first peptide, can constitute the core of the nanoassembly. In one exemplary embodiment, the exemplary HNH, as the second peptide, can encapsulate the exemplary nanoassembly. In one exemplary embodiment, the exemplary MIRGD can include functional elements such as DNA aggregation, endosome escape, nuclear delivery, and tissue penetration. In one exemplary embodiment, the exemplary HNH can include DNA binding and pH-responsive elements. In one exemplary embodiment, the exemplary MIRGD and the exemplary HNH peptide can form highly stable peptideticles in the presence of DNA. These peptideticles can successfully recognize tumor "zip codes," penetrate tumor spheroids under environmental pH regulation, and initiate exemplary cellular uptake, subsequently inducing high levels of gene expression. In one exemplary embodiment, the exemplary peptideticles can efficiently accumulate in tumor tissue while avoiding aggregation in healthy tissue. In one exemplary implementation, exemplary peptideticles can efficiently deliver luciferase genes to tumor tissues by overcoming various challenges under physiological and cellular conditions.

[0091] In one exemplary implementation, a gene delivery platform may face a number of exemplary challenges. Exemplary conventional gene delivery platforms may each have different drawbacks. Exemplary drawbacks that may be common among exemplary conventional platforms may include the lack of a safe, size-adjustable, functionally tunable, organ-targetable, cell-type-targetable, stable, and non-toxic vector that can achieve good delivery efficiency in vivo.

[0092] In one exemplary embodiment, this disclosure relates to methods, materials, and apparatus for designing, biosynthesizing, and characterizing a multifunctional chimeric peptide as a carrier, which addresses, one by one, the exemplary common drawbacks of the aforementioned conventional gene delivery platforms. In one exemplary embodiment, this disclosure relates to methods, materials, and apparatus for designing, developing, and characterizing a gene delivery platform (nanocomplex) comprising an exemplary chimeric peptide and a plasmid containing a target DNA sequence, addressing, one by one, the exemplary common drawbacks of the aforementioned conventional gene delivery platforms. In one exemplary embodiment, features of this disclosure provide targeted solutions to each of the mentioned exemplary drawbacks, and are described in detail in the accompanying drawings.

[0093] In one exemplary embodiment, the safety of the exemplary vector may primarily stem from the fact that the exemplary vector is likely constructed based on peptides and employs an exemplary nature-inspired domain design. In one exemplary embodiment, the exemplary nature-inspired domain design implies that each domain has been utilized in different cellular pathways. In one exemplary embodiment, the safety of the exemplary vector based on chimeric peptides can be assessed through a variety of tests, some of which are conducted in… Figure 9A , Figure 9B , Figure 12A and Figure 12B As shown in the study, the results indicate that it has a high cell viability in cell culture (which can be assessed by imaging) and does not exhibit cytotoxicity in vivo (organs were harvested on day 30 for H&E staining and analysis). Figure 9A Fluorescence microscopy images of HEK293T cells transfected with a nanocomposite containing CTAMPG2H as a chimeric peptide carrier and pDB2 as a reporter plasmid are shown, demonstrating the results of in vitro gene delivery using this platform (peptide-based DNA vaccine), consistent with one or more exemplary embodiments of this disclosure. Figure 9B Fluorescence microscopy images of HEK293T cells transfected with a nanocomposite containing MIRGD as a chimeric peptide carrier and pDB2 as a reporter plasmid are shown, demonstrating the results of in vitro gene delivery using this platform (peptide-based DNA vaccine), consistent with one or more exemplary embodiments of this disclosure.

[0094] In one exemplary embodiment, the size of the support and the nanocomposite can be easily adjusted by adding or omitting different structural domains. Furthermore, size tunability can also be achieved by adjusting the nanocomposite formation conditions (e.g., N / P ratio, time, and temperature). Reference Figure 1 and Figure 6It can be seen that the arrangement of the domains and different exemplary N / P ratios lead to different nanocomposite sizes, which are obtained through dynamic light scattering measurements.

[0095] In one exemplary embodiment, the functionality of the exemplary carrier and the exemplary nanocomposite can be easily tuned by adding or omitting different structural domains, and further modulated by adjusting the nanocomposite formation conditions (e.g., N / P ratio, time, and temperature). In one exemplary embodiment, as described in the examples of this disclosure, by adding an exemplary CTAT domain to a previously designed chimeric peptide backbone named MPG2H, the exemplary carrier and the nanocomposite can be endowed with an alternative cell entry pathway, namely a cell penetration pathway; see reference. Figure 1 , Figure 9A and Figure 10 It is evident that adding the exemplary CTAT domain may improve the exemplary gene transfer efficiency in cell culture. In one exemplary embodiment, the exemplary gene transfer efficiency in cell culture can be assessed by imaging (green fluorescence signal indicates cells successfully transfected with the GFP reporter gene) and luciferase analysis techniques.

[0096] In one exemplary embodiment, the chimeric peptide carrier can be easily redesigned to target a specific organ by adding or omitting different structural domains. In one exemplary embodiment, the exemplary nanocomposite can be developed to achieve the specific size required for targeting a specific organ.

[0097] In one exemplary embodiment, the chimeric peptide vector can be easily redesigned by adding or omitting different domains, and its size can also be adjusted to target specific cell types; see reference. Figure 9A and Figure 9B It can be seen that using the iRGD domain may enable the vector to target cells with RGD-binding integrins (receptors). In one exemplary embodiment, the exemplary chimeric peptide vector can be evaluated by imaging (green fluorescent signal indicates cells successfully transfected with the GFP reporter gene).

[0098] In one exemplary embodiment, the stability of the exemplary carrier and nanocomposite based on the chimeric peptide can be evaluated by a variety of tests, some of which target serum stability, time stability, and temperature stability, see [link to relevant documentation]. Figure 5 and Figure 7 .

[0099] In one exemplary embodiment, the non-toxicity of the exemplary chimeric peptide-based carrier and exemplary nanocomposite can be evaluated through a variety of tests, some of which involve cytotoxicity, see [link to relevant documentation]. Figure 9A , Figure 9B , Figure 12A and Figure 12BThese results showed high cell viability in cell culture (assessed by imaging) and no signs of cytotoxicity in vivo (organs were harvested on day 30 for H&E staining and analysis).

[0100] In one exemplary embodiment, the in vivo delivery rate of the exemplary chimeric peptide-based vector and exemplary nanocomposite can be evaluated through a variety of tests, some of which involve the delivery rate of plasmids containing RBD DNA sequences in a mouse model; see [link to documentation]. Figure 11 As can be seen, a high titer of antibody was produced in the animal model after two injections, and this result was evaluated by ELISA analysis.

[0101] The foregoing description of the features of this disclosure, and how these features provide targeted solutions to each of the aforementioned drawbacks, is for illustrative purposes only and is not intended to exhaustively or limit the features of this technology.

[0102] This disclosure relates to methods, materials, and apparatus for designing, developing, and characterizing a DNA vaccine platform (nanocomplex) comprising an exemplary chimeric peptide and a plasmid containing an exemplary target DNA sequence, and is capable of addressing each of the exemplary common drawbacks of the aforementioned conventional DNA vaccine platforms. Features in this disclosure responsible for addressing each of the aforementioned exemplary drawbacks have been described in detail with reference to the accompanying drawings.

[0103] In one exemplary embodiment, the design and biosynthesis of the two components of the exemplary platform (the chimeric peptide-based vector and the plasmid containing the target DNA sequence), as well as the development of the exemplary nanocomposite, can employ well-established methods that are efficient in both time and cost and can be easily used to achieve relatively simple large-scale production; see reference. Figure 13 and Figure 14 This demonstrates an exemplary method for expressing and purifying chimeric peptides, as well as the materials used.

[0104] In one exemplary implementation, the ability of the exemplary platform to induce a favorable immune response can be assessed through a variety of tests, some of which involve humoral immunity against SARS-CoV-2; see reference. Figure 11 and Figure 15 It can be seen that the animal model produced high-titer antibodies after two injections (assessed by ELISA analysis), and the functionality of the produced antibodies was also assessed by virus neutralization assays.

[0105] In one exemplary embodiment, the methods of this disclosure have broad applicability in multiple fields because the exemplary platforms introduced can be easily designed to provide targeted solutions to challenges faced in gene delivery research, including DNA vaccines, cancer vaccines, therapeutic vaccines, infectious diseases, and immunotherapy. These challenges include the lack of an ideal carrier platform that can form stable and safe nanocomposites across different nanoscale ranges and is tunable to target different cell lines and organs to achieve good delivery efficiency and expression levels.

[0106] In one exemplary embodiment, an exemplary platform can be used to target and deliver a target gene in vitro to a cell line, as described in the detailed description of a specific embodiment; see reference Figure 9B and Figure 10 As can be seen, the exemplary platform demonstrates good gene transfer efficiency in cells with RGD-binding integrins (receptors), and this result was evaluated using imaging (green fluorescence signal showing cells transfected with the GFP reporter gene) and luciferase analysis. The presentation also includes a PEI complex using PEI as a vector and psiCHECK plasmid as a reporter plasmid (as a positive control), as well as untreated cells and cells treated only with the naked psiCHECK plasmid (without any vector) (as negative controls).

[0107] In one exemplary embodiment, the exemplary platform can be used for non-targeted in vitro delivery of the target gene to a cell line, as described in the detailed description of the specific embodiment; see reference Figure 9A and Figure 10 This demonstrates that the platform exhibits good gene transfer efficiency in cell culture. The results were evaluated using imaging (green fluorescent signal indicates cells successfully transfected with the GFP reporter gene) and luciferase analysis. Figure 10 A graph is shown depicting the transfection efficiency of HEK293T cells using a nanocomposite containing CTATMPG2H and MiRGD as chimeric peptide carriers and psiCHECK plasmid as a reporter plasmid. This illustrates the results of in vitro gene delivery using this platform (peptide-based DNA vaccine), consistent with one or more exemplary embodiments of this disclosure. Figure 10As shown, image 1020 is a blank sample. Images 1002 and 1004 correspond to untreated cells and naked plasmids (psiCHECK.2), respectively. Image 1006 is PEI, N / P=5. Image 1022 has N / P=12. Image 1008 is CTATMPG2H. Image 1010 is MIRGO, image 1012 is CTATMPG2H, image 1014 is MIRGO, image 1016 is CTATMPG2H, and image 1018 is MIRGO. Image 1024 has N / P=8. Image 1026 has N / P=4.

[0108] In one exemplary embodiment, an exemplary platform can be used to deliver a target gene in vivo to an animal model, as described in the detailed description of a specific embodiment; see reference Figure 11 As can be seen, the animal model produced high-titer antibodies after two injections, a result evaluated by ELISA analysis. In one exemplary embodiment, the exemplary platform can also be used to deliver the target gene in vivo non-targeted to the animal model.

[0109] In one exemplary embodiment, the application of the exemplary platform in the targeted delivery of a target gene into an animal model can be considered as a DNA vaccine platform, as described in the detailed description of the specific embodiments; see also Figure 11 It can be seen that the animal model produced high-titer antibodies after two injections. This result was evaluated by ELISA, and the functionality of the produced antibodies was also evaluated by virus neutralization assay.

[0110] In one exemplary embodiment, the application of the exemplary platform in the targeted in vivo delivery of the RBD gene to an animal model can be considered as a DNA vaccine platform for COVID-19, as described in the detailed description of the specific embodiments; see also Figure 11 and Figure 15 It can be seen that the animal model can produce high-titer antibodies after two injections. This result was evaluated by ELISA analysis, and the functionality of the produced antibodies was also evaluated by virus neutralization assay. Figure 11 A chart is shown depicting the evaluation results of humoral immune responses during in vivo gene delivery using this platform (peptide-based DNA vaccine), consistent with one or more exemplary embodiments of this disclosure. Figure 11As shown, image 1136 corresponds to sample 1 (peptide control (48.4µg) - MIRGD), image 1134 corresponds to sample 2 (plasmid control (10µg) - PCDNA3.1 containing RBD sequence), image 1132 corresponds to sample 3 (control - no injection), image 1130 corresponds to sample 4 (nanocomplex (48.8µg MIRGD + 10µg PCDNA3.1 containing RBD sequence + 500 mM chloroquine + 20 ng GM-CSF)), and image 1128 corresponds to sample 5 (nanocomplex (48.8µg MIRGD + 10µg PCDNA3.1 containing RBD sequence + 500 mM chloroquine + Freund's complete adjuvant)). Image 1126 corresponds to a serum dilution of 0.001, image 1124 corresponds to a serum dilution of 0.002, image 1122 corresponds to a serum dilution of 0.01; image 1120 corresponds to a dilution of 0.001, image 1118 corresponds to a dilution of 0.002, image 1116 corresponds to a dilution of 0.01; image 1114 corresponds to a dilution of 0.001, image 1112 corresponds to a dilution of 0.002, image 1110 corresponds to a dilution of 0.01; image 1108 corresponds to a dilution of 0.001, image 1106 corresponds to a dilution of 0.002, image 1104 corresponds to a dilution of 0.01; image 1103 corresponds to a dilution of 0.001, image 1102 corresponds to a dilution of 0.002.

[0111] In one exemplary embodiment, the exemplary platform can be used to identify exemplary tumor vascular “zip codes,” enter tumor tissue, and target the exemplary tumor microenvironment for gene delivery. In one exemplary embodiment, the vector (introduced in the form of a multifunctional chimeric peptide), which is part of the exemplary platform, can be readily redesigned (the methods of which have been fully described), for example by adding or omitting multiple domains, thereby enabling targeted or non-targeted gene delivery to a variety of cells and organs in vitro or in vivo. In one exemplary embodiment, the above-described exemplary method can be used to achieve the inhibition or overexpression of a target gene.

[0112] In one exemplary embodiment, an exemplary gene vector (introduced in the form of a plasmid containing the target gene) that is part of the platform can be easily redesigned (the methods of which have been fully described), for example by adding or omitting multiple gene sequences, to achieve targeted or non-targeted gene delivery to multiple cells and organs in vitro or in vivo. In one exemplary embodiment, the exemplary method described above can be used to achieve the suppression or overexpression of the target gene.

[0113] In one exemplary embodiment, the exemplary nanocomposite may include additional constituent elements. For example, chimeric peptides or drugs may be conjugated to one or more molecules to perform multiple functions, such as for therapeutic-diagnostic applications. Overall, the introduced exemplary platform can be customized as needed or remain non-customized to suit a wide range of research areas related to gene delivery.

[0114] In one exemplary embodiment, an exemplary embodiment will now be described more fully in conjunction with the accompanying drawings. Descriptions of multifunctional chimeric peptides, their functions, and the sequence of their domains (these chimeric peptides serve as gene delivery vectors, and more specifically as a key component of DNA vaccine platforms) can be found in [reference needed]. Figure 1 The illustration shows examples of non-limiting domain arrangements. In one exemplary embodiment, an exemplary multifunctional chimeric peptide, serving as a gene delivery vector, may comprise five domains. In one exemplary embodiment, exemplary domains may include a cell penetration domain, an endosome escape domain, a nucleus entry domain, a DNA condensation domain, and a targeting domain.

[0115] In one exemplary embodiment, the exemplary cell penetration domain may include a TAT; in one exemplary embodiment, the exemplary cell penetration domain may be used to provide an exemplary platform with a cell entry method other than the endosome pathway.

[0116] In one exemplary embodiment, the exemplary endosome escape domain may include gp41. In one exemplary embodiment, the exemplary endosome escape domain can be used to endow an exemplary platform with the ability to escape from endosomes, thereby protecting the target gene and promoting subsequent processes required for target gene expression. In one exemplary embodiment, the CTAT domain has also been reported to facilitate endosome escape.

[0117] In one exemplary embodiment, the exemplary nuclear entry domain may include an NLS. In one exemplary embodiment, the exemplary nuclear entry domain may be responsible for delivering a target gene into the nucleus, a step that may be necessary for gene expression and the induction of a favorable immune response.

[0118] In one exemplary embodiment, the exemplary DNA condensation domain may include 2Histon1. In one exemplary embodiment, the exemplary DNA condensation domain may be responsible for binding to DNA to form a nanocomplex capable of protecting a target gene while facilitating gene delivery.

[0119] In one exemplary embodiment, the exemplary targeting domain may include iRGD. In one exemplary embodiment, the exemplary targeting domain may serve as an example to demonstrate the exemplary platform's ability to target cells by targeting multiple cell surface markers.

[0120] The arrangement of functional domains is designed to not only maintain the functionality of each domain, but also to add other capabilities, such as environmental targeting (nanocomposites smaller than 100 nm can enter the lymphatic system).

[0121] Example 1: Building an exemplary DNA vaccine platform

[0122] Figure 13 and Figure 14 The methods and materials used for the expression and purification of chimeric peptides, including MIRGD and CTATMPG2H, are demonstrated.

[0123] Biosynthesis: The MIRGD construct, previously designed by Hosseinkhani and colleagues, was transformed into E. coli BL21 strain and expressed under inducible conditions. Figure 13 )

[0124] Purification: A two-step purification method consisting of Ni-NTA affinity chromatography and dialysis was employed. After expression, cells were collected by centrifugation at 5500 RPM for 6 minutes. The resulting precipitate was resuspended in lysis buffer. Figure 14 The suspension was then subjected to pulsed sonication for 10 minutes using a probe sonicator (10 seconds on, 20 seconds off, for a total of 20 pulses). The suspension was then centrifuged at 12000 RPM for 30 minutes. The supernatant was added to a Ni-NTA chromatography column and incubated for 1 hour. Washing buffer was then used (…). Figure 14 The chromatography column was washed to remove non-target proteins. Then, elution buffer (…) was used. Figure 14 The target peptide was eluted and stored at -20°C. For additional purification and desalting, the purified peptide was dialyzed in PBS buffer containing 3% glycerol at 4°C for 48 hours, with the buffer changed every 6 hours. The further purified peptide was stored at low temperature to prevent aggregation.

[0125] Primer design: Two sets of primers were designed to add another functional domain to the N-terminus of the previously described chimeric peptide (MPG2H).

[0126] PCR amplification: PCR products were obtained under optimized conditions, and then recovered and purified from agarose gel.

[0127] Cloning: The purified PCR product was digested with restriction endonucleases and ligated into the pET28a vector. To verify the cloning results, colony PCR, double enzyme digestion analysis, and sequencing were performed.

[0128] Biosynthesis: After successful cloning validation, the construct was transformed into E. coli Rosetta and expressed under inducible conditions. Figure 13 ). Figure 13 A table is shown that describes the optimized biosynthetic conditions for CTATMPG2H and MIRGD, consistent with one or more exemplary embodiments of this disclosure.

[0129] Purification: A two-step purification method consisting of Ni-NTA affinity chromatography and dialysis was employed. After expression, cells were collected by centrifugation at 5500 RPM for 6 minutes. The resulting precipitate was resuspended in lysis buffer. Figure 14 The suspension was then subjected to pulsed sonication for 10 minutes using a probe sonicator (10 seconds on, 20 seconds off, for a total of 20 pulses). The suspension was then centrifuged at 12000 RPM for 30 minutes. The supernatant was added to a Ni-NTA chromatography column and incubated for 1 hour. Washing buffer was then used (…). Figure 14 The column was washed to remove non-target proteins. Then, elution buffer (…) was used. Figure 14 The desired peptides were eluted and stored at -20°C. For additional purification and desalting, the purified peptides were dialyzed in PBS buffer containing 3% glycerol at 4°C for 48 hours, with the buffer changed every 6 hours. The additionally purified peptides were stored at low temperature to prevent aggregation. Figure 14 A table is shown that describes an optimized purification process for CTATMPG2H and MIRGD, consistent with one or more exemplary embodiments of this disclosure.

[0130] Figure 2 , Figure 3A and Figure 3B The purity and identity of the purified chimeric peptides, as assessed by SDS-PAGE electrophoresis and Western blotting, are shown, along with the secondary structures of the two chimeric peptides, as assessed by circular dichroism spectroscopy. Figure 3A A circular dichroism spectrum is shown to depict the secondary structures of MIRGD and CTATMPG2H, consistent with one or more exemplary embodiments of this disclosure. Figure 3BA circular dichroism (CD) spectral reference image of a protein secondary structure is shown, consistent with one or more exemplary embodiments of this disclosure. In one exemplary embodiment, image 302 shows a schematic diagram of MIRGD. In one exemplary embodiment, image 304 shows a schematic diagram of CTATMPG2H. In one exemplary embodiment, image 306 shows a CD spectral reference image of an α-helix. In one exemplary embodiment, image 308 shows a CD spectral reference image of a β-sheet. In one exemplary embodiment, image 310 shows a CD spectral reference image of a random coil.

[0131] Example 2: Expression and purification of an exemplary DNA vaccine platform

[0132] Expression and purification analysis: The expression of the target peptide was identified by Western blotting, using an anti-His-tag antibody to recognize the His-tag domains designed at the C-terminus and N-terminus of the peptide. Figure 2 The purification rate was evaluated by 12.5% ​​Tris-Glycine SDS-PAGE and 214 nm densitometric analysis. Figure 2 Results: Based on bioinformatics calculations, the theoretical pI / Mw of MIRGD and CTATMPG2H are approximately 10.93 / 9682.39 and 11.85 / 11589.67, respectively. However, due to the high positive charge of these peptides and their conformational characteristics, the peptide bond band appears at approximately 15 kD in the SDS-PAGE electrophoresis results.

[0133] Example 3: Secondary Structure Analysis of an Exemplary DNA Vaccine Platform

[0134] Secondary structure analysis: The secondary structure of the peptide was determined by circular dichroism spectroscopy (CD) in the far-ultraviolet region (190-250 nm). Figure 3A Results: The results showed that the CTATMPG2H construct exhibited a significant peak in the 200-210 nm region, with another peak at 220 nm. According to... Figure 3B The spectral graphs of the models shown indicate that the CTATMPG2H construct appears to fold into a conformation predominantly α-helical, while MIRGD exhibits a secondary structure composed of β-sheets and random coils.

[0135] Example 4: Testing an exemplary DNA vaccine platform

[0136] The description of implementation methods related to materials, methods, and results relates to the development of a target antigen expression plasmid as a key component of a peptide-based DNA vaccine.

[0137] The target gene suitable for use as a SARS-CoV-2 antigen—expressing RBD (part of the first subunit of the spike protein)—was subcloned into PCDNA3.1. The subcloning process was confirmed by colony PCR, double enzyme digestion, and sequencing.

[0138] The designed plasmid was transformed into E. coli DH5α. After replication in LB medium for 2 days, the plasmid was extracted using a commercially available MN medium-volume plasmid extraction kit.

[0139] In addition, commercially available versions of plasmids carrying GFP (pDB2) and luciferase (psiCHECK2) were used as reporter gene vectors in the optimization process.

[0140] Example 5: Gel electrophoresis experiment of an exemplary DNA vaccine platform

[0141] This paper describes the materials, methods, and results related to the preparation and characterization of nanocomposites used as a platform for peptide-based DNA vaccines, referencing... Figure 4 , Figure 5 , Figure 6 , Figure 7 and Figure 8 These figures show: the formation of nanocomposites at different N / P ratios (assessed by agarose gel migration retardation experiments), where genes lose their migration ability due to binding with chimeric peptides to form nanocomposites; the stability of the formed nanocomposites over different temperature and time ranges (assessed by gel migration retardation experiments), showing that different conditions do not disrupt nanocomposite formation; the particle size and zeta potential of the formed nanocomposites (assessed by dynamic light scattering); the stability of the formed nanocomposites in the presence of serum nucleases (assessed by gel migration retardation experiments), showing that the presence of serum nucleases does not reduce the quality of genes after binding with chimeric peptides to form nanocomposites; and images of the nanocomposites assessed by transmission electron microscopy (TEM). Figure 4 An agarose gel image is shown to demonstrate the formation of the peptide-based DNA vaccine nanocomposite used, consistent with one or more exemplary embodiments of this disclosure. In one exemplary embodiment, image 402 shows an agarose gel containing CTATMPG2H. Image 404 shows an agarose gel containing MIRGD. Image 406 shows N / P=1. Image 408 shows N / P=2. Image 410 shows N / P=4. Image 412 shows N / P=8. Image 414 shows a plasmid. Image 416 shows N / P=8. Image 418 shows N / P=4. Image 420 shows N / P=2. Image 422 shows N / P=1. Figure 5Images of agarose gels are shown to demonstrate the stability of the peptide-based DNA vaccine nanocomplexes under different time and temperature conditions, consistent with one or more example embodiments of this disclosure. In one exemplary embodiment, image 502 shows an agarose gel containing CTATMPG2H. Image 504 shows an agarose gel containing MIRGD. Image 506 shows the nanocomplexes stable at 15°C for 30 minutes. Image 508 shows the nanocomplexes stable at 28°C for 24 hours. Image 510 shows the nanocomplexes stable at 37°C for 24 hours. Image 512 shows the stability of the DNA ladder. Image 514 shows the nanocomplexes stable at 37°C for 24 hours. Image 516 shows the nanocomplexes stable at 28°C for 24 hours. Image 518 shows the nanocomplexes stable at 15°C for 30 minutes.

[0142] Figure 6 Hydrodynamic dimensions and zeta potential diagrams of nanocomposites for peptide-based DNA vaccines are shown, consistent with one or more exemplary embodiments of this disclosure. Figure 7 An agarose gel image is shown to demonstrate the stability of the nanocomposite used as a peptide-based DNA vaccine, consistent with one or more exemplary embodiments of this disclosure. In one exemplary embodiment, image 702 shows an agarose gel containing MIRGD. In one exemplary embodiment, image 704 shows an agarose gel containing CTATMPG2H. In one exemplary embodiment, image 706 shows N / P = 0.5. In one exemplary embodiment, image 708 shows N / P = 1. In one exemplary embodiment, image 710 shows N / P = 4. In one exemplary embodiment, image 712 shows N / P = 4 + serum. In one exemplary embodiment, image 714 shows N / P = 4 + SDS. In one exemplary embodiment, image 716 shows plasmid + serum. In one exemplary embodiment, image 718 shows N / P = 4 + SDS. In one exemplary embodiment, image 720 shows N / P = 4 + serum. In one exemplary embodiment, image 722 shows N / P = 4. In one exemplary embodiment, image 724 shows N / P = 1. In one exemplary embodiment, image 726 shows N / P = 0.5. Figure 8 Transmission electron microscopy (TEM) images are shown to demonstrate the formation of nanocomposites and to show the morphology and size of nanocomposites used as peptide-based DNA vaccines, consistent with one or more exemplary embodiments of this disclosure. In one exemplary embodiment, images 802, 804, and 806 show TEM images of nanocomposites of different sizes.

[0143] Pre-defined amounts of peptides were mixed in HBG and PBS buffers and then gently pipetted with equal volumes of plasmids (PBDb2 and PCDNA3.1+RBD) at various N:P ratios, followed by incubation at 15°C for 30 minutes. The formation of the nanocomplexes was assessed using a 1% agarose gel migration retardation assay, and the electrophoretic migration of the complexes was observed using a UV imager. Figure 4 )

[0144] Results: According to the gel electrophoresis results, both peptides were able to form stable complexes with plasmids and inhibit plasmid migration on agarose gels.

[0145] Example 6: Time and Temperature Stability Analysis of an Exemplary DNA Vaccine Platform

[0146] Temporal and temperature stability analysis: After incubating each sample with different N:P ratios at 28℃ and 37℃ for 24 hours, retardation experiments were performed to evaluate their stability. Figure 5 )

[0147] Results: According to gel electrophoresis analysis, both nanocomposites (MIRGD+ plasmid and CTATMPG2H+ plasmid) remained completely intact and stable after incubation at 37℃ and 28℃ for 24 hours.

[0148] Example 7: Hydrodynamic dimensions, zeta potential, and distribution analysis of an exemplary DNA vaccine platform

[0149] Hydrodynamic size, zeta potential, and distribution analysis: At 25°C, the average particle size, particle size distribution, and zeta potential of various nanocomposites with different N:P ratios were determined using a Zetasizer Nano ZS instrument. Figure 6 )

[0150] Results: Depending on the time, temperature, purification process, and preparation technology of the nanocomposite, various particle sizes and zeta potentials can be obtained and controlled according to experimental objectives. The particle size of the nanocomposite can be controlled within the range of 50-1000 nm, and the zeta potential can be tuned within the range of -10 to +20 mV. The nanocomposite used for mouse immunization experiments was optimized to approximately 100 nm and +10 mV. The positively charged surface and nanoscale particle size of the nanocomposite are two key factors promoting platform adhesion to cells and entry into cells.

[0151] Example 8: Serum stability analysis of an exemplary DNA vaccine platform

[0152] Serum stability: The stability of the nanocomposite in the presence of serum was assessed using a gel retardation assay. The nanocomposite was incubated with 10% FBS at 37°C for 60 minutes; SDS was added to the sample to allow DNA to dissociate from the nanocomposite. The samples were then subjected to electrophoresis on a 1% agarose gel, and the results were observed using UV chromatography. Figure 7 )

[0153] Results: According to gel electrophoresis analysis, both nanocomposites (MIRGD+ plasmid and CTATMPG2H+ plasmid) showed good ability to protect the plasmids from degradation in the presence of serum nucleases.

[0154] Transmission electron microscopy (TEM): The nanocomposite was prepared at an N / P ratio of 10 and dropped onto a Cu Mesh 300 mesh coated with a Formvar carbon film. It was then stained with 2% uranium acetate for 2 minutes at room temperature. Imaging was subsequently performed using a transmission electron microscope (TEM, Zeiss-EM10C, 80 kV). Figure 8 ) Example 9: Gene delivery efficiency analysis of an exemplary DNA vaccine platform

[0155] Examples of materials, methods, and results for in vitro gene delivery using this platform (peptide-based DNA vaccines) can be found in [reference needed]. Figure 9A , Figure 9B and Figure 9C Gene delivery efficiency was evaluated using imaging (green fluorescence signal indicating cells successfully transfected with the GFP reporter gene) and luciferase assays. The results showed that this platform had good gene transfer efficiency compared to PEI (positive control), which is the gold standard gene vector, and was also superior to untreated cells and cells treated with naked genes directly without vectors (negative control). Figure 9C Fluorescence microscopy images of Hek293T cells transfected using a polyethyleneimine (PEI) complex containing PEI as a vector and pDB2 plasmid as a reporter plasmid are shown, with the conditions serving as a positive control, consistent with one or more exemplary embodiments of this disclosure. Figure 9C Untreated cells and cells without any vectors, only with the naked pDB2 plasmid added, were also shown as negative controls. Figure 9A As shown, image 902 is a fluorescence micrograph of CTATMPG2H-N / P:4, image 904 is a fluorescence micrograph of CTATMPG2H-N / P:8, and image 906 is a fluorescence micrograph of CTATMPG2H-N / P:12. Figure 9BAs shown, image 908 is a fluorescence micrograph of MIRGD-N / P:4, image 910 is a fluorescence micrograph of MIRGD-N / P:8, and image 912 is a fluorescence micrograph of MIRGD-N / P:12. Figure 9C As shown, image 914 is a fluorescence micrograph of PEI-N / P:8, image 916 is a fluorescence micrograph of PEI-N / P:5, image 918 is a fluorescence micrograph of the untreated sample, and image 920 is a fluorescence micrograph of the naked plasmid.

[0156] HEK293T cells were seeded at a density of 60,000 in 48-well plates in DMEM containing 10% FBS and PenStrep antibiotic, and incubated for 24 hours. One hour before transfection, cells were pre-incubated in serum-free medium. Subsequently, cells were transfected using nanocomplexes with different N:P ratios and with or without chloroquine. Four hours later, cells were incubated again in fresh medium for 48 hours. Cell bioimaging was performed using fluorescence microscopy. Transfection efficiency was also analyzed using luciferase assays. Cells were lysed using hypotonic lysis buffer containing luciferin. The luciferase activity of lysed cells was measured under ATP-containing conditions, and the readings were taken using a luminescence analyzer. Figure 10 )

[0157] Use MIRGD ( Figure 9B ) and CTATMPG2H ( Figure 9A Transfection experiments were performed using PEI as a gene vector. PEI was also used as a positive control. Figure 9C In addition, untreated cells and cells treated with only naked plasmids were considered negative controls. Figure 9C ).

[0158] Results: Both peptides showed significantly higher transfection efficiency compared to PEI, which was the gold standard. Gene delivery efficiency was assessed using complementary methods (luciferase assay and imaging) under various N / P ratio conditions.

[0159] Example 10: In vivo gene delivery analysis of an exemplary DNA vaccine platform

[0160] This embodiment describes the materials, methods, and results used to evaluate the immune response when using this platform (peptide-based DNA vaccine) for in vivo gene delivery. In particular, by referring to... Figure 11 and Figure 15The humoral immune response of animal models after using this platform (peptide-based DNA vaccine) was evaluated. The results showed that the animal models produced high titers of antibodies after two injections (assessed by ELISA), and the functionality of the produced antibodies was verified by a virus neutralization assay.

[0161] Immunogenicity assessment of the exemplary vaccine candidate (nanocomplex) was performed in inbred BALB / c mice (female, 6–8 weeks old). The mouse model received intramuscular injections (IM) of the nanocomplex containing 10 µg of the designed plasmid and 500 mM chloroquine on days 0 and 15. Concurrently, groups containing only the plasmid or only the peptide served as control groups. Additionally, 20 ng GM-CSF was used as an adjuvant. Blood and organ samples were collected on day 30.

[0162] Antibody titers were determined by ELISA: Antibody titers in animal serum samples were determined using an ELISA method. Multi-well 96-well RBD pre-coated plates were incubated with animal serum samples at different dilutions at 37°C for 2 hours. The plates were then washed 5 times and incubated at 37°C for 1 hour with HRP-labeled goat anti-mouse IgG antibody (lowest cross-reactivity, BioLegend, San Diego, CA, USA). The plates were washed 5 times again, and then TMB / H2O2 substrate was added. The reaction was terminated with 1 M HCl, and the absorbance was read at 450 nm using a microplate reader. Figure 11 )

[0163] Results: Significant differences in antibody titers were observed between the main experimental group and the control group. The main experimental group consisted of animal models that received the nanocomposite (48.8 µg MIRGD + 10 µg PCDNA3.1 containing the RBD sequence + 500 mM chloroquine) supplemented with 20 ng GM-CSF or Freund's Complete Adjuvant. The control group included animal models that received only 10 µg PCDNA3.1 containing the RBD sequence, only 48.8 µg MIRGD, or no treatment.

[0164] Based on the above results, the proposed DNA vaccine platform demonstrates good ability in in vivo gene delivery, can induce humoral immune response, and produce high titers of anti-RBD antibodies.

[0165] Example 11: Virus neutralization test (VNT) of an exemplary DNA vaccine platform

[0166] Virus neutralization assays were conducted at the Amirabad Virology Laboratory (Tehran, Iran). Briefly, BALB / c mouse serum was serially diluted twofold in 2% DMEM. The serum was derived from mice treated with a nanocomposite (48.8 µg MIRGD + 10 µg PCDNA3.1 containing the RBD sequence + 500 mM chloroquine + 20 ng GM-CSF). Subsequently, 100 TCID45 was added to the diluted serum. 50 The SARS-CoV-2 strain (Wuhan strain, isolated from an infected patient) was incubated at 37°C for 60 minutes. The mixture was then added to VeroE6 cells and incubated further at 37°C with 5% CO2. Finally, the cytopathic effect (CPE) was observed and recorded. Figure 15 )

[0167] Results: Serum samples at different dilutions (1 / 2, 1 / 4, and 1 / 8) were tested and found to inactivate the virus and prevent CPE, indicating that the antibodies produced by the animal model in response to this DNA vaccine platform are functionally effective. It can be inferred that further optimization of the target plasmid sequence could potentially yield higher virus neutralization test (VNT) titers.

[0168] Based on these results, it can be concluded that the proposed DNA vaccine platform can not only induce a good humoral immune response by generating high-titer antibodies (ELISA results) through the introduction of the plasmid (PCDNA3.1+RBD), but these antibodies also have practical functionality. Figure 15 A table showing the results of a virus neutralization experiment demonstrates the functionality of the antibodies detected in the serum of animal models inoculated with the nanocomposite, consistent with one or more exemplary embodiments of this disclosure. The nanocomposite used comprises 48.8 µg MIRGD + 10 µg PCDNA3.1 containing the RBD sequence + 500 mM chloroquine + 20 ng GM-CSF. The results indicate that this DNA vaccine platform possesses the ability to elicit a humoral immune response.

[0169] Example 12: Cytotoxicity assessment of an exemplary DNA vaccine platform

[0170] This embodiment describes the materials, methods, and results used to assess the safety of this platform (peptide-based DNA vaccine) during in vivo gene delivery. Specifically, by referring to... Figure 12A and Figure 12B The cytotoxicity of this platform (a peptide-based DNA vaccine) in animal models was evaluated. Results showed high cell viability in cell culture imaging assessment, with no signs of cytotoxicity observed in vivo: organs were collected on day 30 for H&E staining and analysis. Figure 12AImages of H&E stained sections of mouse lungs, heart, and spleen are shown, consistent with one or more exemplary embodiments of this disclosure. Figure 12B Images of H&E-stained sections of mouse kidneys and livers are shown, also consistent with one or more exemplary embodiments of this disclosure. Figure 12A As shown, image 1202 is the control sample, image 1204 is the sample using MIRGD, image 1206 is the sample using pcDNA3.1+RBD, and image 1208 is the sample using the nanocomposite. Figure 12B As shown, image 1210 is the control sample, image 1212 is the sample using MIRGD, image 1214 is the sample using pcDNA3.1+RBD, and image 1216 is the sample using the nanocomposite.

[0171] To assess the biosafety of the candidate platform, mouse organs from all experimental groups were collected, fixed, and subjected to H&E staining and analysis (including heart, lungs, liver, kidneys, spleen, and lymph nodes).

[0172] Results: Control group: Liver: Aggregation of polymorphonuclear and mononuclear inflammatory cells was observed, leading to cholangitis. Kidney: Vacuoleic degeneration and cast formation were observed in some renal tubular epithelial cells, along with some diffusely distributed lymphocytes. Spleen: Some neutrophils were observed. Heart: Mild monocytic myocarditis. Lung: Alveolar collapse, hemorrhage, hemosiderin deposition, fibrin deposition, partial alveolar dilation, and infiltration of polymorphonuclear and mononuclear inflammatory cells were observed, leading to moderate pneumonia. Lymph nodes: Some neutrophils were observed.

[0173] MIRGD: Liver: Some hepatocytes show cytoplasmic loss due to glycogen storage, and small foci composed of necrotic hepatocytes, neutrophils, and mononuclear inflammatory cells are visible. Kidneys: Casts are visible, vacuolar degeneration of some renal tubular epithelial cells is observed, and scattered or diffuse mononuclear inflammatory cells are present. Spleen: Hemorrhage (hematoma) and some polymorphonuclear inflammatory cells are visible. Heart: Mild lymphocytic myocarditis. Lungs: Alveolar collapse, congestion, hemosiderin deposition, fibrin deposition, and infiltration of some mononuclear and polymorphonuclear inflammatory cells are visible, leading to alveolitis and bronchiolitis.

[0174] Plasmid (PCDNA3.1, containing RBD sequence): Liver: Small aggregates of inflammatory cells are visible. Kidneys: Casts, vacuolar degeneration of some renal tubular epithelial cells, and scattered mononuclear inflammatory cells are visible. Spleen: Some polymorphonuclear inflammatory cells are visible. Heart: Mild monocytic myocarditis. Lungs: Alveolar collapse, hemorrhage, hemosiderin deposition, a small amount of fibrin deposition, and infiltration of some mononuclear and polymorphonuclear inflammatory cells are visible. Lymph nodes: Normal.

[0175] Nanocomposite (MIRGD + PCDNA3.1 containing RBD sequence + 500 mM chloroquine + 20 ng GM-CSF): Liver: Small aggregates of inflammatory cells are visible. Kidneys: Casts, vacuolar degeneration of some renal tubular epithelial cells, and diffuse mononuclear inflammatory cells are present. Spleen: Some neutrophils are visible. Heart: Mild lymphocytic myocarditis. Lungs: Alveolar collapse, edema, a small amount of fibrin deposition, perivascular cuff infiltration, and moderate infiltration of mononuclear and polymorphonuclear inflammatory cells, leading to interstitial pneumonia. Lymph nodes: Scattered polymorphonuclear inflammatory cells are visible.

[0176] Note: Most histopathological findings showed lesions caused by an inflammatory response, and the severity was almost identical. Other observed changes either occurred repeatedly in all sections or were of little significance.

[0177] No significant signs of cytotoxicity were observed.

[0178] In addition, the group injected with the nanocomposite (48.8µg MIRGD + 10µg PCDNA3.1 containing RBD sequence + 500 mM chloroquine + 20 ng GM-CSF) was followed up for 5 months, and the results showed that the survival rate was 100%.

[0179] The description of the above embodiments is intended for illustrative and explanatory purposes and is not intended to be exhaustive or to limit the scope of this disclosure. Individual elements or features in a particular embodiment are generally not limited to that particular embodiment, but are interchangeable where applicable and can be used in any chosen embodiment, even if not explicitly shown or described herein. Similarly, these elements can be varied in many ways. Such variations should not be considered a departure from the scope of this disclosure, and all such modifications are intended to be included within the scope of this disclosure.

[0180] The above description of the features of this disclosure (purposeful features for addressing the aforementioned disadvantages) is for illustrative and explanatory purposes only and is not intended to exhaust all content or limit the features of the technology.

[0181] Industrial applicability

[0182] This disclosure enables the construction of exemplary nanocomposites using chimeric peptides and provides methods for using such nanocomposites as gene carriers. The specific advantages of constructing and using these exemplary nanocomposites address many challenges associated with DNA vaccines, particularly the difficulty of engineering conventional gene delivery methods in a reasonable manner within a short timeframe, and the inability to achieve the desired functions (e.g., gene transfer efficiency) for different cells and organs. The methods of this disclosure can be applied in multiple fields because the exemplary nanocomposites can be readily designed to address challenges in gene delivery research, including DNA vaccines, cancer vaccines, therapeutic vaccines, infectious diseases, and immunotherapy. These challenges include the lack of ideal carrier platforms, the inability to form stable and safe nanocomposites across different nanoscale ranges, and the inability to design them as needed to target different cell lines and organs to achieve good delivery efficiency and expression levels.

[0183] While the foregoing describes what is considered the best mode and / or other examples, it should be understood that various modifications can be made thereto, and the subject matter of this disclosure can be implemented in many forms and instances, and its related techniques can be applied to numerous scenarios, of which only a portion has been described in this description. The following claims are intended to cover all applications, modifications, and variations that fall within the true scope of this disclosure.

[0184] Unless otherwise stated, all measurements, values, grades, locations, orders of magnitude, dimensions, and other specifications listed in this specification (including the following claims) are approximate and not precise. These values ​​are intended to be within a reasonable range consistent with their associated functions and in accordance with conventional practice in the art.

[0185] The scope of protection is defined solely by the following claims. This scope should be interpreted as broadly as possible, consistent with the ordinary meaning of the language in the claims, and understood in conjunction with this specification and subsequent examination history, and should cover all structural and functional equivalents. Nevertheless, no claim is intended to cover subject matter that does not comply with the requirements of Sections 101, 102, or 103 of the Patent Act, nor should it be interpreted in this way. Any unintended coverage of such subject matter is hereby expressly waived.

[0186] Except as stated above, nothing described or shown in this specification is intended, nor should it be construed, as disclosing to the public any component, step, feature, object, benefit, advantage, or equivalent thereof, whether or not it is stated in the claims.

[0187] It should be understood that the terms and expressions used in this specification, unless otherwise specifically defined, shall be interpreted according to their ordinary meaning in the relevant technical field. Relational terms such as “first” and “second” are used only to distinguish different entities or actions and do not necessarily require or imply any actual relationship or order between these entities or actions. The terms “comprises,” “comprising,” or any variations thereof are intended to indicate non-exclusive inclusion, meaning that when a process, method, product, or apparatus includes the listed elements, it does not mean that only those elements are included, but may also include other elements not expressly listed or inherent to the process, method, product, or apparatus. Elements introduced by “a” or “an,” without further limitation, do not exclude the presence of additional identical elements in the process, method, product, or apparatus.

[0188] This abstract is intended to enable the reader to quickly understand the nature of the technical disclosure. The abstract is submitted on the premise that it will not be used to interpret or limit the scope or meaning of the claims. Furthermore, as can be seen from the foregoing detailed description, various features are combined in different embodiments. This is to make the disclosure more concise and clear, and should not be construed as indicating that the claimed embodiments require more features than expressly recited in each claim. Rather, as the following claims show, the innovation may only exist in a portion of the features of a particular embodiment disclosed. Therefore, the following claims are incorporated herein by reference, each claim standing alone as an independent claim subject.

[0189] Although various embodiments have been described, these descriptions are intended as examples and not as limitations. Those skilled in the art will understand that many other implementations are possible within the scope of these embodiments. While a variety of possible combinations of features are shown and discussed in the accompanying drawings and this detailed description, other combinations of the disclosed features are also possible. Unless specifically limited, any feature in any embodiment may be used in combination with or substituted for any other feature or element in another embodiment. Therefore, it should be understood that any feature shown and / or discussed in this disclosure can be implemented by combining it in appropriate ways. Consequently, the limitations of the embodiments should be borne only by the appended claims and their equivalents. Furthermore, various modifications and variations are possible within the scope of the appended claims.

Claims

1. A method for delivering a drug into living cells, the method comprising: The nanocomplex is administered to the cells of the living organism, the nanocomplex comprising at least one chimeric peptide linked to the drug, wherein the chimeric peptide comprises endosome escape motifs, nuclear entry motifs, and DNA condensation motifs 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, wherein MIRGD contains the same amino acid sequence as SEQ ID NO.2, and CTATMPG2H contains the same amino acid sequence as SEQ ID NO.

3.

4. The method of claim 3, wherein the MIRGD comprises: An endosome escape motif containing the same amino acid sequence as SEQ ID NO.5; A cell nucleus enters the motif, which contains the same amino acid sequence as SEQ ID NO.6; A DNA condensation motif containing the same amino acid sequence as SEQ ID NO. 7; and A targeting motif containing the same amino acid sequence as SEQ ID NO.

8.

5. The method of claim 3, wherein the CAMPG2H comprises: A cell-penetrating motif containing the same amino acid sequence as SEQ ID NO.9; An endosome escape motif containing the same amino acid sequence as SEQ ID NO.5; A cell nucleus enters the motif, which contains the same amino acid sequence as SEQ ID NO. 6; and A DNA condensation motif containing the same amino acid sequence as SEQ ID NO.

7.

6. The method of claim 1, wherein the cargo comprises at least one of the following: nucleic acids, proteins, lipids, macromolecules, carbohydrates, chemical compounds, nanoparticles, microspheres containing diagnostic or therapeutic agents, and combinations thereof.

7. The method of claim 1, wherein the step of delivering the nanocomposite to somatic cells of a living organism comprises vaccinating the test organism to induce an adaptive immune response.

8. The method of claim 1, wherein the step of delivering the nanocomposite to somatic cells comprises in vivo or in vitro gene delivery as a DNA vaccine platform to induce an adaptive immune response.

9. The method of claim 8, wherein the in vivo gene delivery comprises administering a nanocomposite comprising a DNA vaccine platform to induce an adaptive immune response against SARS-CoV-2.

10. The method of claim 9, wherein the in vivo gene delivery comprises the delivery of an RBD-encoding gene containing a SARS-CoV-2 antigen, and the RBD-encoding gene is subcloned into PCDNA3.1 for use in COVID-19 vaccination.

11. The method of claim 8, wherein delivering the nanocomposite to living cells comprises using the nanocomposite as a DNA vaccine platform for gene delivery for one of the following purposes: inducing adaptive immunity against an infectious disease, inducing adaptive immunity for cancer treatment, inducing adaptive immunity for allergy treatment, treating autoimmune diseases, and gene therapy.

12. The method of claim 11, wherein the nanocomposite is used as a DNA vaccine platform for gene delivery to induce adaptive immunity for cancer treatment, including targeted gene delivery to tumor tissue.

13. The method of claim 1, wherein delivering the nanocomposite to somatic cells comprises targeting gene inhibition of any target gene via at least one microRNA, small interfering RNA (siRNA), DNAi, peptide nucleic acid (PNA), or a combination thereof.

14. A plurality of nanocomposites for diagnostic / therapeutic purposes, wherein each nanocomposite comprises: A drug comprising at least one nucleic acid, protein, lipid, macromolecule, carbohydrate, chemical compound, nanoparticle, microsphere comprising a diagnostic or therapeutic agent, or a combination thereof; as well as At least one chimeric peptide linked to the drug, the chimeric peptide comprising an endosome escape motif, a nuclear entry motif, and a DNA condensation motif, linked together in any order.

15. The plurality of nanocomposites of claim 14, wherein the plurality of nanocomposites comprises a DNA vaccine nanoassembly platform capable of inducing a specific immune response against an antigenic peptide encoded by an expression vector, wherein the drug comprises a nucleic acid containing DNA or RNA, wherein the link between the chimeric peptide and the nucleic acid is non-covalent, including electrostatic interactions, and the size of the nanoassembly is tunable in the range of 20 nm to 2000 nm.

16. The plurality of nanocomposites of claim 15, wherein the chimeric peptide comprises MIRGD, CTATMPG2H or a derivative thereof, wherein MIRGD comprises an amino acid sequence identical to that of SEQ ID NO.2, and CTATMPG2H comprises an amino acid sequence identical to that of SEQ ID NO.

3.

17. The plurality of nanocomposites as claimed in claim 16, wherein MIRGD comprises: —Endosome escape motif, which contains the same amino acid sequence as SEQ ID NO.5; —The cell nucleus enters the motif, which contains the same amino acid sequence as SEQ ID NO.6; —A DNA condensation motif containing the same amino acid sequence as SEQ ID NO. 7; and —Targeting motif, which contains the same amino acid sequence as SEQ ID NO.

8.

18. The plurality of nanocomposites as claimed in claim 16, wherein CTAPG2H comprises: —A cell-penetrating motif containing the same amino acid sequence as SEQ ID NO.9; —Endosome escape motif, which contains the same amino acid sequence as SEQ ID NO.5; —The cell nucleus enters the motif, which contains the same amino acid sequence as SEQ ID NO. 6; and —A DNA condensation motif containing the same amino acid sequence as SEQ ID NO.

7.

19. The nanocomposite of claim 14, wherein: —Each of the plurality of nanocomposites has a ζ potential in the range of +1 to +20; as well as —Each of the plurality of nanocomposites has an average particle size in the range of 50 nm to 1000 nm.

20. A method for administering a nanocomposite for diagnostic / therapeutic purposes, comprising: —The formation of the nanocomposite, the formation step comprising: Multiple MIRGD peptides are formed, wherein at least one MIRGD peptide is transferred into bacteria, the MIRGD peptide containing the same amino acid sequence as the amino acid sequence of SEQ ID NO.2; The plurality of MIRGD peptides were purified using Ni-NTA agarose affinity chromatography; and A link is formed between the cargo and at least one of the plurality of MIRGD peptides, the cargo comprising at least one nucleic acid, protein, lipid, polymer, carbohydrate, compound, nanoparticle, microsphere containing a diagnostic or therapeutic agent, or a combination thereof; —and the nanocomposite is injected into the body via intramuscular injection.

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