Improvement of surface-presented antigen expression

Abstract

The present invention relates to a composition comprising a transfection agent and a human endogenous retrovirus (HERV) envelope protein having a mutation in its immunosuppressive domain (ISD) that reduces its immunosuppressive properties, or mRNA encoding at least the immunogenic portion thereof. The present invention also relates to the manufacture of pharmaceuticals and the use of this composition in therapeutic and prophylactic treatments.

Description

Technical Field

[0001] Background of the Invention Although immune cells can find and kill tumor cells, it is clear from the fact that nearly 9 million people die from cancer annually worldwide that this system does not always function. Vaccination approaches to induce a specific immune response against tumor cells are a relatively old topic in cancer immunotherapy but are still under development and only recently have started to yield relevant results. One vaccination strategy involves vaccination with attenuated tumor cells, for example, irradiated autologous or allogeneic tumor cell lines, which often secrete granulocyte macrophage colony-stimulating factor (GM-CSF). In either case, the material injected contains cancer antigens that are likely to be present in the actual tumor. Other vaccination strategies include the administration of peptides or proteins to induce a specific immune response. These antigens are either injected directly in combination with an adjuvant or are encoded by a DNA plasmid or a viral vector.

[0002] Despite the continuous progress of immunotherapy approaches, a wide-acting and highly efficient vaccine has not yet been found. The special reason for this is the immune suppression by the aforementioned tumor cells.

[0003] Endogenous retroviruses (ERVs) are evidence that our distant ancestors were infected with retroviruses from ancient times. Upon infection, viral RNA was reverse-transcribed into parvovirus DNA, which was then integrated into the host genome. Eventually, the provirus was integrated into germline cells, becoming heritable and giving rise to endogenous retroviruses. Over millions of years, this viral DNA was passed down through generations and fixed in the population. Today, every human genome consists of approximately 8% endogenous retroviral DNA, but these are merely remnants of the retroviruses of the past. Through mutations, deletions, and insertions, most of the retroviral genes have been inactivated or completely lost from the genome. Today, fully functional, full-length endogenous retroviruses no longer exist in humans. However, ERVs have undergone replication processes, resulting in multiple copies being integrated into the host genome, each accompanied by different functional proteins. Therefore, in some cases, multiple homologous ERVs still have the potential to produce viral particles. Human ERV type K (HERV-K, HML2) is one of the most recently acquired ERVs in the human genome, and members of this family have retained the full-length open reading frames for almost all viral proteins.

[0004] Numerous studies have revealed the relationship between ERV expression and cancer development and progression. The detection of ERVs in human tumors has opened up new fields in anti-cancer therapy, with prospects for new vaccination strategies. Notable examples of human ERVs (HERVs) include HERV type K (HERV-K), which is associated with prostate cancer, breast cancer, ovarian cancer, lymphoma, melanoma, leukemia, and sarcoma. Further examples include HERV-H, which is expressed in colorectal cancer, and Syncytin-1, which is found in testicular cancer, ovarian cancer, breast cancer, lymphoma, and leukemia.

[0005] It is not always easy to determine whether ERV protein expression is the cause or consequence of tumor development. However, it is known that conditions within cancer cells enable ERV expression. The common hypomethylation state in tumor cells promotes the activation of ERV genes, which are normally silenced by DNA methylation in healthy cells (Downey, RF, et al., Human endogenous retrovirus K and cancer: Innocent bystander or tumorigenic accomplice? Int J Cancer, 2015. 137(6): p. 1249-57. and Gimenez, J., et al., Custom human endogenous retroviruses dedicated microarray identifies self-induced HERV-W family elements reactivated in testicular cancer upon methylation control. Nucleic Acids Res, 2010. 38(7): p. 2229-46). Exogenous factors can also promote ERV expression. Activation of human ERV has been observed, for example, in response to viral infection. HERV-W expression was detected after influenza and herpes simplex virus infection (Nellaker, C., et al., Transactivation of elements in the human endogenous retrovirus W family by viral infection. Retrovirology, 2006. 3: p. 44), while HERV-K was present after Epstein-Barr virus infection (Sutkowski, N., et al., Epstein-Barr virus transactivates the human endogenous retrovirus HERV-K18 that encodes a superantigen. Immunity, 2001. 15(4): p. 579-89).Regardless of the mechanism that leads to ERV expression, cancer cells maintain the activation of these proteins through selective pressure, demonstrating the beneficial effects of ERVs in tumors (Leong, SP, et al., Expression and modulation of a retrovirus-associated antigen by murine melanoma cells. Cancer Res, 1988. 48(17): p. 4954-8).

[0006] ERVs are expressed not only in human tumors but also in mouse cancer cells. This provides a complete model organism for studying the effects of ERVs on tumor progression and testing therapeutic approaches that target ERVs. One ERV model is melanoma-associated retrovirus (MelARV), which is derived from the provirus of murine leukemia virus (MuLV) present in the mouse genome. Most inbred mouse strains contain one or two inactive MuLV copies (Li, M., et al., Sequence and insertion sites of murine melanoma-associated retrovirus. J Virol, 1999. 73(11): p. 9178-86). However, the AKR mouse strain is characterized by having three insertion sites in its genome, producing large amounts of MuLV early in the postnatal period, and causing a high incidence of spontaneous lymphoma. Other mouse strains, such as C57BL / 6, spontaneously produce MuLV particles later in the postnatal period. Several other mouse cancer models also express MuLV / MelARV in a manner similar to human ERV.

[0007] Because the immune system of a viral host is a natural defense mechanism against infection, many viruses, especially retroviruses, have developed strategies to evade this surveillance. One mechanism observed across various viral families [Duch et al., International Publication No. 2013 / 050048] is the development of immunosuppressive domains in envelope proteins (Env), leading to suppression of the immune system at various levels. Immune cells, including natural killer (NK) cells, CD8 T cells, or regulatory T (Treg) cells, can be affected by viruses containing ISD [Schlecht-Louf et al. (2010)].

[0008] Many ERVs contain proteins with immunosuppressive domains (ISDs), and such domains can also be found in the MelARV Env protein (Schlecht-Louf, G., et al., Retroviral infection in vivo requires an immune escape virulence factor encrypted in the envelope protein of oncoretroviruses. Proc Natl Acad Sci USA, 2010. 107(8): p. 3782-7 and Mangeney, M. and T. Heidmann, Tumor cells expressing a retroviral envelope escape immune rejection in vivo. Proc Natl Acad Sci USA, 1998. 95(25): p. 14920-5). The importance of ISD in MuLV or MelARV has been demonstrated by introducing the mouse leukemia virus Env protein into tumor cells that are normally rejected by immune cells (Mangeney, M. and T. Heidmann, Tumor cells expressing a retroviral envelope escape immune rejection in vivo. Proc Natl Acad Sci USA, 1998. 95(25): p. 14920-5). Tumor cells transduced with Env proliferated more rapidly despite the presence of additional exogenous antigens. This observation was explained by the local immunosuppressive effect mediated by the Env protein. ISD affects both the innate and adaptive immune systems, as demonstrated by the inhibition of macrophages, NK cells, and T cells.(Lang, MS, et al., Immunotherapy with monoclonal antibodies directed against the immunosuppressive domain of p15E inhibits tumor growth. Clin Exp Immunol, 1995. 102(3): p. 468-75). Furthermore, effects on regulatory T cell subsets have been suggested, and this effect itself suppresses other immune cells (Mangeney, M., et al., Endogenous retrovirus expression is required for murine melanoma tumor growth in vivo. Cancer Res, 2005. 65(7): p. 2588-91). The detailed mechanism of immunosuppression by ISD is not yet fully understood, but most of its effects appear to be mediated by the CKS-17 peptide within ISD. CKS-17 exerts diverse effects on the immune system, most of which are due to altering cytokine expression (Haraguchi, S., RA Good, and NK Day-Good, A potent immunosuppressive retroviral peptide: cytokine patterns and signaling pathways. Immunol Res, 2008. 41(1): p. 46-55).

[0009] One of the first therapeutic approaches to target tumor cells expressing ERV involved the administration of monoclonal antibodies. Antibodies targeting HERV-K Env were able to reduce tumor growth in breast cancer cell lines. Wang-Johanning et al. showed that the observed effects of anti-HERV-K Env monoclonal antibodies were mediated by changes in the cancer cell cycle and increased apoptosis. Another possible effect of such antibodies, though not tested by Wang-Johanning et al. (Wang-Johanning, F., et al., Immunotherapeutic potential of anti-human endogenous retrovirus-K envelope protein antibodies in targeting breast tumors. J Natl Cancer Inst, 2012. 104(3): p. 189-210), may be the prevention of immunosuppression. Similar to MelARV Env, the HERV-K Env protein contains an ISD and possesses immunomodulatory function (Morozov, VA, VL Dao Thi, and J. Denner, The transmembrane protein of the human endogenous retrovirus-K (HERV-K) modulates cytokine release and gene expression. PLoS One, 2013. 8(8): p. e70399). The approach tested by Wang-Johanning et al. included xenograft tumors in immunodeficient athymic mice. Therefore, the effect of HERV-K could only affect innate immune cells such as NK cells.

[0010] Another part of the adaptive immune response that can help eradicate tumors by targeting ERVs involves T cells. For example, adoptive T cells against the MuLV Env epitope combined with IL-2 were able to eradicate lung metastases of melanoma cells (Yang, JC and D. Perry-Lalley, The envelope protein of an endogenous murine retrovirus is a tumor-associated T-cell antigen for multiple murine tumors. J Immunother, 2000. 23(2): p. 177-83). Similar experiments were carried out in a humanized mouse model of HERV-K. T cells were genetically modified to express a chimeric antigen receptor (CAR) on their surface that recognizes HERV-K Env on cancer cells. Cytotoxic CAR+ T cells were able to lyse tumor cells and prevented not only tumor growth but also metastasis.

[0011] In addition to direct injection of antibodies or T cells, a more practical, less expensive, and more efficient strategy is the induction of an immune response through vaccination. A simple approach is vaccination with virus-encoded antigens. However, this method is rather cumbersome because dendritic cells (DCs) must first be isolated and cultured before being pulsed with defined HLA-restricted peptides and reinjected into mice or patients.

[0012] One sophisticated vaccination strategy involves presenting antigens (e.g., viral envelope proteins) to the immune system on virus-like particles (VLPs) encoded by nucleotides composed of the vaccine. These particles are non-infectious because they do not contain viral nucleic acids. Nevertheless, VLPs are highly immunogenic, and the presented proteins are presented in a natural context. For example, the viral Env protein incorporated into a VLP is presented on a virus-like surface, which facilitates correct folding and conformation. In addition to the advantages of potent immunogenicity, vaccination strategies using VLPs also have practical advantages. Therefore, VLPs are relatively easy to produce, as they can be constructed from a single protein or a small number of proteins and production can be carried out in cell culture. Bayer et al. (2010) showed that only the combination of encoded antigen and capsid-presenting antigen can enhance the level of functional antibody. This observation was consistent with the fact that presentation of adenovirus on the capsid facilitated cross-linking of B cell receptors, while the encoded antigen was required for the essential CD4+ T cell response that promoted B cell affinity maturation. With this vaccination strategy, Bayer et al. were able to reduce the viral load of F-MLV after challenge. However, no signs of an increased CD8+ T cell response to the target antigen were observed. Shoji et al. investigated in-situ formation of Gag-based VLPs, focusing primarily on the optimization of adenovirus-based HIV vaccines. In their study, such a setting showed the highest immune response compared to other presentation strategies that did not promote in-situ formation of VLPs [Shoji et al., 2012].

[0013] For vaccination against viruses or virus-related diseases (e.g., cancers expressing ERVs), the entire Env protein should ideally be presented to the immune system to ensure an immune response to all protein targets. However, because the Env protein contains an immunosuppressive domain (ISD), the vaccine itself has immunosuppressive properties, which is undesirable for immunization approaches. To circumvent this drawback, mutations have been introduced into the ISD to maintain the natural conformation of the target protein while simultaneously preventing immunosuppression. Similarly, U.S. Patent Application Publication No. 2012189647 relates to a mutant envelope protein resulting from a mutation in the immunosuppressive domain of the transmembrane subunit of the wild-type envelope protein.

[0014] Schlecht-Louf et al. were among the first to test inactivating mutations in the ISD of viral proteins [Schlecht-Louf et al. (2010)]. Based on a comparative study between immunosuppressive syncytin-2 and non-immunosuppressive syncytin-1 [Mangeney et al. (2007)], Schlecht-Louf et al. identified mutations that inactivate ISD activity without compromising the overall structure and functionality of the Env protein. This mutation strategy was applied to proteins of other viral origins (e.g., HTLV and XMRV) and more extensively tested on friend mouse leukemia virus (F-MLV). This study not only revealed ISD-mediated suppression of NK and T cells but also showed that attenuated live F-MLV viruses containing mutant ISDs in the Env protein functioned as a vaccine against the same virus with WT ISD sequences. This protection was attributed to increased antibody levels as well as a T cell response to the F-MLV epitope. Their findings were ultimately reflected in International Publication No. 2011 / 092199 of a patent application focusing on heterotropic mouse leukemia virus-associated virus (XMRV) associated with human prostate cancer and chronic fatigue syndrome. Therefore, International Publication No. 2011 / 092199 relates, in particular, to ISD mutations in XMRV and the utilization of such ISD variant viruses in vaccination strategies.

[0015] Another application of the ISD mutation is described in the international publication 2014 / 195510 of the patent application. In this case, the ISD mutation was introduced into feline immunodeficiency virus (FIV) to maintain natural conformation while reducing viral immunosuppression. International publication 2014 / 195510 describes that specific mutations increased the antibody response to the FIV Env protein when administered in a vaccination approach, when conjugated to MBP, or when transduced into transplanted tumor cells. Therefore, international publication 2014 / 195510 relates to the ISD mutation of FIV Env and the use of such mutant proteins in vaccination approaches against infection with FIV or other lentiviruses.

[0016] Another approach addressing a broader spectrum of ISD mutations in viral Env proteins is described in the international publication of a patent application, International Publication 2013 / 050048. In particular, International Publication 2013 / 050048 relates to the generation of antigens by first identifying ISDs in enveloped RNA viruses and then mutating these domains to reduce immunosuppression upon vaccination. The ISD identification strategy is based on the following four parameters: 1) the peptide is located in the fusion protein of the enveloped RNA virus; 2) the peptide is able to interact with the membrane; 3) there is high homology in the primary structure (sequence) of the peptide within any of the order, family, subfamily, genus, or species of the virus; and 4) the location on the surface of the fusion protein in a given conformation is a feature of the immunosuppressive domain revealed by 3D structure or antibody staining. After identifying potential ISDs in the target viral Env, the immunosuppressive function was verified, followed by the introduction of mutations into the ISDs, which confirmed at least a 25% reduction in immunosuppression. Overall, International Publication No. 2013 / 050048 describes the identification of ISDs in enveloped RNA viruses, the generation of ISD mutant peptides, and the use of the aforementioned peptides as vaccines and the production of antibodies.

[0017] While strategies have been employed to use adenoviruses to encode and present viral antigens by mutating the ISD in the viral Env protein, past vaccination strategies employing ISD mutations have primarily aimed at preventing viral infection [Schlecht-Louf et al. 2010; International Publication 2011 / 092199; International Publication 2014 / 195510; US Patent Application Publication 20110305749; International Publication 2014 / 195510]. Therefore, the need to disrupt tolerance to self-antigens still exists. Such disruption of tolerance can be facilitated by particles densely coated with surface proteins (such as VLPs). Dense coating with membrane-associated viral glycoproteins increases with increased cell surface expression before the proteins are incorporated into virus-like particles budding from the cell surface. An additional advantage of robust cell surface expression is its ability to directly stimulate B cell recognition (see also Ferapontov, A., Omer, M., Baudrexel, I. et al. Antigen footprint governs activation of the B cell receptor. Nat Commun 14, 976 (2023)).

[0018] Furthermore, while systems for in situ synthesis of virus-like particles have been used for some time [Luo et al. (2003); Sohji et al. (2011); Andersson et al. (2016); Andersson & Holst (2016); Andersson et al. (2017)], alternative methods for improving antigen presentation other than using VLPs are needed in situations where VLPs are not applicable. Moreover, efficient systems are needed that enable the efficient production of antigens or antigen-VLPs, such as HERV-K VLPs. Ideally, establishing such systems requires the prior effective introduction of the system into host cells or organisms. Therefore, there is a need to provide improved delivery methods and means for administering polynucleotides to induce an immune response in a target and / or to treat diseases involving the expression of proteins, such as cancer marker proteins, in the target cells.

[0019] Summary of the Invention As outlined above, with respect to ERVs, for example, efficient surface presentation of endogenous retroviral antigens is required for targeted vaccination against endogenous tumor potential and / or tumor progression. The present invention aims to produce such effective vaccines for the prevention and / or treatment of diseases caused by endogenous retroviruses. The compositions of the present invention, comprising the mRNA of the present invention, provide improved surface presentation of the encoded antigen to host cells, integration into virus-like particles, and increased stimulation of the immune system.

[0020] Accordingly, in one embodiment, the present invention relates to a composition comprising a transfection agent and mRNA encoding at least a human endogenous retrovirus (HERV) envelope protein or an immunogenic portion thereof, wherein the HERV envelope protein comprises an immunosuppressive domain (ISD), and the HERV Env protein comprises a mutant immunosuppressive domain (ISD) that reduces immunosuppressive properties and / or enables efficient presentation on the cell surface compared to a wild-type ISD. In one embodiment, the composition comprises lipid nanoparticles (LNPs) containing the aforementioned mRNA.

[0021] In another embodiment, the present invention relates to a composition of the present invention for use as a pharmaceutical.

[0022] In another embodiment, the present invention relates to the use of compositions for the manufacture of pharmaceuticals.

[0023] In yet another embodiment, the present invention relates to compositions for use in preventing or treating diseases, preferably for immunizing a subject from disease.

[0024] In yet another embodiment, the present invention relates to the use of compositions for the manufacture of pharmaceuticals for the prophylactic and / or therapeutic treatment of a disease, preferably for the manufacture of pharmaceuticals for immunizing a subject from a disease. In yet another embodiment, the present invention relates to a method for treating and / or preventing a disease, preferably for immunizing a subject from a disease, comprising administering a subject (preferably in a therapeutically effective amount) of the composition of the present invention. In one embodiment, the disease is preferably selected from the group consisting of cancer, HIV and / or related diseases, rheumatic diseases, neurodegenerative diseases, age-related diseases, diseases associated with HERV reactivation, chronic inflammation, multiple sclerosis, ALS associated with TDP-43, Alzheimer's disease associated with Tau expression, ALS, sarcopenia, kidney disease and Alzheimer's disease.

[0025] In yet another aspect, the present invention relates to a composition according to the present invention for use in preventing or delaying aging and / or cellular senescence. In yet another aspect, the present invention relates to a composition according to the present invention for use in the manufacture of a medicament for preventing or delaying aging and / or cellular senescence.

[0026] In another aspect, the present invention relates to a pharmaceutical composition comprising a composition of the present invention and a pharmaceutically acceptable excipient.

[0027] In another aspect, the present invention relates to a DNA molecule encoding the mRNA contained in the composition of the present invention.

[0028] Yet another aspect of the present invention relates to virus-like particles (VLPs) comprising a HERV envelope protein defined according to the present invention described herein.

[0029] Detailed Description Definitions Administration: As used herein, "administration" refers to a method of delivering a composition to a subject or patient. The method of administration can be selected to target (e.g., specifically deliver) delivery to a particular region or system of the body. For example, administration can be parenteral (e.g., subcutaneous, intradermal, intravenous, intraperitoneal, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional, or intracranial injection, and any suitable infusion technique), oral, transdermal or intradermal, intradermal, rectal, vaginal, topical (e.g., by powder, ointment, cream, gel, lotion, and / or drops), mucosal, nasal, buccal, enteral, intravitreal, intratumoral, sublingual, intranasal; by intratracheal instillation, bronchial instillation, and / or inhalation; as an oral spray and / or powder, nasal spray, and / or aerosol, and / or via a portal catheter. Preferred means of administration are intramuscular, intravenous, intradermal or subcutaneous.

[0030] And / or (and / or): As used herein, the term "and / or (and / or)" shall always include the meanings of "and (and)", "or", and "all or any other combination of the elements connected by the aforementioned terms".

[0031] Generally, about: As used herein, the terms "generally" or "about" apply to one or more values of interest and refer to values similar to the specified reference value. In certain embodiments, the terms "generally" or "about" refer to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or smaller) of the specified reference value, unless otherwise specified or clear from the context (except where such numbers exceed 100% of the possible values). For example, when used in the context of the amount of a given compound in the lipid component of an LNP, "about" may mean ±5% of the specified value. For example, an LNP containing a lipid component having about 40% of a given compound may contain 30 - 50% of the compound.

[0032] Comprise: Throughout this specification and the following claims, unless the context otherwise requires, the word "comprise", and variations such as "comprises" and "comprising", are to be understood to mean including the stated integer or step or group of integers or steps but not to mean excluding other integers or steps or group of integers or steps. As used herein, the term "comprising" can be replaced by the term "containing" or "including", and sometimes, as used herein, can be replaced by the term "having".

[0033] Conjugated: As used herein, the term “conjugated” means, when used in reference to two or more sites, that those sites are physically related to each other, either directly or through one or more additional sites acting as linkers, or that in forming a structure, the structure is stable enough that the sites remain physically related under the conditions in which the structure is used, for example, physiological conditions. In some embodiments, two or more sites may be conjugated by direct covalent chemical bonds. In other embodiments, two or more sites may be conjugated by ionic or hydrogen bonds.

[0034] Contact: As used herein, the term “contact” means to establish a physical connection between two or more entities. For example, contacting a cell with an mRNA or lipid nanoparticle composition means that the cell and the mRNA or lipid nanoparticles share a physical connection. Methods for contacting cells with external entities in vivo, in vitro, and ex vivo are well known in the field of biology. In exemplary embodiments of this disclosure, the step of contacting mammalian cells with a composition (e.g., the transfection agent or nanoparticles or pharmaceutical composition of this disclosure) is carried out in vivo. For example, contact between a lipid nanoparticle composition and cells that may be placed in an organism (e.g., a mammal) (e.g., mammalian cells) can be carried out by any suitable route of administration (e.g., parenteral administration to an organism, including intravenous, intramuscular, intradermal, and subcutaneous administration). For cells present in vitro, a composition (e.g., a transfection agent, e.g., lipid nanoparticles) and cells can be brought into contact, for example, by adding the composition to the cell culture medium, which may involve or result in transfection. Furthermore, multiple cells can be brought into contact with the transfection agent or nanoparticle composition.

[0035] To deliver: As used herein, the term “to deliver” means to provide an entity to a destination. For example, delivering a therapeutic and / or prophylactic agent to a target may include administering a composition containing the therapeutic and / or prophylactic agent to the target (e.g., by an intravenous, intramuscular, intradermal, or subcutaneous route). Administering a composition to a mammal or mammalian cells may include bringing one or more cells into contact with the composition.

[0036] Encapsulation: As used herein, the term “encapsulation” means to surround, enclose, or contain. In some embodiments, a compound, polynucleotide (e.g., mRNA), or other composition may be fully encapsulated, partially encapsulated, or substantially encapsulated. For example, in some embodiments, mRNA of the Disclosure may be encapsulated in lipid nanoparticles, such as liposomes.

[0037] Effective dose: An "effective dose" of a drug is an amount sufficient to produce a beneficial or desired outcome, such as a clinical outcome, and therefore, the "effective dose" depends on the context in which it is applied. For example, in the context of the amount of target cell delivery enhancing lipids in a lipid composition (e.g., LNP) of this disclosure, an effective dose of target cell delivery enhancing lipids is an amount sufficient to produce a beneficial or desired outcome compared to a lipid composition (e.g., LNP) lacking target cell delivery enhancing lipids. Non-limiting examples of beneficial or desired outcomes produced by a lipid composition (e.g., LNP) include increasing the percentage of transfected cells and / or increasing the expression level of proteins encoded by nucleic acids associated with / encapsulated in the lipid composition (e.g., LNP). In the context of administering lipid nanoparticles containing target cell delivery enhancing lipids so that an effective dose of lipid nanoparticles is taken up by target cells of interest, an LNP containing an effective dose of target cell delivery enhancing lipids is an amount sufficient to produce a beneficial or desired outcome compared to an LNP lacking target cell delivery enhancing lipids. Non-limiting examples of beneficial or desirable outcomes in a subject include increasing the percentage of transfected cells, increasing the expression level of proteins encoded by nucleic acids associated with / encapsulated in LNPs containing target cell delivery-enhancing lipids, and / or increasing the in vivo prophylactic or therapeutic effect of nucleic acids associated with / encapsulated in LNPs containing target cell delivery-enhancing lipids or the proteins they encode, compared to LNPs lacking target cell delivery-enhancing lipids. In some embodiments, LNPs containing a therapeutically effective amount of target cell delivery-enhancing lipids are sufficient to treat, improve symptoms of, diagnose, prevent, and / or delay the onset of infections, diseases, disorders, and / or conditions when administered to subjects suffering from or susceptible to such conditions. In another embodiment, an effective amount of lipid nanoparticles is sufficient to result in the expression of a desired protein at a value of at least about 5%, 10%, 15%, 20%, 25% or more of target cells.For example, an effective amount of LNP containing target cell delivery-enhancing lipids may be such that a single intravenous injection results in the transfection of at least 5%, 10%, 15%, 20%, 25%, 30%, or 35% of target cells.

[0038] Expression: As used herein, “expression” of a nucleic acid sequence means one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and / or 3' end processing); (3) translation of RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein.

[0039] Ex vivo: As used herein, the term “ex vivo” refers to an event occurring outside of an organism (e.g., an animal, plant, or microorganism, or their cells or tissues). Ex vivo events can occur in an environment that has been minimally altered from its natural (e.g., in vivo) environment.

[0040] Fragment: As used herein, “fragment” means a part of a protein. For example, a protein fragment may include a polypeptide obtained by digesting a full-length protein isolated from cultured cells, or a polypeptide obtained by recombinant DNA technology. A protein fragment may be, for example, a part of a protein that contains one or more functional domains such that the protein fragment retains the functional activity of the protein.

[0041] Isolated: As used herein, the term “isolated” means a substance or entity that has been separated from at least some of the components to which it was associated (whether in nature or in a laboratory setting). Isolated substances may have varying levels of purity relative to the substances to which they were associated. Isolated substances and / or entities may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components to which they were originally associated. In some embodiments, isolated agents have a purity of about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99%. As used herein, a substance is “pure” if it substantially contains no other components.

[0042] Kozak Sequence: The term “Kozak sequence” (also known as “Kozak consensus sequence”) refers to a translation initiation enhancer element for increasing the expression of a gene or open reading frame, located at the 5'UTR in eukaryotes. The Kozak consensus sequence was originally defined as the sequence GCCRCC, where R=purine, based on the analysis of the effect of a single mutation surrounding the start codon (AUG) on the translation of the precursor proinsulin gene (Kozak (1986) Cell 44:283-292). The polynucleotides disclosed herein include Kozak consensus sequences, or their derivatives or modifications. (For examples of translation enhancer compositions and methods of use thereof, see U.S. Patent No. 5,807,707 by Andrews et al. (which is incorporated herein by reference in its entirety); U.S. Patent No. 5,723,332 by Chernajovsky (which is incorporated herein by reference in its entirety); and U.S. Patent No. 5,891,665 by Wilson (which is incorporated herein by reference in its entirety)).

[0043] Metastasis: As used herein, the term “metastasis” refers to the process by which cancer spreads from the site where it first originated as a primary tumor to distant locations within the body. Secondary tumors resulting from this process may also be called “metastases.”

[0044] Modified: As used herein, “modified” or “modified” means an altered state, or change in composition or structure, of a molecule (e.g., a polynucleotide, e.g., mRNA) of the Disclosure. Molecules (e.g., polynucleotides) can be modified in a variety of ways, including chemically, structurally, and / or functionally. For example, a polynucleotide may be structurally modified by the incorporation of one or more RNA elements, which include sequences and / or RNA secondary structures that provide one or more functions (e.g., translational regulatory activity). Thus, a polynucleotide of the Disclosure may consist of one or more modifications (e.g., one or more chemical, structural, or functional modifications, including any combination thereof). In one embodiment, an mRNA molecule of the Disclosure may be modified, for example, by the introduction of non-natural nucleosides and / or nucleotides, since it relates to natural ribonucleotides A, U, G, and C. Non-standard nucleotides, such as cap structures, differ in chemical structure from A, C, G, and U ribonucleotides but are not considered “modified.”

[0045] mRNA: As used herein, “mRNA” refers to messenger ribonucleic acid. mRNA may be naturally occurring or unnatural. For example, mRNA may contain modified and / or unnaturally occurring components such as one or more nucleic acid bases, nucleosides, nucleotides, or linkers. mRNA may contain a cap structure, a chain termination nucleoside, a stem-loop, a poly(A) sequence, and / or a polyadenylation signal. mRNA may have a nucleotide sequence encoding a polypeptide. mRNA may also have a nucleotide sequence encoding multiple (e.g., at least two or at least three) different polypeptides. Polypeptides can be produced by translation of mRNA, for example, in vivo translation of mRNA in mammalian cells. Traditionally, the basic components of an mRNA molecule include at least a coding region, a 5'-untranslated region (5'UTR), a 3'UTR, a 5' cap, and a poly(A) sequence. In the context of the present invention, in the mRNA sequences disclosed herein (particularly in the tables outlined below), the symbol "T" is interpreted to represent uracil in the aforementioned mRNA (or, if indicated, modified uracil such as N1-methylpseudridine).

[0046] Nanoparticles: As used herein, “nanoparticles” refers to particles having any one structural feature on a scale of less than about 1000 nm that exhibit novel properties compared to a bulk sample of the same material. Typically, nanoparticles have any one structural feature on a scale of less than about 500 nm, less than about 200 nm, or less than about 600 nm. Also, typically, nanoparticles have any one structural feature on a scale of about 50 nm to about 500 nm, about 50 nm to about 200 nm, or about 70 nm to about 120 nm. In exemplary embodiments, nanoparticles are particles having one or more dimensions on the order of about 1 to 1000 nm. In other exemplary embodiments, nanoparticles are particles having one or more dimensions on the order of about 10 to 500 nm. In other exemplary embodiments, nanoparticles are particles having one or more dimensions on the order of about 50 to 200 nm. For spherical nanoparticles, for example, they would have a diameter between about 50 to 100 or 70 to 120 nanometers. Nanoparticles, in most cases, behave as a single unit in terms of their transport and properties. Novel properties that distinguish nanoparticles from their corresponding bulk materials typically manifest at a size scale of less than 1,000 nm, or around 100 nm, although it should be noted that nanoparticles can have larger sizes, for example, oval or tubular particles. While the size of most molecules fits within the outline above, individual molecules are not usually referred to as nanoparticles.

[0047] Nucleic acids: As used herein, the term “nucleic acids” is used in its broadest sense and, unless otherwise specified, encompasses any compound and / or substance containing polymers of nucleotides. These polymers are often called polynucleotides. It also includes the chemical derivatization of nucleic acids on nucleotide bases, sugars, or phosphates, and nucleic acids including non-natural nucleotides and nucleotide analogs. Examples of nucleic acids or polynucleotides in this disclosure include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), DNA-RNA hybrids, RNAi inducers, RNAi agents, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, RNA that induces triple helix formation, threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), Loc nucleic acid (LNA, including LNA having a β-D-ribo configuration, α-LNA having an α-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2'-amino functional group, and 2'-amino-α-LNA having a 2'-amino functional group), or hybrids thereof.

[0048] Nucleic acid bases: As used herein, the term “nucleic acid base” (alternatively “nucleotide base” or “nitrogen base”) refers to purine or pyrimidine heterocyclic compounds found in nucleic acids, including any naturally occurring derivatives or analogs of purines and pyrimidines that confer improved properties (e.g., binding affinity, nuclease resistance, chemical stability) to nucleic acids or parts or segments thereof. Adenine, cytosine, guanine, thymine, and uracil are nucleic acid bases primarily found in naturally occurring nucleic acids. Other natural, unnatural, and / or synthetic nucleic acid bases may also be incorporated into nucleic acids as known in the art and / or as described herein.

[0049] Nucleoside / Nucleotide: As used herein, the term “nucleoside” refers to a compound containing a sugar molecule (e.g., ribose in RNA or deoxyribose in DNA) or a derivative or analog thereof, which is covalently bonded to a nucleic acid base (e.g., purine or pyrimidine) or a derivative or analog thereof (also referred herein as “nucleoside base”) but lacks an internucleoside linking group (e.g., a phosphate group). As used herein, the term “nucleotide” refers to a nucleoside covalently bonded to an internucleoside linking group (e.g., a phosphate group), or a derivative, analog, or modification thereof that confers improved chemical and / or functional properties (e.g., binding affinity, nuclease resistance, chemical stability) to a nucleic acid or a portion or segment thereof.

[0050] Open Reading Frame: As used herein, the term “open reading frame” (abbreviated as “ORF”) refers to a segment or region of an mRNA molecule that encodes a polypeptide. An ORF contains a sequence of non-overlapping in-frame codons, beginning with a start codon and ending with a stop codon, and is translated by a ribosome.

[0051] Patient: As used herein, “patient” means a person who may seek or may require treatment, is receiving treatment, is scheduled to receive treatment, or is receiving care from a professional trained for a particular disease or condition. In certain embodiments, patient is a human patient. In some embodiments, patient is a person suffering from, for example, an autoimmune disease as described herein.

[0052] Pharmaceutically acceptable: The expression "pharmaceutically acceptable" is used herein to mean a compound, material, composition, and / or dosage form that, within the bounds of sound medical judgment, is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reaction, or other problems or complications, and that is commensurate with a reasonable benefit / risk ratio.

[0053] Pharmacopoeia-acceptable excipients: “Pharmacopoeia-acceptable excipients” means any component other than the compounds described herein (e.g., a vehicle capable of suspending or dissolving the active compound) that is substantially non-toxic and non-inflammatory to the patient. Examples of excipients include anti-adhesion agents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colorants), emollients, emulsifiers, fillers (diluents), film-forming agents or coatings, fragrances, aromas, lubricants (flow improvers), preservatives, printing inks, adsorbents, suspending agents or dispersants, sweeteners, and hydration water. Examples of excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, dibasic calcium phosphate, calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0054] pharmaceutically acceptable salts: As used herein, “pharmaceutically acceptable salts” means derivatives of the disclosed compounds that are modified by converting an existing acidic or base moiety of the parent compound into its salt form (for example, by reacting a free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic salts of basic residues such as amines; and alkali or organic salts of acidic residues such as carboxylic acids. Typical acid addition salts include acetate, acetic acid, adipine, alginate, ascorbate, aspartate, benzenesulfonate, benzenesulfonic acid, benzoate, bisulfate, borate, butyrate, camphorate, camphor sulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptoneate, hexanoate, hydrobromide, hydrochloride, hydroiodide, and 2-hydroxyethanesulfonate. Examples of alkali metal salts include, but are not limited to, lactobionates, lactates, laurates, lauryl sulfates, malates, maleates, malons, methanesulfons, 2-naphthalenesulfons, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectins, persulfates, 3-phenylpropionates, phosphates, picrates, pivaphosphates, propions, stearates, succinates, sulfates, tartrates, thiocyans, toluenesulfons, undecanoates, and valersates. Representative alkali metal salts or alkaline earth metal salts include, but are not limited to, sodium, lithium, potassium, calcium, magnesium, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine. Examples of pharmaceutically acceptable salts of the present disclosure include conventional non-toxic salts of the parent compound formed from non-toxic inorganic or organic acids. These pharmaceutically acceptable salts may be synthesized from a parent compound containing a basic or acidic moiety by conventional chemical methods.Generally, such salts can be prepared by reacting the free acidic or base form of these compounds with a stoichiometric amount of a suitable base or acid in water, an organic solvent, or a mixture thereof, generally preferred in non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile. A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, PH Stahl and CG Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which is incorporated herein by reference in whole.

[0055] Polyadenyle / Poly-A Tail / Sequence: “Polyadenyle sequence,” “Poly-A sequence,” or “Poly-A tail” refers to a sequence of adenyl residues typically located at the 3' end of an RNA molecule. Such sequences can be added during RNA transcription. Typically, the poly-A sequence is added to the free 3' end of the RNA by template-independent RNA polymerase after transcription in the nucleus. Artificially, poly-A can be added by transcription from a DNA template containing complementary repeating thymidyl residues. Alternatively, the mRNA described herein may contain a polyadenylation signal, which is defined herein as a signal that transmits polyadenylation to the (transcribed) RNA by a specific protein factor. The poly-A sequence is important for the nuclear export, translation, and stability of mRNA, and it shortens over time, eventually leading to enzymatic mRNA degradation.

[0056] Polypeptide: As used herein, the terms “polypeptide” or “polypeptide of interest” refer to polymers of amino acid residues typically linked by peptide bonds, which may be produced naturally (e.g., isolated or purified) or synthetically.

[0057] Prevent / preventing: As used herein, the terms “prevent” or “preventing” mean partially or completely inhibiting the development of one or more symptoms or characteristics of a particular infection, disease, disorder, and / or medical condition.

[0058] Prevention: As used herein, the term “prevention” means partially or completely inhibiting the development of one or more symptoms or characteristics of a particular infectious disease, illness, disorder, and / or medical condition. The term “preventive” is also used in this sense.

[0059] RNA: As used herein, “RNA” means ribonucleic acid, whether naturally occurring or unnatural. For example, RNA may contain one or more nucleic acid bases, nucleosides, nucleotides, or modified and / or unnaturally occurring components such as linkers. RNA may contain a cap structure, a chain termination nucleoside, a stem-loop, a poly(A) sequence, and / or a polyadenylation signal. RNA may have a nucleotide sequence that codes for a polypeptide of interest. For example, RNA is messenger RNA (mRNA). Translation of mRNA that codes for a particular polypeptide, for example, in vivo translation of mRNA in a mammalian cell, can produce the coded polypeptide.

[0060] RNA elements: As used herein, the term “RNA element” refers to a portion, fragment, or segment of an RNA molecule that provides a biological function and / or has biological activity (e.g., translational regulatory activity). Modification of a polynucleotide by the incorporation of one or more RNA elements, as described herein, provides the modified polynucleotide with one or more desirable functional properties. RNA elements, as described herein, may be naturally occurring, unnaturally occurring, synthetic, modified, or any combination thereof. For example, naturally occurring RNA elements that provide regulatory activity include elements found throughout the transcriptomes of viruses, prokaryotes, and eukaryotes (e.g., humans). RNA elements in certain eukaryotic mRNAs and translated viral RNAs have been shown to be involved in mediating many intracellular functions.Examples of native RNA elements include translation initiation elements (e.g., internal ribosome entry sites (IRES), see Kieft et al., (2001) RNA 7(2): 194-206), translational enhancer elements (e.g., APP mRNA translational enhancer elements, see Rogers et al., (1999) J Biol Chem 274(10):6421-6431), mRNA stability elements (e.g., AU-rich elements (AREs), see Garneau et al., (2007) Nat Rev Mol Cell Biol 8(2): 113-126), translational repressor elements (e.g., Blumer et al., (2002) Mech Dev 110(1-2):97-112), and protein-binding RNA elements (e.g., iron-responsive elements, see Selezneva et al., (2013) J Mol Biol Examples include, but are not limited to, cytoplasmic polyadenylated elements (see 425(18):3301-3310), cytoplasmic polyadenylated elements (see Villalba et al., (2011) Curr Opin Genet Dev 21(4):452-457), and catalytic RNA elements (e.g., ribozymes, see Scott et al., (2009) Biochim Biophys Acta 1789(9-10):634-641).

[0061] Sequence: As used herein, the term “sequence” should generally be understood to include both the amino acid sequence in question and the nucleic acid or nucleotide sequence that encodes it, unless the context requires a more restrictive interpretation. An amino acid sequence is interpreted, depending on the context, to mean an unbranched sequence of a single amino acid or two or more amino acids. A nucleotide sequence is interpreted to mean an unbranched sequence of three or more nucleotides.

[0062] Specific delivery: As used herein, the terms “specific delivery,” “specifically deliver,” or “specifically delivering” mean that the nanoparticles deliver more of the therapeutic and / or prophylactic agent to target cells (e.g., mammalian target cells) compared to off-target cells (e.g., non-target cells). The level at which nanoparticles are delivered to specific cells can be measured by comparing the amount of protein produced in target and non-target cells (e.g., by mean fluorescence intensity using flow cytometry), comparing the percentage of target and non-target cells expressing the protein (e.g., by quantitative flow cytometry), comparing the amount of protein produced in target and non-target cells to the total amount of protein in the aforementioned target and non-target cells, or comparing the amount of therapeutic and / or prophylactic agents in target and non-target cells to the total amount of therapeutic and / or prophylactic agents in the aforementioned target and non-target cells. It will be understood that the ability of nanoparticles to specifically deliver to target cells does not need to be determined in the subject under treatment, but can be determined in alternative systems such as animal models (e.g., mouse or NHP models).

[0063] Substantially: As used herein, the term “substantially” refers to a qualitative state that indicates the overall or nearly overall range or degree of the nature or characteristic of the subject. Those skilled in the art of biology will understand that it is very rare for biological and chemical phenomena to be completed and / or progress toward completeness, or to achieve or avoid absolute results. Therefore, the term “substantially” is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0064] Afflicted: An individual “afflicted” with a disease, disorder, and / or condition is diagnosed with or exhibiting one or more symptoms of the disease, disorder, and / or condition.

[0065] Therapeutic agent: The term “therapeutic agent” refers to any drug that, when administered to a subject, has a therapeutic, diagnostic, and / or preventive effect and / or produces a desired biological and / or pharmacological effect.

[0066] Transfection: As used herein, the term “transfection” refers to a method of introducing a species (e.g., polynucleotides such as mRNA) into a cell.

[0067] Subject: As used herein, the term “subject” means any organism to which the composition relating to this disclosure may be administered, for example, for experimental, diagnostic, preventive, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants. In some embodiments, the subject may be a patient.

[0068] To treat: As used herein, the term “to treat” means to partially or completely reduce, remit, improve, alleviate, delay the onset, inhibit the progression, reduce the severity, and / or reduce the incidence of one or more symptoms or features of a particular infection, disease, disorder, and / or condition. For example, “to treat” cancer may mean to inhibit the survival, growth, and / or spread of the tumor. Treatment may be administered to subjects who do not show signs of the disease, disorder, and / or condition, and / or subjects who show only early signs of the disease, disorder, and / or condition, with the aim of reducing the risk of developing the condition associated with the disease, disorder, and / or condition.

[0069] Unmodified: As used herein, “unmodified” refers to a substance, compound, or molecule before it is altered in any way. “Unmodified” may also refer to the wild-type or native-type biomolecule, but not always. A molecule may undergo a series of modifications, and each modified molecule may serve as an “unmodified” starting molecule for subsequent modifications.

[0070] Variant: As used herein, the term “variant” means a molecule having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity of the wild-type molecule, as measured, for example, by assays accepted in the art.

[0071] Wild-type / WT: "Wild-type" (or "WT") refers to the genotype or phenotype of a feature or sequence in its natural form, i.e., the original or unmutated form of a feature. When referred to in particular in the context of an immunosuppressive domain (ISD), this wild-type ISD is active, i.e., it suppresses the immune response. The activity or inactivation of an ISD may be determined as described elsewhere herein.

[0072] Percentage Identity or % Identity: This term refers to the percentage of nucleotides or amino acids that are identical in the optimal alignment between two nucleotide or amino acid sequences being compared. Typically, comparing two sequences requires an optimal alignment step, which can be done manually or by utilizing local homology algorithms such as those described by Smith and Waterman, 1981, Ads App. Math. 2, 482; Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443; Pearson and Lipman, 1988, Proc. Natl Acad. Sei. USA 85, 2444; or by using computer programs that employ these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). Then, one or more corresponding local regions of the two sequences are identified. Next, the number of identical positions shared by the two sequences is determined, and this is divided by the length of the reference sequence to calculate the percentage identity across the entire sequence. This result is then multiplied by 100. In other words, percentage sequence identity defines the total number of identical amino acids (when comparing amino acid sequences) or nucleotides (when comparing nucleotide sequences) in the query sequence across the entire length of the reference sequence. The program "BLAST 2 sequences" is an exemplary tool for performing this calculation and is available on the website http: / / www.ncbi.nlm.nih.gov / blast / bl2seq / wblast2.cgi.

[0073] The 5' / 3' untranslated region / UTR is, as used herein, the "3'" or "3' end" of a nucleic acid is the end with a free hydroxyl group, and the "5'" or "5' end" of a nucleic acid is the end with a free phosphate group. When a double-stranded nucleic acid is diagrammed, the 3' end is on the right and the 5' end is on the left. [ka]

[0074] As used herein, “untranslated region” or “UTR” refers to one of two sections on either side of the coding sequence of mRNA that are not part of the protein-coding region. The 5'-terminal UTR is called the 5'UTR (or leader sequence), and the 3'-terminal UTR is called the 3'UTR (or trailer sequence). UTRs may contain RNA elements that control translation and / or transcription, as described elsewhere herein. For example, the 5'UTR facilitates translation initiation by allowing ribosomes to bind to the sequence, and the 3'UTR is known to be involved in post-transcriptional modifications as well as translation termination, for example.

[0075] LNPs containing HERV envelope proteins In a first embodiment, the present invention relates to a composition comprising a transfection agent and mRNA encoding at least a human endogenous retrovirus (HERV) envelope protein or an immunogenic portion thereof, wherein the HERV envelope protein comprises an immunosuppressive domain (ISD), and the HERV Env protein comprises a mutant immunosuppressive domain (ISD) that reduces the immunosuppressive properties compared to a wild-type ISD.

[0076] In one embodiment, the present invention relates to a composition of the present invention or a pharmaceutically acceptable salt thereof.

[0077] Transfection agent The transfection agent of the composition may be any compound, formulation, or mixture that enhances the transport or uptake of nucleic acids into cells, including cells of different tissues. These agents, when applied in an effective amount as defined elsewhere herein, can increase the uptake of a certain amount of nucleic acid. Such an effect is caused by one or more substances comprising the transfection reagent promoting the aforementioned uptake. The proteins or peptides encoded by the introduced nucleic acid can then modulate, induce, or incorporate cellular processes in target cells.

[0078] Transfection agent compositions may be configured depending on other parameters, such as the delivery environment, i.e., in vivo or in vitro, in addition to being adjusted according to, for example, the target cell type and / or the substance to be delivered. Suitable transfection agents include a variety of uptake promoters selected from the non-limiting group consisting of: calcium phosphate; cationic polymers such as DEAE-dextran or polyethyleneimine (PEI); liposome-forming materials or mixtures thereof, e.g., 2,3-dioleoyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propaniminium-trifluoroacetate (DOSPA), dioleoyl-3-trimethylammoniumpropane (DOTMA), or dioleoyloxypropyltrimethylammonium (DOTAP) sperminecarboxamide, and / or helper lipids such as dioleoylphosphatidylethanolamine (DOPE), cholesterol, and polyethylene glycol (PEG)-lipids; non-liposomal agents such as FuGENE®, commercially available from Promega, for example; and dendrimers. Even more preferable are the wide range of commercially available Lipofectamine mixtures (e.g., those from ThermoFisher Scientific).

[0079] Therefore, in one embodiment, the transfection agent is a transfection agent comprising a cationic lipid and / or a cationic polymer.

[0080] In a preferred embodiment, the composition comprises liposomes containing a cationic lipid and the mRNA of the present invention.

[0081] In a more preferred embodiment, the transfection agent comprises DOSPA (2,3-dioleoyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propaniminium trifluoroacetate) and / or DOPE (1,2-dioleoyl-sn-glycerophosphoethanolamine). In the most preferred embodiment, the transfection agent comprises DOSPA (2,3-dioleoyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propaniminium trifluoroacetate) and DOPE (1,2-dioleoyl-sn-glycerophosphoethanolamine) preferably in a molar ratio of 3:1.

[0082] Lipid nanoparticles (LNPs) The transfection agent may be a composition particularly suitable for application to nucleic acids encoding antigens, such as mRNA. Such suitable compositions are known in the art. Such suitable compositions may be lipid compositions having beneficial properties in vivo, such as lipid nanoparticle (LNP) compositions. LNPs have emerged as a suitable nonviral encapsulation delivery vehicle for exogenous mRNA because they increase circulation time in the body and help effectively deliver antigen-encoding nucleotide sequences, such as mRNA, to target sites.

[0083] Therefore, in one embodiment, the composition comprises lipid nanoparticles (LNPs) containing the mRNA of the present invention.

[0084] LNPs include various lipid-based platforms such as liposomes, nanostructured lipid carriers (NLCs), and solid lipid nanoparticles (SLNs). Therefore, in one embodiment, the LNP composition is selected from liposome compositions, nanostructured lipid carrier (NLC) compositions, and solid lipid nanoparticle (SLN) compositions.

[0085] Compositions comprising mRNA and transfection agents described herein can benefit from the use of LNPs as transfection agents, as formulation with LNPs has been observed to reduce adverse responses. In preferred embodiments, LNPs containing lipids known to reduce Toll-like receptor (TLR) agonism are used. Reduced activation of TLR signaling has been observed to play a crucial role as a trigger for innate immune signaling associated with RNA vaccines, and this triggering effect is thought to be amplified by certain lipids used in vaccine formulations that reduce TLR signaling (Tahtinen, S., Tong, AJ., Himmels, P. et al. IL-1 and IL-1ra are key regulators of the inflammatory response to RNA vaccines. Nat Immunol 23, 532-542 (2022)). While we do not wish to be bound by theory, the use of such lipids is predicted to increase immunogenicity and result in a robust innate immune response to mRNA-encoded antigens. Preferred examples include LNPs containing ionizable lipids.Further preferred examples include LNP compositions containing the following lipids: DLin-MC3-DMA(MC3), 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), (4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), Hasset et al. (2019) (Optimization of Lipid Nanoparticles for Intramuscular Administration of mRNA Vaccines. Mol Ther Nucleic Acids. 2019 Apr Lipids H, M, P, Q, and N in 15;15:1-11), 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA) and 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), and DOTMA and DOPE in a 1:1 ratio. Another preferred example of an LNP composition is one comprising at least one lipid selected from the group consisting of (4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol. In a more preferred example, the LNP comprises all of the lipids ALC-0315, ALC-0159, DSPC, and cholesterol. In an even more preferred example, these lipids are used in a ratio of ALC-0315:ALC-0159.DSPC:cholesterol of 46.3:9.4:42.7:1.6.

[0086] Further aspects of the present invention (i), (ii), (iii) and (iv) are: (i) LNPs containing the mRNA of the present invention; (ii) Liposomes containing the mRNA of the present invention; (iii) Polyplex containing the mRNA of the present invention; (iv) lipoplex containing the mRNA of the present invention To provide.

[0087] A further aspect of the present invention relates to a virus-like particle (VLP) comprising a HERV envelope protein as defined herein. Preferably, the envelope protein comprises a mutant ISD having a mutation selected from the group consisting of L522A, A523Q, N524A, Q525A, I526A, N527A, D528A, L529A, R530A, Q531A, T532A, V533A, I534A and W535A, preferably the mutant ISD comprising a sequence according to one of sequence numbers 4 or 8-20 (most preferably the aforementioned mutant ISD comprising or consisting of the sequence LANAINDLRQTVIW (sequence number 4)), and the HERV envelope protein comprises the amino acid sequence: [ka] It further includes, At least one amino acid located in the range of 170 to 210 is replaced by cysteine, or The HERV envelope protein contains cysteine ​​at position 190, more preferably the HERV envelope protein contains the amino acid mutation S190C, and / or the HERV envelope protein contains the sequence VQNWLVEVPTVSPICRFTYHMVSGMSLRP, and / or the HERV envelope protein contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 2, and the HERV envelope protein contains cysteine ​​at the aforementioned position within the HERV envelope protein corresponding to amino acid 190 of SEQ ID NO: 2.

[0088] Lipid content for encapsulation RNA encapsulation is achieved by combining RNA with ionizable lipids at an acidic pH where the lipids are positively charged, thereby ensuring charge-driven interaction with negatively charged nucleic acids. Subsequently, by adjusting the pH to a value higher than the pKa of the ionizable lipids, a generally neutral surface charge suitable for clinical administration can be obtained. Numerous components of LNP compositions suitable for the delivery of nucleic acids such as mRNA are known in the art and will be apparent to those skilled in the art. More general information and numerous examples regarding lipid nanoparticle formulations are provided, for example, in the literature by Barba et al. (2019), Schoenmaker et al. (2021), or MacLachlan (2007) (Barba et al., 2019, Lipid Delivery Systems for Nucleic-Acid-Based-Drugs: From Production to Clinical Applications. Pharmaceutics; 11(8):360; Schoenmaker et al., 2021; mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability. Int J Pharm.; 601:120586; MacLachlan, 2007, Liposomal formulations for nucleic acid delivery. In Antisense Drug Technology; 255-288). Therefore, the embodiments described below are illustrative and not limiting.

[0089] In one embodiment, the LNP composition may contain one or more ionizable lipids, which may preferably include cationic lipids. Unless otherwise evident from the specific context, the term “cationic” means that each structure has a positive charge permanently or not permanently but in response to specific conditions such as pH. Thus, the term “cationic” encompasses both “permanently cationic” and “cationizable.” Suitable cationic lipids for the compositions of the present invention are known in the art and, for example, advantageously, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol) The following can be selected from a non-limiting group: N-(1-(2,3-dioleoyloxy)propyl)N-2-(sperminecarboxamide)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), dioctadecylamideglycylcarboxyspermine (DOGS), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and N-(1,2-dimyristyloxypropane-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE).Additionally, suitable cationic lipids for the compositions of the present invention may be selected from a non-limiting group of commercially available cationic lipids, such as LIPOFECTIN® (a commercially available cationic liposome containing DOTMA and 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), manufactured by GIBCO / BRL, Grand Island, New York); LIPOFECTAMINE® (a commercially available cationic liposome containing N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamide)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and DOPE, manufactured by GIBCO / BRL); and TRANSFECTAM® (a commercially available cationic lipid containing dioctadecylamideglycylcarboxyspermine (DOGS) in ethanol, manufactured by Promega Corp., Madison, Wisconsin).

[0090] The ionizable lipids of this disclosure may include a central amine moiety and at least one biodegradable group. Therefore, in a more preferred embodiment, the ionizable lipid may be an ionizable cationic aminolipid.

[0091] In some embodiments, the lipid-based compositions described herein (e.g., LNPs) include one or more noncationic helper lipids. As used herein, the term “noncationic helper lipid” refers to a lipid comprising at least one fatty acid chain having at least eight carbon atoms and at least one polar head group moiety. In some embodiments, the noncationic helper lipid may be a phospholipid. Generally, a phospholipid comprises a phospholipid moiety and one or more fatty acid moieties. The phospholipid moiety may be selected from the non-limiting group consisting of, for example, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid portion can be selected from a non-limiting group consisting of, for example, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytanic acid, arachidic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. Phospholipids also include phosphingolipids such as sphingomyelin.

[0092] In preferred embodiments, the helper lipid or phospholipid may be neutral. The LNP composition may also contain, for example, a DSPC analog, a DSPC substitute, oleic acid, or an oleic acid analog, a non-phosphatidylcholine (PC) zwitterionic lipid, a DSPC analog, oleic acid, an oleic acid analog, or a 1,2-distearoyl-i77-glycero-3-phosphocholine (DSPC) substitute. In even more preferred embodiments, the helper lipid or phospholipid of the LNP composition may be DSPC.

[0093] In some embodiments, the lipid-based composition (e.g., LNP) may contain one or more structural lipids. In preferred embodiments, the structural lipid may be a sterol. Such sterols may include cholesterol, β-sitosterol, sitostanol, campesterol, stigmasterol, and brassicasterol. In even more preferred embodiments, the aforementioned sterol may preferably be cholesterol.

[0094] In some embodiments, the LNP composition may contain another lipid that is a phospholipid substitute or substitution. Such phospholipid or phospholipid substitute or substitution may be, for example, one or more saturated or (poly)unsaturated phospholipids, or phospholipid substitutes, or combinations thereof. In preferred embodiments, such phospholipid substitutes or substitutions may be PEGylated lipids or PEG lipids. Incorporation of PEGylated lipids provides, for example, steric stabilization of the nanoparticle core-shell. Non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, and PEG-modified 1,2-diacyloxypropane-3-amines. Such lipids are also called PEGylated lipids. In some embodiments, the PEG-lipids include, but are not limited to, PEG-disterylglycerol (PEG-DSG), or PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA), 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), PEG-dipalmitoyl, PEG-dioleil, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-DLPE, PEG-DMPE, PEG-dipalmitoylphosphatidylethanolamine or-choline (PEG-DPPE, PEG-DPPC), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-aminopolyethylene glycol (PEG-DSPE) lipids. In preferred embodiments, the LNP compositions may include PEG-DMG.

[0095] In one embodiment, the LNP composition comprises at least one lipid, which includes (i) an ionizable lipid, preferably an ionizable cationic lipid, more preferably an ionizable cationic aminolipid; (ii) a non-cationic helper lipid or phospholipid, wherein the lipid is preferably neutral, and more preferably is a DSPC; (iii) a sterol or other structural lipid, wherein the sterol is preferably cholesterol; and (iv) at least one lipid selected from the group consisting of PEG lipids, preferably PEG-DMG.

[0096] The lipid components of the LNP composition may be used in appropriate molar ratios with respect to other lipids. The amount of ionizable lipids, preferably cationic lipids, preferably cationic aminolipids, may be, for example, in the range of about 45 mol% to about 50 mol%. The amount of non-cationic helper lipids or phospholipids, preferably neutral lipids, preferably DSPCs, may be, for example, in the range of about 5 mol% to about 15 mol%. The amount of structural lipids, such as sterols, preferably cholesterol, may be, for example, in the range of about 30 mol% to about 45 mol%. The amount of PEG-lipids in the lipid composition of the pharmaceutical compositions disclosed herein may be, for example, in the range of about 0.1 mol% to about 5 mol%.

[0097] Accordingly, in one embodiment, the composition of the present invention, in which the transfection agent is a lipid nanoparticle (LNP) composition, comprises a molar ratio of about 45 mol% to about 50 mol% ionizable lipid, about 5 mol% to about 15 mol% phospholipid, about 30 mol% to about 45 mol% sterol, and about 1 mol% to about 5 mol% PEG lipid. In a preferred embodiment, the LNP may comprise, for example, a molar ratio of 50 mol% ionizable lipid, 10 mol% phospholipid, 38.5 mol% sterol, and 1.5 mol% PEG lipid. In an alternative preferred embodiment, the LNP may comprise, for example, a molar ratio of 46.3 mol% ionizable lipid, 9.4 mol% phospholipid, 42.7 mol% sterol, and 1.6 mol% PEG lipid.

[0098] In another embodiment, the lipid nanoparticle composition may include a targeting moiety, which is a compound or drug capable of targeting the nanoparticles to specific cell, tissue, and / or organ types. Thus, in one embodiment, the lipid nanoparticle composition containing the targeting moiety has the ability to specifically deliver to specific target cells, tissues, and / or organ types.

[0099] In particular, when a composition containing the RNA of the present invention is applied to a subject or patient for prevention or treatment, for example, it may be desirable or useful to further increase the potency or efficacy of the composition of the present invention. Accordingly, in one embodiment, the composition of the present invention further comprises an adjuvant. In a preferred embodiment, the adjuvant may be a cytokine, and more preferably a cytokine selected from the group consisting of INFγ, IL-2, IL-12, GM-CSF, IL-15, and IL-7. The adjuvant may also be introduced as a polynucleotide configured to express one or more of the above-mentioned cytokines in eukaryotic cells.

[0100] Adjuvants may act by a combination of various mechanisms to induce and enhance an immune response, including one or more of the following: sustained release of antigen at the injection site (depot effect), upregulation of (further) cytokines and chemokines, cell recruitment at the composition administration site, increased antigen uptake and presentation by antigen-presenting cells (APCs), activation and maturation of APCs, increased expression of major histocompatibility complex (MHC) class II and costimulatory molecules, enhanced antigen transport to excretory lymph nodes, and activation of inflammasomes.

[0101] Characteristics of mRNA In one embodiment, the mRNA composed of the composition of the present invention contains at least 300 nucleotides, for example, preferably at least 400, 500, 800, 1000, 1500, 2000, more preferably at least 3000, or most preferably at least 4000 nucleotides.

[0102] Modifications to mRNA elements, such as the 5' cap, 5' and 3' untranslated regions (UTRs), coding region, and polyadenylated tail, help reduce excessive mRNA immunogenicity and / or improve mRNA stability and translation efficiency. Therefore, mRNA composed of the compositions of the present invention may have specific functional sequence features that optimize its properties with respect to stability, expression efficiency, and patient tolerance.

[0103] The mRNA molecule may contain an elongated oligo-A sequence or poly-A sequence, i.e., a poly-A tail, at its 3' end. The 3' poly-A facilitates nuclear export and provides RNA stability and translation efficiency of the mRNA. Over time, the poly-A shortens, eventually leading to the initiation of enzymatic mRNA degradation. Therefore, extending the poly-A can provide additional stability. Accordingly, in one embodiment, the composition comprises mRNA containing at least 60 adenosine nucleotides in the 3'-UTR. In a preferred embodiment, the mRNA contains at least 100, more preferably 120, adenosine nucleotides in the 3'-UTR.

[0104] In further embodiments, the mRNA of the present invention may typically contain a poly(C)(polycysteine) tail of about 10 to 200 cytosine nucleotides, preferably about 10 to 100 cytosine nucleotides, more preferably about 20 to 70, or even more preferably about 20 to 60 cytosine nucleotides at its 3' end.

[0105] Codon optimization is another approach to improving gene expression by modifying synonymous codons based on the codon bias of an organism. By introducing mutations into the target gene based on the codon usage bias of the host organism itself, it increases translation efficiency and, consequently, protein expression in the aforementioned organism without altering the protein sequence. Thus, in another embodiment, mRNA is codon-optimized for expression in humans. Human codon usage is known in the art, as provided, for example, by the GenScript Codon Usage Frequency Table(chart)Tool. The mRNA of the present invention can also be codon-optimized for expression in other animals, such as other mammals.

[0106] In some cases, modified nucleic acid bases are introduced into the nucleic acid sequence (e.g., RNA nucleic acids such as mRNA) to improve stability. In one embodiment, the mRNA of the present invention may contain at least one artificially modified nucleotide. In one embodiment, the mRNA of the present invention does not contain an artificially modified nucleotide.

[0107] In one embodiment, the mRNA of the present invention may be modified and stabilized by modifying the guanosine / cytosine (G / C) content of its mRNA sequence, which can be particularly increased compared to the G / C content of the coding region of each wild-type mRNA, i.e., unmodified mRNA. According to a specific embodiment, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, more preferably at least 70%, even more preferably at least 80%, most preferably at least 90%, 95%, or even more preferably 100% of the substitutable codons in the coding region of the mRNA of the present invention or the entire sequence of the wild-type mRNA sequence may be substituted, thereby increasing the GC / C content of the aforementioned sequence.

[0108] In one embodiment, the mRNA of the present invention may be modified, preferably by increasing, the cytosine (C) content of its mRNA sequence, preferably in the coding region of the mRNA, compared to the C content of the coding region of the respective wild-type mRNA, i.e., unmodified mRNA. Preferably, the mRNA sequence may be modified to achieve at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the theoretically possible maximum cytosine content, or at least 90%, or even the maximum cytosine content.

[0109] In some embodiments, the mRNA of the present invention may include, for example, but not limited to, a CAP analog structure; a suitable promoter or subgenome promoter that results in a high translation rate; self-amplifying mRNA features (for example, the mRNA of the present invention may be a self-replicating mRNA), such as a cytomegalovirus promoter, a T7 promoter, or a subgenome SFV promoter; a Kozak consensus sequence (5'-CCCACCATGG-3'); a 3-6 nucleotide spacer between the (T7) promoter sequence and the Kozak sequence, if present; and at least sequence elements selected from the group consisting of stabilizing and / or structural sequence elements in the UTR sequence. Suitable RNA elements have been described in the art and will be apparent to those skilled in the art.

[0110] For example, suitable CAP analog structures for the mRNA of the present invention can be selected from a non-limiting group consisting of vaccinia 2'-O-methyltransferase Cap 1, ARCA anti-reverse CAP analog or β-S-ARCA cap, modified ARCA (e.g., phosphothioate-modified ARCA); m7GpppN, capl (methylation of ribose at the nucleotide adjacent to m7G), cap2 (additional methylation of ribose at the second downstream nucleotide of m7G), cap3 (additional methylation of ribose at the third downstream nucleotide of m7G), cap4 (methylation of ribose at the fourth downstream nucleotide of m7G), inosine, N1-methylguanosine, 2'-fluoroguanosine, 7-deazaguanosine, 8-oxoguanosine, 2-aminoguanosine, LNA-guanosine, and 2-azidoguanosine.

[0111] For example, suitable stabilizing and / or structural sequence elements in the UTR sequence of the mRNA of the present invention may be based on variants of the UTR sequence of a gene, such as the albumin gene, α-globin gene, β-globin gene, tyrosine hydroxylase gene, lipoxygenase gene, or collagen alpha gene, such as the collagen alpha 1 gene, or variants of the UTR sequence of a part thereof. Accordingly, in some preferred embodiments, the mRNA of the present invention may include 5'UTR HBA1, 5'UTR SFV, or 5'UTR 7, 3'UTR HBB; and / or 3'UTR AES mtRNR1. In another embodiment, the 5'-UTR of the mRNA includes or consists of a nucleic acid sequence derived from the 5'-UTR of the ribosomal protein Large gene, or the 5'-UTR of the vertebrate TOP gene.

[0112] The mRNA of the present invention encoding the HERV envelope protein or its immunogenic portion may encode the aforementioned protein as part of a virus-like particle (VLP). VLPs are virus-like molecules composed of one or more different molecules that self-assemble and have the ability to mimic the release, shape, and size of a virus particle, but lack the genetic material to infect a host cell. In the context of the present invention, they can be formed by encoding a viral Gag protein together with the HERV envelope protein. The gag gene is then translated into a polyprotein which mediates the formation of VLPs in the absence of other viral proteins and incorporates the HERV Env onto the surface of the VLP.

[0113] Accordingly, in one embodiment, the composition of the present invention comprises mRNA encoding at least a HERV envelope protein or its immunogenic portion and a gag protein having the mutant ISD of the present invention. In a preferred embodiment, the gag protein may be selected from the same virus as the Env protein or from a different virus.

[0114] Further applicable and potentially beneficial features of VLPs encoded by mRNA will be apparent to those skilled in the art. For example, in one embodiment, the mRNA of the present invention may encode at least a human endogenous retrovirus (HERV) envelope protein having a mutant ISD or its immunogenic portion, a gag protein, and a 2A peptide. Furthermore, the Env protein may be encoded to include, for example, a surface unit (also called SU, gp70), a cleavage site, and / or a transmembrane unit (also called TM, p15E). Furthermore, the transmembrane unit (also called TM, p15E) may include, for example, a fusion peptide, a transmembrane anchor, and / or a cytoplasmic tail.

[0115] In its most preferred embodiment, the mRNA or mRNA construct of the present invention encoding at least the HERV envelope protein or its immunogenic moiety also encodes a Gag protein and may comprise a polyA comprising a 5'UTR HBA, a 3'UTR HBB, and 70 adenine nucleotides. Thus, the mRNA of the present invention may have the structure (5'UTR)HBA1-HERV-K Gag Env ISDmut-(3'UTR)HBB-polyA(70). In another preferred embodiment, the mRNA or mRNA construct of the present invention encoding at least the HERV envelope protein or its immunogenic moiety also encodes a Gag protein and may comprise a polyA comprising the 5'UTR of HBA1, the 3'UTR of AES mtRNR1, and 100 adenine nucleotides, where 30 and 70 adenine nucleotides are preferably suspended by a linker (30-linker-70). Therefore, the mRNA of the present invention may have the structure of HBA1 5'UTR-HERV-K Gag Env ISDmut (preferably ISD mutation Q525A)-AES mtRNR1 3'UTR-polyA (preferably poly(A) is A30-linker-A70).

[0116] In one embodiment, the present invention relates to a DNA molecule encoding mRNA contained in the composition of the present invention.

[0117] HERV envelope protein The present invention provides a platform for presenting antigens to the body's immune system. Therefore, in principle, the mRNA construct can incorporate a code for any type of protein to which an immune response is desired. In one embodiment of the present invention, the mRNA-encoded protein is an endogenous retroviral envelope protein (ERV Env) or an immunogenic protein derived from such a protein. Generally, vaccines are thought to deliver ERV Env to dendritic cells (DCs), which then present the antigen to cells of the adaptive immune system. Presentation on MHC class I induces activation and proliferation of CD8+ T cells. These cytotoxic T lymphocytes (CTLs), specific to the ERV Env antigen, infiltrate tumors and kill cells presenting their respective antigens. Antigen presentation on MHC class II by specialized antigen-presenting cells (APCs) activates CD4+ T cells, followed by co-activation of B cells. Activated B cells that encounter circulating ERV Env target proteins or antigens presented on cells or VLPs release antibodies specific to ERV Env. These antibodies can bind to targets on cancer cells, inducing the destruction and phagocytosis of malignant cells. In this way, ERV-specific antibodies can prevent tumor growth and metastasis. The restoration of immunogenicity in tumor cells enables priming of diverse tumor-specific T cell sets that recognize different tumor-associated antigens and tumor-specific antigens. Newly primed and expanded CTLs invade the tumor and kill malignant cells.

[0118] Retroviruses possess a protein envelope, and therefore, the retroviral genome encodes Env (envelope protein) as one of three major proteins. ERVs are evidence that distant ancestors were infected with retroviruses long ago. Therefore, using envelope proteins as antigens for vaccination is also useful for targeting a wide range of ERVs. While this composition can, in principle, be used to immunize many mammalian species, the ERV protein in one aspect of the present invention is a human endogenous retrovirus (HERV) protein or its immunogenic portion. It is estimated that every human genome consists of approximately 8% endogenous retroviral DNA. However, most of the endogenous retroviral DNA is merely remnants of past retroviruses. Upon infection, viral RNA is reverse transcribed into proviral DNA, which is incorporated into the host genome. Eventually, the provirus is incorporated into germline cells, becomes heritable, and gives rise to an endogenous retrovirus. Over millions of years, this viral DNA has been passed down through generations and fixed in the population. Therefore, a large portion of the human genome may be usable as the antigen-coding portion of mRNA as in the present invention.

[0119] In one embodiment, HERV is selected from the group consisting of HERV-K, HERV-H, HERV-W, HERV-FRD, HERV-E, HERV-9, HERV-FC, HERV-T, HERV-3, HERV-V1, and HERV-V2. More specifically, HERV-K, which is known to be upregulated in prostate cancer, breast cancer, ovarian cancer, lymphoma, melanoma, leukemia, and sarcoma, may be selected in one embodiment from the group consisting of HERV-K108 (=ERVK-6), ERVK-19, HERV-K115 (=ERVK-8), ERVK-9, HERV-K113, ERVK-21, ERVK-25, HERV-K102 (=ERVK-7), HERV-K101 (=ERVK-24), HERV-K110 (=ERVK-18); HERV-K. HERV-H may be selected from the group consisting of, for example, HERV-H19 (=HERV-H_2q24.3) and HERV-H_2q24.1, HERV-W may be selected as, for example, ERVW-1 (=Syncytin-1), and HERV-FRD may be selected as, for example, ERVFRD-1 (=Syncytin-2).

[0120] In another embodiment, the HERV is selected from the group consisting of HERV-9, HERV-FC, HERV-T, HERV-E, HERV-3, HERV-V1, and HERV-V2.

[0121] In the case of mRNA-encoded HERV-9 envelope proteins, preferred mutant immunosuppressive domains (ISDs) that reduce immunosuppressive properties compared to wild-type ISDs include or consist of the sequence LQNCZGLDLLTAEKGGLCTFLGE (SEQ ID NO: 38).

[0122] For mRNA-encoded HERV-FC envelope proteins, the preferred wild-type immunosuppressive domain (ISD) contains or consists of the sequence AQNRRALDLLTADKGGTCLFLGE (SEQ ID NO: 39).

[0123] For mRNA-encoded HERV-T envelope proteins, the preferred wild-type immunosuppressive domain (ISD) contains or consists of the sequence LQNRRGLDLLFLSQGGLCAALGE (SEQ ID NO: 40).

[0124] For mRNA-encoded HERV-E envelope proteins, the preferred wild-type immunosuppressive domain (ISD) contains or consists of the sequence YQNRLALDYLLAAEGGVCGKFNL (SEQ ID NO: 41).

[0125] For mRNA-encoded HERV-3 envelope proteins, the preferred wild-type immunosuppressive domain (ISD) contains or consists of the sequence YQNRLALDYLLAQEGGVCGKFNL (SEQ ID NO: 42). For mRNA-encoded HERV-V1 envelope proteins, the preferred wild-type immunosuppressive domain (ISD) contains or consists of the sequence MNNRLALDYLLAEQGGVCAVISK (SEQ ID NO: 43).

[0126] For mRNA-encoded HERV-V2 envelope proteins, the preferred wild-type immunosuppressive domain (ISD) contains or consists of the sequence MDNRLALDYLLAEQGGVCAVINK (SEQ ID NO: 44).

[0127] Other gamma-retroviral ISD sequences from other HERVs can be identified by multiple sequence alignment.

[0128] In the case of mRNA-encoded HERV-FC envelope proteins, an immunosuppressive domain (ISD) containing or consisting of the sequence AQNRRALDLLTADKGGTCLFLGE (SEQ ID NO: 39) already contains mutations that reduce or inactivate the activity of the ISD, i.e., the sequence AQNRRALDLLTADKGGTCLFLGE (SEQ ID NO: 39) can be directly used in mRNA-encoded HERV-FC envelope proteins described herein.

[0129] The active gamma retrovirus ISD (consisting of 23 amino acids) can be appropriately mutated into a non-immunosuppressive ISD by replacing the acidic amino acid residue at position 14 with a different amino acid, preferably a basic amino acid residue. Additionally, stability can be further improved by inserting an aromatic amino acid residue at position 20 of the ISD (consisting of 23 amino acids).

[0130] In one embodiment, HERV is selected from the group consisting of HERV-9, HERV-T, HERV-E, HERV-3, HERV-V1, and HERV-V2. The mutant immunosuppressive domain (ISD) that reduces immunosuppressive properties compared to the wild-type ISD contains or consists of one of the following sequences, which are 23 amino acids long: LQNCZGLDLLTAEKGGLCTFLGE (SEQ ID NO: 38) for HERV-9, LQNRRGLDLLFLSQGGLCAALGE (SEQ ID NO: 40) for HERV-T, YQNRLALDYLLAAEGGVCGKFNL (SEQ ID NO: 41) for HERV-E, YQNRLALDYLLAQEGGVCGKFNL (SEQ ID NO: 42) for HERV-3, MNNRLALDYLLAEQGGVCAVISK (SEQ ID NO: 43) for HERV-V2, and MDNRLALDYLLAEQGGVCAVINK (SEQ ID NO: 44) in which one or more amino acids are replaced by different amino acids. In a preferred embodiment, the single amino acid at position 14 is replaced by a different amino acid. In a more preferred embodiment, the single amino acid at position 14 is replaced by a basic amino acid. In a more preferred embodiment, the single amino acid at position 14 is replaced with R.

[0131] In a preferred embodiment, the HERV is selected from the group consisting of HERV-9, HERV-T, HERV-E, HERV-3, HERV-V1, and HERV-V2, and the mutant immunosuppressive domain (ISD) that reduces immunosuppressive properties compared to the wild-type ISD contains or consists of one of the following sequences: for HERV-9, LQNCZGLDLLTAERGGLCTFLGE (SEQ ID NO: 46); for HERV-T, LQNRRGLDLLFLSRGGLCAFLGE (SEQ ID NO: 47); for HERV-E, YQNRLALDYLLAARGGVCGFFNL (SEQ ID NO: 48); for HERV-3, YQNRLALDYLLAQRGGVCGFFNL (SEQ ID NO: 49); for HERV-V1, MNNRLALDYLLAERGGVCAFISK (SEQ ID NO: 50); and for HERV-V2, MDNRLALDYLLAERGGVCAFINK (SEQ ID NO: 51).

[0132] In a preferred embodiment, the HERV is selected from the group consisting of HERV-9, HERV-T, HERV-E, HERV-3, HERV-V1, and HERV-V2, and the mutant immunosuppressive domain (ISD) that reduces immunosuppressive properties compared to the wild-type ISD contains or consists of one of the following sequences starting at the 23rd amino acid position: for HERV-9, LQNCZGLDLLTAEKGGLCTFLGE (SEQ ID NO: 38); for HERV-T, LQNRRGLDLLFLSQGGLCAALGE (SEQ ID NO: 40); for HERV-E, YQNRLALDYLLAAEGGVCGKFNL (SEQ ID NO: 41); for HERV-3, YQNRLALDYLLAQEGGVCGKFNL (SEQ ID NO: 42); for HERV-V1, MNNRLALDYLLAEQGGVCAVISK (SEQ ID NO: 43); and for HERV-V2, MDNRLALDYLLAEQGGVCAVINK (SEQ ID NO: 44). A single amino acid at position 14 is replaced with a different amino acid, and a single amino acid at position 20 is replaced with a different amino acid, preferably at position 20, to increase stability.

[0133] In a more preferred embodiment, the mutant immunosuppressive domain (ISD) that reduces immunosuppressive properties compared to the wild-type ISD includes or consists of one of the following sequences starting at the 23rd amino acid position: for HERV-9, LQNCZGLDLLTAEKGGLCTFLGE (SEQ ID NO: 38); for HERV-T, LQNRRGLDLLFLSQGGLCAALGE (SEQ ID NO: 40); for HERV-E, YQNRLALDYLLAAEGGVCGKFNL (SEQ ID NO: 41); for HERV-3, YQNRLALDYLLAQEGGVCGKFNL (SEQ ID NO: 42); for HERV-V1, MNNRLALDYLLAEQGGVCAVISK (SEQ ID NO: 43); and for HERV-V2, MDNRLALDYLLAEQGGVCAVINK (SEQ ID NO: 44). The single amino acid at position 14 is replaced with R, and the single amino acid at position 20 is replaced with F, preferably the 20th position is replaced to increase stability.

[0134] In one embodiment, the present invention relates to a composition comprising a transfection agent and at least a human endogenous retrovirus (HERV) envelope (Env) protein or mRNA encoding an immunogenic portion thereof, wherein the HERV envelope protein comprises an immunosuppressive domain (ISD), and the HERV Env protein comprises a mutant immunosuppressive domain (ISD) that reduces immunosuppressive properties compared to a wild-type ISD. HERV is selected from the group consisting of HERV-9, HERV-FC, HERV-T, HERV-E, HERV-3, HERV-V1, and HERV-V2. The mutant immunosuppressive domain (ISD) that reduces immunosuppressive properties compared to the wild-type ISD is the following sequence consisting of 23 amino acids: for HERV-9, LQNCZGLDLLTAEKGGLCTFLGE (SEQ ID NO: 38); for HERV-FC, AQNRRALDLLTADKGGTCLFLGE (SEQ ID NO: 39); for HERV-T, LQNRRGLDLLFLSQGGLCAALGE (SEQ ID NO: 40); for HERV-E, YQNRLALDYLLAAEGGVCGKFNL (SEQ ID NO: 41); for HERV-3, YQNRLALDYLLAQEGGVCG The present invention relates to a composition comprising or consisting of one of the following: KFNL (SEQ ID NO: 42), MNNRLALDYLLAEQGGVCAVISK (SEQ ID NO: 43) in the case of HERV-V1, and MDNRLALDYLLAEQGGVCAVINK (SEQ ID NO: 44) in the case of HERV-V2, wherein in SEQ ID NOs: 38, 40, 41, 42, 43, and 44, the single amino acid at position 14 is replaced with a different amino acid, preferably R, and the single amino acid at position 20 is replaced with a different amino acid, preferably F.

[0135] In another embodiment, the mRNA-encoded HERV envelope protein contains an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 2 throughout the entire length of SEQ ID NO: 2 (see Table 1).

[0136] In one embodiment, HERV is HERV-K. In several embodiments, it is preferable that the HERV-K Env protein has the HERV-K Env consensus sequence, more preferably a codon-optimized consensus sequence. A particularly preferred amino acid sequence of wild-type HERV-K Env (without ISD mutation) is shown below and designated as Sequence ID No. 1 (see also Table 1): [ka]

[0137] The mRNA encoding the HERV Env protein is preferably constructed so that the encoded protein is expressed in vivo and presented to the immune system to elicit an immunological response.

[0138] Surprisingly, it has been found that replacing one or more amino acid positions of the HERV Env protein with cysteine, preferably by replacing the serine at position 190 of the HERV Env protein with cysteine ​​(S190C), and / or generally increasing the cysteine ​​content, preferably by making the number of cysteines in the HERV Env protein an even number, increases the expression efficiency of the HERV Env protein from the encoding nucleic acid molecule (see Figure 4), which was likely achieved through increased stability of the (transcribed) mRNA and / or translated protein product. For example, the serine at position 190 of the HERV Env protein may have an destabilizing effect, but replacing this position with cysteine ​​to make the number of cysteines in the HERV Env protein an even number of 18 may have a stabilizing effect.

[0139] Therefore, in one embodiment, the HERV envelope protein contains an even number of cysteine ​​molecules. In another embodiment, the HERV envelope protein contains at least 18 cysteine ​​molecules, either substituted or additionally. In another embodiment, the HERV envelope protein has the following amino acid sequence [ka] Includes, At least one amino acid located between positions 170 and 210 is replaced by cysteine.

[0140] In a preferred embodiment, the HERV envelope protein contains cysteine ​​at position 190. In a more preferred embodiment, the HERV envelope protein contains the amino acid mutation S190C.

[0141] In another embodiment, the HERV envelope protein comprises, alternatively or additionally, the sequence VQNWLVEVPTVSPICRFTYHMVSGMSLRP. In another embodiment, the HERV envelope protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 2, and the HERV envelope protein comprises a cysteine ​​at a position within the aforementioned HERV envelope protein corresponding to amino acid 190 of SEQ ID NO: 2.

[0142] In another embodiment, the HERV envelope protein comprises or consists of a sequence selected from the group consisting of SEQ ID NOs: 21-35. In a preferred embodiment, the HERV envelope protein comprises SEQ ID NOs: 22-27, 29-32 and 35 It includes or consists of sequences selected from the group comprising the following.

[0143] In a preferred embodiment, the composition of the present invention comprises mRNA having at least 90% sequence identity, most preferably 100% sequence identity, to any sequence according to SEQ ID NO: 45, 36, or 37. In a more preferred embodiment, the composition of the present invention comprises mRNA having 100% sequence identity to any sequence according to any sequence according to SEQ ID NO: 45, 36, or 37.

[0144] In one embodiment, cell surface expression of the HERV envelope protein (as defined in the previous section) is increased compared to cell surface expression of the HERV envelope protein having the amino acid sequence according to SEQ ID NO: 2.

[0145] A further aspect of the present invention is an mRNA having at least 90% sequence identity with SEQ ID NO: 45, wherein the mRNA encodes a polypeptide comprising HERV-K Gag and HERV-K envelope (Env) protein. The aforementioned HERV Env protein comprises a mutant immunosuppressive domain (ISD) that reduces immunosuppressive properties compared to the wild-type envelope ISD (preferred ISD mutations and mutant ISD sequences are disclosed herein in the context of the present invention—for example, one or more of the ISD mutations L522A, A523Q, N524A, Q525A, N527A, L529A, R530A, Q531A, T532A may be used), and the HERV envelope protein relates to mRNA further comprising cysteine ​​instead of serine at amino acid position 190 within the aforementioned HERV envelope protein. In a preferred embodiment, the above mRNA is present in the composition of the present invention. The use of the above mRNA for producing virus-like particles (VLPs) is also provided.

[0146] A further aspect of the present invention is a polypeptide having at least 90% sequence identity with the polypeptide encoded by SEQ ID NO: 45, wherein the polypeptide comprises HERV-K Gag and HERV-K envelope (Env) protein. The aforementioned HERV Env protein comprises a mutant immunosuppressive domain (ISD) that reduces immunosuppressive properties compared to the wild-type envelope ISD (preferred ISD mutations and mutant ISD sequences are disclosed herein in the context of the present invention—for example, one or more of the ISD mutations L522A, A523Q, N524A, Q525A, N527A, L529A, R530A, Q531A, T532A may be used), and the HERV envelope protein relates to a polypeptide comprising cysteine ​​instead of serine at position 190 within the aforementioned HERV envelope protein. In a preferred embodiment, the aforementioned polypeptide is contained in a virus-like particle (VLP). mRNA encoding the aforementioned polypeptide is also provided. Preferably, the aforementioned mRNA is contained in the composition of the present invention.

[0147] Further aspects of the present invention relate to a polypeptide comprising or comprising a HERV Env protein having at least 95% sequence identity with SEQ ID NO: 2, wherein the Env protein comprises a mutant immunosuppressive domain (ISD) that reduces immunosuppressive properties compared to a wild-type envelope ISD (preferred ISD mutations and mutant ISD sequences are disclosed herein in the context of the present invention—for example, one or more of the ISD mutations L522A, A523Q, N524A, Q525A, N527A, L529A, R530A, Q531A, T532A may be used), and the HERV envelope protein comprises cysteine ​​instead of serine at position 190 of SEQ ID NO: 2. In a preferred embodiment, the polypeptide described above is contained in a virus-like particle (VLP). mRNA encoding the polypeptide described above is also provided. Preferably, the mRNA described above is contained in a composition of the present invention.

[0148] Further aspects of the present invention relate to a polypeptide comprising or consisting of a HERV Env protein, wherein the Env protein comprises a cysteine ​​at position 190, thereby comprising the amino acid sequence VQNWLVEVPTVSPICRFTYHMVSGMSLRP, and the Env protein further comprises a mutant immunosuppressive domain (ISD) that reduces immunosuppressive properties compared to the wild-type Env ISD (preferred ISD mutations and mutant ISD sequences are disclosed herein in the context of the present invention—for example, one or more of the ISD mutations L522A, A523Q, N524A, Q525A, N527A, L529A, R530A, Q531A, T532A may be used). In a preferred embodiment, the polypeptide described above is contained in a virus-like particle (VLP). mRNA encoding the polypeptide described above is also provided. Preferably, the mRNA described above is contained in a composition of the present invention.

[0149] Immunosuppressive domain (ISD) Immunosuppressive domains (ISDs) can be seen as mechanisms utilized by tumors to balance the antitumor immune response, while simultaneously maintaining a pro-tumor inflammatory environment induced by HERV activation, similar to natural viral infections. These ISDs affect both the innate and adaptive immune systems by inhibiting macrophages, NK cells, and T cells. While the detailed mechanisms of ISD-mediated immunosuppression are not yet fully understood, the domain's ability to induce IL-10 secretion upon contact with peripheral blood mononuclear cells, and the ability of mutant ISD domains to reduce NF-κB induction gene expression (surprisingly found in transfection assays), have been observed to be related to the immunosuppressive and / or pro-inflammatory function of ISDs. In particular, the reduction in NF-κB expression by mutant ISD domains may be secondary to immunogenic cell death pathways, resulting in an apparent decrease in NF-κB but actually reflecting enhanced immune stimulation.

[0150] In one embodiment, the mutant ISD of the present invention inhibits the proliferation of human immune cells less than a wild-type ISD that is not mutated and has the amino acid sequence according to Sequence ID No. 3 (see Table 1), and / or has a reduced ability or lack of ability to induce IL-10 secretion from peripheral blood mononuclear cells when the aforementioned cells are brought into contact with the mutant ISD, and / or reduces NF-κB expression.

[0151] For an ISD to be inactivated, its immunosuppressive capacity is preferably reduced by 30% or more compared to the immunosuppression achieved by a wild-type ISD. Preferably, the ISD is further inactivated by 35% or 40% or more, e.g., 45% or more, e.g., 47%, 48%, or 49%, e.g., 50%, compared to the immunosuppression achieved by the original, i.e., unmutated ISD. Preferably, the ISD is also inactivated by 35% or 40% or more, e.g., 45% or more, e.g., 47%, 48%, or 49%, e.g., 50%. Quantification of the level of immunosuppression and / or induction of an inflammatory cellular environment can be performed by quantifying the levels of IL-10, NF-κB, or NF-κB-inducing genes or other molecules secreted by peripheral blood mononuclear cells. Quantification methods are known in the art and include, for example, ELISA or FACS-based methods.

[0152] NF-κB activation / inhibition can be specifically determined by transfecting HEK293T cells with a mixture of a reporter plasmid that expresses luciferase upon NF-κB expression, a DNA plasmid encoding a selected HERV-K Env protein, and lipofectamine. The day after transfection, the cells can be analyzed for luciferase expression by adding a luciferase substrate and then quantifying luminescence. In such assays, HERV-K with the mutant ISD sequence of SEQ ID NO: 4 may reduce basal NF-κB levels by approximately 20%, 25%, 30%, or even more, approximately 40%, or even more than 50% or more.

[0153] The ISD segment can be inactivated by mutation or deletion of one or more amino acids. When inactivation is achieved by mutation, one or more amino acids are typically replaced by different amino acids selected from the other 19 naturally occurring amino acids. Replacing one or two single amino acids at any of the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14 positions of ISD SEQ ID NO: 3 is most appropriate. Those skilled in the art will have sufficient knowledge and experience, through evaluation of initial tests as needed, to determine which amino acids to replace to obtain a satisfactory immune response.

[0154] Therefore, in one embodiment of the present invention, the mutant ISD is an amino acid sequence [ka] Includes or consists of Inactivating the ISD is achieved by replacing one or two single amino acids at any of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, and 14th positions with different amino acids, preferably alanine in each case. In a preferred embodiment, the one or two single amino acids different from the original are selected from naturally occurring amino acids.

[0155] Surprisingly, in this specification, when transfecting cells with a nucleic acid molecule (e.g., DNA) encoding HERV Env, if the HERV Env contains a mutation at any one of the positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the ISD according to Sequence ID No. 3 (LANQINDLRQTVIW), it has been found that the number of transfected cells expressing the encoded HERV Env protein with the mutant ISD is increased compared to the expression in cells transfected with HERV Env having the wild-type ISD (see Figure 4). Therefore, in addition to increased expression of antigenic proteins such as HERV Env, a subsequent increase in the immune response to the antigen may also be achieved when antigenic proteins such as HERV Env are encoded by mutant ISDs containing a mutation at any one of the positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, particularly the experimentally tested mutations L522A, A523Q, N524A, Q525A, I526A, N527A, D528A, L529A, R530A, Q531A, T532A, V533A, I534A, and W535A.

[0156] Therefore, in another embodiment, the ISD mutation is selected from the group consisting of L522A, A523Q, N524A, Q525A, I526A, N527A, D528A, L529A, R530A, Q531A, T532A, V533A, I534A, and W535A. In a preferred embodiment, the mutant ISD contains a sequence according to any one of sequence numbers 4 or 8-20. In a more preferred embodiment, the ISD mutation is selected from L522A, A523Q, N524A, Q525A, N527A, L529A, R530A, Q531A, and T532A. In a still more preferred embodiment, the mutant ISD contains a sequence according to any one of sequence numbers 4, 8-12, 14-17, or 20.

[0157] It may be advantageous to combine the ISD mutations described herein with the stabilizing cysteine ​​substitutions described above. Therefore, in a preferred embodiment, the HERV envelope protein comprises an even number of cysteines and optionally or additionally comprises at least 18 cysteines, and the HERV envelope protein comprises an ISD mutation selected from the group consisting of L522A, A523Q, N524A, Q525A, I526A, N527A, D528A, L529A, R530A, Q531A, T532A, V533A, I534A and W535A, preferably the ISD mutation is selected from the group consisting of L522A, A523Q, N524A, Q525A, N527A, L529A, R530A, Q531A and T532A.

[0158] In another preferred embodiment, the HERV envelope protein is an amino acid sequence [ka] Includes, At least one amino acid located in the range of 170 to 210 is replaced with cysteine, and the HERV envelope protein contains an ISD mutation selected from the group consisting of L522A, A523Q, N524A, Q525A, I526A, N527A, D528A, L529A, R530A, Q531A, T532A, V533A, I534A, and W535A, preferably the ISD mutation is selected from the group consisting of L522A, A523Q, N524A, Q525A, N527A, L529A, R530A, Q531A, and T532A.

[0159] In a more preferred embodiment, the HERV envelope protein contains cysteine ​​at position 190, most preferably the amino acid mutation S190C, and the HERV envelope protein contains an ISD mutation selected from the group consisting of L522A, A523Q, N524A, Q525A, I526A, N527A, D528A, L529A, R530A, Q531A, T532A, V533A, I534A and W535A, and preferably the ISD mutation contains an ISD mutation selected from the group consisting of L522A, A523Q, N524A, Q525A, N527A, L529A, R530A, Q531A and T532A. In one embodiment, the HERV envelope protein contains the amino acid mutation S190C and the ISD mutation Q525A.

[0160] Amino acids are L-amino acids commonly found in naturally occurring proteins. In this disclosure, amino acid residues are indicated according to standard three-letter or one-letter amino acid codes. Any amino acid sequence, including post-translational modified amino acids, may be described as the first translated amino acid sequence, where modifications, such as hydroxylation or glycosylation, are not explicitly indicated in the amino acid sequence. This includes all peptides or proteins that can be represented as sequences modified by linkages, crosslinks, and end caps, non-peptidyl links, etc., all of which are known in the art.

[0161] Those skilled in the art can modify the amino acid sequence by introducing any number or form of mutations or deletions, but currently, it is preferred to replace a single amino acid in the ISD sequence. This modification involves replacing glutamine at position 4 of the ISD sequence with alanine. This change triggers domain inactivation so that the vaccine itself does not produce an immunosuppressive effect. Therefore, in one embodiment, the mutant ISD composed of the RNA of the present invention preferably comprises or consists of the sequence LANAINDLRQTVIW (SEQ ID NO: 4).

[0162] A particularly preferred HERV-K Env sequence, including an ISD-disabling mutation, is shown in SEQ ID NO: 2 (see also Table 1): [ka]

[0163] It is preferable to replace one or more amino acids in the upstream or downstream region of the ISD segment. Such mutations are compensatory mutations aimed at preserving the domain structure so that it can still function against infectious viruses. Therefore, at least one amino acid in the 10-amino acid region upstream or downstream of the ISD may be replaced with a different amino acid. If further compensatory mutations become necessary, those skilled in the art are aware of the effects of such mutations and can select them accordingly.

[0164] Inactivation of HERV ISD can be detected and quantified by detecting the level of immune response in a subject induced by administration of the composition of the present invention. For example, a HERV-specific antibody can be quantified in a blood sample from the subject. Conversely, HERV itself can be labeled using such a specific antibody, and its presentation on the cell surface can be analyzed in both in vitro and in vivo samples using, for example, FACS. Furthermore, the proliferation of immune cells, such as NK cells or T cells, in a sample from an immunized subject (e.g., a mouse), as well as both extracellular and intracellular staining markers of these activated immune cells, can also be analyzed by FACS or other suitable methods. Suitable markers for immune cell activation are obvious to those skilled in the art. For example, activated T cells may be detectable by IFNγ, TNFα, and CD44, and NK cells may be detectable by CD56.

[0165] In this specification, the immunoassays using the mRNA and compositions of the present invention were confirmed by immunizing mice with mRNA encoding HERV-K protein GAG and ENV having mutant ISDs treated with prime and boost regimens (days 0 and 7) (see immunization schedule in Figure 5), followed by tetramer staining to detect and quantify CD8+ T cells specific to HERV-K antigen in mouse spleen tissue, and by detecting specific antibodies in mouse blood samples by ELISA (see Figure 6 or Figure 7, respectively).

[0166] The results in Figure 6 confirm in vivo that immunization with mRNA encoding HERV-K GAG and HERV-K ENV possessing a mutant ENV ISD (e.g., Q525A mutation) induced both CD8+ T cell responses to ENV (shown by Tet18-binding cells) and CD8+ T cell responses to GAG (shown by Tet90-binding cells). Furthermore, the results in Figure 7 confirm that immunization of mice with mRNA encoding HERV-K GAG and ENV possessing an ISD mutation (e.g., Q525A mutation) induced antibody immune responses against both antigen subunits SU and TM in the immunized mice; that is, antibody immune responses against both the HERV-K ENV surface subunit (SU) and the HERV-K ENV transmembrane subunit (TM) were induced. Therefore, mRNA encoding the HERV ENv protein, including mutant ISD, can be effectively administered in vivo to generate a robust immune response to the HERV Env antigen, which is useful as a vaccine against human endogenous retroviruses, particularly HERV-K.

[0167] Another exemplary method for testing the efficacy of immunization and / or immune response induction with compositions containing mRNA encoding HERV Env having an inactive ISD is to assess resistance to tumorigenesis. For example, a tumor challenge and tumor rejection assay may be performed, in which a subject, i.e., an animal, is injected with tumor cells, e.g., tumor cell lines B16F10-GP or CT26 or 4T1 or mouse renal cell carcinoma cells modified to express a portion of the human ERV-K genome, and subsequently treated, i.e., therapeutically vaccinated, with the composition of the present invention. After a period of time, e.g., 1 to 6 weeks, the subject is analyzed for tumor and / or metastasis formation, as well as tumor size and characteristics, e.g., excised tumors are analyzed by HERV Env-specific staining. This makes it possible to determine whether tumorigenesis was reduced or rejected by treatment with the composition of the present invention, and whether the tumor expressed HERV Env.

[0168] Furthermore, as outlined elsewhere in this specification, there are at least two methods for antigen presentation or delivery to initiate an immune response after the composition of the present invention comes into contact with and is taken up by cells: presentation as an MHC class I antigen on the surface of the contacted cell and presented to CD8+ T cells, or presentation as an MHC class II antigen on specialized antigen-presenting cells. Both mechanisms highlight the importance of surface presentation for antigen detection in the immunization process.

[0169] In this context, we surprisingly observed that encoded HERV-K Envs with an ISD containing the point mutation of Sequence ID No. 4 showed improved HERV-K cell surface expression on HEK293 cells in vitro compared to HERV-K Envs without the ISD mutation (see Figure 1). While we do not wish to be constrained by theory, it is expected that the point mutation that inactivates the ISD may also be responsible for the improved surface presentation.

[0170] In addition to the human endogenous retrovirus (HERV) envelope protein or its immunogenic portion, the mRNA of the present invention may also encode further proteins. In this case, the co-encoded portion may, for example, be attracted toward the cell surface when presented together with the HERV Env.

[0171] Accordingly, in one embodiment, the present invention may also relate to a composition comprising a transfection agent and mRNA encoding at least a human endogenous retrovirus (HERV) envelope protein or an immunogenic portion thereof, wherein the aforementioned HERV envelope protein comprises an immunosuppressive domain (ISD), the HERV Env protein comprises a mutant immunosuppressive domain (ISD) that reduces immunosuppressive properties compared to a wild-type ISD, and the aforementioned HERV protein is co-expressed with at least one further protein encoded by the same mRNA. The at least one further protein may be conjugated, i.e., fused, to the HERV envelope protein of the present invention, thereby allowing the further protein to be secreted or presented on the cell surface together with the HERV envelope protein. The at least one further protein may be expressed separately, i.e., may not be fused to the HERV envelope protein of the present invention.

[0172] A person skilled in the art is aware of methods for obtaining fused and unfused co-expressed proteins encoded on the same mRNA strand. In one embodiment, further proteins may be directly conjugated to the HERV envelope protein or conjugated to it via a linker encoded by mRNA. Suitable linkers are known in the art. In one embodiment, at least one further protein may be conjugated to the HERV envelope protein of the present invention via a linker, the aforementioned linker being, for example, a suitable amino acid sequence, particularly preferably consisting of 1 to 30, for example, 1 to 10 amino acid residues. Preferred examples of such amino acid sequences include, but are not limited to, the gly-ser linker.

[0173] In some cases, it may be desirable to induce a stronger immune response or to induce further immunity to an antigen in subjects receiving treatment with the composition of the present invention. Therefore, in one embodiment, the further encoded protein may be an antigen. In one embodiment, the further encoded protein may be an adjuvant.

[0174] In one embodiment, the further encoded protein may be a peptide or protein from a peptide or protein library. Co-presentation may allow the peptide or protein to be presented on the cell surface for selection and / or characterization. Advantageously, proteins are more stable when bound to a matrix than as free molecules, and in this example, the cell surface functions as the matrix.

[0175] By presenting the protein on the cell surface, the preparation or purification of the protein is often unnecessary. Therefore, in one embodiment, the further encoded protein may be a protein or peptide produced within a host cell, i.e., a eukaryotic cell, and such protein or peptide should be obtained with a certain purity.

[0176] Molecules presented on the cell surface, such as peptides or proteins, are freely accessible to substrates or binding partners in activity or binding assays. Therefore, in one embodiment, the further encoded protein may be an enzyme or a catalytic portion of an enzyme, or an antibody or a portion thereof.

[0177] The presented molecules are also readily available for binding in cell purification assays or detection in detection assays. Therefore, in one embodiment, the further encoded protein may be a tag for cell purification, such as an affinity tag or an epitope tag, selected from, but not limited to, the group consisting of CaM-Tag, CBP-Tag, GST-Tag, MBP-Tag; biotinylated tags, such as Avi-Tag, BCCP-Tag, Strep-Tag, His-Tag, FLAG-Tag, Xpress-Tag, T7 epitope tag, and Tap-Tag. In another embodiment, the further encoded protein may be a peptide to be detected, such as a fluorescent tag, such as GFP, YFP, and many others known in the art.

[0178] Cancer cells frequently upregulate surface receptors that promote proliferation and survival. These receptors are effective targets for intervention. One strategy involves delivering toxic receptor-binding agents aimed at killing cancer cells with high receptor levels. Therefore, to enhance the anticancer effect of the treatment using the composition of the present invention, the protein further encoded in one embodiment may be an mRNA-coding agent suitable for killing cancer cells, such as a protein toxin.

[0179] Medical use As shown in the examples, the mRNA of the present invention exhibits a remarkable beneficial effect of significantly improving the cell surface presentation of the encoded antigen, i.e., HERV Env. As stated in the introduction, such efficient antigen surface presentation is crucial for inducing a response from the immune system through contact with the antigen. In parallel with inactivation by mutation of the ISD, the increased surface presentation of the encoded antigen, i.e., HERV Env, is thought to significantly enhance the immunological responsiveness to HERV Env in the subject's body. Therefore, the subject can be immunized with the composition of the present invention, which is effective against the development or progression of HERV-related cancer and / or prompts the subject's body to fight HERV-related cancer through immunological action. Thus, the composition of the present invention, i.e., a transfection agent and mRNA comprising at least a human endogenous retrovirus (HERV) envelope protein or its immunogenic portion and having a mutant immunosuppressive domain (ISD) with reduced immunosuppressiveness compared to wild-type ISD, is suitable for administration in the treatment or prophylaxis. The composition may be administered as a treatment or vaccine, or as part of a treatment or vaccine, to induce a specific immune response against endogenous retrovirus-associated tumor cells and / or to immunize the subject against the development or progression of tumors caused by HERV activity.

[0180] Accordingly, in one embodiment, the present invention relates to compositions of the present invention for use as pharmaceuticals. In another embodiment, the present invention relates to compositions of the present invention for use in the prevention or treatment of disease, preferably for immunizing a subject from disease.

[0181] In yet another embodiment, the present invention relates to compositions of the present invention for the manufacture of pharmaceuticals. In yet another embodiment, the present invention relates to the use of compositions of the present invention for the manufacture of pharmaceuticals for the preventive and / or therapeutic treatment of diseases, preferably for the manufacture of pharmaceuticals for immunizing a subject from a disease.

[0182] In yet another embodiment, the present invention relates to a method for treating and / or preventing a disease, preferably a method for immunizing a subject from the disease, comprising administering a therapeutically effective amount of the composition of the present invention to the subject.

[0183] The pathological conditions caused by ERV activity and / or reactivation are diverse. In one embodiment, the diseases targeted for prophylactic and / or therapeutic treatment with the compositions of the present invention are diseases or disorders associated with ERV reactivation, preferably diseases or disorders associated with HERV reactivation. Therefore, in a preferred embodiment, the diseases targeted for prophylactic and / or therapeutic treatment with the compositions of the present invention are selected from the group consisting of cancer, HIV and / or related disorders, rheumatic diseases, neurodegenerative diseases, age-related diseases, diseases associated with HERV reactivation, chronic inflammation, multiple sclerosis, ALS, sarcopenia, kidney disease, and Alzheimer's disease.

[0184] For example, HIV can reactivate HERVs in human subjects (Jakobsson, Johan, and Michelle Vincendeau. “SnapShot: Human endogenous retroviruses.” Cell 185.2 (2022): 400-400.). Therefore, it is expected that using the composition of the present invention when treating HIV will reduce this HERV reactivation in HIV patients, thereby providing health benefits to HIV patients.

[0185] In one embodiment, if the disease is ALS, it is preferably ALS associated with transcriptional activation response DNA-binding protein 43kDa (TDP-43) and / or its C-terminal fragment, for example, ALS associated with increased ubiquitination, hyperphosphorylation, abnormal localization, and / or accumulation of TDP-43 and / or its C-terminal fragment. In one embodiment, the disease is Alzheimer's disease, preferably Alzheimer's disease associated with tau protein expression, tau protein elevation, tau protein abnormal localization, and / or tau protein aggregation.

[0186] The present invention is particularly suitable for use in the prevention and / or treatment of cancer. For example, the target cancers of HERV-K are prostate cancer, breast cancer, ovarian cancer, lymphoma, melanoma, leukemia, and sarcoma; the target cancer of HERV-H is colorectal cancer; the target cancers of HERV-W are testicular cancer, ovarian cancer, breast cancer, lymphoma, and leukemia; the target cancers of HERV-E are lung cancer and liver cancer. Therefore, the types of cancer treated or prevented by the present invention are not particularly limited, but include prostate cancer, breast cancer, ovarian cancer, lymphoma, melanoma, leukemia, sarcoma, colorectal cancer, testicular cancer, ovarian cancer, breast cancer, lymphoma, lung cancer, and liver cancer. In a preferred embodiment, the cancer is a cancer that expresses HERV. In a more preferred embodiment, the cancers expressing the aforementioned HERV include PD-L1-expressing tumors, cervical cancer, penile cancer, anal cancer, vulvar cancer, vaginal cancer, bladder cancer, breast cancer, clear cell renal cancer, head and neck squamous cell carcinoma, lung squamous cell carcinoma, melanoma, non-small cell lung cancer (NSCLC), ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer (SCLC), triple-negative breast cancer, endometrial cancer, acute lymphoblastic leukemia (ALL), acute myeloid leukemia ( The group is selected from AML, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, Hodgkin lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myeloid leukemia-1 protein (Mcl-1), myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL), and small lymphocytic lymphoma (SLL). Other cancer types may also be appropriate targets.

[0187] Appropriate treatment regimens have been proposed in the art and are obvious to those skilled in the art. For example, under certain conditions, it may be advantageous to treat a patient using a prime-boost regimen. Accordingly, its use in the prevention and / or treatment of cancer may include, for example, the step of priming a subject with the composition of the present invention and boosting it with the composition of the present invention at least five days later.

[0188] The compositions of the present invention are suitable for use in the prevention and / or treatment of cancer, and include the step of post-treating the subject with a different coding antigen, namely the HERV Env protein of the present invention, for example, as a VLP derived from adenovirus, modified vaccinia ankara (MVA), or encoded by another virus, after exposure of the subject to the compositions of the present invention, for example, five days or more after vaccination with the aforementioned compositions.

[0189] The mRNA of the present invention is preferably used as a gene vaccine, particularly in the prevention and / or treatment of diseases, preferably cancer. Alternatively, the nucleic acid molecule can also be used to produce VLPs, particularly HERV-K VLPs, in vitro. The resulting VLPs can then be used in immunotherapy, particularly in the prevention and / or treatment of diseases, preferably cancer. In this context as well, it is understood that the cancers to be treated are cancers that express ERVs.

[0190] In the organs and tissues of aged individuals, activation of endogenous retroviruses occurs, and it has been found that suppression of ERVs reduces cellular senescence, tissue degeneration, and organismal aging (Liu X, et al.; Resurrection of endogenous retroviruses during aging reinforces senescence. Cell. 2023 Jan 19;186(2):287-304.e26). Therefore, reactivation of endogenous retroviruses is a prominent feature and driving force of cellular and tissue aging. Accordingly, the present invention relates, in yet another aspect, to compositions according to the present invention for use in the prevention or delay of aging and / or cellular senescence. In yet another aspect, the present invention relates to compositions according to the present invention for use in the manufacture of pharmaceuticals for the prevention or delay of aging and / or cellular senescence.

[0191] Furthermore, the present invention also relates to a VLP encoded by a nucleic acid molecule encoding a Gag protein and a HERV envelope protein (Env) or its immunogenic portion, wherein the native genomic structure linking Gag and Env is replaced by an activatable linker. Preferably, the aforementioned activatable linker is p2A. More preferably, the HERV is HERV-K. More preferably, the HERV is HERV-K having the amino acid sequence published by Lee et al. (Lee YN, Bieniasz PD (2007) Reconstitution of an Infectious Human Endogenous Retrovirus. PLoS Pathog 3(1): e10). As described above, it is envisioned that such VLPs will be used in immunotherapy. Furthermore, the present invention relates to a nucleic acid molecule or VLP for use in the prevention and / or treatment of a disease. Preferably, the disease is cancer. The cancer is understood to be a cancer that expresses the corresponding HERV.

[0192] Pharmaceutical composition As outlined above, the compositions of the present invention are useful, for example, in the treatment or prevention of diseases. Therefore, in one embodiment, the present invention relates to a pharmaceutical composition comprising the composition of the present invention.

[0193] In some cases, as outlined above, when administering the composition of the present invention in the course of treatment or prevention, further agents such as excipients may be beneficial to improve the therapeutic or preventive efficacy and / or patient tolerance. Accordingly, in another embodiment, the present invention relates to a pharmaceutical composition comprising the composition of the present invention and a pharmaceutically acceptable excipient.

[0194] In some embodiments, pharmaceutically acceptable excipients may be selected as defined elsewhere herein. In preferred embodiments, the pharmaceutical composition of the present invention may contain at least one pharmaceutically acceptable excipient selected from the group consisting of water, sodium chloride, potassium chloride, sucrose, sodium acetate, or physiological saline. [Brief explanation of the drawing]

[0195] [Figure 1] HEK293 cells were transfected with DNA encoding HERV-K GAG-ENV or HERV-K GAG-ENV ISDmut prepared in JetPEI, or with RNA 1(5'UTR)HBA1-HERV-K Gag Env ISDmut-(3'UTR)HBB-poly(A(70)) (SEQ ID NO: 45) prepared in Lipofectamine Messenger MAX. Similar results are expected when HEK293 cells are transfected with RNA having a sequence according to SEQ ID NO: 5. Control samples were left untreated (control NT) or treated with JetPEI (control JetPEI) or Lipofectamine Messenger MAX (control LipoMAX). Cells were stained for HERV-K ENV expression 17 hours after transfection. The graph shows the percentage of HERV K ENV-positive cells among viable cells. [Figure 2] NF-κB activation or inhibition was determined by transfecting HEK293T cells with a mixture of a reporter plasmid expressing luciferase upon NF-κB expression, a DNA plasmid encoding either HERV-K Env protein with complete ISD (WT) or HERV-K Env protein with mutant ISD (ISDmut), and lipofectamine. The day after transfection, cells were analyzed for luciferase expression by quantifying luminescence (RLU: relative luminescence units) as an indicator of NF-κB activation or inhibition. The data shown were normalized to baseline activity. [Figure 3]Figure 3 provides supplementary data to the results shown in Figure 1. HEK293 cells were transfected with either of two RNAs prepared in Lipofectamine Messenger MAX, one of which was "RNA4," encoding the 3'UTR-polyA (30-linker-70) of HBA1 5'UTR-HERV-K Gag Env ISDmut (with ISD mutation Q525A)-AES mtRNR1 and containing N1-methylpseudridine (m1Ψ) modification (SEQ ID NO: 36), and the other was "RNA8," encoding the 3'UTR-polyA (30-linker-70) of HBA1 5'UTR-HERV-K Gag Env (with wild-type ISD without mutation)-AES mtRNR1 and containing N1-methylpseudridine (m1Ψ) (SEQ ID NO: 37). In the control samples, cells were either left untreated (control NT) or treated with Lipofectamine Messenger MAX (control LipoMAX). Cells were stained for HERV-K ENV expression 24 hours after transfection. The graph shows the percentage of HERV K ENV-positive cells among viable cells. [Figure 4]HEK293 cells were transfected with either DNA encoding HERV-K GAG-ENV (with a wild-type ISD without mutations; samples were labeled "WT") or DNA encoding HERV-K GAG ENV ISDmut (with a specific mutation in the ISD), both prepared in JetPEI. The DNA constructs used were identical to those used in the experiments for the data shown in Figure 1 (referencing the same protein-coding sequence as SEQ ID NO: 45), except that the ISD sequences were different, i.e., different ISD mutations / positions were used. The ISD mutations tested for the ISDmut construct were L522A, A523Q, N524A, Q525A, I526A, N527A, D528A, L529A, R530A, Q531A, T532A, V533A, I534A, and W535A (samples were labeled with the corresponding mutations; see also ISD sequences, SEQ ID NOs. 4 and 8-20 in Table 3). Additionally, constructs with the stabilizing mutation S190C combined with the wild-type ISD or with the stabilizing mutation S190C combined with a mutant ISD containing the ISD mutation Q525A were also tested (samples labeled "WT+S190C" and "Q525A+S190C"). In the control samples, cells were either left untreated (labeled "control NT") or treated with JetPEI (labeled "control JetPEI"). Cells were stained for HERV-K ENV expression 24 hours post-transfection. The graph shows the percentage of viable cells that were HERV-K ENV positive. These data were generated using DNA constructs encoding the HERV-K protein; however, similar results are expected for RNA constructs encoding these proteins, particularly since protein expression involves the transcription of DNA into RNA. [Figure 5]This is a schematic diagram of the vaccine regimen in which CB6F1 WT (wild-type) mice were immunized on day 0 (prime) with mRNA encoding the HERV-K protein GAG and ENV ISDmut, including N1-methylpseudridine (m1Ψ) modification (construct according to SEQ ID NO: 36), which can be assembled into a VLP form in which the ISD of the ENV protein is mutated (Q525A mutation) (represented by SEQ ID NO: 36). On day 7, mRNA encoding the same antigen cassette was administered a second time (boost). Mice were euthanized on day 21, and the immune response was analyzed by tetramer staining to detect and quantify HERV-K antigen-specific T cells in spleen tissue samples, or by ELISA to detect specific antibodies in mouse blood samples. The results are shown in Figures 6 and 7. [Figure 6]The immunization schedule is shown in Figure 5, with N=5 mice in each group. This data shows the frequency (%) of tetramer-bound CD8+ T cells in the spleen tissue of immunized mice 21 days after prime immunization (see method described in Altman JD, Moss PA, Goulder PJ, Barouch DH, McHeyzer-Williams MG, Bell JI, McMichael AJ, Davis MM, Phenotypic analysis of antigen-specific T lymphocytes. Science. 1996 Oct 4; 274(5284):94-6). The frequency of each tetramer (Tet) is shown individually. In tetramer staining of splenocytes, tetramers are conjugated with four MHC class I molecules, loaded with a selected antigen-specific peptide (derived from HERV-K), and conjugated with a fluorescent dye. Tetramers bind only to T cells specific to the loaded target antigen peptide. The vaccines used for immunization in this specification induce activation and proliferation of HERV-K antigen-specific T cells, and the proportion of antigen-specific T cells spread after vaccination of mice is detected by tetramer staining. Tetramer Tet18 binds to CD8+ T cells specific to the HERV-K ENV epitope, while tetramers Tet29 and Tet90 bind to CD8+ T cells specific to the HERV-K GAG epitope. Immunization with mRNA encoding HERV-K GAG and HERV-K ENV having a mutated ENV ISD (Q525A mutation) induced a CD8+ T cell response to both ENV (i.e., cells bound to Tet18) and GAG (i.e., cells bound to Tet90). [Figure 7]The immunization schedule is shown in Figure 5, with N=5 mice in each group. The data shows OD values ​​determined by ELISA using serum samples collected from immunized mice 21 days after prime immunization. This signal represents the amount of antibody in the mouse serum sample against each protein subunit. In Figure 7A, the OD value was measured after diluting a serum sample collected 21 days after prime immunization at 1:25. This OD represents the level of antibody against subunit TM (HERV-K ENV transmembrane subunit). In Figure 7B, the OD value was determined after diluting a serum sample collected 21 days after prime immunization at 1:25. This OD represents the level of antibody against subunit SU (HERV-K ENV surface subunit). Immunization using mRNA encoding HERV-K GAG and ENV with ISDmut (Q525A mutation) induced antibody immune responses against both antigen subunits SU and TM in immunized mice.

[0196] Examples Example 1 1.1 Cell culture The HEK293 cell line was generated from human embryonic kidney cultures by transformation with fragmented adenovirus type 5 (Ad5) DNA (available from LGC Standards (ATCC)). Advantages of this cell line include ease of growth and high transfection efficiency. HEK293 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) Glutamax supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin + streptomycin (Pen / Strep). The cell line was maintained at 37°C and 5% CO2 under a humidified atmosphere.

[0197] 1.2 DNA vectors Codon-optimized HERV-K GAG-ENV (SEQ ID NO: 7), or HERV-K GAG-ENV ISDmut (Q525A) (SEQ ID NO: 6), or HERV-K GAG-ENV ISDmut variants L522A (SEQ ID NO: 8), A523Q (SEQ ID NO: 9), N524A (SEQ ID NO: 10), I526A (SEQ ID NO: 11), N527A (SEQ ID NO: 12), D528A (SEQ ID NO: 13), L529A (SEQ ID NO: 14), R530A (SEQ ID NO: 15), Q531A (SEQ ID NO: 16), T532A (SEQ ID NO: 17), V533A (SEQ ID NO: 18), I534A (SEQ ID NO: 19), W535A (SEQ ID NO: 20), or HERV-K GAG-ENV ISDWT (SEQ ID NO: 21) with S190C, or HERV-K GAG-ENV with S190C The DNA vector pO6A19 encoding ISDmut(Q525A) (SEQ ID NO: 25) was synthesized by GenScript. A strong cytomegalovirus promoter and tetracycline operator (TetO) site were located before the target gene, followed by an SV40 polyadenylation signal. The vector's insert containing the target gene encoded the consensus HERV-K GAG gene via a glycine / serine / glycine (GSG) linker, ligated to either the envelope (ENV) or envelope ISDmut (ENV ISDmut)(Q525A), or the ENV ISD variant, or ENV with S190C, or ENV ISDmut(Q525A) with the S190C gene, followed by a 2A peptide (P2A) derived from self-cleaving porcine tesiovirus-1. The synthesized sequence constructs are outlined in the explanatory diagrams in Figures 1 and 4.

[0198] 1.3 DNA Amplification The DNA vector was transformed into PIR-1 competent E. coli by heat shock and amplified overnight in LB medium containing 25 μg / mL kanamycin (the Pir gene encodes the replication protein π, which is necessary for the replication and maintenance of DNA vectors containing R6Kγ origin). The PIR-1 competent E. coli strain has a mutant allele of the pir gene that maintains the donor vector construct at approximately 250 copies per cell. The plasmid was then purified using the Nucleobond Xtra Midi kit according to the manufacturer's protocol (AH Diagnostics).

[0199] 1.4 RNA generation The consensus HERV-K GAG gene was linked to the envelope ISDmut (Env ISDmut) gene via a glycine / serine / glycine (GSG) linker, followed by a 2A peptide (P2A) derived from self-cleaving porcine tesiovirus 1. The synthesized GOI was used as a template by Vectorbuilder and synthesized with a 5'UTR, Kosack sequence, 3'UTR, and poly(A) on both sides (SEQ ID NO: 45). mRNA was produced by Vectorbuilder. The synthesized sequence construct is outlined in the figure caption.

[0200] In addition to the mRNA constructs described above, two more mRNA constructs were synthesized. The consensus HERV-K GAG protein was linked to the envelope (ENV) or envelope ISDmut (ENV ISDmut) protein via a glycine / serine / glycine (GSG) linker, followed by a 2A peptide (P2A) derived from self-cleaving porcine tesiovirus 1, thus encoding the sequence. These synthesized GOIs were used as synthesis templates by Vectorbuilder and synthesized with a 5'UTR, Kozak sequence, 3'UTR, and poly-A tail on both sides, as shown in SEQ ID NOs. 36 and 37. Both mRNA constructs were modified with N1-methylpseudridine (m1Ψ). The synthesized sequence constructs are also outlined in the explanatory diagram in Figure 3.

[0201] 1.5 In vitro transfection HEK293 cells were seeded in 24-well plates at a rate of 2,000,000 cells per well, 23–24 hours prior to transfection. Cells were transfected with either 2.4 μg of mRNA and 5.6 μL of Lipofectamine Messenger MAX per well, or 1 μg of DNA and 2 μL of JetPEI (Polyplus) per well. Transfected cells were incubated at 37°C and 5% CO2 in a humidified atmosphere for 17 hours (Figure 1) or 24 hours (Figures 3 and 4).

[0202] 1.6 Cell surface staining Cells were harvested and washed with PBS containing 1% BSA and 0.1% NaN3. The cells were stained for 30 minutes with HERM 1811-5 (Austral Biologics), a HERV-K ENV-specific antibody conjugated to AlexaFluor647 (Molecular Probes). The cells were washed with PBS and incubated with Viability dye eFluor 780 (eBioscience). The reaction was stopped by adding PBS containing 1% BSA and 0.1% NaN3, and the cells were washed with PBS. The cells were fixed with 2% paraformaldehyde, washed with PBS containing 1% BSA and 0.1% NaN3, and then resuspended in PBS containing 1% BSA and 0.1% NaN3. Samples were subjected to flow cytometry using Fortessa 3 or 5, and the results were analyzed using FlowJo software V10.7.1 or V10.8.1. The results are shown in Figures 1, 3, and 4.

[0203] Example 2 LNP LNPs can be produced by incorporating the nucleotides of the present invention, such as mRNA, according to the methodologies disclosed in, for example, Leung et al. (Leung, et al., 2015, Microfluidic Mixing: A General Method for Encapsulating Macromolecules in Lipid Nanoparticle Systems. The Journal of Physical Chemistry B, 119(28), 8698-8706), Ripoll et al. (Ripoll et al., 2022, Optimal self-assembly of lipid nanoparticles (LNP) in a ring micromixer, Sci Rep 12, 9483), or by other suitable methods known in the art.

[0204] In a preferred embodiment, the LNP is formulated by mixing the mRNA of the present invention with a lipid composition comprising at least one cationic lipid. More preferably, the mRNA is mixed with a lipid composition comprising DOSPA (2,3-dioleoyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propaniminium trifluoroacetate) and DOPE, preferably with a molar ratio of DOSPA to DOPE of 2:1 to 4:1. The mRNA can be introduced into target cells using the formulations outlined in Examples 1.4 and 1.5 above.

[0205] Example 3 NF-κB activation / inhibition was determined by transfecting HEK293T cells in a 96-well plate with a mixture of 50 ng of a reporter plasmid expressing luciferase upon NF-κB expression, 0–50 ng of a DNA plasmid encoding a selected HERV-K Env protein, and 0.6 μl of lipofectamine per well. The total DNA amount was adjusted to 100 ng using an empty plasmid pO6A5tetO empty (IPT22). For the results shown in Figure 2, cells were transfected with the following HERV-K Env plasmids: HERV-K WT IPT24 plasmid with p06A5-(TetO)-CMV-coHERV-K-P2TS and HERV-K ISDmut IP27 plasmid with p06A5-(TetO)-CMV-ISDmut-coHERV-K-P2TS.

[0206] Luciferase expression was analyzed by adding SteadyLite luciferase substrate (PerkinElmer) to cells the day after transfection, and then quantifying the luminescence. Luminescence can be measured by appropriate methods known in the art. For example, it can be carried out according to the method described in Mendez et al. (2020) (Mendez JM, Keestra-Gounder AM. NF-κB-dependent Luciferase Activation and Quantification of Gene Expression in Salmonella Infected Tissue Culture Cells. J Vis Exp. 2020, Jan 12), or essentially according to the protocol derived from the aforementioned protocol. In this assay, HERV-K with the mutant ISD sequence of SEQ ID NO: 4 reduced NF-κB measured by the luciferase assay by approximately 50% compared to cells transfected with the control plasmid. The results are shown in Figure 2.

[0207] Example 4 Example 4.1 Animal experiments and serum separation All animal experiments were conducted in accordance with Danish national guidelines, and the experimental procedures were approved by the Danish National Animal Supervision Agency (Dyreforsφgstilsynet). Female CB6F1 mice were obtained from Envigo at 6-8 weeks of age and were housed at the Panum Institute, University of Copenhagen for at least one week prior to the start of each experiment.

[0208] 4.2 Immunization and Serum Separation Mice were immunized with mRNA-HERV-K GAG-ENV ISDmut containing N1-methylpseudridine (m1Ψ) (SEQ ID NO: 36) in a syngeneic prime-boost regimen. Prime immunization was administered by injection on day 0, followed by boost immunization on day 7. The immune response was analyzed 14 days after the booster, i.e., 21 days after the prime immunization on day 0. The immunization schedule is shown in Figure 5.

[0209] Prior to injection, mRNA was diluted in OptiMEM and mixed with Lipofectamine RNAiMAX in a ratio of mRNA (μg) to RNAiMAX (μl). Each mouse was intravenously (iv) injected with 200 μL of a solution containing 3.5 μg of mRNA. On day 21, blood samples were collected from the animals' cheeks. Serum was isolated from the obtained blood samples by two consecutive centrifugation steps of 800 g and 8°C for 8 minutes each. At the end of the immunization study, the mice were euthanized by cervical dislocation.

[0210] 4.3 Splenocyte suspension The spleens were aseptically removed from euthanized mice (at the end of the immunotherapy study) and transferred to RPMI 1640 GlutaMax (complete RPMI) supplemented with 10% heat-inactivated FBS, 1% penicillin / streptomycin, and 1% sodium pyruvate. The spleens were crushed with a fine mesh (mesh size 70 μm), and then the spleen cells were centrifuged to obtain a single-cell suspension.

[0211] 4.4 Tetramer staining Mouse splenocytes were resuspended in PBS containing 1% BSA, 0.1% NaN3 (FACS buffer), and 50 nM dasatinib, and incubated at 37°C and 5% CO2 for 30 minutes. The splenocytes were then centrifuged and incubated with their respective tetramers. 0.08 μg to 0.25 μg of the corresponding tetramer in FACS buffer containing 50 nM dasatinib was added to 500,000 cells, and incubated at 37°C and 5% CO2 in the dark for 15 minutes. - TET18:H2Kd MHC class I monoclonal antibody peptide TYHMVSGMSL, labeled with brilliant violet BV421 provided by Immunitrack; - TET29: Peptide QNVDYNQL containing the H2Kb MHC class I monoclonal antibody, labeled with allophycocyanin phosphor (APC) provided by Immunitrack; - EPYPDFVARL peptide containing the TET90:H2Kb MHC class I monoclonal antibody, labeled with phycoerythrin phosphor (PE) provided by Immunitrack.

[0212] 50 nM dasatinib surface antibodies—anti-CD8b antibody CD8b-BV510, anti-CD4 antibody CD4-PE-Cy7, and anti-B220 / CD45R antibody B220-PerCP-Cy5.5—along with a viability dye (Efluor780) in FACS buffer were added to a tetramer mix and incubated at 4°C in the dark for 20 minutes. The cells were then washed and fixed with 1% paraformaldehyde (PFA) at 4°C in the dark for 15 minutes. Tetramer-stained samples were subjected to flow cytometry on a Fortessa 3 and analyzed using FlowJo software V10.7.1. The results are shown in Figure 6.

[0213] 4.5 ELISA A MaxiSorp (NUNC) flat-bottom plate was coated overnight at 4°C with HERV-K ENV (envelope protein) transmembrane subunit (TM) or HERV-K surface subunit (SU) protein at a concentration of 2 μg / mL in PBS. The plate was washed three times with washing buffer (PBS + 354 mM NaCl + 0.1% Tween 20, pH 7.2), and then blocked for 1 hour at room temperature (approximately 21°C) with blocking buffer (PBS + 354 mM NaCl + 5 g / L BSA + 0.05% Tween 20, pH 7.2). After removing the blocking buffer and washing the plate three times (with washing buffer), 100 μL / well of serum sample diluted 1:25 with blocking buffer was added to the wells and incubated for 1 hour at room temperature (approximately 21°C). The plate was washed three times with washing buffer, and then horseradish peroxidase (HRP) conjugate polyclonal anti-mouse / IgG secondary antibody (Dako, P0260) was added, diluted 1:2000 with blocking buffer, and incubated at room temperature (approximately 21°C) for 1 hour. After the three washing steps with washing buffer, 50 μL of 3,3',5,5'-tetramethylbenzidine (TMB) PLUS 2 (Kem-en-tec, 4395A), a chromogenic substrate for horseradish peroxidase, was added. The color reaction was stopped after 6 minutes by adding 450 μL of 0.2 M H2SO4.

[0214] The color intensity in each well was determined by quantification of absorbance (OD) at a wavelength of 450 nm (using a SpectraMax microplate reader). Background signals measured for samples obtained from non-immunized mice were subtracted from each sample. The results are shown in Figure 7.

[0215] This specification has described various aspects and embodiments in conjunction with various embodiments. However, by examining the drawings, disclosures, and appended claims, a person skilled in the art can understand and perform other variations of the disclosed embodiments when carrying out the subject matter described in the claims. In the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude the plural. A single processor or other unit may perform the functions of multiple items described in the claims. The mere fact that certain means are described in different dependent claims does not indicate that a combination of these means cannot be used advantageously. Computer programs may be stored / distributed on suitable media such as optical recording media or solid-state media supplied together with or as part of other hardware, but they may also be distributed in other forms, such as via the Internet or other wired or wireless communication systems.

[0216] Sequences are disclosed in the text of the specification and in the appendix sequence listing in accordance with WIPO standard ST.25. Sequence numbers designated by specific numbers must be identical in both the text of the specification and the appendix sequence listing. For example, Sequence ID 1 defines the same sequence in both the text of the specification and the appendix sequence listing. In the event of any discrepancy between sequence definitions in the text of the specification and sequence definitions in the appendix sequence listing (for example, Sequence ID 1 in the text of the specification corresponds to Sequence ID 2 in the appendix sequence listing), references to specific sequences in the application, particularly in specific embodiments, shall be understood as references to sequences in the text of the application, not to the appendix sequence listing. In other words, any discrepancies between sequence definitions / designations in the text of the specification and the appendix sequence listing shall be resolved by modifying the appendix sequence listing to match the sequences and their designations disclosed in the text of the application, including the specification, examples, drawings, and claims.

[0217] Example 5 The compositions according to the present invention can be prepared according to Examples 1.4 and 2 outlined above.

[0218] In preferred embodiments, this composition or any other composition described herein may be administered to patients suffering from age-related diseases. Retroviruses, including HERV, are known to be reactivated in older adults, causing disease symptoms (see Zlotorynski, E. Younger endogenous retroviruses make us older. Nat Rev Mol Cell Biol 24, 165 (2023)).

[0219] Therefore, it is expected that administering the composition of the present invention to such patients will alleviate these symptoms. Those skilled in the art can test multiple doses to find an amount sufficient to alleviate these symptoms.

[0220] [Table 1]

[0221] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6]

[0222] [Table 3-1] [Table 3-2]

[0223] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0224] [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4]

[0225] In sequences 5, 45, 36, and 37, T can be replaced with m1Ψ (N1-methylpseudolidine). This may have an mRNA stabilizing effect.

[0226] The sequence of Sequence ID No. 5 may include the start nucleotide sequence GGG, which precedes the 5'UTR at the DNA level.

[0227] Sequences 36 and 37 may include the start nucleotide sequence GGGA that precedes the 5'UTR at the DNA level.

[0228] The sequence of Sequence ID No. 45 may include the start nucleotide sequence GGGAG, which precedes the 5'UTR at the DNA level.

Claims

1. A composition comprising a transfection agent and mRNA encoding at least a human endogenous retrovirus (HERV) envelope protein or an immunogenic portion thereof, wherein the HERV envelope protein comprises an immunosuppressive domain (ISD), and the HERV envelope protein comprises a mutant immunosuppressive domain (ISD) that reduces the immunosuppressive properties compared to a wild-type ISD.

2. The composition according to claim 1, wherein the composition comprises lipid nanoparticles (LNPs) containing the mRNA.

3. The composition according to claim 1, wherein the transfection agent is a transfection agent comprising a cationic lipid and / or a cationic polymer, and preferably the composition comprises a liposome containing a cationic lipid and the mRNA.

4. The LNP composition is at least one lipid, (i) Ionizable lipids, preferably ionizable cationic lipids, more preferably ionizable cationic amino lipids; (ii) Noncationic helper lipids or phospholipids, wherein the lipid is preferably neutral, and more preferably the lipid is a DSPC; (iii) Sterols or other structural lipids, wherein the sterol is preferably cholesterol; and (iv) PEG lipids, preferably PEG-DMG The composition according to claim 2, comprising at least one lipid selected from the group consisting of the following.

5. The composition according to claim 3, wherein the transfection agent is a composition comprising DOSPA and / or DOPE, preferably DOSPA and DOPE.

6. The composition according to any one of claims 1 to 5, wherein the mRNA contains at least 60 adenosine nucleotides in the 3'-UTR.

7. The composition according to any one of claims 1 to 6, wherein the mRNA is codon-optimized for expression in humans.

8. The composition according to any one of claims 1 to 7, wherein the mRNA comprises at least 300 bases.

9. The composition according to any one of claims 1 to 8, wherein the mRNA does not contain artificially modified bases.

10. The aforementioned human endogenous retrovirus (HERV) is selected from the group consisting of HERV-K, HERV-H, HERV-W, HERV-FRD, HERV-E, HERV-9, HERV-FC, HERV-T, HERV-3, HERV-V1, and HERV-V2. The aforementioned HERV-K is preferably HERV-K108 (=ERVK-6), ERVK-19, HERV-K115 (=ERVK-8), ERVK-9, HERV-K113, ERVK-21, ERVK-25, HERV-K102 (=ERVK-7), HERV-K101 (=ERVK-24), and HERV-K110 (=ERVK From the group consisting of -18), HERV-H is selected from the group consisting of HERV-H19 (=HERV-H_2q24.3) and HERV-H_2q24.1, HERV-W is selected as ERVW-1 (=Syncytin-1), and HERV-FRD is selected as ERVFRD-1 (=Syncytin-2). The composition according to any one of claims 1 to 9.

11. The composition according to any one of claims 1 to 10, wherein the HERV envelope protein comprises an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of Sequence ID No. 2 over the entire length of Sequence ID No.

2.

12. The composition according to any one of claims 1 to 11, wherein, compared to a wild-type ISD that is not mutated and has an amino acid sequence according to SEQ ID NO: 3, the mutant ISD does not inhibit the proliferation of human immune cells to a greater extent and / or has a reduced ability or lacks the ability to induce IL-10 secretion from peripheral blood mononuclear cells when the cells are brought into contact with the mutant ISD, and / or reduces NF-κB expression.

13. The aforementioned mutant ISD has the following amino acid sequence: 【Chemistry 1】 Includes or consists of In any of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, and 14th positions, one or two single amino acids are replaced with different amino acids, preferably alanine in each case, thereby inactivating the ISD. The composition according to any one of claims 1 to 12.

14. (a) The human endogenous retrovirus (HERV) is HERV-K, and the ISD mutation is selected from the group consisting of L522A, A523Q, N524A, Q525A, I526A, N527A, D528A, L529A, R530A, Q531A, T532A, V533A, I534A and W535A, preferably the mutant ISD contains a sequence according to one of sequence numbers 4 or 8 to 20. More preferably, the ISD mutation is selected from L522A, A523Q, N524A, Q525A, N527A, L529A, R530A, Q531A and T532A, and even more preferably, the mutant ISD contains a sequence according to any one of sequence numbers 4, 8-12, 14-17 or 20, or (b) The human endogenous retrovirus (HERV) is a HERV selected from the group consisting of HERV-9, HERV-FC, HERV-T, HERV-E, HERV-3, HERV-V1, and HERV-V2, and the mutant immunosuppressive domain (ISD) that reduces immunosuppressive properties compared to the wild-type ISD is the following sequence starting from the 23rd amino acid position: - In the case of HERV-9, LQNCZGLDLLTAEKGGLCTFLGE (Sequence ID 38); - In the case of HERV-FC, AQNRALDLLTADKGGTCLFLGE (Sequence ID 39); - In the case of HERV-T, LQNRRGLDLLFLSQGGLCAALGE (Sequence ID 40); - In the case of HERV-E, YQNRALDYLLLAAAEGGVCGKFNL (Sequence ID 41); - In the case of HERV-3, YQNRLALDYLLAQEGGVCGKFNL (Sequence ID 42); - For HERV-V1, MNNRLALDYLLLAEQGGVCAVISK (SEQ ID NO: 43); or - For HERV-V2, MDNRLALDYLAEQGGVCAVINK (Sequence ID 44) It includes one of the following or consists of them: In sequence numbers 38, 40, 41, 42, 43, and 44, one or more amino acids are replaced with different amino acids, preferably the amino acid at position 14 is replaced with a different amino acid, more preferably R, and preferably the single amino acid at position 20 is replaced with a different amino acid, more preferably F. The composition according to any one of claims 1 to 13.

15. The composition according to any one of claims 1 to 14, wherein the mutant ISD preferably comprises or consists of the sequence LANAINDLRQTVIW (SEQ ID NO: 4).

16. The HERV envelope protein contains an even number of cysteines, and / or The HERV envelope protein contains at least 18 cysteine ​​molecules, and / or The aforementioned HERV envelope protein has the following amino acid sequence: 【Chemistry 2】 Includes, At least one amino acid located between positions 170 and 210 is replaced by cysteine. Preferably, the HERV envelope protein contains cysteine ​​at position 190, more preferably, the HERV envelope protein contains the amino acid mutation S190C and / or the HERV envelope protein contains the sequence VQNWLVEVPTVSPICRFTYHMVSGMSLRP and / or the HERV envelope protein contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 2, and the HERV envelope protein contains cysteine ​​at a position within the HERV envelope protein corresponding to amino acid 190 of SEQ ID NO:

2. The composition according to any one of claims 1 to 15.

17. The composition according to any one of claims 1 to 16, wherein the HERV envelope protein comprises or consists of a sequence selected from the group consisting of SEQ ID NOs: 21 to 35, preferably a sequence selected from the group consisting of SEQ ID NOs: 22 to 27, 29 to 32 and 35.

18. The composition according to any one of claims 1 to 17, wherein the composition further comprises an adjuvant, the adjuvant is preferably a cytokine, more preferably a cytokine selected from the group consisting of INFγ, IL-2, IL-12, GM-CSF, IL-15, and IL-7, or a polynucleotide configured to express one or more of the cytokines in eukaryotic cells.

19. The composition according to any one of claims 1 to 18, wherein the composition comprises mRNA having at least 90% sequence identity, most preferably 100% sequence identity, with any one of the sequences according to Sequence ID No. 45, 36, or 37.

20. The composition according to claim 16 or 17, wherein the cell surface expression of the HERV envelope protein as defined in claim 16 or 17 is increased compared to the cell surface expression of the HERV envelope protein having the amino acid sequence according to SEQ ID NO:

2.

21. A composition according to any one of claims 1 to 19, for use as a pharmaceutical product.

22. Use of the composition according to any one of claims 1 to 19 for the manufacture of a pharmaceutical product.

23. A composition according to any one of claims 1 to 19, for use in the prevention or treatment of a disease, preferably for immunizing a subject from a disease.

24. Use of the composition according to any one of claims 1 to 19 for the manufacture of a pharmaceutical product for the preventive and / or therapeutic treatment of a disease, preferably for the manufacture of a pharmaceutical product for immunizing a subject from a disease.

25. A method for treating or preventing a disease in a patient who requires treatment or prevention of the disease, A method comprising administering to the patient a pharmaceutically acceptable liquid composition comprising a therapeutically effective amount of the composition according to any one of claims 1 to 19.

26. The disease is preferably selected from the group consisting of cancer, HIV and / or related diseases, rheumatic diseases, neurodegenerative diseases, age-related diseases, diseases associated with HERV reactivation, chronic inflammation, multiple sclerosis, ALS, sarcopenia, kidney disease, and Alzheimer's disease. Preferably, ALS is associated with a transcriptional activation response DNA-binding protein 43 kDa (TDP-43) and / or its C-terminal fragment, and / or Preferably, Alzheimer's disease is associated with the expression of tau protein. The composition for use according to claim 23 or the use of the composition according to claim 23.

27. Preferably, the cancer is a cancer that expresses HERV, More preferably, PD-L1 expressing tumors, cervical cancer, penile cancer, anal cancer, vulvar cancer, vaginal cancer, bladder cancer, breast cancer, clear cell renal cancer, head and neck squamous cell carcinoma, lung squamous cell carcinoma, melanoma, non-small cell lung cancer (NSCLC), ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer (SCLC), triple-negative breast cancer, endometrial cancer, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia The following are selected from the group consisting of hematopoietic lymphoma (CLL), chronic myeloid leukemia (CML), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, Hodgkin lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myeloid leukemia-1 protein (Mcl-1), myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL), and small lymphocytic lymphoma (SLL): The composition for use or use of the composition according to claim 26.

28. A composition according to any one of claims 1 to 19, for use in preventing or delaying aging and / or cellular senescence.

29. A composition according to any one of claims 1 to 19, for use in the manufacture of a pharmaceutical product for the prevention or delay of aging and / or cellular senescence.

30. A pharmaceutical composition comprising the composition according to any one of claims 1 to 19, comprising a pharmaceutically acceptable excipient.

31. A DNA molecule encoding mRNA contained in the composition according to any one of claims 1 to 19.

32. A virus-like particle (VLP) comprising the HERV envelope protein as defined in any one of claims 1 to 19.

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