Improved expression of surface display antigens

Abstract

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

Description

BACKGROUND While immune cells are capable of recognizing and killing tumor cells, this system is not always functional, as evidenced by the nearly 9 million people worldwide who die from cancer each year. Vaccination methods to induce specific immune responses against tumor cells are a relatively early subject in cancer immunotherapy, but are still under development and have only recently begun to yield relevant results. One vaccination strategy is vaccination with attenuated tumor cells, such as irradiated autologous tumors or allogeneic tumor cell lines, which often secrete granulocyte-macrophage colony-stimulating factor (GM-CSF). In both cases, the injected material encompasses cancer antigens likely to be present in the actual tumor. Other vaccination strategies include administration of peptides or proteins to induce specific immune responses. These antigens are injected directly in combination with adjuvants, or are encoded by DNA plasmids or viral vectors.

[0002] Despite the continuous improvement of immunotherapeutic methods, there is still a lack of broadly acting and highly effective vaccines. A particular reason for this is the previously described immunosuppression by tumor cells.

[0003] Endogenous retroviruses (ERVs) are evidence of ancient ancestors of retroviral infections. After infection, viral RNA is reverse transcribed into proviral DNA, which integrates into the host genome. Eventually, the provirus integrates into cells of the germline and becomes heritable, creating endogenous retroviruses. Over millions of years, the viral DNA is passed down and becomes fixed in the population. Today, every human genome consists of about 8% endogenous retroviral DNA, but these are only remnants of previous retroviruses. Due to mutations, deletions and insertions, most retroviral genes become inactive or disappear completely from the genome. Today, there are no longer any functional full-length endogenous retroviruses in humans. However, ERVs undergo a replication process, resulting in several copies with different functional proteins integrating into the host genome. Thus, in some cases, a large number of homologous ERVs still have the potential to produce viral particles. The human ERV type K (HERV-K, HML2) is one of the most recently acquired ERVs in the human genome, and members of this family retain full-length open reading frames for almost all viral proteins.

[0004] Different studies have highlighted a link between ERV expression and cancer development and progression. The detection of ERVs in human tumors opens up a new field in anticancer therapy, with the hope of new vaccination strategies. A prominent example of human ERVs (HERVs) is HERV type K (HERV-K), which is associated with prostate cancer, breast cancer, ovarian cancer, lymphoma, melanoma, leukemia and sarcoma. A further example is HERV-H, which is expressed in colorectal cancer, and syncytin-1, which is expressed in testicular cancer, ovarian cancer, breast cancer, lymphoma and leukemia.

[0005] Determining whether expression of ERV proteins is a cause or a consequence of tumor development is not easy. However, conditions within cancer cells are known to allow expression of ERVs. The generally low methylation state in tumor cells promotes activation of ERV genes, which are normally silenced in healthy cells by DNA methylation (Downey, R. F., 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). Additionally, exogenous factors can promote ERV expression. Activation of human ERVs has been observed, for example, due to viral infection. HERV-W expression was detected after infection with influenza virus and herpes simplex virus (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 emerged 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 leading to ERV expression, cancer cells maintain activation of these proteins through selection pressure, indicating a beneficial effect of ERVs in tumors (Leong, S. P., et al., Expression and modulation of a retrovirus-associated antigen by murine melanoma cells . Cancer Res, 1988. 48(17): p. 4954-8. Not only human tumors are associated with ERV proteins, but murine cancer cells also express ERVs. This provides a perfect model organism to study the effect of ERVs on tumor progression and to test ERV-targeting therapeutic approaches. One ERV model is the melanoma-associated retrovirus (MelARV), which originates from a provirus of murine leukemia virus (MuLV) present in the mouse genome. Most inbred mouse strains contain one or two copies of inactivated MuLV (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 has three insertions in its genome and is characterized by a massive production of MuLV early in life, leading to a frequent occurrence of spontaneous lymphomas. Other mouse strains, such as C57BL / 6, only produce MuLV particles spontaneously later in life. Several other murine cancer models similarly express MuLV / MelARV, similar to human ERVs.

[0006] As the immune system of the viral host is the natural defense mechanism against infection, many viruses and especially retroviruses have developed strategies to evade this surveillance. One mechanism that can be seen in different viral families [Duch et al., WO2013 / 050048] is the development of an immunosuppressive domain (ISD) in the envelope protein (Env), causing immune system suppression at different levels. Immune cells including natural killer (NK) cells, CD8 T cells or regulatory T (Treg) cells can be affected by viruses containing an ISD [Schlecht-Louf et al. (2010)].

[0007] Many ERVs contain proteins with an immunosuppressive domain (ISD) and such domains can also be found in MelARV Env proteins (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 U S A, 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 U S A, 1998. 95(25): p. 14920-5.) The importance of ISD in MuLV or MelARV has been shown by introducing murine leukemia virus Env proteins 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 U S A, 1998. 95(25): p. 14920-5.) Despite the additional foreign antigen, Env-transduced tumor cells grew faster. This observation is explained by a local immunosuppressive effect mediated by the Env protein. ISD affects both the innate and the adaptive immune system, as shown by the identical suppression of macrophages, NK cells and T cells (Lang, M.S., 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). Moreover, an effect on regulatory T cell subsets has been suggested, which in turn suppress 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 fully understood, but the effect seems to be mainly mediated by the CKS-17 peptide within the ISD. CKS-17 has different effects on the immune system, mainly by altering cytokine expression (Haraguchi, S., R.A. Good, and N.K. Day-Good, A potent immunosuppressive retroviral peptide: cytokine patterns and signaling pathways . Immunol Res, 2008. 41(1): p. 46-55.)..

[0008] One of the first therapeutic approaches targeting tumor cells expressing ERVs included the administration of monoclonal antibodies. Thus, antibodies targeting HERV-K Env were able to reduce tumor growth of breast cancer cell lines. Wang-Johanning et al. showed that the observed effect of anti-HERV-K Env monoclonal antibodies was mediated by alterations in the cancer cell cycle and an increase in apoptosis. The effect of the antibodies 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) can be the prevention of immunosuppression. Like MelARV Env, HERV-K Env proteins contain ISD and have immunomodulatory functions (Morozov, V.A., V.L. 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. Thus, the effect of HERV-K can only affect innate immune cells, such as NK cells.

[0009] Another part of the adaptive immune response that can be helped to eradicate tumors by targeting ERVs includes T cells. For example, adoptively transferred T cells against MuLV Env epitopes in combination with IL-2 were able to eradicate lung metastases of melanoma cells (Yang, J.C. and D. Perry-Lalley,The envelope protein of an endogenous murine retrovirus is a tumor-associated T-cell antigen for multiple murine tumors Similar experiments were performed in a humanized mouse model for 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 prevent metastasis and tumor growth.

[0010] In addition to the direct injection of antibodies or T cells, a more practical, less expensive and efficient strategy is the induction of an immune response by vaccination. One simple approach is the vaccination with a virus-encoded antigen. However, this approach is rather cumbersome, as DCs have to be isolated and cultured first, which are then pulsed with defined HLA restricted peptides and reinjected into mice or patients.

[0011] A clever vaccination strategy is the presentation of antigens (e.g. viral envelope proteins) on virus-like particles (VLPs) to the immune system, which are encoded in the nucleotides comprised by the vaccine. These particles do not contain viral nucleic acids and are therefore non-infectious. However, VLPs are highly immunogenic and the displayed proteins are presented in a native context. For example, viral Env proteins integrated into VLPs are presented on a virus-like surface, which facilitates correct folding and conformation. In addition to the advantage of strong immunogenicity, the vaccination strategy with VLPs also includes practical benefits. For example, VLPs are relatively easy to produce, as they are built from only a single or few proteins and production can be performed in cell culture. Bayer et al. (2010) showed that only the combination of encoded antigen and capsid-presented antigen was able to increase the level of functional antibodies. This observation was attributed to the fact that while presentation on the adenoviral capsid helps to cross-link B cell receptors, the encoded antigen is required for the necessary CD4+ T cell response that facilitates affinity maturation of B cells. With this vaccination strategy, Bayer et al. were able to reduce viral load of F-MLV after challenge. However, no indication of an increased CD8+ T cell response against the target antigen could be observed. Shoji et al. focused mainly on the optimization of an adenovirus-based HIV vaccine and investigated the in situ formation of VLPs based on the Gag protein. In their study, such a background showed the highest immune response compared to other display strategies that did not promote the in situ formation of VLPs [Shoji et al., 2012].

[0012] For vaccination against a virus or a virus-related disease, such as a cancer expressing an ERV, the entire Env protein should ideally be displayed to the immune system to ensure an immune response against the complete protein target. However, as the Env protein contains an ISD, the vaccine itself has an immunosuppressive capacity which is undesirable for the immunization method. To circumvent this drawback, mutations are introduced into the ISD to maintain the native conformation of the target protein while preventing immunosuppression. Similarly, US2012189647 relates to mutated envelope proteins resulting from mutations in the immunosuppressive domain of the transmembrane subunit of the wild-type envelope protein.

[0013] One of the first to test inactivating mutations in the ISD of viral proteins was Schlecht-Louf et al. [Schlecht-Louf et al. (2010)]. Based on a comparative study between the immunosuppressive syncytin-2 and the non-immunosuppressive syncytin-1 [Mangeney et al. (2007)], Schlecht-Louf et al. identified mutations that abolish ISD activity without eliminating the general structure and functionality of the Env protein. This mutation strategy was applied to other virus-originated proteins (e.g. HTLV and XMRV) and more extensively tested for Friend murine leukemia virus (F-MLV). The study not only revealed the inhibition of both NK cells and T cells by the ISD, but also showed that a live-attenuated virus of F-MLV containing the mutated ISD in the Env protein acts as a vaccine against the same virus with the WT ISD sequence. The protection was due to increased antibody levels and T cell responses against F-MLV epitopes. Their findings were eventually embodied in patent application WO 2011 / 092199, which focuses on the xenotropic murine leukemia virus-related virus (XMRV) that has been associated with human prostate cancer and chronic fatigue syndrome. Thus, WO 2011 / 092199 relates to ISD mutations in XMRV in particular and the use of such ISD mutated viruses for vaccination strategies.

[0014] Another application of ISD mutations is described in patent application WO 2014 / 195510. In this case, mutations in the ISD were introduced in the feline immunodeficiency virus (FIV) in order to reduce the immunosuppression by the virus while still maintaining its native conformation. WO 2014 / 195510 describes that specific mutations increase the antibody response against the FIV Env protein when administered in a vaccination method, bound to MBP or transduced in implanted tumor cells. Thus, WO 2014 / 195510 relates to mutations in the ISD of the FIV Env and the use of such mutated proteins in a vaccination method against infection by FIV or other lentiviruses.

[0015] Another approach to ISD mutations in viral Env proteins of broader spectrum is described in patent application WO 2013 / 050048. In particular, WO 2013 / 050048 relates to the generation of antigens that reduce immunosuppression during vaccination by first identifying ISDs in enveloped RNA viruses, and subsequently mutating these domains. 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 membranes, 3) the high homology in the primary structure (sequence) of the peptide exists within the order, family, subfamily, genus or species of the virus, 4) the location at the surface of the fusion protein in a given conformation is a characteristic of an immunosuppressive domain revealed by 3D structure or antibody staining. After identification of potential ISDs in the viral Env of interest, the immunosuppressive function is verified, and subsequently mutations are introduced in the ISDs, and a reduction of at least 25% of the immunosuppression is confirmed. In summary, WO 2013 / 050048 describes the identification of ISDs in enveloped RNA viruses, the generation of ISD mutated peptides, and the utilization of said peptides as vaccines and the generation of antibodies.

[0016] Although previous strategies to mutate ISDs in viral Env proteins using adenoviruses encoding and displaying viral antigens, previous vaccination strategies employing ISD mutations primarily aimed at preventing viral infection [Schlecht-Louf et al. 2010; WO 2011 / 092199; WO 2014 / 195510; US20110305749; WO 2014 / 195510]. Therefore, there is still a need to break tolerance against self-antigens. Such breaking of tolerance can be facilitated by dense surface protein-coated particles, such as VLPs. The dense coating by membrane-associated viral glycoproteins increases with increasing cell surface expression before protein incorporation into budding virus-like particles from the cell surface. Additional benefits of robust cell surface expression include the 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)).

[0017] Furthermore, in situ synthesis systems for virus-like particles have previously been used [Luo et al. (2003); Sohji et al. (2011); Andersson et al. (2016); Andersson & Holst (2016); Andersson et al. (2017)], but for situations where VLPs are not applicable, alternative methods for improved antigen display are needed in addition to the use of VLPs. Furthermore, there is also a need for efficient systems allowing efficient production of antigens or antigen-VLPs, such as HERV-K VLPs. Establishment of such systems ideally requires that the system is previously efficiently introduced into a host cell or organism. Thus, there is a need to provide improved delivery methods and means for administration of polynucleotides to elicit an immune response in a subject and / or to treat a disease involving expression of a protein, e.g. a cancer marker protein, in cells of the subject. SUMMARY As outlined above, with respect to ERVs, there is a need for efficient surface display of endogenous retroviral antigens, e.g. for vaccination of a subject against endogenous tumor potential and / or tumor progression. The present invention contemplates the generation of such efficient 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 for improved surface display of the encoded antigens on host cells, incorporation into virus-like particles and increased stimulation of the immune system.

[0019] Thus, in one aspect, the present invention relates to a composition comprising a transfection agent and an mRNA encoding at least one human endogenous retrovirus (HERV) envelope protein or an immunogenic portion thereof; wherein the HERV envelope protein comprises an immunosuppressive domain (ISD); wherein the HERV Env protein comprises a mutated immunosuppressive domain (ISD) which reduces its immunosuppressive properties and / or allows it to be more efficiently presented on the cell surface as compared to the wild-type ISD. In one embodiment, the composition comprises a lipid nanoparticle (LNP) comprising the mRNA.

[0020] In another aspect, the present invention relates to the composition of the present invention for use as a medicament.

[0021] In another aspect, the present invention relates to the use of the composition for the manufacture of a medicament.

[0022] In yet another aspect, the present invention relates to the composition for use in the prevention or treatment of a disease, preferably for immunizing a subject against a disease.

[0023] In yet another aspect, the present application relates to the use of a composition according to the present application for the manufacture of a medicament for the prophylactic and / or therapeutic treatment of a disease, preferably for immunizing a subject against a disease. In yet another aspect, the present application relates to a method of treating and / or preventing a disease, preferably immunizing a subject against a disease, comprising administering (preferably a therapeutically effective amount) of a composition according to the present application to the subject. In one embodiment, the disease is preferably selected from the group consisting of: cancer, HIV and / or related conditions, rheumatic diseases, neurodegenerative diseases, aging-related diseases, diseases associated with HERV reactivation, 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.

[0024] In yet another aspect, the present application relates to a composition according to the present application for use in the prevention or delay of aging and / or cellular senescence. In yet another aspect, the present application relates to a composition according to the present application for use in the manufacture of a medicament for the prevention or delay of aging and / or cellular senescence.

[0025] In another aspect, the present application relates to a pharmaceutical composition comprising a composition of the present application, comprising a pharmaceutically acceptable excipient.

[0026] In another aspect, the present application relates to a DNA molecule encoding the mRNA comprised in a composition of the present application.

[0027] A further aspect of the present application relates to a virus-like particle (VLP) comprising a HERV envelope protein as defined according to the present application as described herein. DETAILED DESCRIPTION DEFINITIONS administering: As used herein, “administering” refers to the method of delivering a composition to a subject or patient. The method of administration can be selected to target delivery (e.g., specific delivery) to a particular region or system of the body. For example, administration can be parenteral (e.g., subcutaneous, intradermal, intravenous, intraperitoneal, intramuscular, intraarticular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional, or intracranial injection, as well as any suitable infusion techniques), oral, transdermal or intradermal, interdermal, rectal, intravaginal, topical (e.g., by powder, ointment, cream, gel, lotion, and / or drops), mucosal, nasal, buccal, enteral, vitreous, intratumoral, sublingual, intranasal; by intratracheal instillation, intrabronchial instillation, and / or inhalation; as oral sprays and / or powders, nasal sprays and / or aerosols, and / or through a portal vein catheter. Preferred means of administration are intramuscular, intravenous, intradermal, or subcutaneous.

[0029] and / or:The term "and / or," where used herein, includes the meaning of "and," "or," and "any or all of the other combinable elements, in addition to the various combinations of the elements themselves.

[0030] about, approximately: As used herein, the term "about" or "approximately," when applied to one or more values of interest, indicates that the value range for the value in question is 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 than or less than) of the stated reference value, unless otherwise stated or otherwise evident from the context (unless such number exceeds 100% of the possible values). For example, when used in the context of an amount of a given compound in a lipid component of an LNP, "about" can mean + / - 5% of the stated value. For example, an LNP comprising a lipid component with about 40% of a given compound can include 30-50% of the compound.

[0031] comprising: Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise," and variations such as "comprises" and "comprising," will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. As used herein, the term "comprising" can be replaced by the term "containing" or "including" or, at times as used herein, by the term "having."

[0032] conjugated: As used herein, the term "conjugated," when used in reference to two or more moieties, means that the moieties are physically bound or linked to one another, either directly or via one or more additional moieties that serve as a linker, to form a structure that is sufficiently stable such that the moieties remain physically bound under the conditions in which the structure is used (e.g., physiological conditions). In some embodiments, two or more moieties can be conjugated by direct covalent chemical bonding. In other embodiments, two or more moieties can be conjugated by ionic bonding or hydrogen bonding.

[0033] contacting:As used herein, the term“contacting” means establishing a physical connection between two or more entities. For example, contacting a cell with an mRNA or a lipid nanoparticle composition means that the cell shares a physical connection with the mRNA or the lipid nanoparticle. Methods of contacting a cell with an external entity in vivo, in vitro, and ex vivo are well known in the biological arts. In exemplary embodiments of the disclosure, the step of contacting a mammalian cell with a composition (e.g., a transfection agent or a nanoparticle or a pharmaceutical composition of the disclosure) is performed in vivo. For example, contacting a lipid nanoparticle composition with a cell (e.g., a mammalian cell) that can be disposed within a living organism (e.g., a mammal) can be performed by any suitable route of administration (e.g., parenteral administration to the organism, including intravenous, intramuscular, intradermal, and subcutaneous administration). For cells that exist in vitro, the composition (e.g., a transfection agent, such as a lipid nanoparticle) and the cell can be contacted, for example, by adding the composition to the culture medium of the cell, and can involve or result in transfection. Moreover, more than one cell can be contacted with a transfection agent or a nanoparticle composition.

[0034] delivering: As used herein, the term“delivering” means providing an entity to a destination. For example, delivering a therapeutic and / or prophylactic agent to a subject can involve administering a composition comprising the therapeutic and / or prophylactic agent to the subject (e.g., by intravenous, intramuscular, intradermal, or subcutaneous routes). Administering a composition to a mammal or a mammalian cell can involve contacting one or more cells with the composition.

[0035] encapsulating: As used herein, the term“encapsulating” means enclosing, surrounding, or encompassing. In some embodiments, a compound, polynucleotide (e.g., mRNA), or other composition can be completely encapsulated, partially encapsulated, or substantially encapsulated. For example, in some embodiments, an mRNA of the disclosure can be encapsulated in a lipid nanoparticle, such as a liposome.

[0036] effective amount:An“effective amount” of an agent is an amount that is sufficient to achieve a beneficial or desired result, e.g., a clinical result, and as such, an“effective amount” depends on the context in which it is being applied. For example, in the context of an amount of a target cell delivery-enhancing lipid in a lipid composition (e.g., an LNP) of the disclosure to deliver to a target cell, an effective amount of a target cell delivery-enhancing lipid is an amount that is sufficient to achieve a beneficial or desired result as compared to a lipid composition (e.g., an LNP) lacking the target cell delivery-enhancing lipid. Non-limiting examples of beneficial or desired results achieved by a lipid composition (e.g., an LNP) include increasing the percentage of transfected cells and / or increasing the expression level of a protein encoded by a nucleic acid associated / encapsulated with the lipid composition (e.g., an LNP). In the context of administering a lipid nanoparticle containing a target cell delivery-enhancing lipid such that an effective amount of the lipid nanoparticle is taken up by a target cell in a subject, an effective amount of an LNP containing a target cell delivery-enhancing lipid is an amount that is sufficient to achieve a beneficial or desired result as compared to an LNP lacking the target cell delivery-enhancing lipid. Non-limiting examples of beneficial or desired results in a subject include increasing the percentage of transfected cells, increasing the expression level of a protein encoded by a nucleic acid associated / encapsulated with an LNP containing a target cell delivery-enhancing lipid, and / or increasing the prophylactic or therapeutic effect of a nucleic acid or a protein encoded thereby associated / encapsulated with an LNP containing a target cell delivery-enhancing lipid in vivo as compared to an LNP lacking the target cell delivery-enhancing lipid. In some embodiments, a therapeutically effective amount of an LNP containing a target cell delivery-enhancing lipid is sufficient to treat, ameliorate symptoms of, diagnose, prevent, and / or delay onset of an infection, disease, disorder, and / or condition when administered to a subject suffering from or susceptible to the infection, disease, disorder, and / or condition. In another embodiment, an effective amount of a lipid nanoparticle is sufficient to result in expression of a desired protein in at least about 5%, 10%, 15%, 20%, 25%, or more of target cells. For example, an effective amount of an LNP containing a target cell delivery-enhancing lipid can be an amount that results in transfection of at least 5%, 10%, 15%, 20%, 25%, 30%, or 35% of target cells following a single intravenous injection.

[0037] expressing: As used herein, “expression” of a nucleic acid sequence refers to one or more of the following: (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 an RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein.

[0038] ex vivo: As used herein, the term“ex vivo” refers to an event that occurs outside of an organism (e.g., an animal, a plant, or a microorganism or a cell or tissue thereof). An ex vivo event can be performed in an environment that is minimally altered from the native (e.g., in vivo) environment.

[0039] fragment: As used herein, "fragment" refers to a portion. For example, a fragment of a protein can include a polypeptide obtained by digesting a full-length protein isolated from cultured cells or a polypeptide obtained by recombinant DNA technology. A fragment of a protein can be a portion of a protein that includes one or more functional domains such that the fragment of the protein retains the functional activity of the protein.

[0040] isolated: As used herein, the term "isolated" refers to a substance or entity that has been separated from at least some of the components with which it was associated (whether in nature or in the context of an experiment). Isolated substances can have varying levels of purity with respect to the substances with which they have been associated. Isolated substances and / or entities can 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 with which they were originally associated. In some embodiments, an isolated reagent is more than 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 more than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components.

[0041] Kozak sequence: The term "Kozak sequence" (also referred to as "Kozak consensus sequence") refers to a translational initiation enhancer element to enhance expression of a gene or open reading frame, and in eukaryotes, the element is positioned in the 5' UTR. The Kozak consensus sequence was originally defined as the sequence GCCRCC, where R = purine, following analysis of the effects of single mutations around the start codon (AUG) on preproinsulin gene translation (Kozak (1986) Cell 44:283-292). The polynucleotides disclosed herein comprise a Kozak consensus sequence, or a derivative or modification thereof. (See, U.S. Patent No. 5,807,707 to Andrews et al., incorporated by reference in its entirety herein; U.S. Patent No. 5,723,332 to Chernajovsky, incorporated by reference in its entirety herein; U.S. Patent No. 5,891,665 to Wilson, incorporated by reference in its entirety herein, for examples of translational enhancer compositions and methods of use thereof.) transferring: As used herein, the term "metastasis" means the process by which a cancer spreads from the location at which it first appears as a primary tumor to a distant location in the body. The secondary tumors that arise as a result of this process can be referred to as "metastases."

[0042] modified:As used herein, “modified” or “modification” refers to a change or alteration in the composition or structure of a molecule (e.g., a polynucleotide, e.g., an mRNA) of the disclosure. A molecule (e.g., a polynucleotide) can be modified in various ways including chemically, structurally, and / or functionally. For example, a polynucleotide can be structurally modified by the incorporation of one or more RNA elements, wherein the RNA elements comprise a sequence and / or an RNA secondary structure that provides one or more functions (e.g., a translational modulatory activity). Thus, a polynucleotide of the disclosure can be composed of one or more modifications (e.g., can include one or more chemical modifications, structural modifications, or functional modifications, including any combination thereof). In one embodiment, an mRNA molecule of the disclosure is modified by the introduction of a non-natural nucleoside and / or nucleotide, e.g., when it refers to the natural ribonucleotides A, U, G, and C. Although non-canonical nucleotides such as cap structures differ in chemical structure from A, C, G, U ribonucleotides, they are not considered “modified.”

[0043] mRNA: As used herein, “mRNA” refers to messenger ribonucleic acid. An mRNA can be naturally occurring or non-naturally occurring. For example, an mRNA can include modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, or linkers. An mRNA can include a cap structure, a chain-terminating nucleoside, a stem loop, a poly-A sequence, and / or a polyadenylation signal. An mRNA can have a nucleotide sequence that encodes a polypeptide. An mRNA can also have a nucleotide sequence that encodes multiple (e.g., at least two or at least three) different polypeptides. Translation of an mRNA, e.g., in vivo translation of an mRNA within a mammalian cell, can result in a polypeptide. 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 (particularly in the tables outlined below), the symbol “T” in the mRNA sequences disclosed herein is interpreted to mean uracil (or a modified uracil such as N1-methyl pseudouridine, as indicated) in the mRNA.

[0044] nanoparticle:As used herein, "nanoparticle" refers to a particle having any one structural feature on a scale of less than about 1000 nm that exhibits novel properties compared to bulk samples of the same material. Conventionally, a nanoparticle has any one structural feature on a scale of less than about 500 nm, less than about 200 nm, or about l00 nm. Additionally, conventionally, a nanoparticle has 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, a nanoparticle is a particle having one or more dimensions on the order of about l-l000 nm. In other exemplary embodiments, a nanoparticle is a particle having one or more dimensions on the order of about l0-500 nm. In other exemplary embodiments, a nanoparticle is a particle having one or more dimensions on the order of about 50-200 nm. For example, a spherical nanoparticle has a diameter of, e.g., about 50-100 or 70-120 nanometers. Nanoparticles most often behave as units in their transport and properties. It should be noted that the novel properties that distinguish a nanoparticle from a corresponding bulk material typically develop at size scales less than l000 nm or at sizes of about 100 nm, but a nanoparticle can have larger sizes, e.g., for particles that are elliptical, tubular, etc. While the size of most molecules fits the above generalization, various molecules are not generally referred to as nanoparticles.

[0045] nucleic acid: As used herein, the term "nucleic acid" is used in its broadest context and encompasses any compound and / or substance comprising a polymer of nucleotides, unless otherwise indicated. These polymers are often referred to as polynucleotides. Also included are chemical modifications of nucleic acids at the nucleotide bases, sugars, or phosphates, as well as nucleic acids containing non-natural nucleotides and nucleotide analogs. Exemplary nucleic acids or polynucleotides of the present disclosure include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), DNA-RNA hybrids, RNAi inducers, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNAs, RNAs that induce triple helix formation, threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNAs with beta-D-ribo configuration, a-LNA (diastereomer of LNA) with a-L-ribo configuration, 2'-amino-LNA with 2'-amino functionalization, and 2'-amino-a-LNA with 2'-amino functionalization), or hybrids thereof.

[0046] nucleobase:As used herein, the term "nucleobase" (alternatively "nucleotide base" or "nitrogenous base") refers to a purine or pyrimidine heterocyclic compound found in nucleic acids, including any derivative or analog of a naturally occurring purine and pyrimidine that imparts improved properties (e.g., binding affinity, nuclease resistance, chemical stability) to a nucleic acid or a portion or segment thereof. Adenine, cytosine, guanine, thymine, and uracil are the nucleobases predominantly found in natural nucleic acids. Other natural, non-natural, and / or synthetic nucleobases can also be incorporated within a nucleic acid, as known in the art and / or described herein.

[0047] 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, covalently linked to a nucleobase (e.g., purine or pyrimidine), or a derivative or analog thereof (also referred to herein as a "nucleobase"), but lacking an internucleoside linking group (e.g., a phosphate group). As used herein, the term "nucleotide" refers to a nucleoside, or any derivative, analog, or modification thereof, covalently bonded to an internucleoside linking group (e.g., a phosphate group), that imparts improved chemical and / or functional properties (e.g., binding affinity, nuclease resistance, chemical stability) to a nucleic acid or a portion or segment thereof.

[0048] open reading frame: As used herein, the term "open reading frame," abbreviated "ORF," refers to a segment or region in an mRNA molecule that encodes a polypeptide. An ORF comprises a contiguous stretch of non-overlapping, in-frame codons starting with an initiation codon and ending with a termination codon, and is translated by a ribosome.

[0049] patient: As used herein, the term "patient" refers to a subject who can seek or need treatment, who is under treatment, who will undergo treatment, or who is under the care of a trained professional for a particular disease or condition. In particular embodiments, the patient is a human patient. In some embodiments, the patient is a patient having an autoimmune disease, e.g., as described herein.

[0050] pharmaceutically acceptable: The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0051] pharmaceutically acceptable excipient:As used herein, the phrase "pharmaceutically acceptable excipient" refers to any ingredient other than the compounds described herein (e.g., a vehicle able to suspend or dissolve the active compound) and having the properties of being substantially non-toxic and non- inflammatory in a patient. Excipients can include, for example: antiadherents, antioxidants, binders, coatings, tableting aids, disintegrants, dyes (colorants), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow promoters), lubricants, preservatives, printing inks, sorbents, suspending or dispersing agents, sweeteners, and waters of hydration. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, crosscarmellose, crosslinked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium carboxymethyl starch, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0052] pharmaceutically acceptable salt:As used herein, "pharmaceutically acceptable salt" refers to a derivative of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety into its salt form (e.g., 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 acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Representative acid addition salts include acetate, acetic acid, adipate, alginate, ascorbate, aspartate, besylate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Pharmaceutically acceptable salts of the present disclosure include the conventional non-toxic salts of the parent compounds formed, for example, from non-toxic inorganic or organic acids. As used herein, the term "pharmaceutically acceptable salt" of a compound means a salt that is not biologically or otherwise undesirable. Pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in, inter alia, Remington's Pharmaceutical Sciences, 17th Ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P.H. Stahl and C.G. 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 its entirety.

[0053] polyadenylate / poly-A tail / sequence: "Polyadenosine sequence," "poly A sequence," or "poly A tail" refers to a sequence of adenosine residues that an RNA molecule typically has positioned at the 3' end. Such a sequence can be attached during RNA transcription. Poly A sequences are typically attached to the free 3' end of an RNA after nuclear transcription by a template-independent RNA polymerase. Artificially, poly A can be attached by transcription from a DNA template containing complementary repeated thymidine residues. Alternatively, an mRNA as described herein can comprise a polyadenylation signal, which is defined herein as a signal that transmits polyadenylation to (transcribed) RNA by specific protein factors. Poly A sequences are important for nuclear export, translation, and stability of an mRNA, and are shortened over time and eventually lead to enzymatic degradation of the mRNA.

[0054] polypeptide: As used herein, the term "polypeptide" or "polypeptide of interest" refers to a polymer of amino acid residues typically linked by peptide bonds, which can be naturally occurring (e.g., isolated or purified) or synthetically produced.

[0055] prevent / preventing: As used herein, "prevent" or "preventing" refers to the partial or complete inhibition of the onset of one or more symptoms or features of a particular infection, disease, disorder, and / or condition.

[0056] prophylactic: As used herein, the term "preventive" refers to the partial or complete inhibition of the onset of one or more symptoms or features of a particular infection, disease, disorder, and / or condition. "Preventive" is also used in this sense.

[0057] RNA: As used herein, "RNA" refers to a ribonucleic acid, which can be naturally occurring or non-naturally occurring. For example, an RNA can include modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, or linkers. An RNA can include a cap structure, a chain-terminating nucleoside, a stem loop, a poly A sequence, and / or a polyadenylation signal. An RNA can have a nucleotide sequence that encodes a polypeptide of interest. For example, an RNA can be a messenger RNA (mRNA). Translation of an mRNA that encodes a particular polypeptide, such as in vivo translation of an mRNA within a mammalian cell, can produce the encoded polypeptide.

[0058] RNA element: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 a biological activity (e.g., a translational regulatory activity). By modifying a polynucleotide with the introduction of one or more RNA elements (such as those described herein), the modified polynucleotide is provided with one or more desirable functional properties. As described herein, an RNA element can be naturally occurring, non-naturally occurring, synthetic, engineered, 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, particularly eukaryotic mRNA and translated viral RNA, have been shown to be involved in mediating a variety of functions in cells. Exemplary natural RNA elements include, but are not limited to, translational initiation elements (e.g., internal ribosome entry sites (IRES), see Kieft et al. (2001) RNA 7(2): 194-206), translational enhancer elements (e.g., the APP mRNA translational enhancer element, 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 repression elements (see, 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 425(18):3301-3310), cytoplasmic polyadenylation elements (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).

[0059] sequence: As used herein, the term "sequence" shall be generally understood to include both the relevant amino acid sequence as well as the nucleic acid sequence or nucleotide sequence encoding the same, unless a more limited interpretation is required by the context. Depending on the context, an amino acid sequence is interpreted to mean either a single amino acid or an unbranched sequence of two or more amino acids. A nucleotide sequence is interpreted to mean an unbranched sequence of three or more nucleotides.

[0060] specifically delivering:As used herein, the term“specific delivery,”“specifically deliver,” or“specifically delivering” means that a nanoparticle delivers more (e.g., at least 10% more, at least 20% more, at least 30% more, at least 40% more, at least 50% more, at least 1.5-fold more, at least 2-fold more, at least 3-fold more, at least 4-fold more, at least 5-fold more, at least 6-fold more, at least 7-fold more, at least 8-fold more, at least 9-fold more, at least 10-fold more) of a therapeutic and / or prophylactic agent to a target cell of interest (e.g., a mammalian target cell) as compared to a non-target cell (e.g., a non-target cell). The level of delivery of a nanoparticle to a particular cell can be measured by comparing the amount of protein produced in a target cell relative to a non-target cell (e.g., by mean fluorescence intensity using flow cytometry), comparing the fraction (%) of target cells expressing a protein relative to non-target cells (e.g., by quantitative flow cytometry), comparing the amount of protein produced in a target cell relative to a non-target cell to the amount of total protein in the target cell relative to the non-target cell, or comparing the amount of therapeutic and / or prophylactic agent in a target cell relative to a non-target cell to the amount of total therapeutic and / or prophylactic agent in the target cell relative to the non-target cell. It will be appreciated that the ability of a nanoparticle to specifically deliver to a target cell need not be determined in a subject receiving treatment, it can be determined in a surrogate, e.g., an animal model (e.g., a mouse or NHP model).

[0061] substantially: As used herein, the term“substantially” refers to a qualitative term used to describe an overall or relative degree or range of a property or characteristic of interest. Those skilled in the biological arts understand that biological and chemical phenomena rarely, if ever, achieve absolute or complete or realized or avoided outcomes. The term“substantially” is therefore used herein to capture the potential lack of complete in many biological and chemical phenomena.

[0062] suffering from: An individual“having” a disease, disorder, and / or condition has been diagnosed with or displays one or more symptoms of the disease, disorder, and / or condition.

[0063] therapeutic agent: The term“therapeutic agent” refers to any agent that has a therapeutic, diagnostic, and / or prophylactic effect, and / or elicits a desired biological and / or pharmacological effect when administered to a subject.

[0064] transfecting: As used herein, the term“transfection” refers to a method of introducing a substance (e.g., a polynucleotide, such as mRNA) into a cell.

[0065] subject: As used herein, the term "subject" refers to any organism to which a composition according to the present disclosure can be administered, e.g., for experimental, diagnostic, prophylactic, 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, a subject can be a patient.

[0066] treatment: As used herein, the term "treat" refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of, reducing severity of, and / or reducing incidence of one or more symptoms or features of a particular infection, disease, disorder, and / or condition. For example, "treating" a cancer can refer to inhibiting survival, growth, and / or spread of a tumor. Treatment can be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition, and / or to a subject who exhibits only early signs of a disease, disorder, and / or condition, for the purpose of decreasing risk of developing a pathological condition associated with the disease, disorder, and / or condition.

[0067] unmodified: As used herein, "unmodified" refers to any substance, compound, or molecule prior to any change. Unmodified can, but does not necessarily, refer to the wild-type or native form of a biomolecule. A molecule can undergo a series of modifications, whereby each modified molecule can serve as the "unmodified" starting molecule for subsequent modifications.

[0068] variant: As used herein, the term "variant" refers to a molecule having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity of a wild-type molecule, e.g., as measured by an art-recognized assay.

[0069] wild type / WT: "Wild-type" (or "WT") refers to the natural form of a genotype or phenotype of a feature or sequence, i.e., the original form of a feature or unmutated form of a sequence. In particular, when referred to in the context of an immunosuppressive domain (ISD), such wild-type ISD is active, i.e., it suppresses an immune response. Activity or inactivity of an ISD can be determined as described elsewhere herein.

[0070] percent identity or % identity:This term refers to the percentage of identical nucleotides or amino acids in the optimal alignment between two nucleotide sequences or amino acid sequences to be compared. The comparison of two sequences generally requires the step of optimal alignment, which can be performed manually or with the aid of: by the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, and by the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85, 2444, or using computer programs of said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). One or more local regions of correspondence in the two sequences are then identified with each other. The percentage identity of the entire sequence is then calculated by determining the number of identical positions common to both sequences, dividing by the length of the reference sequence. The result is then multiplied by 100. In other words, the 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 over the entire length of the reference sequence. The program "BLAST 2 sequences" is an exemplary tool available on the website http: / / www.ncbi.nlm.nih.gov / blast / bl2seq / wblast2.cgi to perform such a calculation.

[0071] 5' / 3' untranslated region / UTR: 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. In graphical representations of double-stranded nucleic acids, the 3' end is on the right and the 5' end is on the left hand side: 5' end 5' - P - NNNNNNN - OH - 3' 3' end 3' - HO - NNNNNNN - P - 5' As used herein, "untranslated region" or "UTR"either of the two segments, one on each side of the coding sequence of the mRNA, which are not part of the protein coding region. On the 5' end, the UTR is called 5' UTR (or leader sequence), on the 3' side, the UTR is called 3' UTR (or trailer sequence). UTRs can contain RNA elements that modulate translation and / or transcription, as described elsewhere herein. For example, the 5' UTR facilitates the initiation of translation by allowing the ribosome to bind to the sequence, while the 3' UTR is known to be involved in, for example, translation termination as well as post-transcriptional modification.

[0072] LNP comprising a HERV envelope protein In a first aspect, the present application relates to a composition comprising a transfection agent and an mRNA encoding at least one human endogenous retrovirus (HERV) envelope protein or an immunogenic part thereof; wherein the HERV envelope protein comprises an immunosuppressive domain (ISD); wherein the HERV Env protein comprises a mutated immunosuppressive domain (ISD) which reduces its immunosuppressive properties compared to the wild type ISD.

[0073] In one embodiment, the present application can relate to a composition of the present application or a pharmaceutically acceptable salt thereof.

[0074] Transfection agent The transfection agent of the composition can for example be any compound, formulation or mixture that enhances the transport or uptake of nucleic acids into cells, including cells of different tissues. When applied in an effective amount as defined elsewhere herein, these agents can increase the amount of nucleic acid uptake. Such effects are caused by one or more substances comprised by the transfection agent that facilitate said uptake. The protein or peptide encoded by the introduced nucleic acid can then modulate, induce or integrate into the cellular processes of the target cell.

[0075] The transfection agent composition can be composed adjusted, for example, to the targeted cell type and / or to the substance to be delivered, and depending on other parameters such as the delivery environment, i.e. in vivo or in vitro. Suitable transfection agents comprise a range of different uptake-promoting substances selected from the non-limiting group consisting of calcium phosphate; cationic polymers such as DEAE-dextran or polyethylenimine (PEI); liposome-forming substances or mixtures thereof such as cationic lipids like 2,3-dioleoyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1 -propaniminium trifluoroacetate (DOSPA), dioleoyl-3-trimethylammonium propane (DOTMA) or dioleoyloxypropyl-trimethylammonium (DOTAP), and / or helper lipids like dioleoylphosphatidylethanolamine (DOPE), cholesterol and polyethylene glycol (PEG) lipids; non-liposomal agents such as FuGENE® commercially available from Promega; and dendrimers. Further preferred are a broad range of commercially available Lipofectamine mixtures (e.g. from ThermoFisher Scientific).

[0076] Thus, in one embodiment, the transfection agent is a transfection agent comprising a cationic lipid and / or a cationic polymer.

[0077] In a preferred embodiment, the composition comprises a liposome comprising a cationic lipid and the mRNA of the application.

[0078] In a more preferred embodiment, the transfection agent comprises DOSPA (2,3-dioleoyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1 -propaniminium trifluoroacetate) and / or DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine). In a most preferred embodiment, the transfection agent comprises DOSPA (2,3-dioleoyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1 -propaniminium trifluoroacetate) and DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) in a preferred molar ratio of 3:1.

[0079] Lipid nanoparticles (LNP) The transfection agent can be a composition particularly suitable for applying a nucleic acid encoding an antigen, such as an mRNA, to a subject. Such suitable compositions are known in the art. Such suitable compositions can be lipid compositions with in vivo beneficial properties, such as lipid nanoparticle (LNP) compositions. LNPs increase the circulation time in vivo and are effective in helping to deliver nucleotide sequences encoding an antigen, such as an mRNA, to the target site and have thus emerged as suitable non-viral encapsulation delivery vehicles for exogenous mRNA.

[0080] Thus, in one embodiment, the composition comprises a lipid nanoparticle (LNP) comprising the mRNA of the application.

[0081] LNP include various lipid-based platforms, such as liposomes, nanostructured lipid carriers (NLCs), and solid lipid nanoparticles (SLNs). Thus, in one embodiment, the LNP composition is selected from the group consisting of a liposome composition, a nanostructured lipid carrier (NLC) composition, and a solid lipid nanoparticle (SLN) composition.

[0082] Compositions comprising mRNA and a transfection agent as described herein can benefit from the use of LNP as the transfection agent, as it has been observed that formulation in LNP can reduce adverse responses. In preferred embodiments, LNP comprising lipids known to exhibit reduced Toll-like receptor (TLR) agonism are used. It has been observed that reduced activation of TLR signaling plays a key role in triggering RNA vaccine-related innate signaling, and that the triggering effect is believed 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)). Without wishing to be bound by theory, it is predicted that the use of such lipids will increase immunogenicity, resulting in a robust innate immune response to mRNA-encoded antigens. Preferred examples include LNP comprising ionizable lipids. Further preferred examples include LNP compositions comprising the following lipids: DLin-MC3-DMA (MC3), 9-heptadecenyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2- hexyldecanoate) (ALC-0315), Hasset et al. (2019) (https: / / doi.org / 10.1016 / j.jconrel.2019.01.030). Optimization of Lipid Nanoparticles for Intramuscular Administration of mRNA Vaccines.Any one of lipids H, M, P, Q, and N in Mol Ther Nucleic Acids. 2019 Apr 15; 15: 1-11), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), and 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), and DOTMA and DOPE at a ratio of 1:1. Another preferred example of an LNP composition comprises at least one lipid selected from the group consisting of (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 2-[(polyethylene glycol)-2000]-N,N-tetracosanamide (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 at a ratio of 46.3 : 9.4 : 42.7 : 1.6 ALC-0315 : ALC-0159 : DSPC : cholesterol.

[0083] Further aspects (i), (ii), (iii), and (iv) of the present application provide for the following: (i) an LNP comprising an mRNA of the present application; (ii) a liposome comprising an mRNA of the present application; (iii) a polyplex comprising an mRNA of the present application; (iv) a lipoplex comprising an mRNA of the present application.

[0084] Yet a further aspect of the present application relates to a virus-like particle (VLP) comprising a HERV envelope protein as defined according to the present application as described herein. Preferably, the envelope protein comprises a mutated 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 wherein the mutated ISD comprises a sequence according to any one of SEQ ID NOs: 4 or 8-20 (most preferably the mutated ISD comprises or consists of the sequence LANAINDLRQTVIW (SEQ ID NO: 4)), and wherein the HERV envelope protein further comprises the amino acid sequence: At least one amino acid at a position in the range of 170 to 210 has been replaced by a cysteine; or The HERV envelope protein contains a cysteine ​​residue at position 190, and more preferably, the HERV envelope protein contains an 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, wherein the HERV envelope protein contains a cysteine ​​residue at the position corresponding to amino acid 190 of SEQ ID NO: 2 within the HERV envelope protein.

[0085] Lipid content used for encapsulation RNA encapsulation is achieved by combining RNA with lipids at an acidic pH, where the ionizable lipids are positively charged, thus ensuring charge-driven interactions with the negatively charged nucleic acids. The pH is then adjusted above the pKa of the ionizable lipids, resulting in a near-neutral surface charge suitable for clinical administration. A variety of suitable components for LNP compositions for delivering nucleic acids such as mRNA are known in the art and will be apparent to those skilled in the art. Further general information and numerous examples of lipid nanoparticle formulations are available in the art, for example in publications 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 The embodiments described below are provided in InAntisense Drug Technology (255-288). Therefore, the embodiments described below are merely examples and are not intended to be limiting.

[0086] In one embodiment, the LNP composition can comprise one or more ionizable lipids. The one or more ionizable lipids can preferably comprise a cationic lipid. The term "cationic" means that the respective structure carries a permanent or non-permanent (but in response to certain conditions such as pH) positive charge, unless a different meaning is clear from the specific context. Thus, the term "cationic" covers both "permanent cationic" and "cationizable". Suitable cationic lipids for the compositions of the present application are known in the art and can for example advantageously be selected from the non-limiting group consisting of N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N-(N',N' dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1-(2,3-dioleoyloxy)propyl) N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), octadecylamidoglycylcarboxyspermine (DOGS), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE). Additionally, suitable cationic lipids for the compositions of the present application can for example be selected from the non-limiting group of commercial formulations of cationic lipids consisting of LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and 1,2-dioleoyl-sn-3 phosphoethanolamine (DOPE), from GIBCO / BRL, Grand Island, N.Y.); LIPOFECTAMINE® (commercially available cationic liposomes comprising N-(1-(2,3 dioleoyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and DOPE, from GIBCO / BRL); and TRANSFECTAM® (commercially available cationic liposomes comprising an ethanolic solution of octadecylamidoglycylcarboxyspermine (DOGS), from Promega Corp., Madison, Wis.).

[0087] The ionizable lipids of the present disclosure can comprise a central amine moiety and at least one biodegradable group. Thus, in an even more preferred embodiment, the ionizable lipid can be an ionizable cationic amino lipid.

[0088] In some embodiments, the lipid-based compositions described herein (e.g., LNPs) comprise one or more non-cationic helper lipids. As used herein, the term “non-cationic helper lipid” refers to a lipid comprising at least one fatty acid chain of at least 8 carbons in length and at least one polar head group moiety. In some embodiments, the non-cationic helper lipid can be a phospholipid. Generally, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties. For example, the phospholipid moiety can be, for example, selected from the non-limiting group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lyso-phosphatidylcholine, and sphingomyelin. The fatty acid moieties can be, for example, selected from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, a-linolenic acid, erucic acid, phytanic acid, eicosanoic acid, arachidonic 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 phosphosphingolipids, such as sphingomyelin.

[0089] In a preferred embodiment, the helper lipid or phospholipid can be neutral. For example, the LNP composition can further comprise 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- sn-glycero-3-phosphocholine (DSPC) substitute. In an even more preferred embodiment, the helper lipid or phospholipid of the LNP composition can be DSPC.

[0090] In some embodiments, the lipid-based compositions (e.g., LNPs) can comprise one or more structural lipids. In a preferred embodiment, the structural lipid can be a sterol. Such sterols can include cholesterol, beta-sitosterol, dihydrobrassicasterol, campesterol, stigmasterol, and brassicasterol. In an even more preferred embodiment, the sterol can preferably be cholesterol.

[0091] In some embodiments, the LNP composition can comprise another lipid, which is a phospholipid substitute or replacement. The phospholipid or phospholipid substitute or replacement may, for example, be one or more saturated or (poly)unsaturated phospholipids, or a phospholipid substitute, or a combination thereof. In a preferred embodiment, such phospholipid substitute or replacement can be a pegylated or PEG lipid. Incorporation of pegylated lipids results in steric stabilization of, for example, the nanoparticle core-shell. Non-limiting examples of PEG-lipids include PEG-modified phosphatidylethanolamines and phosphatidic acids, 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 referred to as pegylated lipids. In some embodiments, the PEG-lipid includes, but is not limited to, PEG-distearyl glycerol (PEG-DSG), or PEG-1,2-dimyristyl- oxypropyl-3-amine (PEG-c-DMA), 1,2-dimyristyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), PEG-dipalmetoleyl, PEG-dioleoyl, PEG-distearyl, PEG-diglycamide (PEG-DAG), PEG-DLPE, PEG-DMPE, PEG-dipalmitoyl phosphatidyl ethanolamine or phosphatidyl choline (PEG-DPPE, PEG-DPPC), 1,2-distearyl-sn-glycero-3-phosphoethanolamine-N- aminopolyethylene glycol (PEG-DSPE) lipids. In a preferred embodiment, the LNP composition can comprise PEG-DMG.

[0092] In one embodiment, the LNP composition comprises at least one lipid selected from the group consisting of: (i) an ionizable lipid, preferably an ionizable cationic lipid, more preferably an ionizable cationic amino lipid; (ii) a non-cationic helper lipid or a phospholipid, wherein the lipid is preferably neutral, more preferably wherein the lipid is DSPC; (iii) a sterol or other structural lipid, wherein the sterol is preferably cholesterol; and (iv) a PEG lipid, preferably PEG-DMG.

[0093] The lipid components of the LNP composition can be used in suitable molar ratios with respect to the other lipids. The amount of ionizable lipid, preferably cationic lipid, preferably cationic amino lipid may, for example, range from about 45 mole % to about 50 mole %. The amount of non-cationic helper lipid or phospholipid, preferably neutral lipid, preferably DSPC may, for example, range from about 5 mole % to about 15 mole %. The amount of structural lipid, for example sterol, preferably cholesterol may, for example, range from about 30 mole % to about 45 mole %. The amount of PEG-lipid in the lipid composition of the pharmaceutical compositions disclosed herein may, for example, range from about 0.1 mole % to about 5 mole %.

[0094] Thus, in one embodiment of the composition of the application, wherein the transfection agent is a lipid nanoparticle (LNP) composition, the LNP composition comprises a molar ratio of about 45 mole % to about 50 mole % of an ionizable lipid, about 5 mole % to about 15 mole % of a phospholipid, about 30 mole % to about 45 mole % of a sterol, and about 1 mole % to about 5 mole % of a PEG lipid. In a preferred embodiment, the LNP composition can for example comprise a molar ratio of 50 mole % of an ionizable lipid, 10 mole % of a phospholipid, 38.5 mole % of a sterol and 1.5 mole % of a PEG lipid. In an alternative preferred embodiment, the LNP can for example comprise a molar ratio of 46.3 mole % of an ionizable lipid, 9.4 mole % of a phospholipid, 42.7 mole % of a sterol and 1.6 mole % of a PEG lipid.

[0095] In another embodiment, the lipid nanoparticle composition can comprise a targeting moiety, which is a compound or agent that can target the nanoparticle to a specific cell, tissue and / or organ type. Thus, in one embodiment, the lipid nanoparticle composition comprising a targeting moiety has the ability to specifically deliver to a specific target cell, tissue and / or organ type.

[0096] It can be desirable or useful to further increase the efficacy or potency of the composition of the application, in particular for example when applying the composition comprising the RNA of the application to a subject or patient for prophylaxis or treatment. Thus, in one embodiment, the composition of the application further comprises an adjuvant. In a preferred embodiment, the adjuvant can be a cytokine, and more preferably a cytokine selected from the group consisting of INFy, IL-2, IL-12, GM-CSF, IL-15 and IL-7. The adjuvant can also be introduced as a polynucleotide configured to express one or more of the above-mentioned cytokines in a eukaryotic cell.

[0097] The adjuvant can act through a combination of various mechanisms to elicit and boost the immune response, including one or more of the following: sustained release of antigen at the site of injection (depot effect), upregulation of (further) cytokines and chemokines, cell recruitment at the site of administration of the composition, increased antigen uptake and presentation to antigen presenting cells (APCs), activation and maturation of APCs, increased expression of major histocompatibility complex (MHC) class II and costimulatory molecules, promotion of antigen transport to draining lymph nodes, and activation of inflammasomes.

[0098] mRNA characteristics In one embodiment, the mRNA comprised by the composition of the application comprises at least 300 nucleotides, such as preferably at least 400, 500, 800, 1000, 1500, 2000, more preferably at least 3000 nucleotides, or most preferably at least 4000 nucleotides.

[0099] Modifications of mRNA elements, such as the 5' cap, the 5' and 3' untranslated regions (UTRs), the coding region, and the poly-A tail, help to reduce excessive mRNA immunogenicity and / or improve mRNA stability and translation efficiency. Thus, the mRNA comprised by the composition of the application can have certain functional sequence features, which optimize its properties with respect to stability, expression efficiency, and tolerability in a patient.

[0100] The mRNA molecule can comprise an oligo- or poly-A sequence, i.e. a poly-A tail, elongated 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 is shortened, eventually leading to the initiation of enzymatic degradation of the mRNA. Elongating the poly-A can thus provide additional stability. Thus, in one embodiment, the composition comprises an mRNA comprising at least 60 adenosine nucleotides at the 3'-UTR. In a preferred embodiment, the mRNA comprises at least 100, more preferably 120 adenosine nucleotides at the 3'-UTR.

[0101] According to a further embodiment, the mRNA of the application can comprise a poly(C) (poly-cysteine) tail of typically 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 the 3' end.

[0102] Codon optimization is another approach to alter synonymous codons to improve gene expression by the host organism's codon bias preference. Based on the host organism's own codon usage bias, mutations are introduced into the gene of interest to increase translation efficiency in the organism and thus protein expression without changing the sequence of the protein. Thus, in another embodiment, the 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 application can also be codon optimized for expression in other animals, such as for example other mammals.

[0103] In some cases, modified nucleobases in nucleic acids are introduced into a nucleic acid sequence (e.g., an RNA nucleic acid, such as an mRNA nucleic acid) to improve stability. In one embodiment, the mRNA of the application can comprise at least one artificially modified nucleotide. In one embodiment, the mRNA of the application does not comprise artificially modified nucleotides.

[0104] In one embodiment, the mRNA of the application can be modified by altering the guanosine / cytosine (G / C) content of the mRNA sequence, with a modified and thus stabilized, in particular increased, G / C content compared to the G / C content of the coding region of the respective wild-type mRNA (i.e. unmodified mRNA). According to one specific embodiment, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, more preferably at least 70%, even more preferably at least 80% and most preferably at least 90%, 95% or even 100% of the substitutable codons in the coding region of the mRNA of the application or the entire sequence of the wild-type mRNA sequence can be substituted, thereby increasing the GC content of said sequence.

[0105] In one embodiment, the mRNA of the application can be modified by modifying, preferably increasing, the cytosine (C) content of the mRNA sequence, preferably of the coding region of the mRNA. Preferably, the mRNA sequence can be modified such that at least 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80%, or at least 90% of the theoretically possible maximum cytosine content or even the maximum cytosine content is reached.

[0106] In some embodiments, the mRNA of the application may, for example and without limitation, comprise at least one sequence element selected from the group consisting of: a CAP mimic structure; a suitable promoter or subgenomic promoter that generates a high translation rate; a self-amplifying mRNA feature (e.g. the mRNA of the application can be a self-replicating mRNA), such as for example a cytomegalovirus promoter, a T7 promoter or a subgenomic SFV promoter; a Kozak consensus sequence (5'-CCACCATGG-3'); a spacer of 3 to 6 nucleotides between the (T7) promoter sequence and the Kozak sequence, if present; a stabilizing and / or structural sequence element in the UTR sequence. Suitable RNA elements are described in the art and will be apparent to the person of ordinary skill in the relevant art.

[0107] For example, suitable CAP mimicking structures with respect to the mRNA of the present application can be selected from the non-limiting group consisting of: Vaccinia virus 2'-O-methyltransferase Cap 1, ARCA anti-reverse CAP analog or beta-S-ARCA cap, modified ARCA (e.g. phosphorothioate modified ARCA); m7GpppN, cap1 (methylation of ribose of adjacent nucleotides of m7G), cap2 (additional methylation of ribose of second nucleotide downstream of m7G), cap3 (additional methylation of ribose of third nucleotide downstream of m7G), cap4 (methylation of ribose of fourth nucleotide downstream of m7G), inosine, N1-methylguanosine, 2'-fluoroguanosine, 7-deazaguanosine, 8-oxoguanosine, 2-aminoguanosine, LNA-guanosine, and 2-azidoguanosine.

[0108] For example, suitable stabilizing and / or structural sequence elements in the UTR sequence of the mRNA of the present application can be based on variants of the UTR sequence of a gene, such as variants of the UTR of the albumin gene, the a-globin gene, the b-globin gene, the tyrosine hydroxylase gene, the lipoxygenase gene or the collagen a gene, such as the collagen a1 gene or parts thereof. Thus, in some preferred embodiments, the mRNA of the present application can comprise a 5'UTR HBA1, a 5'UTR SFV, or a 5'UTR 7, a 3'UTR HBB; and / or a 3'UTR AES mtRNR1. In another embodiment, the mRNA 5'-UTR can comprise or consist 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.

[0109] The mRNA of the present application encoding a HERV envelope protein or an immunogenic part thereof can also encode said protein as part of a virus-like particle (VLP). VLPs are molecules that closely resemble viruses composed of one or more different molecules, with the ability to self-assemble and mimic the release, form and size of a viral particle, but lack the genetic material to infect a host cell. In the context of the present application, they can be formed by encoding a viral Gag protein together with a 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, incorporating the HERV Env on the surface of the VLP.

[0110] Thus, in one embodiment, the composition of the present application comprises an mRNA encoding at least one HERV envelope protein or an immunogenic part thereof having the mutated ISD of the present application and a gag protein. In a preferred embodiment, the gag protein can be selected from the same or a different virus as the Env protein.

[0111] Further applicable and potentially beneficial features of the VLP to be encoded by the mRNA will be clear to the person skilled in the relevant art. For example, in one embodiment, the mRNA of the present application can encode at least one human endogenous retrovirus (HERV) envelope protein or an immunogenic portion thereof with a mutated ISD, a gag protein and a 2A peptide. Furthermore, the Env protein may, for example, be encoded to comprise a surface unit (SU, also referred to as gp70), a cleavage site and / or a transmembrane unit (TM, also referred to as p15E). In addition, the transmembrane unit (TM, also referred to as p15E) may, for example, comprise a fusion peptide, a transmembrane anchor and / or a cytoplasmic tail.

[0112] In a most preferred embodiment, the mRNA or mRNA construct of the present application encoding at least one HERV envelope protein or an immunogenic portion thereof, can also encode a Gag protein and can comprise a 5’UTR HBA, a 3’UTR HBB and a polyA comprising 70 adenine nucleotides. Thus, the mRNA of the present application can have the following 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 application encoding at least one HERV envelope protein or an immunogenic portion thereof, can also encode a Gag protein and can comprise a 5’UTR of HBA1, a 3’UTR of AES mtRNR1 and a polyA comprising 100 adenine nucleotides, wherein the 30thand 70thadenine nucleotides are preferably interrupted by a linker (30-linker-70). Thus, the mRNA of the present application can have the following structure: 5’UTR of HBA1- HERV-K Gag Env ISDmut (preferably with ISD mutation Q525A) - 3’UTR of AES mtRNR1 - polyA (preferably poly(A) is A30-linker-A70).

[0113] In one aspect, the present application relates to a DNA molecule encoding the mRNA comprised in the composition of the present application.

[0114] HERV envelope protein The present invention provides a platform for the display of antigens to the immune system of a body. Thus, in principle, the coding of any type of protein against which an immune response is desired can be incorporated into the mRNA construct. In one aspect of the invention, the mRNA encoded protein is an endogenous retroviral envelope protein (ERV Env) or an immunogenic protein derived from such a protein. It is believed that the vaccine directs the ERV Env to dendritic cells (DCs) which 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 for the antigen of the ERV Env infiltrate tumors and kill cells displaying the respective antigen. Antigen on MHC class II activates CD4+ T cells through presentation by professional antigen presenting cells (APCs) which subsequently co-activate B cells. Activated B cells that encounter the target protein ERV Env in the circulation or antigens displayed on cells or VLPs release antibodies specific for the ERV Env. These antibodies are able to bind their target on cancer cells, inducing destruction and phagocytosis of the malignant cells. In this way, ERV specific antibodies are able to prevent tumor growth and metastasis. The immunogenicity of tumor cells allows for the elicitation of a multitude of tumor specific T cells which recognize different tumor associated and tumor specific antigens. Newly elicited and expanded CTLs infiltrate tumors and kill malignant cells.

[0115] Retroviruses are enveloped with a protein envelope and, thus, the retroviral genome encodes Env (envelope protein) as one of the three major proteins. ERVs are evidence of a remote ancestral infection of a retrovirus by a remote ancestor. Thus, the use of the envelope protein as an antigen in vaccination can also be used to target a broad range of ERVs. While the present composition can in principle be used to immunize a plurality of mammalian species, in one aspect of the invention, the ERV protein is a human endogenous retrovirus (HERV) protein or an immunogenic portion thereof. It has been estimated that each human genome consists of about 8% of endogenous retroviral DNA. However, most of the endogenous retroviral DNA is just a relic of a former retrovirus. Upon infection, the viral RNA is reverse transcribed into proviral DNA which integrates into the host genome. Eventually, the provirus integrates into cells of the germline and becomes heritable, creating the endogenous retrovirus. Over millions of years, the viral DNA is passed on from generation to generation and becomes fixed in the population. It follows that a large portion of the human genome potentially can be used as an antigen coding portion for an mRNA such as the one of the present invention.

[0116] In one embodiment, the 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, can in one embodiment be selected 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 can for example be selected from the group consisting of HERV-H19 (=HERV-H_2q24.3), HERV-H_2q24.1; HERV-W can for example be selected as ERVW-1 (=Syncytin-1); and HERV-FRD can for example be selected as ERVFRD-1 (=Syncytin-2).

[0117] 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.

[0118] For a HERV-9 envelope protein encoded by mRNA, a preferred mutated immunosuppressive domain (ISD) that reduces the immunosuppressive properties compared to the wild-type ISD comprises or consists of the sequence LQNCZGLDLLTAEKGGLCTFLGE (SEQ ID NO: 38).

[0119] For a HERV-FC envelope protein encoded by mRNA, a preferred wild-type immunosuppressive domain (ISD) comprises or consists of the sequence AQNRRALDLLTADKGGTCLFLGE (SEQ ID NO: 39).

[0120] For a HERV-T envelope protein encoded by mRNA, a preferred wild-type immunosuppressive domain (ISD) comprises or consists of the sequence LQNRRGLDLLFLSQGGLCAALGE (SEQ ID NO: 40).

[0121] For a HERV-E envelope protein encoded by mRNA, a preferred wild-type immunosuppressive domain (ISD) comprises or consists of the sequence YQNRLALDYLLAAEGGVCGKFNL (SEQ ID NO: 41).

[0122] For mRNA-encoded HERV-3 envelope proteins, a preferred wild-type immunosuppressive domain (ISD) comprises or consists of the sequence YQNRLALDYLLAQEGGVCGKFNL (SEQ ID NO: 42).

[0123] For mRNA-encoded HERV-V1 envelope proteins, a preferred wild-type immunosuppressive domain (ISD) comprises or consists of the sequence MNNRLALDYLLAEQGGVCAVISK (SEQ ID NO: 43).

[0124] For mRNA-encoded HERV-V2 envelope proteins, a preferred wild-type immunosuppressive domain (ISD) comprises or consists of the sequence MDNRLALDYLLAEQGGVCAVINK (SEQ ID NO: 44).

[0125] Further gammaretroviral ISD sequences of other HERVs can be identified by multiple sequence alignment.

[0126] For mRNA-encoded HERV-FC envelope proteins, an immunosuppressive domain (ISD) comprising or consisting of the sequence AQNRRALDLLTADKGGTCLFLGE (SEQ ID NO: 39) already comprises a mutation that results in a less active or inactive ISD, i.e. the sequence AQNRRALDLLTADKGGTCLFLGE (SEQ ID NO: 39) can be used directly in HERV-FC envelope proteins encoded by mRNA as described herein.

[0127] An active gammaretroviral ISD (consisting of 23 amino acid positions) can be mutated to a non-immunosuppressive ISD by replacing the acidic amino acid residue at position 14 with a different amino acid, e.g. preferably a basic amino acid residue. Additionally, an insertion of an aromatic amino acid residue at position 20 of the ISD (consisting of 23 amino acid positions) can further improve stability.

[0128] In one embodiment, the HERV is selected from the group consisting of HERV-9, HERV-T, HERV-E, HERV-3, HERV-V1 and HERV-V2; wherein the immunosuppressive domain (ISD) comprising a mutation that reduces its immunosuppressive properties compared to a wild-type ISD comprises one of the following sequences consisting of 23 amino acid positions or consists thereof: LQNCZGLDLLTAEKGGLCTFLGE (SEQ ID NO: 38) with respect to HERV-9, LQNRRGLDLLFLSQGGLCAALGE (SEQ ID NO: 40) with respect to HERV-T, YQNRLALDYLLAAEGGVCGKFNL (SEQ ID NO: 41) with respect to HERV-E, YQNRLALDYLLAQEGGVCGKFNL (SEQ ID NO: 42) with respect to HERV-3, MNNRLALDYLLAEQGGVCAVISK (SEQ ID NO: 43) with respect to HERV-V1, and MDNRLALDYLLAEQGGVCAVINK (SEQ ID NO: 44) with respect to HERV-V2, wherein one or more amino acids are replaced by a different amino acid. In a preferred embodiment, the single amino acid at position 14 is replaced by a different amino acid. In an even more preferred embodiment, the single amino acid at position 14 is replaced by a basic amino acid. In an even more preferred embodiment, the single amino acid at position 14 is replaced by R.

[0129] 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, wherein the immunosuppressive domain (ISD) comprising a mutation that reduces its immunosuppressive properties compared to a wild-type ISD comprises one of the following sequences or consists thereof: LQNCZGLDLLTAERGGLCTFLGE (SEQ ID NO: 46) with respect to HERV-9, LQNRRGLDLLFLSRGGLCAFLGE (SEQ ID NO: 47) with respect to HERV-T, YQNRLALDYLLAARGGVCGFFNL (SEQ ID NO: 48) with respect to HERV-E, YQNRLALDYLLAQRGGVCGFFNL (SEQ ID NO: 49) with respect to HERV-3, MNNRLALDYLLAERGGVCAFISK (SEQ ID NO: 50) with respect to HERV-V1, and MDNRLALDYLLAERGGVCAFINK (SEQ ID NO: 51) with respect to HERV-V2, 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, wherein the mutated immunosuppressive domain (ISD) reducing its immunosuppressive properties compared to the wild type ISD comprises one of the following sequences consisting of 23 amino acid positions or consists of them: LQNCZGLDLLTAEKGGLCTFLGE (SEQ ID NO: 38) with respect to HERV-9, LQNRRGLDLLFLSQGGLCAALGE (SEQ ID NO: 40) with respect to HERV-T, YQNRLALDYLLAAEGGVCGKFNL (SEQ ID NO: 41) with respect to HERV-E, YQNRLALDYLLAQEGGVCGKFNL (SEQ ID NO: 42) with respect to HERV-3, MNNRLALDYLLAEQGGVCAVISK (SEQ ID NO: 43) with respect to HERV-V1, and MDNRLALDYLLAEQGGVCAVINK (SEQ ID NO: 44) with respect to HERV-V2, wherein the single amino acid at position 14 is replaced by R and wherein the single amino acid at position 20 is replaced by F, preferably wherein position 20 is exchanged to increase stability.

[0130] 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, wherein the mutated immunosuppressive domain (ISD) reducing its immunosuppressive properties compared to the wild type ISD comprises one of the following sequences consisting of 23 amino acid positions or consists of them: LQNCZGLDLLTAEKGGLCTFLGE (SEQ ID NO: 38) with respect to HERV-9, LQNRRGLDLLFLSQGGLCAALGE (SEQ ID NO: 40) with respect to HERV-T, YQNRLALDYLLAAEGGVCGKFNL (SEQ ID NO: 41) with respect to HERV-E, YQNRLALDYLLAQEGGVCGKFNL (SEQ ID NO: 42) with respect to HERV-3, MNNRLALDYLLAEQGGVCAVISK (SEQ ID NO: 43) with respect to HERV-V1, and MDNRLALDYLLAEQGGVCAVINK (SEQ ID NO: 44) with respect to HERV-V2, wherein the single amino acid at position 14 is replaced by R and wherein the single amino acid at position 20 is replaced by F, preferably wherein position 20 is exchanged to increase stability.

[0131] In one embodiment, the present application relates to a composition comprising a transfection agent and an mRNA encoding at least one human endogenous retrovirus (HERV) envelope (Env) protein or an immunogenic portion thereof; wherein the HERV envelope protein comprises an immunosuppressive domain (ISD); wherein the HERV Env protein comprises a mutated immunosuppressive domain (ISD) which reduces its immunosuppressive properties compared to the wild-type ISD, wherein the HERV is selected from the group consisting of HERV-9, HERV-FC, HERV-T, HERV-E, HERV-3, HERV-V1 and HERV-V2; wherein the mutated immunosuppressive domain (ISD) which reduces its immunosuppressive properties compared to the wild-type ISD comprises one or consists of the following sequence consisting of 23 amino acid positions: LQNCZGLDLLTAEKGGLCTFLGE (SEQ ID NO: 38) with respect to HERV-9, AQNRRALDLLTADKGGTCLFLGE (SEQ ID NO: 39) with respect to HERV-FC, LQNRRGLDLLFLSQGGLCAALGE (SEQ ID NO: 40) with respect to HERV-T, YQNRLALDYLLAAEGGVCGKFNL (SEQ ID NO: 41) with respect to HERV-E, YQNRLALDYLLAQEGGVCGKFNL (SEQ ID NO: 42) with respect to HERV-3, MNNRLALDYLLAEQGGVCAVISK (SEQ ID NO: 43) with respect to HERV-V1, and MDNRLALDYLLAEQGGVCAVINK (SEQ ID NO: 44) with respect to HERV-V2, wherein the single amino acid at position 14 is replaced by a different amino acid and preferably by R in SEQ ID NOs: 38, 40, 41, 42, 43 and 44, and preferably wherein the single amino acid at position 20 is replaced by a different amino acid and preferably by F.

[0132] In another embodiment, the mRNA encodes a HERV envelope protein comprising an amino acid sequence which is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical over its entire length to the amino acid sequence of SEQ ID NO: 2 (see Table 1).

[0133] In one embodiment, the HERV is HERV-K. In some embodiments, it is preferred that the HERV-K Env protein has a 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 is designated SEQ ID NO: 1 (see also Table 1): MNPSEMQRKAPPRRRRHRNRAPLTHKMNKMVTSEEQMKLPSTKKAEPPTWAQLKKLTQLATKYLENTKVTQTPESMLLAALMIVSMVVSLPMPAGAAAANYTYWAYVPFPPLIRAVTWMDNPIEVYVNDSVWVPGPIDDRCPAKPEEEGMMINISIGYRYPPICLGRAPGCLMPAVQNWLVEVPTVSPISRFTYHMVSGMSLRPRVNYLQDFSYQRSLKFRPKGKPCPKEIPKESKNTEVLVWEECVANSAVILQNNEFGTIIDWAPRGQFYHNCSGQTQSCPSAQVSPAVDSDLTESLDKHKHKKLQSFYPWEWGEKGISTPRPKIVSPVSGPEHPELWRLTVASHHIRIWSGNQTLETRDRKPFYTVDLNSSLTVPLQSCVKPPYMLVVGNIVIKPDSQTITCENCRLLTCIDSTFNWQHRILLVRAREGVWIPVSMDRPWEASPSVHILTEVLKGVLNRSKRFIFTLIAVIMGLIAVTATAAVAGVALHSSVQSVNFVNDWQKNSTRLWNSQSSIDQK LAN QINDLRQTVIW MGDRLMSLEHRFQLQCDWNTSDFCITPQIYNESEHHWDMVRRHLQGREDNLTLDISKLKEQIFEASKAHLNLVPGTEAIAGVADGLANLNPVTWVKTIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRERAMMTMAVLSKRKGGNVGKSKRDQIVTVSV.

[0134] The mRNA encoding the HERV Env protein is preferably constructed so as to allow the encoded protein to be expressed in vivo and presented to the immune system to elicit an immune response.

[0135] Surprisingly, it was found that the expression efficiency of a HERV Env protein from a coding nucleic acid molecule is increased by replacing one or more amino acid positions in the HERV Env protein by a cysteine, and preferably by replacing the serine at position 190 of the HERV Env protein by a cysteine (S190C), and / or by increasing the cysteine content in the HERV Env protein in general, preferably to an even number of cysteines, possibly by an increased stability of the (transcribed) mRNA and / or the translated protein product. For example, the serine at position 190 of the HERV Env protein can have a destabilizing effect, while replacing this position by a cysteine to obtain an even number of 18 cysteines in the HERV Env protein can have a stabilizing effect. Figure 4

[0136] Thus, in one embodiment, the HERV envelope protein comprises an even number of cysteines. In another embodiment, the HERV envelope protein can alternatively or additionally comprise at least 18 cysteines. In another embodiment, the HERV envelope protein comprises the amino acid sequence wherein at least one amino acid at a position in the range of positions 170 to 210 has been replaced by a cysteine.

[0137] In a preferred embodiment, the HERV envelope protein comprises a cysteine at position 190. In a more preferred embodiment, the HERV envelope protein comprises the amino acid mutation S190C.

[0138] In another embodiment, the HERV envelope protein can alternatively or additionally comprise the sequence VQNWLVEVPTVSPICRFTYHMVSGMSLRP. In another embodiment, the HERV envelope protein can alternatively or additionally comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2, wherein the HERV envelope protein comprises a cysteine at a position within the HERV envelope protein corresponding to amino acid 190 of SEQ ID NO: 2.

[0139] 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 or consists of a sequence selected from the group consisting of SEQ ID NOs: 22-27, 29-32 and 35

[0140] ​​In a preferred embodiment, the composition of the application comprises an mRNA having at least 90% sequence identity, and most preferably 100% sequence identity, to any sequence according to any one of SEQ ID NO: 45, 36 or 37. In an even more preferred embodiment, the composition of the application comprises an mRNA having 100% sequence identity to any sequence according to any one of SEQ ID NO: 45, 36 or 37.

[0141] In one embodiment, the cell surface expression of a HERV envelope protein (as defined in the previous paragraph) is increased compared to the cell surface expression of a HERV envelope protein having an amino acid sequence according to SEQ ID: 2.

[0142] A further aspect of the application relates to an mRNA having at least 90% sequence identity to SEQ ID NO: 45, wherein the mRNA encodes a polypeptide comprising a HERV-K Gag and a HERV-K envelope (Env) protein; and wherein the HERV Env protein comprises a mutated immunosuppressive domain (ISD) which reduces its immunosuppressive properties compared to a wild-type Env ISD (preferred ISD mutations and mutated ISD sequences are disclosed herein in the context of the application - for example, one or more of the ISD mutations L522A, A523Q, N524A, Q525A, N527A, L529A, R530A, Q531A, T532A can be used); and wherein the HERV envelope protein further comprises a cysteine instead of a serine at amino acid position 190 within the HERV envelope protein. In a preferred embodiment, the above-mentioned mRNA is in a composition of the application. Also provided is the use of the above-mentioned mRNA to construct a virus-like particle (VLP).

[0143] A further aspect of the application relates to a polypeptide having at least 90% sequence identity to a polypeptide encoded by SEQ ID NO: 45, wherein the polypeptide comprises a HERV-K Gag and a HERV-K envelope (Env) protein; and wherein said HERV Env protein comprises a mutated immunosuppressive domain (ISD) which reduces its immunosuppressive properties compared to the wild-type Env ISD (preferred ISD mutations and mutated ISD sequences are disclosed herein in the context of the present application - for example, one or more of the ISD mutations L522A, A523Q, N524A, Q525A, N527A, L529A, R530A, Q531A, T532A can be used); and wherein said HERV envelope protein comprises a cysteine instead of a serine at position 190 within said HERV envelope protein. In a preferred embodiment, the above polypeptide is comprised in a virus-like particle (VLP). Also provided is an mRNA encoding the above polypeptide. Preferably, said mRNA is comprised in a composition of the present application.

[0144] A further aspect of the present application relates to a polypeptide comprising or consisting of a HERV Env protein having at least 95% sequence identity to SEQ ID NO: 2, wherein said Env protein comprises a mutated immunosuppressive domain (ISD) which reduces its immunosuppressive properties compared to the wild-type Env ISD (preferred ISD mutations and mutated ISD sequences are disclosed herein in the context of the present application - for example, one or more of the ISD mutations L522A, A523Q, N524A, Q525A, N527A, L529A, R530A, Q531A, T532A can be used); and wherein said HERV envelope protein comprises a cysteine instead of a serine at position 190 of SEQ ID NO: 2. In a preferred embodiment, the above polypeptide is comprised in a virus-like particle (VLP). Also provided is an mRNA encoding the above polypeptide. Preferably, said mRNA is comprised in a composition of the present application.

[0145] A further aspect of the application relates to a polypeptide comprising or consisting of a HERV Env protein, wherein the Env protein comprises a cysteine at position 190, such that the Env protein comprises the amino acid sequence VQNWLVEVPTVSPICRFTYHMVSGMSLRP; and wherein the Env protein further comprises a mutated immunosuppressive domain (ISD) which reduces its immunosuppressive properties compared to the wild-type Env ISD (preferred ISD mutations and mutated ISD sequences are disclosed herein in the context of the application - for example, one or more of the ISD mutations L522A, A523Q, N524A, Q525A, N527A, L529A, R530A, Q531A, T532A can be used). In a preferred embodiment, the above polypeptide is comprised in a virus-like particle (VLP). Also provided is an mRNA encoding the above polypeptide. Preferably, the mRNA is comprised in a composition of the application.

[0146] Immunosuppressive domain (ISD) The immunosuppressive domain (ISD) can be seen as a mechanism used by the tumor to balance the anti-tumor immune response while maintaining the pro-tumoral inflammatory microenvironment induced by HERV activation, similar to a natural viral infection. The ISD affects both the innate and adaptive immune system due to the same suppression of macrophages, NK cells and T cells. Although the detailed mechanism of immunosuppression by the ISD is not fully understood, the ability of the ISD to induce IL-10 secretion from peripheral blood mononuclear cells upon contact, and the ability of a mutated ISD domain to reduce NF-κΒ-induced gene expression (which was discovered serendipitously in a transfection assay), have been observed to be associated with the immunosuppressive and / or pro-inflammatory microenvironment function of the ISD. In particular, the reduced NF-κΒ expression due to the mutated ISD domain can be secondary to an immunogenic cell death pathway, resulting in a pronounced reduction of NF-κΒ, but reflecting an enhanced immune stimulation.

[0147] In one embodiment, the mutated ISD of the application suppresses the proliferation of human immune cells less than a wild-type ISD which is not mutated and which has an amino acid sequence according to SEQ ID NO: 3 (see Table 1), and / or has a reduced or lost ability to induce IL-10 secretion from peripheral blood mononuclear cells upon contacting said cells with said mutated ISD, and / or reduces NF-κΒ expression.

[0148] To inactivate the ISD, the immunosuppressive capacity is preferably reduced by 30% or more compared to the immunosuppression achieved by the wild type ISD. Preferably, the ISD is inactivated even by 35% or 40% or more, such as 45% or more, such as 47, 48 or 49 or more, such as 50% compared to the immunosuppression performed by the original, i.e. non-mutated ISD. Quantification of the level of immunosuppression and / or induction of an inflammatory cell environment can be performed by quantifying the level of IL-10, NF-κΒ or NF-κΒ-inducible 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.

[0149] NF-κΒ activation / inhibition can be determined specifically by transfecting HEK293T cells with a mixture having a reporter plasmid expressing luciferase upon NF-κΒ expression, the selected HERV-K Env protein encoding DNA plasmid and lipofectamine. The day after transfection, the cells can be analyzed with respect to luciferase expression by adding luciferase substrate followed by quantification of luminescence. In such assays, HERV-K having the mutated ISD sequence of SEQ ID NO: 4 can, for example, reduce the basal NF-κΒ level by about 20%, 25%, 30%, or even about 40%, or even up to 50% or even more.

[0150] The ISD segment can be inactivated by mutation or deletion of one or more amino acids. In case inactivation is performed by mutation, one or more amino acids are exchanged for a different amino acid, typically selected from the other 19 naturally occurring amino acids. Most suitably, one or two single amino acids at any one of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14 of ISD SEQ ID NO: 3 are replaced. The person skilled in the art is in possession of sufficient knowledge and experience, optionally by evaluation of preliminary experiments, which amino acids to exchange to direct them towards a satisfactory immune response.

[0151] Thus, in one embodiment of the application, the mutated ISD comprises or consists of the following amino acid sequence: wherein one or two single amino acids at any one of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14 are replaced by a different amino acid, and preferably in each case by an alanine, to inactivate the ISD. In a preferred embodiment, the one or two single amino acids which differ from the original are selected from the naturally occurring amino acids.

[0152] In the present context, it was surprisingly found that when cells are transfected with a nucleic acid molecule (e.g. DNA) encoding a HERV Env, wherein the ISD according to SEQ ID NO: 3 (LANQINDLRQTVIW) comprises a mutation at any one of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, the number of transfected cells expressing the encoded HERV Env protein with the mutated ISD is increased compared to the expression in cells transfected with a HERV Env having a wild-type ISD (see Figure 4 ). Thus, when an antigen protein, e.g. a HERV Env, is encoded with a mutated ISD comprising a mutation at any one of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, e.g. in particular the experimentally tested mutations L522A, A523Q, N524A, Q525A, I526A, N527A, D528A, L529A, R530A, Q531 A, T532A, V533A, I534A and W535A, an increased expression of the encoded antigen protein, e.g. a HERV Env, and a subsequent increase of the immune response against the antigen can be achieved.

[0153] Thus, in another embodiment, the ISD mutation is selected from L522A, A523Q, N524A, Q525A, I526A, N527A, D528A, L529A, R530A, Q531 A, T532A, V533A, I534A and W535A. In a preferred embodiment, the mutated ISD comprises a sequence according to any one of SEQ ID NOs: 4 or 8-20. In an even more preferred embodiment, the ISD mutation is selected from L522A, A523Q, N524A, Q525A, N527A, L529A, R530A, Q531 A and T532A. In an even more preferred embodiment, the mutated ISD comprises a sequence according to any one of SEQ ID NOs: 4, 8-12, 14-17 or 20.

[0154] It can be advantageous to combine the ISD mutations described herein with a stabilizing cysteine substitution as further described above. Thus, in a preferred embodiment, the HERV envelope protein comprises an even number of cysteines, and alternatively or additionally comprises at least 18 cysteines, and the HERV envelope protein comprises an ISD mutation selected from L522A, A523Q, N524A, Q525A, I526A, N527A, D528A, L529A, R530A, Q531A, T532A, V533A, I534A, and W535A, preferably the ISD mutation is selected from L522A, A523Q, N524A, Q525A, N527A, L529A, R530A, Q531A, and T532A.

[0155] In another preferred embodiment, the HERV envelope protein comprises the amino acid sequence wherein at least one amino acid at a position in the range of positions 170 to 210 has been replaced by a cysteine, and the HERV envelope protein comprises an ISD mutation selected from L522A, A523Q, N524A, Q525A, I526A, N527A, D528A, L529A, R530A, Q531A, T532A, V533A, I534A, and W535A, preferably the ISD mutation is selected from L522A, A523Q, N524A, Q525A, N527A, L529A, R530A, Q531A, and T532A.

[0156] In an even more preferred embodiment, the HERV envelope protein comprises a cysteine at position 190, and most preferably the amino acid mutation S190C, and the HERV envelope protein comprises an ISD mutation selected from L522A, A523Q, N524A, Q525A, I526A, N527A, D528A, L529A, R530A, Q531A, T532A, V533A, I534A, and W535A, preferably the ISD mutation is selected from L522A, A523Q, N524A, Q525A, N527A, L529A, R530A, Q531A, and T532A. In one embodiment, the HERV envelope protein comprises the amino acid mutation S190C and the ISD mutation Q525A.

[0157] Amino acids are those L-amino acids commonly found in naturally occurring proteins. Amino acid residues are indicated in the present disclosure according to the standard three-letter or one-letter amino acid code. Any amino acid sequence containing post-translationally modified amino acids can be described as the originally translated amino acid sequence, with modifications such as hydroxylation or glycosylation not explicitly shown in the amino acid sequence. Any peptide or protein that can contain linkages, crosslinks, and end caps, non-peptidyl linkages, etc. that can be represented as sequence modifications are included herein, all as known in the art.

[0158] While the skilled person is able to modify the amino acid sequence by performing any number or form of mutation or deletion, it is currently preferred to exchange a single amino acid of the ISD sequence. This modification involves replacing the glutamine at position 4 of the ISD with an alanine. This change triggers inactivation of the domain in order to prevent the vaccine itself from producing an immunosuppressive effect. Thus, in one embodiment, the mutated ISD comprised by the RNA of the present application preferably comprises or consists of the sequence LANAINDLRQTVIW (SEQ ID NO: 4).

[0159] A particularly preferred HERV-K Env sequence containing the ISD inactivation mutation is shown in SEQ ID NO: 2 (see also Table 1): MNPSEMQRKAPPRRRRHRNRAPLTHKMNKMVTSEEQMKLPSTKKAEPPTWAQLKKLTQLATKYLENTKVTQTPESMLLAALMIVSMVVSLPMPAGAAAANYTYWAYVPFPPLIRAVTWMDNPIEVYVNDSVWVPGPIDDRCPAKPEEEGMMINISIGYRYPPICLGRAPGCLMPAVQNWLVEVPTVSPISRFTYHMVSGMSLRPRVNYLQDFSYQRSLKFRPKGKPCPKEIPKESKNTEVLVWEECVANSAVILQNNEFGTIIDWAPRGQFYHNCSGQTQSCPSAQVSPAVDSDLTESLDKHKHKKLQSFYPWEWGEKGISTPRPKIVSPVSGPEHPELWRLTVASHHIRIWSGNQTLETRDRKPFYTVDLNSSLTVPLQSCVKPPYMLVVGNIVIKPDSQTITCENCRLLTCIDSTFNWQHRILLVRAREGVWIPVSMDRPWEASPSVHILTEVLKGVLNRSKRFIFTLIAVIMGLIAVTATAAVAGVALHSSVQSVNFVNDWQKNSTRLWNSQSSIDQK LANAINDLRQTVIW MGDRLMSLEHRFQLQCDWNTSDFCITPQIYNESEHHWDMVRRHLQGREDNLTLDISKLKEQIFEASKAHLNLVPGTEAIAGVADGLANLNPVTWVKTIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRERAMMTMAVLSKRKGGNVGKSKRDQIVTVSV.

[0160] It can be preferred to exchange one or more amino acids in the region upstream or downstream of the ISD segment. The mutation is a compensatory mutation that is expected to preserve the structure of the domain, so that it can still act on the infectious virus. Thus, at least one amino acid in the region of 10 amino acids upstream or downstream of the ISD can be exchanged for a different amino acid. In case further compensatory mutations should be required, the skilled person will know the effect of such mutations and be able to select accordingly.

[0161] HERV ISD inactivation can be detected and quantified via the level of immune response in the subject, induced by administration of the composition of the application. For example, HERV-specific antibodies can be quantified in blood samples from the subject. Conversely, such specific antibodies can be used to label the HERV itself, to analyze its presentation on the cell surface in both in vitro and in vivo obtained samples, e.g. using FACS. Furthermore, the proliferation of immune cells, e.g. NK cells and T cells, in samples from immunized subjects, e.g. mice, can also be analyzed via FACS or other suitable methods, as well as both extracellular and intracellular staining markers of these activated immune cells. Suitable markers of immune cell activation will be clear to the skilled person. For example, activated T cells can be detected via IFNy, TNFa and CD44, while NK cells can be detected via CD56.

[0162] In this context, the immune effect of using the mRNA of the application and the composition of the application was confirmed by immunizing mice treated in a prime and boost regimen (day 0 and day 7) with mRNA encoding HERV-K proteins GAG and ENV having a mutated ISD (cf. immunization regimen in Figure 5 ), and subsequently performing tetramer staining to detect and quantify CD8+ T cells specific for HERV-K antigens in the spleen tissue of the mice, as well as detecting specific antibodies in blood samples of the mice by ELISA (cf. respectively Figure 6 or Figure 7 ).

[0163] Figure 6The results in Example 2 confirm that in vivo immunization with mRNA encoding HERV-K GAG and HERV-K ENV with a mutated ISD, e.g., mutated Q525A, induces CD8+ T cell responses to both ENV (demonstrated by cell binding Tet18) and GAG (demonstrated by cell binding Tet90). Further, Figure 7 The results in Example 2 confirm that in vivo immunization with mRNA encoding HERV-K GAG and HERV-K ENV with a mutated ISD, e.g., mutated Q525A, induces CD8+ T cell responses to both ENV (demonstrated by cell binding Tet18) and GAG (demonstrated by cell binding Tet90). Further,

[0164] Another exemplary method to test the effectiveness of immunization and / or elicitation of an immune response by the compositions of the application, which comprise mRNA encoding HERV Env with an inactivated ISD, is to evaluate resistance to tumor formation. For example, tumor challenge and tumor rejection assays can be performed in which a subject, i.e., an animal, is injected with tumor cells, e.g., cells of the tumor cell line B16F10-GP or CT26 or 4T1 or murine kidney cancer cells engineered to express portions of the human ERV-K genome, and subsequently treated, i.e., therapeutically vaccinated, with a composition of the application. After a period of time, e.g., 1 to 6 weeks, the subject is analyzed for tumor and / or metastasis formation and tumor size and tumor properties, e.g., by dissection of tumors for HERV Env-specific staining. Thus, it can be determined whether tumor formation is reduced or rejected by treatment with a composition of the application, and whether tumors express HERV Env.

[0165] Further, as outlined elsewhere herein, upon cell contact and uptake of the compositions of the application, there are at least two ways in which antigen display or antigen provision occurs to initiate an immune response: as MHC class I antigen presentation to CD8+ T cells on the surface of the contacted cell, or as MHC class II antigen on professional antigen presenting cells. Both of these mechanisms underscore the importance of surface display for antigen detection in the immune process.

[0166] In this context, the inventors surprisingly observed that an encoded HERV-K Env with an ISD containing a point mutation of SEQ ID NO: 4 showed improved HERV-K cell surface display on HEK293 cells in vitro compared to a HERV-K Env without the ISD mutation (see Figure 1 ) Without wishing to be bound by theory, it is expected that the point mutation rendering the ISD inactive is also the reason for the improved surface display.

[0167] In addition to the human endogenous retrovirus (HERV) envelope protein or immunogenic portion thereof, the mRNA of the application can also encode a further protein. The co-encoded portion can then for example undergo a pulling effect towards the cell surface for display together with the HERV Env.

[0168] Thus, in one embodiment, the application can also relate to a composition comprising a transfection agent and a mRNA encoding at least one human endogenous retrovirus (HERV) envelope protein or immunogenic portion thereof; wherein the HERV envelope protein comprises an immunosuppressive domain (ISD), wherein the HERV Env protein comprises a mutated immunosuppressive domain (ISD) which reduces its immunosuppressive properties compared to the wild-type ISD, and wherein the HERV protein is co-expressed with at least one further protein encoded by the same mRNA. The at least one further protein can be conjugated, i.e. fused, to the HERV envelope protein of the application, which can enable the further protein to be secreted or displayed at the cell surface together with the HERV envelope protein. The at least one further protein can also be expressed separately, i.e. not fused to the HERV envelope protein of the application.

[0169] The skilled person will know how to obtain co-expressed proteins, fused and non-fused, encoded on the same mRNA strand. In one embodiment, the further protein can be linked directly to the HERV envelope protein, or linked thereto via a linker also encoded by the mRNA. Suitable linkers are known in the art. In one embodiment, the at least one further protein can be conjugated to the HERV envelope protein of the application via a linker, wherein the linker is for example a suitable amino acid sequence, particularly preferably having 1 to 30, for example 1 to 10 amino acid residues. Preferred examples of such amino acid sequences include, but are not limited to, a gly-ser linker.

[0170] In some cases, it can be desirable to generate a stronger immune response in a subject receiving treatment by the composition of the application, or to induce immunity against a further antigen in a subject. Thus, in one embodiment, the further encoded protein can be an antigen. In one embodiment, the further encoded protein can be an adjuvant.

[0171] In one embodiment, the further encoded protein can be a peptide or protein from a peptide or protein library. Co-display can allow peptide or protein presentation at the cell surface for screening and / or characterization. Advantageously, proteins are more stable when attached to a matrix than as free molecules and in the present case the cell surface acts as a matrix.

[0172] By displaying proteins on the cell surface, in many cases, the production or purification of the protein also becomes superfluous. Thus, in one embodiment, the further encoded protein can be a protein or peptide produced in the host cell, i.e. the eukaryotic cell, wherein the protein or peptide is obtained in a certain purity.

[0173] Molecules displayed at the cell surface, e.g. peptides or proteins, are freely accessible to substrates or binding partners in activity or binding assays. Thus, in one embodiment, the further encoded protein can be an enzyme or catalytic part of an enzyme or an antibody or a part thereof.

[0174] The displayed molecules are also freely accessible to binding in cell purification assays or detection in detection assays. Thus, in one embodiment, the further encoded protein can be a tag for cell purification, e.g. an affinity tag or epitope tag, e.g. selected from but not limited to the following: CaM tag, CBP tag, GST tag, MBP tag; biotinylation tags, e.g. 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 can be a peptide to be detected, e.g. a fluorescent tag, e.g. as GFP, YFP and many others known in the art.

[0175] Cancer cells frequently upregulate surface receptors that promote growth and survival. These receptors constitute effective targets for intervention. One strategy involves the delivery of toxic receptor-binding agents, with the goal of killing those cancer cells with high receptor levels. Thus, in order to enhance the anti-cancer effect of the treatment by using the composition of the present application, in one embodiment, the further encoded protein can be an mRNA encoding an agent suitable for killing cancer cells, e.g. a protein toxin.

[0176] Medical use As shown in the examples, the mRNA of the present application exhibit a surprising beneficial effect of greatly improving the cell surface display of the encoded antigen, i.e. HERV Env. As described in the introductory section, such efficient antigen surface presentation is key for eliciting a response from the immune system by contact with the antigen. The increased surface display of the encoded antigen, i.e. HERV Env, in conjunction with the mutation of the ISD that renders it inactive, necessarily greatly facilitates the immunoreactivity of the subject's body against the HERV Env. Thus, a subject can be immunized with a composition of the present application that is effective against the development or progression of a HERV-related cancer, and / or that facilitates the subject's body's fight against a HERV-related cancer by the immunological action. The composition of the present application, i.e. a composition comprising a transfection agent and an mRNA that encodes at least one human endogenous retrovirus (HERV) envelope protein or an immunogenic portion thereof, with a mutated immunosuppressive domain (ISD) having reduced immunosuppressive properties compared to the wild-type ISD, is thus suitable for administration in therapy or prophylaxis. The composition can be administered as a therapy or vaccine, or as part of a therapy or vaccine, in order to induce a specific immune response against endogenous retrovirus-related tumor cells, and / or to immunize a subject against tumor development or progression caused by HERV activity.

[0177] Thus, in one aspect, the present application relates to a composition of the present application for use as a medicament. In another aspect, the present application relates to a composition of the present application for use in the prophylaxis or treatment of a disease, preferably for immunizing a subject against a disease.

[0178] In yet another aspect, the present application relates to a composition of the present application for the manufacture of a medicament. Yet another aspect of the present application relates to the use of a composition of the present application for the manufacture of a medicament for the prophylactic and / or therapeutic treatment of a disease, preferably for immunizing a subject against a disease.

[0179] In yet another aspect, the present application relates to a method of treating and / or preventing a disease, preferably immunizing a subject against a disease, comprising administering to the subject a therapeutically effective amount of a composition of the present application.

[0180] There is a wide range of pathological conditions caused by the activity and / or reactivation of ERVs. In one embodiment, the disease which is the subject of prophylactic and / or therapeutic treatment by the composition of the application is an ERV reactivation-related disease or disorder, preferably a HERV reactivation-related disease or disorder. Thus, in a preferred embodiment, the disease which is the subject of prophylactic and / or therapeutic treatment by the composition of the application is selected from the group consisting of: cancer, HIV and / or related disorders, rheumatic diseases, neurodegenerative diseases, aging-related diseases, diseases associated with HERV reactivation, chronic inflammation, multiple sclerosis, ALS, sarcopenia, kidney diseases and Alzheimer’s disease.

[0181] For example, HIV can reactivate HERVs in a human subject (Jakobsson, Johan and Michelle Vincendeau. "SnapShot: Human endogenous retroviruses." Cell 185.2 (2022): 400-400.). Thus, when the composition of the application treats HIV, it is expected that this HERV reactivation in HIV patients is reduced, thus providing a health benefit to HIV patients.

[0182] In an embodiment wherein the disease is ALS, it is preferably ALS associated with trans-activated response DNA binding protein 43 kDa (TDP-43) and / or C-terminal fragments thereof, for example ALS associated with increased ubiquitination, hyperphosphorylation, mislocalization and / or accumulation of TDP-43 and / or C-terminal fragments thereof. In an embodiment wherein the disease is Alzheimer’s disease, it can preferably be Alzheimer’s disease associated with Tau protein expression, elevated Tau protein, mislocalization of Tau protein and / or Tau protein aggregation.

[0183] The present application is particularly suitable for the prevention and / or treatment of cancer. For example, the target cancer for HERV-K is prostate cancer, breast cancer, ovarian cancer, lymphoma, melanoma, leukemia and sarcoma; the target cancer for HERV-H is colorectal cancer; the target cancer for HERV-W is testicular cancer, ovarian cancer, breast cancer, lymphoma and leukemia; and the target cancer for HERV-E is lung cancer and liver cancer. Thus, the type of cancer treated or prevented by the present application is not particularly limited and includes 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 HERV-expressing cancer. In an even more preferred embodiment, the HERV-expressing cancer is selected from the group consisting of: a tumor expressing PD-L1, cervical cancer, penile cancer, anal cancer, vulvar cancer, vaginal cancer, bladder cancer, breast cancer, clear cell kidney cancer, head / 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 (CLL), chronic myeloid leukemia (CML), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, Hodgkin's lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myeloid cell leukemia-1 protein (Mcl-1), myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL), and small lymphocytic lymphoma (SLL). Other cancer types can also present suitable targets.

[0184] Suitable treatment regimens are presented in the art and will be clear to the person skilled in the art. For example, under certain conditions, it can be advantageous to treat a patient using a prime-boost regimen. Thus, the use in the prevention and / or treatment of cancer can for example comprise the step of priming a subject with a composition of the application for at least 5 days, followed by boosting with a composition of the application.

[0185] The composition of the application is also suitable for the prevention and / or treatment of cancer, which comprises the step of treating a subject for 5 days or more after exposure of the subject to a composition of the application, for example after vaccination with said composition, said composition having a different encoded antigen, i.e. a HERV Env protein of the application, for example as a VLP derived from an adenovirus, Modified Vaccinia Ankara (MVA) or other virus encoded VLP.

[0186] It is preferred that the mRNA of the present application is used as a genetic vaccine, in particular in the prevention and / or treatment of a disease, preferably a cancer. Alternatively, the nucleic acid molecule can also be used for the production of VLPs, in particular HERV-K VLPs, in vitro. The resulting VLPs can then be used for immunotherapy, in particular in the prevention and / or treatment of a disease, preferably a cancer. It is understood that also in this context the cancer to be treated is a cancer expressing the respective HERV.

[0187] It was found that activation of endogenous retroviruses occurs in organs and tissues of elderly individuals and that repression of ERVs mitigates cellular senescence, tissue degeneration, and biological aging (Liu X, et al.; Resurrection of endogenous retroviruses during aging reinforces senescence. Cell. 2023 Jan 19;186(2):287-304.e26). Thus, reactivation of endogenous retroviruses is a hallmark and driver of cellular senescence and tissue aging. Thus, in yet another aspect, the present application relates to a composition according to the present application for use in the prevention or slowing of aging and / or cellular senescence. In yet another aspect, the present application relates to a composition according to the present application for use in the manufacture of a medicament for the prevention or slowing of aging and / or cellular senescence.

[0188] Further, the present application also relates to a VLP encoded by a nucleic acid molecule encoding a Gag protein and a HERV envelope protein (Env) or an immunogenic portion thereof, wherein the natural genomic structure connecting Gag and Env has been replaced by an operable linker. Preferably, the operable linker is p2A. It is further preferred that the HERV is HERV-K. More preferably, the HERV is HERV-K, the amino acid sequence of which is disclosed by Lee et al. (Lee YN, Bieniasz PD (2007) Reconstitution of an Infectious Human Endogenous Retrovirus. PLoS Pathog 3(1): e10). As mentioned above, the use of such VLPs in immunotherapy is envisaged. Furthermore, the present application relates to a nucleic acid molecule or a VLP for use in the prevention and / or treatment of a disease. It is preferred that the disease is a cancer. It is understood that the cancer is a cancer expressing the respective HERV.

[0189] Pharmaceutical composition As outlined above, the composition of the present application can be used, for example, in the treatment or prevention of a disease. Thus, in one embodiment, the present application relates to a pharmaceutical composition comprising the composition of the present application.

[0190] In some cases, when the compositions of the present invention are administered for treatment or prevention as outlined above, further agents such as excipients may be beneficial to improve treatment or prevention efficacy and / or patient tolerability. Therefore, in another aspect, the present invention relates to pharmaceutical compositions comprising the compositions of the present invention and comprising pharmaceutically acceptable excipients.

[0191] In some embodiments, pharmaceutically acceptable excipients may be selected as defined elsewhere herein. In some preferred embodiments, the pharmaceutical compositions of the present invention may comprise at least one pharmaceutically acceptable excipient selected from water, sodium chloride, potassium chloride, sucrose, sodium acetate, or saline solution. Brief description of the attached diagram Figure 1 HEK293 cells were transfected with DNA encoding HERV-K GAG-ENV or HERV-K GAG-ENV ISDmut formulated in JetPEI, or RNA 1 (5'UTR) HBA1-HERV-K Gag Env ISDmut-(3'UTR) HBB-polyA(70)) (SEQ ID NO: 45) formulated in Lipofectamine Messenger MAX. The same results were expected when HEK293 cells were transfected with RNA having the 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 post-transfection. The figure shows % of HERV-K ENV-positive cells in live cells.

[0193] Figure 2 NF-κB activation or inhibition was determined by transfecting HEK293T cells with a mixture containing a reporter plasmid expressing luciferase after NF-κB expression, a DNA plasmid encoding either the HERV-K Env protein with intact ISD (WT) or the HERV-K Env protein with mutant ISD (ISDmut), and lipofectamine. Cells were analyzed for luciferase expression by quantitative luminescence (RLU, relative light units) as a readout for NF-κB activation or inhibition the day after transfection. The data presented are normalized to baseline activity.

[0194] Figure 3 : Figure 3 yes Figure 1Supplemental data to the results shown in Figure 2. HEK293 cells were transfected with DNA encoding HERV-K GAG-ENV (with wild-type ISD without mutations, sample labeled “WT”) or DNA encoding HERV-K GAG-ENV ISDmut (with ISD containing specific mutations), each formulated in JetPEI. The DNA constructs used were identical to the ones described in relation to the data shown in Figure 1 (refer to the same protein-coding sequences in SEQ ID NOs: 45 and 46), except for the different ISD sequences, i.e. different ISD mutations / positions were used. The ISD mutations tested for the ISDmut constructs were L522A, A523Q, N524A, Q525A, I526A, N527A, D528A, L529A, R530A, Q531A, T532A, V533A, I534A, and W535A (samples labeled according to the mutation; see also ISD sequences in Table 3, SEQ ID NOs: 4 and 8-20). In addition, constructs with the wild-type ISD combined with or combined with a mutant ISD containing the mutation Q525A were tested (samples labeled “WT + S190C” and “Q525A + S190C”). In control samples, cells were not treated (sample labeled “Control NT”) or cells were treated with JetPEI (sample labeled “Control JetPEI”). Twenty-four hours after transfection, cells were stained for HERV-K ENV expression. The figure shows the % of HERV-K ENV positive cells in live cells. 5'UTR of HBA1 - HERV-K Gag Env ISD mut (with ISD mutation Q525A) - 3'UTR of AES mtRNR1 - polyA (30-linker-70) “RNA 4” (SEQ ID NO: 36), or DNA encoding “RNA 8” (SEQ ID NO: 37) with N1-methylpseudouridine (mlY) modifications, each formulated in Lipofectamine Messenger MAX. In control samples, cells were not treated (Control NT) or treated with Lipofectamine Messenger MAX (Control LipoMAX). Twenty-four hours after transfection, cells were stained for HERV-K ENV expression. The figure shows the % of HERV-K ENV positive cells in live cells. 5'UTR of HBA1 - HERV-K Gag Env (with wild type ISD without mutation) - 3'UTR of AES mtRNR1 - polyA (30-linker-70) “RNA 4” (SEQ ID NO: 36), or DNA encoding “RNA 8” (SEQ ID NO: 37) with N1-methylpseudouridine (mlY) modifications, each formulated in Lipofectamine Messenger MAX. In control samples, cells were not treated (Control NT) or treated with Lipofectamine Messenger MAX (Control LipoMAX). Twenty-four hours after transfection, cells were stained for HERV-K ENV expression. The figure shows the % of HERV-K ENV positive cells in live cells.

[0195] Figure 4 HEK293 cells were transfected with DNA encoding HERV-K GAG-ENV (with wild-type ISD without mutations, sample labeled “WT”) or DNA encoding HERV-K GAG-ENV ISDmut (with ISD containing specific mutations), each formulated in JetPEI. The DNA constructs used were identical to the ones described in relation to the data shown in Figure 1 (refer to the same protein-coding sequences in SEQ ID NOs: 45 and 46), except for the different ISD sequences, i.e. different ISD mutations / positions were used. The ISD mutations tested for the ISDmut constructs were L522A, A523Q, N524A, Q525A, I526A, N527A, D528A, L529A, R530A, Q531A, T532A, V533A, I534A, and W535A (samples labeled according to the mutation; see also ISD sequences in Table 3, SEQ ID NOs: 4 and 8-20). In addition, constructs with the wild-type ISD combined with or combined with a mutant ISD containing the mutation Q525A were tested (samples labeled “WT + S190C” and “Q525A + S190C”). In control samples, cells were not treated (sample labeled “Control NT”) or cells were treated with JetPEI (sample labeled “Control JetPEI”). Twenty-four hours after transfection, cells were stained for HERV-K ENV expression. The figure shows the % of HERV-K ENV positive cells in live cells. Figure 1 HEK293 cells were transfected with DNA encoding HERV-K GAG-ENV (with wild-type ISD without mutations, sample labeled “WT”) or DNA encoding HERV-K GAG-ENV ISDmut (with ISD containing specific mutations), each formulated in JetPEI. The DNA constructs used were identical to the ones described in relation to the data shown in Figure 1 (refer to the same protein-coding sequences in SEQ ID NOs: 45 and 46), except for the different ISD sequences, i.e. different ISD mutations / positions were used. The ISD mutations tested for the ISDmut constructs were L522A, A523Q, N524A, Q525A, I526A, N527A, D528A, L529A, R530A, Q531A, T532A, V533A, I534A, and W535A (samples labeled according to the mutation; see also ISD sequences in Table 3, SEQ ID NOs: 4 and 8-20). In addition, constructs with the wild-type ISD combined with or combined with a mutant ISD containing the mutation Q525A were tested (samples labeled “WT + S190C” and “Q525A + S190C”). In control samples, cells were not treated (sample labeled “Control NT”) or cells were treated with JetPEI (sample labeled “Control JetPEI”). Twenty-four hours after transfection, cells were stained for HERV-K ENV expression. The figure shows the % of HERV-K ENV positive cells in live cells.

[0196] Figure 5 : Schematic representation of the vaccine protocol, where CB6F1 WT (wild type) mice were immunized at day 0 (priming) with mRNA encoding for HERV-K proteins GAG and ENV ISDmut with N1 -methylpseudouridine (mlY) modification (construct according to SEQ ID NO: 36) that can assemble into VLPs format, where the ISD of the ENV protein is mutated (mutation Q525A), represented by (SEQ ID NO: 36). At day 7, mice received a second dose of mRNA encoding for the same antigen cassette (boost). Mice were euthanized at day 21 for immune response analysis by detection and quantification of T cells specific for HERV-K antigens in spleen tissue samples by tetramer staining, or specific antibodies in mouse blood samples by ELISA. Results are shown in Figure 6 and 7 .

[0197] Figure 6 : Immunization schedule in Figure 5The data show that N=5 mice / group. The data show the frequency (%) of tetramers (using the 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. Oct 4, 1996; 274(5284):94-6) binding to CD8+ T cells in spleen tissue of immunized mice on day 21 post-immunization. The frequency of each tetramer (Tet) is shown separately. For tetramer staining on spleen cells, the tetramer consists of four conjugated MHC class I molecules loaded with a selected antigen-specific peptide (from HERV-K) and conjugated with a fluorescent dye. The tetramer binds only to T cells specific to the loaded target antigen peptide. Because the vaccine used in this study induced the activation and expansion of HERV-K antigen-specific T cells, tetramer staining was used to detect the fractionation of antigen-specific T cells expanded after vaccine administration to mice. Tet18 tetramers bound to CD8+ T cells specific to the HERV-K ENV epitope, while Tet29 and Tet90 tetramers bound to CD8+ T cells specific to the HERV-K GAG epitope. Immunization with mRNA encoding HERV-K GAG and HERV-K ENV with a mutated ENV ISD (mutant Q525A) induced CD8+ T cell responses against ENV (i.e., cells bound by Tet18) and GAG (i.e., cells bound by Tet90).

[0198] Figure 7 Immunization schedule Figure 5 The data shows N=5 mice / group. The data are presented as OD values ​​determined by ELISA performed on serum samples harvested from immunized mice on day 21 post-primary immunization, and the signal represents the number of antibodies against the respective protein subunits in the mouse serum samples. Figure 7 In A, OD values ​​were determined from a 1:25 dilution of serum samples harvested on day 21 post-primary immunization, where OD represents the antibody level against subunit TM (HERV-K ENV transmembrane subunit). Figure 7In B, the OD value was determined in a 1 :25 dilution of serum samples harvested on day 21 after the first immunization, wherein the OD represents the level of antibodies against the subunit SU (HERV-K ENV surface subunit). Immunization with mRNA encoding HERV-K GAG and ENV with ISDmut (mutated Q525A) induced an antibody immune response against both antigen subunits SU and TM in the immunized mice. Examples

[0199] Example 1 1.1 Cell culture The HEK293 cell line derived from human embryonic kidney cultures was generated by transformation with sheared adenovirus type 5 (Ad5) DNA (available from LGC Standards (ATCC)). Advantages of this cell line include ease of growth and efficient transfection. HEK293 cells were maintained in Dulbecco's Modified Eagle's 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 with 5% C02 in a humidified atmosphere.

[0200] 1.2 DNA vector DNA vectors pO6A19 encoding the following were synthesized by GenScript: 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 with S190C (SEQ ID NO: 21), or HERV-K GAG-ENV ISDmut (Q525A) with S190C (SEQ ID NO: 25). The targeted genes were preceded by a strong cytomegalovirus promoter and tetracycline operator (TetO) sites, and followed by an SV40 polyadenylation signal. The insert with the gene of interest for the vector encoded the consensus HERV-K GAG gene linked via a glycine / serine / glycine (GSG) linker to the envelope (ENV), envelope ISDmut (ENV ISDmut) (Q525A), or ENV ISD variants, or ENV with S190C, or ENV ISDmut (Q525A) with S190C gene, followed by a self-cleaving porcine teschovirus-1 2A peptide (P2A). The synthetic sequence constructs are outlined in the figure descriptions of Figure 1 and 4 .

[0201] 1.3 Amplification of DNA DNA vectors were transformed by heat shock into PIR-1 competent E. coli and expanded overnight in LB media containing 25 ug / mL kanamycin. (The Pir gene encodes replication protein π, which is required for replication and maintenance of DNA vectors containing the R6K gamma origin. The PIR-1 competent E. coli strain contains a mutant allele of the pir gene that maintains donor vector constructs at ~250 copies / cell.) Plasmids were then purified using Nucleobond Xtra Midi Kit following the manufacturer’s protocol (AH diagnostics).

[0202] 1.4 RNA generation The consensus HERV-K GAG gene was linked via a glycine / serine / glycine (GSG) linker to the Envelope ISDmut (EnvISDmut) gene, followed by a self-cleaving porcine teschovirus-1 2A peptide (P2A). The synthetic GOI was used as a template by Vectorbuilder and synthesized with flanking 5’ UTR, Kozak sequence, 3’ UTR, and polyA (SEQ ID NO: 45). The mRNA was manufactured by Vectorbuilder. The synthetic sequence construct is outlined in the figure description.

[0203] In addition to the mRNA construct described above, two additional mRNA constructs were synthesized. The consensus HERV-K GAG protein was encoded linked via a glycine / serine / glycine (GSG) linker to the Envelope (ENV) or Envelope ISDmut (ENV ISDmut) protein, followed by a self-cleaving porcine teschovirus-1 2A peptide (P2A). These synthetic GOIs were used as a synthesis template by Vectorbuilder and synthesized with flanking 5’ UTR, Kozak sequence, 3’ UTR, and polyA tail, outlined in SEQ ID NO: 36 and 37. Both of these mRNA constructs were modified with N1 -methylpseudouridine (m1Ψ). The synthetic sequence construct is also outlined in the figure description of Figure 3 .

[0204] 1.5 In vitro transfection HEK293 cells were seeded at 200,000 cells per well in 24-well plates 23-24 hours prior to transfection. Cells were transfected with 2.4 ug mRNA and 5.6 µL of Lipofectamine Messenger MAX or with 1 ug and 2 µL JetPEI (Polyplus) DNA per well. Transfected cells were incubated at 37°C with 5% CO2 in a humidified atmosphere for 17 hours Figure 1 ) or 24 hours Figure 3and Figure 4 ).

[0205] 1.6 Staining of cell surface Cells were harvested and washed with PBS containing 1% BSA and 0.1% NaN3. Cells were stained for 30 minutes with the HERV-K ENV-specific antibody HERM 1811-5 (Austral Biologics) conjugated with AlexaFluor647 (Molecular probes). Cells were washed with PBS and incubated with the viability dye eFluor 780 (eBioscience). The reaction was terminated by adding PBS containing 1% BSA and 0.1% NaN3, and the cells were washed with PBS. 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 run for flow cytometry on Fortessa 3 or 5, and results were analyzed using FlowJo software V10.7.1 or V10.8.1. Results are shown in [image / image / etc.]. Figure 1 , 3 and 4 in.

[0206] Example 2 LNP LNPs can be produced by obtaining the nucleotides of the present invention, such as mRNA, and following, 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 The method disclosed in Sci Rep 12, 9483 or another suitable method known in the art.

[0207] In a preferred embodiment, 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-(sperminecarbamoylamino)ethyl]-N,N-dimethyl-1-propaneonium trifluoroacetate) and DOPE, wherein preferably DOSPA and DOPE are in a molar ratio of 2:1 to 4:1. The formulations as outlined in Examples 1.4 and 1.5 above can be used to deliver mRNA into target cells.

[0208] Example 3 NF-κΒ activation / inhibition was determined by transfection of HEK293T cells in 96-well plates with a mix of 50 ng of a reporter plasmid expressing luciferase after NF-κΒ expression, 0-50 ng of the selected DNA plasmid encoding HERV-K Env protein, and 0.6 μΐ lipofectamine / well. The total DNA amount was adjusted to 100 ng using empty plasmid p06A5tetO empty (IPT22). For Figure 2 Based on the results shown in Table 1, 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.

[0209] The day after transfection, cells were analyzed for luciferase expression by addition of SteadyLite luciferase substrate (PerkinElmer) followed by quantification of luminescence. Luminescence can be measured by suitable methods known in the art. The experiments can be performed, e.g., essentially as described by 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. January 12, 2020 Figure 2 Figure 5 ) or according to a protocol derived from said protocol. In the assay, HERV-K with the mutated ISD sequence of SEQ ID NO: 4 exhibited about 50% reduction of NF-κΒ compared to cells transfected with control plasmid, as measured in the luciferase assay. The results are shown in Figure 6

[0210] Example 4 4.1 Animal procedures and serum isolation All animal procedures were performed in accordance with the Danish National Guidelines and the experimental procedures were approved by the National Animal Experimental Inspectorate (Dyreforsøgstilsynet), Denmark. Female CB6F1 mice were obtained from Envigo at 6-8 weeks of age and housed at the Panum Institute, University of Copenhagen for at least one week before any experiments.

[0211] 4.2 Immunization and serum isolation ​Mice were immunized with mRNA-HERV-K GAG-ENV ISDmut (SEQ ID NO: 36) containing N1-methylpseudouridine (m1Ψ) in a homologous priming-boost regimen. The primary immunization was administered on day 0, followed by a booster immunization on day 7. Immune responses were analyzed 14 days after the booster, i.e., day 21 following the primary immunization on day 0. The immunization schedule is shown below. Figure 7 middle.

[0212] Prior to injection, the mRNA was diluted in OptiMEM and mixed with Lipofectamine RNAiMAX at a ratio of 1:2 µg mRNA to µL RNAiMAX. Each mouse received a dose of 3.5 µg mRNA in 200 µL of the solution via intravenous (iv) injection. On day 21, blood samples were obtained from bleeding from the mouse's cheek. Serum was separated from the obtained blood samples by two consecutive centrifugation steps, each at 800g and 8°C for 8 minutes. At the end of the immunization study, the mice were euthanized by cervical dislocation.

[0213] 4.3 Spleen cell suspension Spleens were aseptically removed from euthanized mice (at the end of the immunization study) and transferred to RPMI 1640 GlutaMax (complete RPMI) supplemented with 10% heat-inactivated FBS, 1% Pen / Strep, and 1% sodium pyruvate. Single-cell suspensions were obtained by squeezing the spleen through a fine mesh (70 µm mesh) followed by centrifugation of the spleen cells.

[0214] 4.4 Tetramer staining Mouse spleen cells 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 min. The spleen cells were then centrifuged and incubated with the respective tetramers, wherein 0.08 µg to 0.25 µg of the relevant tetramer in FACS buffer containing 50 nM dasatinib was added to 500,000 cells in the dark at 37°C and 5% CO2 for 15 min.

[0215] - TET18: The peptide TYHMVSGMSL with an H2Kd MHC class I monoclonal antibody, labeled with bright purple BV421 provided by Immunitrack; - TET29: Contains the H2Kb MHC class I monoclonal antibody peptide QNVDYNQL, labeled with an allophycocyanin fluorescent dye (APC) provided by Immunitrack; and - TET90: peptide EPYPDFVARL with H2Kb MHC class I monoclonal antibody, labeled with phycoerythrin fluorochrome (PE) provided by Immunitrack.

[0216] In FACS buffer with 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, and viability dye (Efluor780) were added on top of the tetramer mix and incubated for 20 min at 4°C in the dark. Cells were then washed and fixed with 1% paraformaldehyde (PFA) for 15 min at 4°C in the dark. Fixed samples from tetramer staining were run on a Fortessa 3 flow cytometer and analyzed using FlowJo software V10.7.1. Results are shown in ​

[0217] 4.5 ELISA MaxiSorp (NUNC) flat bottom plates were coated overnight at 4°C with HERV-K ENV (envelope protein) transmembrane subunit (TM) or HERV-K surface subunit (SU) protein at 2 pg / mL in PBS. Plates were washed three times with wash buffer (PBS + 354 mM NaCl + 0.1% Tween20, pH 7.2) and then blocked for 1 hour at room temperature (approximately 21 °C) using blocking buffer (PBS + 354 mM NaCl + 5 g / L BSA + 0.05% Tween20, pH 7.2). After removal of the blocking buffer and washing the plates three times with wash buffer, 100 pL / well of serum sample diluted 1 :25 in blocking buffer was added to the wells, followed by a 1 hour incubation at room temperature (approximately 21 °C). Plates were washed three times with wash buffer and horseradish peroxidase (HRP)-conjugated polyclonal anti-mouse / IgG secondary antibody (Dako, P0260) was added at a dilution of 1 :2000 in blocking buffer and the samples were incubated for 1 hour at room temperature (approximately 21 °C). After three wash steps with wash buffer, 50 pL of 3,3',5,5'-tetramethylbenzidine (TMB) PLUS 2 chromogenic substrate for horseradish peroxidase (Kem-en-tec, 4395A) was added. After 6 minutes, the colorimetric reaction was stopped by adding 50 pL of 0.2 M H2SO4.

[0218] ​The color intensity in each well was determined by quantification of the optical density (OD) at a wavelength of 450 nm (in a SpectraMax Microplate Reader). The background signal measured for samples from non-immunized mice was subtracted for each sample. The results are shown in ​

[0219] Various aspects and implementations have been described in conjunction with the various embodiments. However, other variations to the disclosed implementations can be understood and effected within the scope of the claimed subject matter, from an inspection of the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite articles "a" or "an" do not exclude a plurality. A single processor or other unit can fulfill the functions of several items recited in the claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. A computer program can be stored / distributed on a suitable medium, such as an optical storage medium or a solid-state storage medium supplied together with or as part of other hardware, but can also be distributed in other forms, such as via the Internet or other wired or wireless telecommunication systems.

[0220] The sequences are disclosed according to WIPO Standard ST.25 in the body of the specification and in a separate sequence listing. A SEQ ID no. specified with a specific number should be the same in the body of the specification and in the separate sequence listing. For example, SEQ ID no.: 1 should define the same sequence in both the body of the specification and in the separate sequence listing. If there is a discrepancy between the sequence definitions in the body of the specification and in the separate sequence listing (if, for example, SEQ ID no.: 1 in the body of the specification erroneously corresponds to SEQ ID no.: 2 in the separate sequence listing), in the application, in particular in the specific embodiments, a reference to a specific sequence should be understood as a reference to the sequence in the body of the specification and not to the separate sequence listing. In other words, a discrepancy between the sequence definitions / names in the body of the specification and in the separate sequence listing is resolved by correcting the separate sequence listing to the sequences and their names disclosed in the body of the application, which includes the specification, examples, drawings, and claims.

[0221] Example 5 The composition according to the application can be produced by the following examples 1.4 and 2 as outlined above.

[0222] ​In a preferred embodiment, the composition or any other composition of the application described herein can be administered to a patient suffering from an age-related disease. It is known that retroviruses including HERV can be reactivated in the elderly and cause disease symptoms (see Zlotorynski, E. Younger endogenous retroviruses make us older. Nat Rev Mol Cell Biol 24, 165 (2023)).

[0223] Accordingly, administration of the composition of the application to such patients is expected to improve these symptoms. The skilled person can test several doses to find the amount sufficient to improve these symptoms.

[0224] Table 1: Peptide sequences of HERV-K Env with mutated ISD, wild-type ISD and ISD variants .

[0225] Table 2: Nucleotide sequence of mRNA encoding HERV-K Env (ISD coding sequence underlined) and Gag .

[0226] Table 3: Peptide sequences of different mutated ISD variants and HERV-K Env and different mutated ISD variants .

[0227] Table 4: Peptide sequences of HERV-K Env with different ISD variants and stabilizing mutation S190C .

[0228] Table 5: Nucleotide sequence of mRNA constructs for transfection / immunization .

[0229] In any of the sequences according to SEQ ID NO: 5, 45, 36 and 37, T can be replaced by mlY (N1 -methyl pseudouridine). This can have an mRNA stabilizing effect.

[0230] The sequence of SEQ ID NO: 5 can comprise at the DNA level an initial nucleotide sequence GGG preceding the 5' UTR.

[0231] The sequences of SEQ ID NO: 36 and 37 can comprise at the DNA level an initial nucleotide sequence GGG preceding the 5' UTR.

[0232] The sequence of SEQ ID NO: 45 can comprise at the DNA level an initial nucleotide sequence GGGAG preceding the 5' UTR.

Claims

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

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

3. The composition of claim 1, wherein the transfection agent is a transfection agent comprising cationic lipids and / or cationic polymers, and preferably wherein the composition comprises liposomes comprising cationic lipids and the mRNA.

4. The composition according to claim 2, wherein the LNP composition comprises at least one lipid selected from: (i) Ionizable lipids, preferably ionizable cationic lipids, more preferably ionizable cationic amino lipids; (ii) Non-cationic auxiliary lipids or phospholipids, wherein the lipids are preferably neutral, and more preferably DSPCs; (iii) sterols or other structural lipids, wherein the sterols are preferably cholesterol; and (iv) PEG lipids, preferably PEG-DMG.

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 the preceding claims, wherein the mRNA comprises at least 60 adenosine nucleotides at the 3'-UTR.

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

8. The composition according to any one of the preceding claims, wherein the mRNA comprises at least 300 nucleotides.

9. The composition according to any one of the preceding claims, wherein the mRNA does not contain artificially modified nucleotides.

10. The composition according to any one of the preceding claims, wherein the human endogenous retrovirus (HERV) is selected from HERV-K, HERV-H, HERV-W, HERV-FRD, HERV-E, HERV-9, HERV-FC, HERV-T, HERV-3, HERV-V1, and HERV-V2. Furthermore, the HERV-K is preferably selected from 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-18); HERV-H is selected from HERV-H19 (=HERV-H_2q24.3) and HERV-H_2q24.1; HERV-W is selected as ERVW-1 (=syncytokine-1); and HERV-FRD is selected as ERVFRD-1 (=syncytokine-2).

11. The composition according to any one of the preceding claims, wherein the HERV envelope protein comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 2 over its entire length.

12. The composition according to any one of the preceding claims, wherein the mutated ISD, compared with a wild-type ISD that is not mutated and has the amino acid sequence according to SEQ ID NO: 3, has less inhibition of human immune cell proliferation, and / or has a reduced or lost ability to induce IL-10 secretion from peripheral blood mononuclear cells when the cells are contacted with the mutated ISD, and / or reduced NF-κB expression.

13. The composition according to any one of the preceding claims, wherein the mutated ISD comprises or is composed of the following amino acid sequence: One or two single amino acids at any of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14 are replaced by different amino acids, preferably alanine in each case, so as to inactivate ISD.

14. The composition according to any one of the preceding claims, wherein (a) The human endogenous retrovirus (HERV) is HERV-K, and the ISD mutation is selected from L522A, A523Q, N524A, Q525A, I526A, N527A, D528A, L529A, R530A, Q531A, T532A, V533A, I534A, and W535A, preferably wherein the mutated ISD comprises a sequence according to any one of SEQ ID NO: 4 or 8-20; Even more preferably, the ISD mutation is selected from L522A, A523Q, N524A, Q525A, N527A, L529A, R530A, Q531A, and T532A, and even more preferably, the mutated ISD comprises a sequence according to any one of SEQ ID NO: 4, 8-12, 14-17, or 20; or (b) The human endogenous retrovirus (HERV) is a HERV selected from HERV-9, HERV-FC, HERV-T, HERV-E, HERV-3, HERV-V1, and HERV-V2, and the mutated immunosuppressive domain (ISD) that reduces the immunosuppressive properties compared to the wild-type ISD contains one or more of the following sequences consisting of 23 amino acid positions: Regarding HERV-9, LQNCZGLDLLTAEKGGLCTFLGE (SEQ ID NO: 38); Regarding HERV-FC, AQNRRALDLLTADKGGTCLFLGE (SEQ ID NO: 39); Regarding HERV-T, LQNRRGLDLLFLSQGGLCAALGE (SEQ ID NO: 40); YQNRLALDYLLAAEGGVCGKFNL (SEQ ID NO: 41) regarding HERV-E; YQNRLALDYLLAQEGGVCGKFNL (SEQ ID NO: 42) regarding HERV-3; Regarding HERV-V1, MNNRLALDYLLAEQGGVCAVISK (SEQ ID NO: 43); or Regarding HERV-V2, MDNRLALDYLLAEQGGVCAVINK (SEQ ID NO: 44), In SEQ ID NO: 38, 40, 41, 42, 43 and 44, one or more amino acids are replaced by different amino acids, preferably wherein the amino acid at position 14 is replaced by a different amino acid, and more preferably replaced by R, and preferably wherein the single amino acid at position 20 is replaced by a different amino acid, and more preferably replaced by F.

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

16. The composition according to any one of the preceding claims, wherein the HERV envelope protein comprises an even number of cysteine ​​residues, and / or The HERV envelope protein contains at least 18 cysteine ​​residues, and / or The HERV envelope protein contains the following amino acid sequence: At least one amino acid at a position in the range of 170 to 210 has been replaced by a cysteine. Preferably, the HERV envelope protein contains a cysteine ​​residue at position 190, and more preferably, the HERV envelope protein contains an 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, wherein the HERV envelope protein contains a cysteine ​​residue at the position corresponding to amino acid 190 of SEQ ID NO: 2 within the HERV envelope protein.

17. The composition according to any one of the preceding claims, wherein the HERV envelope protein comprises or is composed of sequences selected from or consisting of sequences selected from SEQ ID NO: 21-35, preferably selected from SEQ ID NO: 22-27, 29-32 and 35 sequence.

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

19. The composition according to any one of the preceding claims, wherein the composition comprises mRNA having at least 90% sequence identity, and most preferably 100% sequence identity, with respect to any sequence according to any one of SEQ ID NO: 45, 36 or 37.

20. The composition according to any one of claims 16 or 17, wherein the cell surface expression of the HERV envelope protein according to claim 16 or 17 is increased compared with the cell surface expression of the HERV envelope protein having the amino acid sequence according to SEQ ID:

2.

21. The composition according to any one of claims 1 to 19, used as a pharmaceutical agent.

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

23. The composition according to any one of claims 1 to 19, for the prevention or treatment of a disease, preferably for immunizing a subject against a disease.

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

25. A method for treating or preventing a disease in a patient with this need, the method comprising: The patient is given a pharmaceutically acceptable liquid composition comprising a therapeutically effective amount of the composition according to any one of claims 1 to 19.

26. Use of the composition according to claim 23 or the composition according to claim 23, wherein the disease is preferably selected from the following: cancer, HIV and / or related conditions, 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 transactivation response DNA-binding protein 43 kDa (TDP-43) and / or its C-terminal fragment, and / or Preferably, Alzheimer's disease is associated with Tau protein expression.

27. Use of the composition or composition according to claim 26, Preferably, the cancer is a cancer that expresses HERV. More preferably selected from the following: tumors expressing PD-L1, cervical cancer, penile cancer, anal cancer, vulvar cancer, vaginal cancer, bladder cancer, breast cancer, clear cell renal cell carcinoma, head / 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 (CLL), chronic myeloid leukemia (CML), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, Hodgkin's lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myeloid leukemia-1 protein (Mcl-1), myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL), and small lymphocytic lymphoma (SLL).

28. The composition according to any one of claims 1 to 19, for preventing or slowing aging and / or cellular senescence.

29. The composition according to any one of claims 1 to 19, used to manufacture a medicament for preventing or slowing aging and / or cellular senescence.

30. A pharmaceutical composition comprising the composition according to any one of claims 1 to 19, wherein the composition comprises 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 according to any one of claims 1 to 19.

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