WO2021186042A1 - Plasmid comprising alopecia antigen - Google Patents

Abstract

The present invention relates to plasmids encoding an alopecia associated antigen and cytokines TGF-, IL-2, and IL-10 useful for treatment, prevention and/or delay onset of e.g. alopecia especially alopecia areata.

Description

PLASMID COMPRISING ALOPECIA ANTIGEN

TECHNICAL FIELD

The present invention relates a plasmid comprising an alopecia associated antigen, a method for making said plasmid, and the plasmid for use in the delaying onset, prevention and/or treatment of alopecia.

INCORPORATION-BY-REFERENCE OF THE SEQUENCE LISTING

The Sequence Listing, entitled “200002W001_ST25” is 57.547 bytes, was created on 19-Mar-2021 and is incorporated herein by reference.

BACKGROUND

Alopecia Areata (AA) is an autoimmune disease affecting the hair follicles, resulting in hair loss and is affecting 1.7-2% of the US population and is thus, one of the most common autoimmune diseases linked to T cells. It is thought to be a cell-mediated autoimmune disorder in which the body attacks its own anagen hair follicles. There is no cure for the disorder today, but it is usually treated with corticosteroids and the use of Janus Kinase (JAK) inhibitors has shown some promise in clinical trials. Thus, there is a need for improved treatment.

Autoimmune diseases in general are characterized by leukocyte recognition of ‘self antigens’, i.e. molecular structures belonging to host tissues. Alopecia is an example of an autoimmune disease, and literature indicates that leukocytes can be found in patients that show reactivity against said ‘self antigens’ (J Invest Dermatol. 2016 Aug;136(8):1617-1626, J Proteome Res. 2010 Oct 1;9(10):5153-63). The association of such ‘self antigens’ and the leukocytes that recognize them derives from in vitro experimentation, showing that a fraction of blood leukocytes react to the presence of the ‘self antigen’. This observation does not establish the ‘self antigen’ as a cause of disease or a target for therapeutic intervention.

DNA immuno-therapy vaccines have previously been used against other T cell driven diseases e.g. type 1 diabetes (WO/2018/083111). According to traditional vaccine approaches, purified protein/antigen is injected in a person/patient/animal in order to stimulate immune responses specifically to that protein/antigen. This vaccine approach tends to impact primarily antibody production, while the T cells tend not to be significantly affected, other than to generate T cell memory of the antigen. Traditional vaccine approaches are thus not considered suitable in connection with treatment and/or prevention of T cell driven diseases such as alopecia e.g. Alopecia Areata (AA), as activation of T cells, especially NKG2D⁺ CD8⁺ T cells, are considered the causative agent of this disease. Experimental approaches with tolerogenic, protein-based vaccines have targeted primarily antibody producing B cells rather than disease relevant T cells.

DNA based vaccines, in contrast to protein-based vaccines, are usually plasmids encoding particular antigens - these plasmids are taken up by cells in the host’s body (“transfected”). These transfected host cells then process the antigen into small fragments (T cell epitopes) for presentation to circulating T cells. As T cells can only detect these small antigen fragments and not whole proteins, this approach preferentially leads to a modification of T cell responses, especially for CD8⁺ T cells (or cytotoxic T cells), the key drivers of e.g.

AA pathology. Thus, DNA vaccines, rather than protein vaccines, are suitable for inducing T cell responses. While no DNA vaccines are currently available for human use, there are some stimulatory plasmid DNA vaccines licensed for veterinary use, inducing immunity to Equine Infectious Anemia Virus, West Nile Virus, and certain canine cancers.

In contrast to stimulatory DNA vaccines, tolerogenic DNA immuno-therapy vaccines are intended to suppress immune reactivity towards an antigen, rather than activating immune responses against it. These vaccines do not stimulate immunity against the encoded antigen, or change the type of stimulation (as e.g. antigenic desensitization vaccination approaches for allergies does), but instead cause depletion, and/or lack of function, and/or death of self-reactive T cells. In order to do so, the antigen must be presented to the immune system without co-stimulation or inflammatory effects, which would otherwise prime stimulatory immune responses. This approach of presenting an antigen to be ignored by the immune system, or tolerized against, could be of value in treating autoimmune diseases, as the specific mechanism of the disease would thus be targeted rather than systemically suppressing the entire immune response. A tolerogenic DNA immuno-therapy vaccine is thus a mild method of modulating undesired immune responses.

The end goal of an AA-specific tolerogenic DNA immuno-therapy vaccine is to preserve hair follicles. This may occur through prevention or delay of onset of the disease.

While DNA based vaccines are known to be safe, none of the (stimulatory or tolerogenic) DNA vaccines that have been tested in clinical studies have sufficient potency as a stand-alone approach for treatment. Tolerogenic DNA vaccines known in the art showed little efficacy and typically required highly artificial systems to induce the desired effects. There is thus a need in the art for tolerogenic DNA immuno-therapy vaccines with significantly increased potency, without compromising the safety profile and preferably also without requiring an inconvenient administration regimen. SUMMARY

The present invention relates to a multi-cistronic vector/plasmid which co expresses/encodes a cellularly retained alopecia associated antigen, such as an antigen comprising fragments of gp100 and MARTI, as well as secreted immune modifiers such as TGF-b, IL-10, and IL-2. The present invention furthermore relates to DNA immuno-therapy vaccines comprising such plasmids as well as such pharmaceutical formulations and kits thereof. The present invention finally relates to the medicinal use of such products in the treatment of alopecia, such as Alopecia Areata (AA) as well as methods for producing such plasmids.

The plasmids/DNA immuno-therapy vaccines herein have therapeutic potential in treatment of autoimmune diseases that are mainly T cell driven, such as alopecia including Alopecia Areata (AA).

In one aspect, the present invention provides a plasmid which encodes: i. an alopecia associated antigen; ii. TGF-b; iii. IL-10, and iv. IL-2

Also or alternatively, in a further aspect, the present invention provides a DNA immuno-therapy vaccine comprising a plasmid encoding i) an alopecia associated antigen, ii) TGF-b, iii) IL-10, and iv) IL-2.

Also or alternatively, in a further aspect, the present invention provides a pharmaceutical composition comprising a plasmid encoding i) an alopecia associated antigen, ii) TGF-b, iii) IL-10, and iv) IL-2.

Also or alternatively, in a further aspect, the present invention provides the use of a plasmid encoding i) an alopecia associated antigen, ii) TGF-b, iii) IL-10, and iv) IL-2 in delaying, preventing or treating alopecia.

In one aspect, the present invention shows that therapeutic administration of an alopecia associated ‘self antigen’, e.g. in form of the present plasmid, alter the course of disease in alopecia. This is surprising in that it constitutes the first evidence that alopecia per se is treatable by the concept of antigen-specific immuno-therapy. Similar approaches in other autoimmune disease models for multiple sclerosis and rheumatoid arthritis failed, confirming that going from identification of ‘self antigens’ to therapy using those self antigens is not a trivial step.

In another aspect, the present invention defines specific immuno-dominant antigen that confers protective benefit against alopecia. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Circular plasmid map of mouse-derived plasmid.

Figure 2. Circular plasmid map for a proposed human plasmid.

Figure 3. Incidence of hair loss amongst C3H/HeJ mice injected with AA lymph node cells at week 0 and during dosing for 13 weeks with PBS, an empty plasmid, or AA plasmid.

Figure 4. Incidence of hair loss amongst C3H/HeJ mice injected with AA lymph node cells at week 0 and during dosing for 13 weeks with PBS, an empty plasmid, or AA plasmid and following withdrawal to week 22.

Figure 5. Presence of disease-driving CD8 T cells at study end in spleen and skin-draining lymph nodes in mice with and without hair loss.

DESCRIPTION

The inventor of the present invention has herein provided a single vector which drives expression of multiple secreted cytokines, as well as a cellularly retained alopecia associated antigen, from a single promoter/multi-cistronic mRNA.

DNA immuno-therapy vaccination with a single vector encoding all components of the therapy in a single cell is highly preferred over immuno-therapy vaccination with a mixture of separate vectors/plasmids each driving expression of single components, as random transfection of cells with different vectors does not guarantee expression of all components, or even any specific ratio of components, from a given, specific transfected cell.

Transfection of a single multi-cistronic plasmid/vector results in a specifically engineered local environment/micro-environment around the transfected cell. In this way, combinations of immuno-modulators can be added to the antigen such that they potentiate the desired immunologic effect of single T cells without the requirement of high systemic immuno-modulator doses that could otherwise cause adverse events and broad immunosuppression.

This local restriction of immuno-modulator production of host cells transfected with the DNA immuno-therapy vaccine allows for the safe use of highly potent cytokine hormones, which are synergistic for modification of T cell responses, but cannot be dosed either frequently enough for effect, and/or titrated to give the desired response, without unacceptable adverse events. For example, Interleukin-10 (IL-10) and Transforming Growth Factor-betal (TGF-bI) are both known to be able to induce regulatory T cells (Tregs) from naive CD4⁺ T cells. However, the IL-IO/TGF-bI combination provides a synergistic effect (15 to 20 fold more efficacious) in inducing Tregs than either of the two cytokines alone (US6083919 A) and this combination furthermore results in immune tolerance in a broader population of target cells than either cytokine alone (Zeller JC, Panoskaltsis-Mortari A, Murphy WJ, et. al. 1999 J Immunol. 163(7):3684-91).

Additionally, lnterleukin-2 (IL-2) is known to both expand and stabilize Tregs but may on the other hand also contribute to inflammatory responses. The combination of IL-2 and IL-10, however, results in suppressive Tregs rather than inflammatory stimulation. As circulating T cells encounter cells that are transfected with the DNA immuno-therapy vaccine herein, they are temporarily exposed to sub-optimal concentrations of IL-10 and IL-2. The circulating T cells are slightly biased toward tolerance, and if they are also reactive toward the co-expressed antigen (e.g. an alopecia associated antigen) they will bind to the transfected cell and thus receive a longer duration of immuno-modulator exposure and in addition they will also receive another signal that programs/re-educates them for suppressive effects. In this way, those T cells which are responsive to the encoded antigen are selectively re-educated to a suppressive phenotype when they encounter the transfected cell.

The plasmids/vectors/DNA immuno-therapy vaccines herein are thus designed for induction of antigen specific Tregs accumulating at sites of autoimmunity to dampen disease (e.g. the hair follicles in AA) rather than to directly impact disease through the expressed cytokine hormones.

In addition to an alopecia associated antigen, the vector/operon/plasmid herein encodes at least two cytokines (e.g. TGF-bI and IL-10) which together synergistically suppress antigen presenting cells, as well as T cell function, and drive induction of Tregs.

This effect is enhanced if it also occurs in combination with effective exposure to antigen.

In one embodiment, TGF-bI is in a constitutively active form that does not require processing or an inflammatory environment for function. While Tregs can be produced from naive T cells via exposure to antigen and TGF-bI, Tregs are, however, “plastic” meaning that they can de-differentiate and convert into Th17 effector cells and then cause more, not less, autoimmune destruction. The combination of IL-10 with TGF-bI, in addition to being a more potent immuno-modulator, suppresses the environment that would produce pathogenic Th17 cells rather than Tregs.

In one embodiment, the multi-cistronic vector herein also encodes IL-2 in addition to the alopecia associated antigen, TGF-bI, and IL-10. IL-2 expands Treg numbers and stabilizes their phenotype (prevents Treg cells from de-differentiation into effector T cells) and thus increases their functional lifespan in inflamed target tissues.

These three cytokines (TGF-bI, IL-10, and IL-2), in combination with antigen, thus have well-known synergistic effects for inducing tolerance by the following mechanisms: (i) significantly enhanced generation of antigen-specific suppressive Tregs, (ii) longer Treg lifespan, and (iii) greater efficacy per individual Treg cell in suppressing inflammation/auto reactivity. However, the required concentrations of systemically infused purified cytokine would have a number of serious, or maybe even lethal, side effects, such as: (i) lethal fibrosis from excess TGF-bI, (ii) flu-like symptoms, (iii) capillary leak syndrome from excess IL-2, (iv) broad immunosuppression leading to chronic infections, (v) enhanced tumor development as well as (vi) anemia from excess IL-10.

By co-expressing these cytokines from the same vector/plasmid, and therefore by the same cell presenting the alopecia associated antigen to the immune system, the vector achieves the desired local environment for tolerance induction without systemic action and corresponding side-effects that would otherwise result from high-dose purified cytokine administration.

Injection of “nakedYbare” plasmid/vector DNA (vector and buffer alone) has a very low uptake and transfection rate - fewer than one in about 100,000 plasmid molecules transfects a cell, while the rest are degraded and thus without any biological effect. This extremely low inefficiency of transfection provides a safety mechanism for distributing and limiting the transfected cells.

Administration of systemically active quantities of any of these cytokines, either by administration of mature proteins or by high-efficiency viral vector transduction, would be difficult, if not impossible, to titrate for a safe and effective dose. Limiting the total exposure to a very small systemic dose distributed in a few high expressing micro-environments leads to a highly advantageous safety and efficacy profile.

The combination of alopecia associated antigen and these three cytokines herein produces an efficient protection from development of AA. Due to the low transfection efficiency of the bare DNA plasmid/vector injection, very few cells produce these recombinant proteins and there is thus no detectable change in serum cytokine levels from plasmid/vector encoded cytokines - and therefore no detectable immune stimulation or immuno-suppression toward any other antigens than the alopecia associated antigen encoded by the plasmid/vector. This results in a desirable safety profile.

Normally, DNA vaccines perform poorly in connection with subcutaneous (s.c.) injection and are therefore typically administered using intra-muscular injection (often with electroporation) or alternatively using intradermal jet injection requiring a cumbersome device as well as significant maintenance and calibration. As most side effect issues with intra muscular injection are adjuvant- related (injection site irritation) they are therefore not a concern for the bare DNA immuno-therapy vaccine format herein. Additionally, the volumes injected are usually relatively small and therefore do not cause significant muscle distension and pain. In one embodiment, the volumes injected are 1 ml or less. In another embodiment, the volumes injected are approximately 0.6 or 0.5 ml. Regardless, the multi cytokine plasmid/vector provided herein unexpectedly appears to provide protection from AA even when administered through the s.c. route, thereby allowing multiple potential dosing formats for patients.

In addition to providing local synergy, by encoding all three or four of the translated products by a single plasmid/vector and a single promoter, the regulatory burden and drug substance release criteria are furthermore simplified with the provision of the multi-cistronic plasmid herein.

In contrast, if each of the protein products is produced from a separate plasmid, then the synergistic value of co-expression from the same transfected cell would then potentially be lost or reduced as each plasmid/vector transfection would be an independent event, likely targeting different cells. If the three to four recombinant proteins are produced from two, three, or four individual plasmids/vectors, any synergistic effects in the local environment of the transfected cell are potentially lost; in addition, several individual clinical trials would thus be necessary (one for each plasmid and each combination). Producing all proteins from a single plasmid/vector and single mRNA relieves the requirements to test multiple individual molecules and determining ideal co-packaging ratios inherent to a multiple plasmid/vector format.

Any vector formats suitable for the present invention can be used herein, such as plasmids (replicating or passive), mini-circles, linear vectors (MiLVs), viral vectors (both integrating [e.g. lentiviral] and non-integrating [e.g. adenoviral]), cosmids, bacterial artificial chromosomes (BACs), human artificial chromosomes (HACs), etc.

Furthermore, any permissible transfection enhancement method can be used herein: e.g. electroporation, sonoporation (ultrasound enhancement, with or without microbubble contrast enhancement), lipid/polymer aggregates, hydrodynamics (pressure via high injection volume), bio-ballistics/gene-gun (deposition through skin via compressed gas), etc.

In one embodiment, non-replicating episomal plasmid DNA is used herein due to: i) multiple copies of mRNA derived from a single plasmid transfection, and ii) extended stability and function of plasmid nucleic acids over mRNA and other DNA vector formats. Thus, while both mRNA and DNA-based expression systems can provide intracellular delivery and co localization, plasmid-based systems provide greater control and persistence of dosing.

In one embodiment, plasmids/vectors encode at least four protein elements: i) an alopecia associated antigen, ii) TGF beta 1 (TGF-bI), iii) Interleukin-10 (IL-10), and iv) lnterleukin-2 (IL-2).

In one embodiment, the alopecia associated antigen comprises gp100 and/or MARTI, or functional fragments of gp100 and/or MARTI In one embodiment, the alopecia associated antigen comprises one or more of fragments gp100(149-167), gp 100(204-222), gp100(275-293) and/or MARTI (22-40), such as e.g. SEQ ID NO: 7.

In one embodiment, TGF-bI is in an activated form, such as a constitutively active form.

Expression of four proteins from one plasmid/vector is possible e.g. if the desired sequences are separated either with A) separate promoters, B) an IRES (Internal Ribosome Entry Site) sequences which recruit a new ribosome to translate each segment, or C) viral 2A sequences (e.g. FMDV 2A/F2A or TaV 2A sequences) which are translated and induce a ribosomal pause/skip which results in production of separate polypeptides from a single open reading frame. However, in practice, each of these strategies is complex and difficult to enable.

Expression of four independent proteins from a single plasmid/vector is most easily achieved by having a separate promoter for each gene. However, this format has significant disadvantages in that it A) results in a very large, unstable, and hard to produce plasmid due to the excess length of multiple promoters, B) results in unpredictable behaviour of the translated proteins relative to each other (they are no longer produced in fixed ratios to each other), C) each promoter may be independently silenced, leading to selective expression of some genes but not others required for full efficacy, and D) a lack of regulatory simplicity. In contrast, IRES elements and 2A sequences operate on the mRNA and translation levels and reproducibly co-express fixed ratios of each protein from a single promoter.

Each of the four classes of IRES elements has different co-factor requirements for function as well as different sequence requirements for the downstream gene to be translated. For instance, the EMCV (EndoMyoCarditis Virus) IRES is a 630 base pair type 1 IRES which utilizes all eukaryotic translation initiation factors while the CrPv (Cricket Paralysis virus) IRES is a 200 base pair type 4 IRES that has no required cofactors but utilizes a non-standard initiation codon.

When IRES elements from different classes are utilized, they interfere with each other such that each type of IRES element can only be used once in each plasmid, and when used together, different types of IRES elements attenuate each other (decrease in efficacy) in ways that are difficult to predict.

Furthermore, shuffling the gene/I RES combinations result in unpredictable ratios of translated products as the interactions of the genes with the IRES elements are not static but context dependant on the flanking nucleotide sequences. In addition, IRES elements impose restrictions on the first few amino acid positions at or immediately following initiation. For instance, the CrPv IRES requires that the first amino acid be an alanine rather than the standard methionine and the EMCV IRES cannot tolerate P, W, C, R, or K amino acids within the first three codons. In one embodiment, to accommodate the N-terminal amino acid restrictions imposed by the EMCV IRES, the DNA vaccine contains a three Alanine extension to the N-terminal of the IL-10 gene.

In addition, each IRES element comprises a substantial number of base pairs, ranging from 230 bp to over 700 bp; the inclusion of multiple IRES elements thus increases the size and complexity of plasmids/vectors to the extent that many become unstable and difficult to be industrially produced due to spontaneous deletions and recombinations.

Further, due to the high degree of secondary structure that IRES elements impart on the transcribed mRNAs that contain them, they increase the probability of activating pathogen recognition receptors (Dabo S, Meurs EF. 2012 Viruses 4(11):2598-635) in the transfected cell and producing stimulatory effects counter to the tolerance induction that is intended.

2A sequences, unlike IRES elements, do not interact with each other and therefore provide stable and consistent performance. However, they are translated themselves and therefore affect the folding, function, and stability of the final translated protein products. All 2A sequences result in a significant C-terminal fusion (19-22 aa) onto the 5’ end of the sequences to be separated and also begin the 3’ sequence with a proline. Some proteins are permissive of these modifications and some are not, leading to practical restrictions to the use of 2A sequences. For instance, the Interleukin- 10 product is permissive of the 2A tail but both lnterleukin-2 and TGF-bI mis-fold and lose function if expressed upstream of a 2A tag. Therefore, while it is possible to express several independent proteins separated by 2A sequences, two of the four proteins herein cannot terminate in 2A tags and therefore other strategies must be utilized. As each type of 2A amino acid sequence modifies ribosomal function during protein translation, it will have different efficiencies in the two core properties of the 2A family namely (i) separation of the juxtaposed gene products and (ii) processivity (re-initiation) into the second gene product. Different 2A sequences have different efficiencies at generating the ribosomal pause that breaks the peptide backbone (resulting in the two separate proteins) as well as different efficiencies at re-initiating the peptide synthesis of the second gene product.

The ability of the 2A sequences to separate protein products and re-initiate protein translation are dependent on the 2A amino acid sequence (Donnelly ML, Hughes LE, Luke G, et. al. 2001 J Gen Virol. 82(Pt 5): 1027-41). Small variations in 2A amino acid sequences result in significantly different mixes of separated and fused flanking gene products, ranging from under 5% (>95% fused) to completely separated (0% fused or 100% separated).

Furthermore, the inventor has herein discovered that adjacent amino acid sequences encoding the two flanking protein products also affect efficiency of re-initiation and separation of the 2A sequences, leading to significant deviations from reported results. Re-initiation efficiency thus varies depending on the type of 2A amino acid sequence used as well as the environment provided by the adjacent amino acid sequences, and thus the ratio of the pre-2A gene product and separation of the proteins will be determined by both the 2A amino acid sequence used and its context.

In one embodiment, “FMDV 2A” or “F2A” is inserted between the alopecia associated antigen encoding sequence and the TGF-bI encoding sequence herein; resulting in 100% separation, as well as a 1:1 ratio, of the protein products.

In another embodiment, “TaV 2A” may be inserted between the IL-10 encoding sequence and the IL-2 encoding sequence herein, resulting in about 50% separate products as well as a 10 to 6 ratio of the protein products. Each transfected cell thus delivers a relatively low dose of lnterleukin-2, that is incapable of stimulating effector T cells, and a higher dose of Interleukin- 10 to bias the T cells toward the Treg phenotype. Since the production of fused IL-10/IL-2 is disadvantageous, attempts to engineer increased cleavage efficiency of the TaV 2A segment were made. An attempt to precede the 2A segment with an “insulator segment”, which is an element that extends the translated region upstream of the TaV 2A to reduce upstream sequence impact on the 2A element, did not improve separation. In a different attempt to solve the fusion problem, an upstream uncoupler segment with a translated protein sequence of GSG was added; however, this approach resulted only in an incremental improvement of cleavage efficiency.

As such, cytokine fusions, resulting from separation of the IL-10 and IL-2 encoding genes by a TaV 2A, are likely to be immunogenic. In a further embodiment, the vector/plasmid herein has a “P2A” segment.

Separation of the IL-10 and IL-2 encoding genes by a P2A results in complete or near- complete separation of the protein products as well as a ratio of at least twice as much (or maybe even up to four or five times as much) IL-10 compared to IL-2.

In order to address the shortcomings of the IRES-only and 2A-only systems described above, the four cDNA sequences herein (antigen, TGF-bI, IL-10, IL-2) are arranged in pairs before and after a single IRES. Each pair is further separated by a 2A sequence, which induces ribosomal skipping and production of independent proteins from each sequence in the polyprotein pair. As TGF-bI and IL-2 may not be on the N-terminal side of the fusion, one of them must terminate at the central IRES site and the other one must end the translated portion of the mRNA sequence.

The chronology/sequence of expressed proteins and IRES/2A elements herein may therefore be as follows: (i) Alopecia associated antigen, (ii) F2A, (iii) TGF beta 1, (iv) IRES,

(v) IL-10, (vi) P2A, and (vii) IL-2. As a consequence, all four proteins can be independently expressed from a single operon/gene segment in a stable and predictable fashion. As each of these proteins is expressed from a single mRNA, the ratios of each product are fixed - it is not possible to generate an excess of IL-2 over IL-10 for instance.

Besides using a combination of IRES and 2A elements for separation of encoded genes, an alternative solution herein could be use of a bidirectional promoter to generate 2 mRNAs - these mRNAs would each encode a pair of proteins rather than all four in one mRNA molecule. Equivalent arrangements may therefore be constructed utilizing pairs of expression cassettes appropriately arranged around a bidirectional mammalian promoter and utilizing separating 2A sequences and/or IRES elements. This approach is, however, associated with disadvantages, primarily due to the large size of bi-directional promoters but also a potential increased regulatory burden having separate mRNA elements included in one medicinal product. Preferred embodiments herein therefore utilize a single promoter and a combination of IRES and 2A elements rather than a bidirectional promoter.

An exemplary EMCV IRES elements is (SEQ ID NO: 30): TAAACGCGTCGAGCATGCATCTAGGGCGGCCAATTCCGCCCCTCTCCCCCCCACCCCT CTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGC GTTT GTCTAT AT GTT ATTTTCCACC AT ATT GCCGTCTTTTGGCAAT GT G AGGGCCCGG AA ACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAA T GCAAGGTCT GTT GAAT GTCGT GAAGG AAGCAGTTCCT CTGG AAGCTT CTT G AAG ACAA ACAACGTCT GT AGCGACCCTTT GT AGACAGCGGAACCCCCCACCTGGCGAT AGATGCC TCT GCGGCCAAAAGCCACGT GT AT AAGAT ACACCTGCAAAGGCGGCACAACCCCAGT G CCACGTT GT GAGTT GG AT AGTT GTGG AAAGAGT CAAAT GGCTCTCCT CAAGCGTATT CA ACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCC TCGGTGCACAT GCTTT ACAT GT GTTT AGT CGAGGTT AAAAAACGTCT AGGCCCCCCGAA CCACGGGGACGTGGTTTTCCTTT GAAAAACACGAT GAT AAT AT G

In order for the alopecia associated antigen(s) to be processed and presented to the immune system in a local environment where plasmid-encoded cytokines are secreted, the antigen(s), whether under the form of protein or peptide fragments, must be retained within, and not released from, the transfected cell.

However, while any translated antigen product(s) would not be actively secreted, they could be released during lysis due to necrosis resulting from attack by CD8⁺ T cells. Additionally, cytoplasmic retention of antigen only allows for processing via the proteasome and presentation via the MHC class I pathway, which detects intracellular pathogens via CD8⁺ T cells. As CD4⁺ T cells are significant contributors to pro-inflammatory cytokines and most, if not all, autoimmunity suppressing Tregs are CD4⁺, broadening the presentation of antigen to include MHC class II, which is recognized by CD4⁺ T cells, may be advantageous.

MHC class II processing and CD4+ T cell stimulation normally do not include intracellular antigen, as access to this pathway is via endocytosis of extra cellular antigen. Normally, protein products produced within a transfected cell are only presented via the default intracellular/proteasomal processing pathway and MHC class I, resulting in CD8+ T cell effects but not CD4+ T cell effects. In order to target both CD4+ and CD8+ T cells for immunomodulation the preferred embodiment also includes factors leading to MHC class II presentation.

In principle, to induce MHC class II presentation, the antigen can be fused to any partner that directs the fusion to an endosomal compartment, but there are functional differences in activity and exposure. Transferrin receptor, also known as iron transporting protein receptor, fusions cycle from the plasma membrane/extracellular space to the endosome and therefore may also expose other immune cells to whole antigen, such as B cells, macrophages, etc. Limpll/SCARB fusions target directly to the endosome, but preferentially to the early endosome and sometimes result in over processing and total destruction of the antigen li (CD74) fusions, utilizing the same chaperone signal that MHC class II uses for late endosome localization, deliver the antigen and MHC class II to the same vesicles at the same developmental stage and maximize the likelihood of effectively presenting antigen in the context of MHC class II.

In one embodiment, the plasmid DNA vaccine is used herein. The plasmid is grown/replicated for example in E. coli, and isolated/purified from the media, and subsequently formulated in liquid formulations e.g. water, saline, PBS liquid formulations, or as a lyophilized powder for intradermal jet injection, intranasal administration, or inhalation. In one embodiment, the plasmid herein is formulated in an aqueous pharmaceutical formulation optionally comprising stabilizers. Any suitable microbial system may be utilized for plasmid production.

Stabilizers in the formulation include, but are not limited to, chelating agents, such as EDTA, EGTA, or DPTA for scavenging Mg⁺⁺ and Fe⁺⁺⁺ which may otherwise be involved in degradation of DNA, and/or citrate, which protects the plasmid from non-specific degradation effects. In one embodiment, the plasmid herein may be formulated in isotonic PBS or alternatively TRIS + citrate + EDTA. Such plasmids have the advantages of being stable, easy to produce and being safe and convenient in use.

In another embodiment, delivery agents, such as virus, lipids, liposomes, co packaging etc., could be added in connection with the present invention. However, the use of delivery agents herein may have potential problems with immunity, viral integration, etc.

Definitions

Alopecia associated antigen: the DNA immuno-therapy vaccine herein encodes an alopecia associated antigen. In one embodiment, the alopecia associated antigens herein are derived from e.g. gp100 and/or MARTI The antigen may comprise a combination of gp100, and MART 1 , or a combination of one or more functional fragments of gp100 and MARTI to form an alopecia minigene, e.g. SEQ ID NO: 7. Such fragments can be recognized by the T cell component of the immune system. A non-limiting example of an alopecia minigene comprises fragments of gp100 and MARTI, e.g. fragments selected from gp100(149-167), gp100(204-222), gp100(275-293) and/or MART1(22-40).

Antigen targeting: In one embodiment, antigen herein is endosomally targeted. Antigens herein include whole protein, secretion-deficient pre-proteins, or a functional or immuno-dominant peptide fragment thereof.

For example, alopecia associated antigen herein is an antigen for use in immune modulatory therapy. It should therefore not be fully processed/matured or secreted in order to make sure that it is presented on MHC molecules to circulatory T cells. The DNA immuno therapy vaccine herein does therefore not result in increased levels of peptides encoded by the antigen in the blood but rather results in an increased presentation of antigens to the immune system, in particular the T cells.

Therefore, alopecia associated antigen herein can be small immuno-dominant peptide encoding fragments (e.g. gp100 149-167, including shifted register peptides displaying equivalent T cell epitopes), whole alopecia associated antigens, which lacks the required secretion sequence but otherwise intact.

Non-limiting examples of alopecia associated antigens herein include:

Human MARTI full length (SEQ ID NO: 1):

MPREDAHFIYGYPKKGHGHSYTTAEEAAGIGILTVILGVLLLIGCWYCRRRNGYRALMDKSL HVGTQCALTRRCPQEGFDHRDSKVSLQEKNCEPVVPNAPPAYEKLSAEQSPPPYSP Human gp100 full length (SEQ ID NO: 2):

MDLVLKRCLLHLAVIGALLAVGATKGSQVWGGQPVYPQETDDACIFPDGGPCPSGSWSQK

RSFVYVWKTWGQYWQVLGGPVSGLSIGTGRAMLGTHTMEVTVYHRRGSRSYVPLAHSSS

AFTITDQVPFSVSVSQLRALDGGNKHFLRNQPLTFALQLHDPSGYLAEADLSYTWDFGDSS

GTLISRALVVTHTYLEPGPVTAQVVLQAAIPLTSCGSSPVPGTTDGHRPTAEAPNTTAGQVP

TTEVVGTTPGQAPTAEPSGTTSVQVPTTEVISTAPVQMPTAESTGMTPEKVPVSEVMGTTL

AEMSTPEATGMTPAEVSIVVLSGTTAAQVTTTEWVETTARELPIPEPEGPDASSIMSTESITG

SLGPLLDGTATLRLVKRQVPLDCVLYRYGSFSVTLDIVQGIESAEILQAVPSGEGDAFELTVS

CQGGLPKEACMEISSPGCQPPAQRLCQPVLPSPACQLVLHQILKGGSGTYCLNVSLADTNS

LAVVSTQLIMPGQEAGLGQVPLIVGILLVLMAVVLASLIYRRRLMKQDFSVPQLPHSSSHWLR

LPRIFCSCPIGENSPLLSGQQV

Human fragment gp 100(149- 167) (SEQ ID NO: 3)

FVYVWKTWGQYWQVLGG PV

Human fragment gp 100(204-222) (SEQ ID NO: 4):

SSAFTITDQVPFSVSVSQL

Human fragment gp100(275-293) (SEQ ID NO: 5):

VVTHTYLEPGPVTAQVVLQ

Human fragment MARTI (22-40) (SEQ ID NO: 6):

TTAEEAAGIGILTVILGVL

A non-limiting example of an Alopecia minigene is SEQ ID NO: 7 as used herein in Example 1:

FVYVWKTWGQYWQVLGGPVSSAFTITDQVPFSVSVSQLVVTHTYLEPGPVTAQV

VLQTTAEEAAGIGILTVILGVL

Alopecia associated antigens herein may thus accumulate in the cytosol of the transfected host cell and can thus be presented via MHC class I, or be released upon cytolysis.

Endosomal targeting resulting in MHC class presentation may be accomplished

herein via fusion of the antigen sequence with leader sequences which form transmembrane segments with cytoplasmic ΎCC0” sequences, in which Y is tyrosine, X is any amino acid, and 0 is a bulky hydrophobic amino acid such as tryptophan or isoleucine, “[DE]XXXL[LI]” where D and E are aspartic or glutamic acid respectively, while L and I are leucine and isoleucine respectively, or “DXXLL” endosomal/lysosomal sorting signals, which are underlined in the following exemplary sequences. Protein domains that include these signals therefore target or cycle to the endosome/lysosome include: transferrin receptor, Limpll, or CD74, also known as Invariant chain, MHC II chaperone, or li, or any similar domain.

Examples of endosomal targeting domains herein include, but are not limited to:

Mouse CD74/lnvariant chain (li) endosomal targeting domain (SEQ ID NO: 38):

MDDQRDLISNHEQLPILGNRPREPERCSRGALYTGVSVLVALLLAGQATTAYFLYQ QQGRLDKLTITSQNLQLESLRMKLP, comprising mli-pre (CD74) (SEQ ID NO: 36): MDDQRDLISNHEQLPILGNRPREPER and li-post (CD74) (SEQ ID NO: 37): CSRGALYTGVSVLVALLLAGQATTAYFLYQQQGRLDKLTITSQNLQLESLRMKLP

Human CD74/lnvariant chain (li) endosomal targeting domain is (SEQ ID NO: 41): MAHRRRSRSCREDQKPVMDDQRDLISNNEQLPMLGRRPGAPEQVQQRSLVHGVLHFSDS PSRRPSYHRLLSVPTARQTRQTDNHKPEPSAGVSADEAA comprising CD74 pre (SEQ ID NO: 39): MAHRRRSRSCREDQKPVMDDQRDLISNNEQLPMLGRRPGAPE and CD74 post (SEQ ID NO: 40):

QVQQRSLVHGVLHFSDSPSRRPSYHRLLSVPTARQTRQTDNHKPEPSAGVSADEAA

Alopecia Areata: Alopecia Areata (AA) is a common autoimmune disease resulting from the attack of autoreactive CD8+ T cells on hair follicle, causing patches of hair loss. The severity can vary from patches (AA patchy) to total body hair loss (AA universalis).

Tolerogenic DNA vaccine: DNA-based immuno-therapy vaccines/vectors/plasmids herein are designed to switch off or down-regulate the part of the immune system responsible for destroying normal healthy “self’ cells and thus prevent or ameliorate T cell- based autoimmunity.

The term “DNA immuno-therapy vaccine” as used herein is intended to mean a compound or composition comprising a DNA molecule and which is administered to a subject in order to reduce the risk of said subject developing Alopecia Areata or treating AA.

In some embodiments, DNA based immuno-therapy vaccines herein are plasmids/vectors encoding particular alopecia associated antigens. Following vaccination, these plasmids are taken up by, in other words, transfected into antigen presenting cells in the host’s body. The “transfected” host cells then produce the antigen and present small fragments of the antigen to the immune system, in particular the T cells. This approach leads to a modification of specific T cell responses to the encoded antigen as well as minimal modification to immune responses to other (non-encoded or “irrelevant”) antigens. Only a very few host cells are typically transfected with the DNA vaccine plasmid/vector herein, meaning that likely fewer than one out of hundred thousand, one out of five hundred thousand, or even fewer than one out of a million plasmid/vector molecules eventually enter a host cell. DNA vaccines herein thus represent a very mild and specific approach for modulating immune responses to antigens such as alopecia associated antigens in AA patients or patients at risk of developing AA.

Plasmid: A plasmid is a small DNA molecule that is most commonly found in bacteria as small, circular, double-stranded DNA molecules. Artificial plasmids are widely used as vectors in molecular cloning, serving to drive the replication of recombinant DNA sequences within host organisms. Plasmids can be engineered to be suitable for use as immuno-therapy DNA vaccines. Plasmids are considered replicons, a unit of DNA capable of replicating autonomously within a suitable host. Plasmids can be transmitted from one bacterium to another bacterium, which could be of the same or different bacterial species via three main mechanisms: transformation, transduction, and conjugation. DNA vaccine plasmids can be taken up by a host cell by passive transformation - usually at a relatively low rate. The plasmids herein replicate efficiently - but do not drive protein expression - in bacteria. The plasmids herein furthermore drive protein expression - but not replication of plasmid - in humans and other mammals, e.g. mice. In one embodiment, a pVAX1 vector (Invitrogen/LifeTechnologies) is used as a scaffold herein for inserting the elements that are part of the present invention. Other suitable vector scaffolds herein include any vector backbone containing a eukaryotic promoter element, a prokaryotic high copy origin of replication, and a selection system for plasmid maintenance.

Selection gene and selection system: In one aspect, DNA immuno-therapy vaccines herein comprise a selection gene/selection marker for manufacturing purposes. The selectable marker herein is e.g. a gene that confers resistance to a cell toxin - e.g. an antibiotic such as ampicillin, kanamycin, chloramphenicol, streptomycin, etc.

Other types of suitable selection systems herein include e.g. conditional lethal silencing systems (e.g. CcdA/CcdB or ParD/ParE Hok/Sok type systems), or sequences that complements a genomic defect in the production cell strain and thus permits growth of an otherwise inviable host (e.g. dapD- or pyrF- auxotrophic complementation, infA- translation initiation complementation, etc.)

Production cells harbouring the plasmid/DNA vaccine, which includes the selection marker, will survive when exposed to the toxin/antibiotic/condition, while those that have failed to take up plasmid sequences will die. As such, in one embodiment, DNA vaccines herein comprises the nucleic acid sequence encoding a selection marker in order to provide for higher yield/purity and more efficient production/replication in production cells, such as E.coli.

While antibiotic selection is a common laboratory strategy there may be advantages associated with anti biotic- free selection systems - e.g. in relation to more efficient regulatory processes. While vectors which do not contain a selection mechanism such as minicircles, synthetic linear vectors, etc., can also be used herein, these implementations are associated with certain drawbacks in production, in particular due to increased production and quality control costs.

Examples of complementation (“rescue”) strategies are known in the prior art, however these strategies suffer from various disadvantages.

Metabolic complementation systems such as dapD [lysine biosynthesis] or pyrF [uridine biosynthesis] systems, often result in “cross-feeding” during high density E.coli production, where a plasmid-containing bacterium will produce and secrete an excess of the required compound and thereby “relaxing” the selection pressure for neighbouring bacteria without the plasmid.

Another example of a suitable selection system herein are plasmids encoding essential proteins, such as infA, encoding IF1/lnitiation Factor 1 which is required for protein synthesis. In this selection system, cross-feeding does not occur because the infA protein is not secreted. However, it is not possible to further modify the plasmid or expand plasmid- deficient cells as there is no way to exogenously complement the required protein/infA (J Bacteriol. 1994 Jan; 176(1): 198-205 and J Biotechnol. 2004 Jul 1;111(1):17-30).

In order to circumvent the disadvantages associated with the infA selection system, an alternative selection system has been provided herein with a temperature-sensitive translation switch (or “thermosensor”) from the invasion protein gene prfA of L monocytogenes (Cell. 2002 Sep 6; 110(5):551 -61 ). By placing the hairpin forming portion of an RNA “thermosensor” sequence upstream of the E.coli genomic copies of infA via standard recombination technology, expression thereof becomes regulated via control of the fermentation temperature, enabling slow growth of plasmid free cells at 37°C, and rapid cell death at temperatures <30°C. Transformation of the engineered thermo sensitive E.coli production strain with plasmids expressing wt infA thus allow full normal growth rates at all temperatures, allowing for plasmid-free expansion at 37°C as well as stringent selection for plasmid at 30°C. Additionally, this system generates no selective pressure for wt E.coli to retain the plasmid and it is thus lost within 8 hours in culture - ensuring no environmental persistence of the therapeutic plasmid. Such method has been disclosed in WO20 18/083116. wt E.coli infA nucleotide sequence (SEQ ID NO: 14):

AT GGCCAAAGAAG ACAAT ATT GAAATGCAAGGT ACCGTT CTT G AAACGTT GCCT AAT ACCAT GTTCCGCGT AGAGTT AGAAAACGGT CACGTGGTT ACTGCACACATCTCCGG T AAAATGCGCAAAAACT ACATCCGCATCCT G ACGGGCG ACAAAGT G ACT GTT G AACT GA CCCCGTACGACCTGAGCAAAGGCCGCATTGTCTTCCGTAGTCGCTGA wt E.coli IF1 protein sequence resulting from translation of the infA gene (SEQ ID NO: 15)):

MAKEDNIEMQGTVLETLPNTMFRVELENGHVVTAHISGKMRKNYIRILTGDKVTVEL

TPYDLSKGRIVFRSR

E.coli production cell lines used herein for production of DNA immuno-therapy vaccine plasmids may thus harbour the following thermo sensitive prfA nucleotide sequence: wt L. monocytogenes prfA (“thermo sensor hairpin”) nucleotide sequence (Shine Dalgarno underlined, ATG start bolded - (SEQ ID NO: 18)):

TGT AAAAAACAT CATTT AGCGT G ACTTTCTTT CAACAGCT AACAATT GTT GTT AC TGCCTAATGTTTTTAGGGTATTTTAAAAAAGGGCGATAAAAAACGATTGGGGGATGAGAA ATGAACGCTCAA wt L. monocytogenes prfA protein sequence (fused upstream of E.coli IF1 - resulting from translation of SEQ ID NO: 19):

MNAQ

In addition, the E.coli production cell lines may also carry a modified infA nucleotide sequence e.g. (not including initial ATG; SEQ ID NO: 16):

GCCAAAGAAGACAAT ATT GAAAT GCAAGGT ACCGTTCTT GAAACGTTGCCT AAT ACCAT GTTCCGCGT AGAGTT AGAAAACGGTCACGTGGTTACTGCACACAT CTCCGGTAA AAT GCGCAAAAACT ACATCCGCATCCT G ACGGGCG ACAAAGT GACT GTT G AACT GACCC CGT ACGACCT GAGCAAAGGCCGCATT GTCTTCCT GAGTCGC

The resulting modified E.coli IF1 protein with R69L shown in bold (initial methionine/M not included; SEQ ID NO: 17):

AKEDNIEMQGTVLETLPNTMFRVELENGHVVTAHISGKMRKNYIRILTGDKVTVELT

PYDLSKGRIVFLSR

Origin of replication (“Ori”): The origin of replication, also called the replication origin, is a particular sequence in a genome at which replication of the DNA strand is initiated. In one embodiment, origin of replication sites herein includes the “pUC Ori” which allows replication in the bacterial E.coli production cell line - but not in the mammalian host cells, i.e. , cells from the body of the vaccinated subject/person/patient. Other suitable bacterial replication origins herein include but are not limited to: R6K, pBR322, ColE1, pMB1 , 15A, pSC101, etc. In one aspect, the origin of replication herein is a high copy version which yields a high plasmid/biomass ratio for more efficient production. Vectors which do not contain an origin of replication, such as minicircles, synthetic linear vectors, etc., can also be used herein. As a non-limiting example, the pUC Ori used herein includes (SEQ ID NO: 31):

T AAAACTT CATTTTT AATTT AAAAGGAT CTAGGT G AAGATCCTTTTT GAT AAT CT C ATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAA GAT CAAAGG ATCTT CTT G AG ATCCTTTTTTTCT GCGCGT AAT CTGCTGCTT GCAAACAAA AAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTC CGAAGGT AACT GGCTTCAGCAGAGCGCAGAT ACCAAAT ACT GTT CTTCT AGT GT AGCCG T AGTT AGGCCACCACTTCAAGAACTCT GT AGCACCGCCT ACAT ACCT CGCT CT GOT AAT CCT GTT ACCAGT GGCTGCT GCCAGT GGCGAT AAGTCGT GTCTT ACCGGGTTGGACTCA AGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACA CAGCCCAGCTT GGAGCGAACGACCT ACACCGAACT GAGAT ACCT ACAGCGT GAGCT AT GAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCA GGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTT ATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCA GGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGGCT TTT GCT GGCCTTTTGCT CACAT GTT CTT

Promoter: A promoter is a region of DNA that initiates transcription of a particular gene. Promoters are located near the transcription start sites of genes, on the same strand and upstream on the DNA, which towards the 5' region of the sense strand. For the transcription to take place, the RNA polymerase must attach to the DNA near a gene. Promoters contain specific DNA sequences, such as response elements, that provide a secure initial binding site for RNA polymerase and for transcription factors that recruit RNA polymerase. Transcription factors have specific activator or repressor sequences that attach to specific promoters and regulate gene expression. Promoters thus represent critical elements that can work in concert with other regulatory regions, such as enhancers, silencers, boundary elements/insulators, to direct the level of transcription of a given gene. A classical promoter drives the production of a single messenger RNA (mRNA), whereas bidirectional promoters herein drive the production of two mRNAs immediately adjacent to the promoter, both upstream and downstream of the promoter.

In one embodiment, eukaryotic promoters are used herein. Eukaryotic promoters do not necessarily obey the one gene/one promoter rule, such as several viral promoters as well as promoters that exhibit broad expression (i.e. do not have narrow cell type specificities such as neuron-only expression). Examples of promoters herein that are capable of driving broad transcription of large multi-gene mRNA molecules include: the viral CMV immediate- early (IE) and SV40 promoters; endogenous EF1a, PGK1, Ubc, and beta actin promoters; and synthetic promoters such as the CAG hybrid promoter. Many other suitable mammalian promoters exist and more are being designed via synthetic biology efforts. Any promoter that results in the desired expression characteristics in human cells may be used in the DNA immuno-therapy vaccine plasmids herein. A non-limiting example of a suitable CMV promoter and enhancer as used herein includes (SEQ ID NO: 32):

TT GACATT GATT ATT G ACT AGTT ATT AAT AGT AAT CAATT ACGGGGTCATT AGTT CATAGC CCAT AT AT GGAGTTCCGCGTT ACAT AACTT ACGGT AAATGGCCCGCCTGGCT GACCGCC CAACG ACCCCCGCCCATT G ACGTCAAT AAT GACGT AT GTTCCC AT AGT AACGCCAAT AG GGACTTTCCATT GACGTCAATGGGTGGACT ATTT ACGGT AAACTGCCCACTTGGCAGT A CATCAAGT GT ATCAT AT GCCAAGT ACGCCCCCT ATT GACGTCAAT GACGGT AAAT GGCC CGCCT GGCATT ATGCCCAGT ACAT GACCTT AT GGGACTTTCCT ACTTGGCAGT ACATCT ACGT ATT AGTCAT CGCT ATT ACCAT GGT GAT GCGGTTTT GGCAGT ACATCAATGGGCGT GGAT AGCGGTTT GACTCACGGGGATTTCCAAGTCT CCACCCCATT GACGT CAATGGGA GTTTGTTTTGGCAC

Enhancers: Enhancers are DNA elements that increase the efficiency of promoters in producing mRNA transcripts. The enhancers herein may be matched (e.g. SV40 enhancer/CMV promoter) or unmatched. Any suitable enhancer/promoter combination for eukaryotic function can be used herein.

Eukaryotic translation start: The eukaryotic translation start sequence is usually referred to as the “Kozak” consensus sequence. The Kozak sequence on an mRNA molecule is recognized by the ribosome as the translational start site, from which a protein is encoded. The eukaryotic ribosome requires this sequence, or a variation thereof, to initiate protein translation. Kozak sequences are degenerate or variable and rarely match consensus sequences. In fact, consensus Kozak sequences are typically less efficient than wild type variants isolated from mammalian mRNAs. While weak Kozak sequences are regularly isolated from native mRNAs and likely play a role in translational control of low abundance proteins, DNA immuno-therapy vaccines herein preferably encode a medium or high efficiency Kozak sequence. Examples of useful Kozak sequences herein comprise the following nucleotide sequence: gccRccATGG (SEQ ID NO: 20), where lower case bases are the most common nucleotides but may vary while upper case nucleotides are fixed (R is the lUPAC uncertainty code for A or G bases), and the ATG indicates the translational start site of Methionine codon at position +1. Endosome sorting signal: An endosome is a membrane-bounded compartment inside eukaryotic cells. Some proteins can be transported to endosomes and therein be degraded into peptide fragments. The peptide fragments can bind to MHC molecules present in the endosome to form MHC/peptide complexes, which can subsequently be transported to the cell surface in order to be presented to circulating T cells, particularly CD4⁺ T cells.

Sorting of proteins to endosomes is mediated by signals present within the cytosolic domains of the proteins. The endosomal signals are usually short linear amino acid sequences. Antigens herein are preferably targeted to the endosomes using an endosome sorting signal, such as e.g. UCC0, [DE]XXXL[LI], or DXXLL endosomal/lysosomal sorting signals. Endosome sorting signals include various naturally occurring or synthetic endosomal sorting signals. Examples herein include the endosome sorting signals present on Cd74/invariant chain/li, Limpll/SCARB, or transferrin receptor. Any endosomal targeting domain which is pharmaceutically acceptable and provides the desired function may be utilized. Fusion of such endosomal targeting domains to the antigens directs them to the endosomal compartment upon translation for increased efficacy. Endosomal sorting of antigens confers processing and presentation to the immune system in MHC class II complexes, in addition to constitutive presentation in MHC class I complexes, for more complete and robust induction of tolerance and possible expansion of Tregs (which cannot be accomplished via MHC class l/antigen complexes). In one embodiment, tolerogenic DNA vaccines herein encode a fusion of the antigen with the CD74/invariant chain/li to drive endosomal targeting and presentation of the antigen via MHC class II.

Introns: Introns are non-coding sequences within an mRNA. It is known that some introns significantly increase translation and function of mRNA. Accordingly, the inclusion of intron sequences may also be used herein. Standard introns, such as beta-globin, or any intron obeying mammalian splicing conventions, such as MCM7, may be utilized. In one embodiment, DNA immuno-therapy vaccine vectors herein comprise sequences encoding one or more introns. A non-limiting example of a mouse beta-globin intron used herein includes (SEQ ID NO: 33):

GTT AACTT AAT GAGACAGAT AGAAACTGGTCTT GT AGAAACAGAGT AGTCGCCTGCTTTT CTGCCAGGTGCTGACTTCTCTCCCCTGGGCTTTTTTCTTTTTCTCAG. A non-limiting exemplary human beta-globin intron includes (SEQ ID NO: 34):

GT AAGTT AAT GAGACAGAT AG AAACT GGTCTT GT AGAAACAGAGT AGTCGCCT GCTTTT C T GCC AGGTGCT G ACTT CT CTCCCCTGGGCTTTTTT CTTTTT CTCAG .

In another embodiment, DNA immuno-therapy vaccine vectors herein do not possess sequences encoding introns. Ribosomal pause tag: In connection with the present invention, it may be an advantage to include one or more ribosomal pause tag sequence(-s) between the protein coding sequences in the DNA immuno-therapy vaccine vector/plasmid herein in order to separate protein products.

An example is the viral “FMDV 2A tag” (Foot-and-mouth disease virus 2A tag) or just “F2A”. The translated amino acid sequence of F2A is VKQTLNFDLLKLAGDVESNPGP - (SEQ ID NO: 21). F2A tag is capable of pausing and reinitiating the ribosome. The ratio of translated product before and after the F2A tag is close to 1 : 1 and the resulting protein products are normally completely separated. These types of ribosome tags have previously been used in connection with co-expression of two different domains, e.g. heavy chain and light chain in recombinant antibody production.

Another example of a ribosomal pause tag sequence herein is the viral sequence tag “T aV 2A” ( Thosea asigna virus 2A - translated amino acid sequence of TaV 2A: RAEGRGSLLTCGDVEENPGP (SEQ ID NO: 22). The ratio of translated product before/upstream and after/downstream of this tag is reported to be 50:1 (or close to). The inventor of the present invention has made the surprising discovery that while this type of tag can be used to control expression levels in cases where it is vital that one translated product absolutely dominates another, the separation of flanking cytokine products is less than 50% relative to the sequences disclosed in literature and the expression ratio is thus about 10:6.

In connection with the present invention, a 2A type of ribosomal pause tag sequence should preferably result in different expression levels of two proteins encoded by the same vector/plasmid. Expression of small amounts of a pleiotropic cytokine (such as IL-2) relative to an anti-inflammatory cytokine, such as IL-10, is desirable herein and fused products are not desirable.

A further non-limiting example of a ribosomal pause tag amino acid sequence herein includes the viral sequence “P2A” ( Porcine teschovirus-1 2A, ATNFSLLKQAGDVEENPGP - (SEQ ID NO: 23)). P2A sequences function appropriately when inserted between IL-10 and IL-2 herein, resulting in near complete separation with an expression ratio of >5:1 between IL-10 and IL-2, e.g. a ratio of 2:1.

Alternatively, proteinase sensitive sequences, allowing for endogenous cleavage between plasmid expressed poly proteins, may be used herein. A furin sensitive sequence (recognizing RAKR motifs) or carboxypeptidase sensitive sequence (recognizing RRRR, RKRR, or RRKR motifs) may be used herein for separating protein products. However, the inventor of the present invention has made the surprising discovery that neither furin nor carboxy peptidase cleavage sequences result in separated products herein - thus leading to secretion of undesired IL-10/IL-2 fusion proteins.

Interleukin-2 (IL-2) is a cytokine that plays an important role as a growth factor that induces the proliferation of CD8+ and CD4+ T cells. Its other biological roles include the induction of the proliferation of Natural Killer cells (NK), the increment of cytolytic activity, or the promotion of antibody production and B-cell proliferation. IL-2 is instrumental to the generation of CD4+ regulatory T cells expressing transcription factor FOXP3, which act to mitigate or suppress the actions of effector T cells and thus provide immune tolerance. As a result, many therapeutic agents based on biomolecules and recombinant chimeric proteins have been developed to date in order to harness the beneficial effects of IL-2 and treat different autoimmune diseases (Orozco Valencia A et al. Int Immunopharmacol. 2020 Feb 11 ;81 : 106296)

Non-limiting examples of IL-2 sequences as used herein include:

Mouse IL-2 (SEQ ID NO: 12):

MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLS

RMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFIS

NIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQ

Human IL-2 (SEQ ID NO: 13):

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQ

CLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRW

ITFCQSIISTLT

Interleukin- 10 (IL-10) is a cytokine with potent anti-inflammatory properties that plays a crucial part in curbing host immune response to pathogens, thereby preventing damage to the host and preserving normal tissue homeostasis. Consequently, exacerbated immunopathology and tissue damage can ensue in situations where expression of IL-10 is impaired or abolished. Many different cell types can be the source of IL-10, including T helper cells, monocytes, macrophages and dendritic cells, primarily. However, other immune cell types are capable of producing IL-10 in certain contexts including B cells, cytotoxic T cells, NK cells, mast cells, and granulocytes like neutrophils and eosinophils. Furthermore, non-immune effector types such as epithelial cells and keratinocytes are also capable of producing IL-10 in response to infection or tissue damage (Iyer S. S. and Cheng G.; Crit Rev Immunol. 2012 ; 32(1): 23-63). As an anti inflammatory cytokine IL-10 down-regulates the expression of T helper 1 cytokines, MHC class II and costimulatory molecules on macrophages. Additionally, IL-10 is thought to contribute in influencing the balance of T helper 1 versus T helper 2 cytokines, which can impact pathogenesis of autoimmune diseases.

Non-limiting examples of IL-10 sequences as used herein include:

Mouse IL-10 (SEQ ID NO: 10):

AAAPGSALLCCLLLLTGMRISRGQYSREDNNCTHFPVGQSHMLLELRTAFSQVKTFFQTKD

QLDNILLTDSLMQDFKGYLGCQALSEMIQFYLVEVMPQAEKHGPEIKEHLNSLGEKLKTLRM

RLRRCHRFLPCENKSKAVEQVKSDFNKLQDQGVYKAMNEFDIFINCIEAYMMIKMKS

Human IL-10 (SEQ ID NO: 11):

MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQ LDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVM PQAENQDPDIKAHVNSLGENLKTLRLR LRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

TGF-b/3/31 (Transforming growth factor beta/31): TGF-3 is a secreted protein that controls proliferation, cellular differentiation, and other functions in most cells. TGF-3 is a very potent cytokine with significant effects on cell fate and phenotype in a context- dependent manner, e.g. depending upon the other cytokine signals received contemporaneously. Endogenous TGF-3 is produced in a latent form associated with the outer membrane surface of the producing cell and requires activation (e.g. by inflammatory macrophages expressing CD36 and plasmin proteinase) for maturation and release of the active form. In one embodiment, TGF-3 herein is a modified form that is constitutively active. This is achieved by replacing the cysteines at positions 223 and 225 with amino acids incapable of forming disulfide bridges. For example, serine or valine are used to replace cysteines at positions 223 and 225. This results in an active pro-protein structure that is released into the local microenvironment.

Human endogenous TGF-31sequence - SEQ ID NO: 26: MPPSGLRLLLLLLPLLWLLVLTPGRPAAGLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPS QGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFK QSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLL APSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDL ATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGW KWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYV GRKPKVEQLSNMIVRSCKCS.

Modified human TGF-31 sequence that is constitutively active and secreted (substitutions in relation to wt TGF-31 shown with bold and underline) - SEQ ID NO: 27: MPPSGLRLLLLLLPLLWLLVLTPGRPAAGLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPS QGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFK QSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLL

APSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHVSVDSRDNTLQVDINGFTTGRRGDL

ATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGW

KWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYV

GRKPKVEQLSNMIVRSCKCS.

Another modified human TGF-bI sequence that may be used is SEQ ID NO: 28:

MPPSGLRLLLLLLPLLWLLVLTPGRPAAGLSTCKTI DM ELVKRKRI EAI RGQI LSKLRL ASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEI YDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYL SNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHSSSDSRDNTLQVDINGFTTGR RGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRK DLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPL PIVYYVGRKPKVEQLSNMIVRSCKCS.

A non-limiting example of a constitutively active and secreted modified human TGF- b1 sequence that may be used is SEQ ID NO: 29:

MPPSGLRLLPLLLPLPWLLVLTPGRPAAGLSTCKTIDMELVKRKRIEAIRGQILSKLR

LASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESADPEPEPEADYYAKEVTRVLMVDRNNA

IYEKTKDISHSIYMFFNTSDIREAVPEPPLLSRAELRLQRLKSSVEQHVELYQKYSNNSWRYL

GNRLLTPTDTPEWLSFDVTGVVRQWLNQGDGIQGFRFSAHSSSDSKDNKLHVEINGISPKR

RGDLGTIHDMNRPFLLLMATPLERAQHLHSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRK

DLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASASPCCVPQALEPL

PIVYYVGRKPKVEQLSNMIVRSCKCS.

Terminator sequence: a transcription terminator is a section of a nucleic acid sequence that marks the end of a gene during transcription. Release of the transcriptional complex frees RNA polymerase and related transcriptional machinery to begin transcription of new mRNAs. Additionally, the same cellular factors add a non-templated “poly-A tail” which significantly enhances the lifetime and functionality of the mRNA. Non-limiting examples of suitable transcription terminators herein includes the “bGH_PA” terminator, GCCTT CAGTT GCCAGCCATCT GTT GTTTGCCCCTCCCCCGTGCCTTCCTT GACCCTGGA AGGT GCC ACTCCCACT GTCCTTTCCT AAT AAAAT G AGG AAATT GCATCGCATT GTCT GAG TAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTG GGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTC (SEQ ID NO: 24) or

GCCTT CT AGTT GCCAGCCATCT GTT GTTTGCCCCTCCCCCGTGCCTTCCTT GACCCTGG AAGGTGCCACTCCC ACT GTCCTTTCCT AAT AAAAT G AGGAAATTGCATCGCATT GT CT GA GTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATT GGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTC (SEQ ID NO: 25).

Any acceptable terminator sequence may be utilized herein. Variations include use of two different flanking terminator sequences in the instance of bidirectional promoters producing two oppositely-oriented mRNAs.

In one embodiment, the plasmid of the invention has the sequence as set out in SEQ ID NO: 8, full (non-annotated) plasmid sequence:

GACTCTTCGCGAT GT ACGGGCCAGAT AT ACGCGTT GACATT GATT ATT GACT AG TT ATT AAT AGT AAT CAATT ACGGGGTCATT AGTTCAT AGCCCAT AT AT GGAGTTCCGCGT TACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTG ACGTCAAT AAT G ACGT AT GTTCCCAT AGT AACGCCAAT AGGG ACTTT CCATT G ACGT CAA T GGGT GGACT ATTT ACGGT AAACT GCCCACTTGGCAGT ACATCAAGT GT ATCAT ATGCC AAGT ACGCCCCCT ATT GACGTCAAT GACGGT AAAT GGCCCGCCTGGCATT AT GCCCAGT ACAT G ACCTT ATGGG ACTTTCCT ACTTGGCAGT ACAT CTACGT ATT AGT CATCGCT ATT A CCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACG GGG ATTTCCAAGTCTCCACCCCATT G ACGTCAATGGG AGTTT GTTTTGGCACC AAAAT C AACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAG GCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACT GCTT ACTGGCTT ATCGAAATT AAT ACGACTCACT AT AGGGAGACCCAAGCTGGCT AGCG TTT AAACTT AAGCTTGGT ACCGAGCTCGGATCCACT AGTCCAGT GTGGT GGAATTCTGC ACTGCAGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCAC GCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGC CGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGG AGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTA GTT CT CTCGTT AACTT AAT GAG ACAGAT AG AAACTGGT CTT GT AG AAACAG AGT AGTCGC CTGCTTTTCTGCCAGGTGCTGACTTCTCTCCCCTGGGCTTTTTTCTTTTTCTCAGGTTGA AAAGAAGAAGACGAAGAAGACGAAGAAGACAAACCGTCGTCGACGCCACCATGGATGA CCAACGCGACCTCATCTCT AACCAT GAACAGTTGCCCAT ACT GGGCAACCGCCCT AGAG AGCCAGAAAGGTGCAGCCGTGGAGCTCTGTACACCGGTGTCTCTGTCCTGGTGGCTCT GCTCTTGGCTGGGCAGGCCACCACTGCTTACTTCCTGTACCAGCAACAGGGCCGCCTA GACAAGCTGACCATCACCTCCCAGAACCTCCAGCTGGAGAGCCTCAGGATGAAGCTGC CATTCGTGTACGTGTGGAAGACCTGGGGCCAGTACTGGCAGGTGCTGGGCGGCCCCG TGAGCAGCGCCTTCACCATCACCGACCAGGTGCCCTTCAGCGTGAGCGTGAGCCAGCT GGTGGTGACCCACACCTACCTGGAGCCCGGCCCCGTGACCGCCCAGGTGGTGCTCCA GACCACCGCCGAGGAGGCCGCCGGCATCGGCATCCTGACCGTGATCCTGGGCGTGCT

GGGAAGCGGAGT GAAGCAGACGTT GAATTTT GATTT GTT GAAGTTGGCGGGGGAT GT G

GAGAGCAATCCGGGGCCGATGCCACCTTCCGGCTTAAGGCTTCTTCCGCTCTTACTGC

CCTTGCCTTGGCTTTTAGTGCTGACCCCAGGTCGGCCAGCCGCTGGTCTGTCTACCTGT

AAAACT ATT GAT AT GGAATTGGTT AAGCGT AAGCGCATT GAGGCAAT ACGAGGACAGAT

CTTGTCAAAGCTCCGACTTGCATCGCCCCCTTCTCAGGGTGAGGTACCGCCAGGACCT

TTGCCCGAGGCAGTTCTAGCCCTTTATAATTCAACCCGCGACAGAGTCGCAGGCGAAA

GCGCCGACCCAGAACCT GAACCT GAGGCT GACT ACT ACGCCAAGGAGGT AACAAGGGT

CTT GATGGTGG ACAGGAACAAT GCAAT AT ACGAG AAAACCAAAGACATTT CT C ACT CAAT

AT AT AT GTTTTTT AACACTTCCG ACATT AG AGAAGCCGTTCCAGAACC ACCCTTGCT AAG

CCGTGCCGAGCTGCGACTTCAACGCCTGAAGTCCAGTGTCGAGCAACACGTGGAGCTG

T AT CAAAAAT ATTCCAACAAT AGTTGG AG AT ATCT GGGAAATCGGCTCCT AACACCT ACT

GACACGCCTGAGTGGCTGTCTTTCGACGTTACTGGGGTGGTACGACAGTGGCTGAACC

AAGGCGATGGAATCCAGGGATTTAGGTTCTCGGCGCATAGCTCCAGCGACAGTAAAGA

CAACAAACTACATGTCGAGATTAATGGGATCTCACCAAAAAGGCGCGGGGATCTGGGG

ACCATT CACG AT AT GAACCGGCCCTTCCT CCTGCT C ATGGCC ACTCCACTGGAG AG AGC

CCAGCATCTGCATAGCTCACGGCATCGGCGTGCTCTCGATACAAACTACTGCTTTAGCT

CT ACAGAG AAAAATT GCTGCGT G AG ACAGCT CT AT ATCGATTT CAGG AAAG AT CTGGGC

T GGAAGT GGAT ACACGAACCGAAGGGCT ATCACGCT AACTTTT GCCTCGGGCCCT GCC

CTTACATCTGGAGTCTGGATACACAGTACAGTAAGGTCCTCGCGCTCTACAACCAGCAC

AACCCCGGAGCTAGCGCTTCTCCTTGTTGTGTGCCTCAGGCTCTGGAACCGCTGCCCA

TCGT GT ACT ACGT GGGCAGAAAGCCCAAGGTGGAACAGCT GTCCAACATGATCGT GCG

CAGCTGTAAGTGTTCCTGATAAACGCGTCGAGCATGCATCTAGGGCGGCCAATTCCGC

CCCTCTCCCCCCCACCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCT

T GG AAT AAGGCCGGT GT GCGTTT GTCTAT AT GTT ATTTTCC ACCAT ATTGCCGTCTTTT G

GCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCT

TTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTC

T GGAAGCTTCTT GAAGACAAACAACGT CT GT AGCGACCCTTTGCAGGCAGCGGAACCC

CCCACCT GGCGACAGGTGCCTCTGCGGCCAAAAGCCACGT GT AT AAGAT ACACCT GCA

AAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAAT

GGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTG

T ATGGGATCT GATCTGGGGCCTCGGTGCACATGCTTT ACAT GT GTTT AGTCGAGGTT AA

AAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGAT

AATATGGCTGCCGCTCCCGGCAGCGCCCTGCTGTGCTGCCTGCTGCTGCTGACCGGCA

TGAGAATCAGCAGAGGCCAGTACAGCAGAGAGGACAACAACTGCACCCACTTCCCCGT GGGCCAGAGCCACAT GCT GCTGGAGCT GAGAACCGCCTTCAGCCAGGT GAAGACCTTC TTCCAGACCAAGGACCAGCTGGACAACATCCTGCT GACCGACAGCCT GATGCAGGACT TCAAGGGCTACCTGGGCTGCCAGGCCCTGAGCGAGATGATCCAGTTCTACCTGGTGGA GGTGATGCCCCAGGCCGAGAAGCACGGCCCCGAGATCAAGGAGCACCTGAACAGCCT GGGCGAGAAGCT GAAGACCCT GAGAAT GAGACT GAGAAGAT GCCACAGATTCCTGCCC TGCGAGAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAGCGACTTCAACAAGCTCCAG GACCAGGGCGTGTACAAGGCCATGAACGAGTTCGACATCTTCATCAACTGCATCGAGG CCT ACAT GAT GATCAAGAT GAAGAGCGGGAGCGGCGCTACT AACTTCAGCCTGCT GAA GCAGGCTGGAGACGTGGAGGAGAACCCTGGACCT AT GT ACAGT ATGCAGCT AGCTTCC TGCGTAACGTT AACCTTGGT GTT ACTT GT GAAT AGTGCCCCCACAT CTT CATCG ACCT CT TCAAGCACCGCCGAAGCTCAACAGCAGCAACAGCAACAACAGCAGCAGCAACAGCACC T GGAACAACTGCTT ATGGACCTTCAGGAACTCCTCAGCCGGATGGAAAACT AT CGAAAT TTGAAACTCCCGAGAATGCTAACCTTCAAGTTTTACCTCCCAAAGCAGGCCACAGAACTT AAGGACCTTCAGTGTCTGGAGGATGAGCTCGGACCGCTGCGTCATGTCCTGGACCTGA CCCAGT CT AAAAGCTTCCAGCTGGAGG AT GCT G AGAACTT CAT CAGCAACAT AAG AGTT ACT GTCGT G AAGCT G AAAGGTT CAG ACAACACCTTCGAGT GCCAATTT GAT GACG AGTC CGCAACT GTGGTT GACTTT CTGCGC AG ATGG AT CGCATTTT GT CAGTCC ATT ATTT CT AC TTCCCCTCAGTGACTCGAGGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACT GTGCCTTCAGTTGCCAGCCATCT GTT GTTTGCCCCTCCCCCGTGCCTTCCTT GACCCT G GAAGGTGCCACTCCCACT GTCCTTTCCT AAT AAAAT G AGG AAATTGCATCGCATT GTCTG AGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGAT TGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTACTGGGC GGTTTT AT GGACAGCAAGCGAACCGGAATT GCCAGCTGGGGCGCCCTCT GGT AAGGTT GGGAAGCCCT GCAAAGT AAACT GGATGGCTTTCTCGCCGCCAAGGATCT GATGGCGCA GGGGATCAAGCTCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGGCCAAAGAAGA CAAT ATT GAAAT GC AAGGT ACCGTT CTT G AAACGTT GCCT AAT ACCAT GTTCCGCGT AG A GTT AGAAAACGGTCACGT GGTTACT GCACACAT CTCCGGT AAAAT GCGCAAAAACT ACA TCCGCATCCT GACGGGCGACAAAGT GACT GTT GAACT GACCCCGT ACGACCT GAGCAA AGGCCGCATT GT CTTCCGT AGTCGCT GAT AAATT ATT AACGCTT ACAATTTCCT GAT GCG GT ATTTT CTCCTT ACGCAT CTGTGCGGT ATTT CACACCGCAT ACAGGTGGCACTTTTCGG GG AAAT GT GCGCGG AACCCCT ATTT GTTT ATTTTT CT AAAT ACATT CAAAT ATGTATCCGC T CAT G AG ACAAT AACCCT GAT AAATGCTT CAAT AAT AGCACGTGCT AAAACTT CATTTTT A ATTT AAAAGG AT CTAGGT G AAG ATCCTTTTT GAT AAT CT CAT GACCAAAATCCCTT AACGT GAGTTTTCGTTCCACT GAGCGTCA GACCCCGT AG AAAAG AT C AAAGG AT CTT CTT GAGA TCCTTTTTTT CTGCGCGT AAT CTGCT GCTT GCAAACAAAAAAACCACCGCT ACCAGCGGT GGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCA

GAGCGCAGAT ACCAAAT ACT GTTCTTCT AGT GT AGCCGT AGTT AGGCCACCACTTCAAG

AACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGC

CAGTGGCGAT AAGTCGT GTCTT ACCGGGTT GGACTCAAGACGAT AGTT ACCGGAT AAG

GCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACG

ACCT ACACCGAACT GAGAT ACCT ACAGCGT GAGCT AT GAGAAAGCGCCACGCTTCCCG

AAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCA

CGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCA

CCTCT GACTT GAGCGTCGATTTTT GT GATGCTCGTCAGGGGGGCGGAGCCT AT GGAAA

AACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGGCTTTTGCTGGCCTTTTGCTCACAT

GTTCTT

In a second embodiment, the plasmid of the invention has the sequence SEQ ID NO: 9: full (non-annotated) plasmid sequence:

GACTCTTCGCGAT GT ACGGGCCAGAT AT ACGCGTT GACATT GATT ATT GACT AG TT ATT AAT AGT AAT CAATT ACGGGGTCATT AGTTCAT AGCCCAT AT AT GGAGTTCCGCGT TACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTG ACGTCAAT AAT G ACGT AT GTTCCCAT AGT AACGCCAAT AGGG ACTTT CCATT G ACGT CAA T GGGT GGACT ATTT ACGGT AAACT GCCCACTTGGCAGT ACATCAAGT GT ATCAT ATGCC AAGT ACGCCCCCT ATT GACGTCAAT GACGGT AAAT GGCCCGCCTGGCATT AT GCCCAGT ACAT G ACCTT ATGGG ACTTTCCT ACTTGGCAGT ACAT CTACGT ATT AGT CATCGCT ATT A CCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACG GGG ATTTCCAAGTCTCCACCCCATT GACGTCAATGGG AGTTT GTTTTGGCACC AAAAT C AACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAG GCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACT GCTT ACTGGCTT ATCGAAATT AAT ACGACTCACT AT AGGGAGACCCAAGCTGGCT AGCG TTT AAACTT AAGCTTGGT ACCGAGCTCGGAT CCACT AGTCCAGT GTGGT GGAATTCTGC ACTGCAGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCAC GCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGC CGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGG AGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTA GGT AAGTT AAT GAG AC AG AT AG AAACTGGTCTT GTAG AAACAG AGT AGTCGCCTGCTTT TCTGCCAGGTGCT GACTT CT CT CCCCTGGGCTTTTTT CTTTTT CT CAGGTT G AAAAG AAG AAGACG AAG AAGACG AAG AAG ACAAACCGTCGTCG ACT GCC ATGCGCCGCT GATT AAC GCCGCCACCATGGCCCACCGACGCAGATCCAGAAGCTGCCGTGAGGACCAGAAGCCC GT GAT GGAT GATCAGAGGGACCTT ATCTCT AACAAT GAACAACTGCCAATGCTCGGCAG ACGGCCTGGGGCCCCGGAGAGCAAGTGCAGCAGAGGAGCCTTGTACACGGGGTTCTC

CATTTTAGTGACTCTCCTTCTCGCCGGCCAAGCTACCACCGCCTACTTTCTGTACCAACA

GCAAGGCAG ACT AG ACAAACT G ACAAT CACAAGCCAG AACCTT CAGCTGGAGT CTCTGC

GGATGAAGCTGCCCTTCGTGTACGTGTGGAAGACCTGGGGCCAGTACTGGCAGGTGCT

GGGCGGCCCCGTGAGCAGCGCCTTCACCATCACCGACCAGGTGCCCTTCAGCGTGAG

CGTGAGCCAGCTGGTGGTGACCCACACCTACCTGGAGCCCGGCCCCGTGACCGCCCA

GGTGGTGCTCCAGACCACCGCCGAGGAGGCCGCCGGCATCGGCATCCTGACCGTGAT

CCTGGGCGT GCTGGGAAGCGGAGT GAAGCAGACGTT GAATTTT GATTT GTT GAAGTT G

GCGGGGGATGTGGAGAGCAATCCGGGGCCGATGCCCCCTAGTGGCCTCAGACTTTTG

TTATTGTTATTACCGCTTTTATGGCTCTTGGTGCTGACACCGGGCCGTCCGGCTGCTGG

CTT GTCG ACTT GT AAG ACAATT GAT AT GG AATTGGT G AAACG AAAACGG ATT GAGGCCA

TCCGAGGACAGATTTTGAGCAAGCTGCGGCTTGCCTCGCCACCCTCGCAAGGGGAAGT

CCCACCCGGACCTCT ACCAGAAGCAGTCCT AGCGCT GT ACAACAGT ACAAGAGAT AGA

GTGGCCGGGGAATCCGCAGAACCAGAGCCTGAGCCTGAAGCCGATTATTATGCAAAGG

AAGT GACT AGGGTCCT GAT GGTCG AG ACCC AT AACG AAAT CT ACG ACAAATT CAAACAA

AGTACCCACTCTATCTACATGTTCTTCAACACCAGTGAGCTAAGAGAAGCCGTGCCCGA

ACCTGTGCTTCTTTCCCGCGCAGAACTCCGCCTCTTGAGACTCAAATTGAAAGTTGAAC

AACACGT AGAGCTTT ACCAGAAAT ACT CT AAT AATT CATGGCG AT AT CTTT CT AATCGTCT

CCTCGCCCCATCTGACAGCCCTGAATGGCTCTCCTTCGACGTTACGGGAGTTGTGCGC

CAGTGGCTCAGCAGAGGCGGAGAGATAGAGGGCTTTCGGCTGAGCGCACATAGCTCTA

GCGACTCAAGGGACAACACATTGCAAGTGGAT ATT AACGGTTTT ACAACT GGACGGAGA

GGGGACCTGGCGACCATCCACGGCAT GAAT AGACCTTTCCTGCT GCT GAT GGCT ACTC

CCCTGGAGAGGGCACAGCACTTACAGTCTTCCAGACACCGGCGCGCCCTGGATACAAA

CT ACT GCTTCAGCTCCACCGAAAAGAACT GTTGCGTGCGGCAGCT GT ACATT GACTTCA

GAAAGGATCTGGGCTGGAAGTGGATTCATGAGCCCAAGGGGTATCATGCCAACTTCTG

TCTTGGGCCATGCCCATACATCTGGTCACTGGATACCCAGTACTCCAAAGTTCTGGCCT

T GT ACAATCAACACAACCCT GGAGCTTCCGCCGCTCCTTGCT GT GTGCCCCAAGCCCT A

GAGCCCCT GCCCATCGTTT ATT AT GTCGGACGCAAGCCCAAAGT AGAACAGCT ATCAAA

T AT GATCGT GAGAAGCTGCAAGT GT AGCT GAT AAACGCGTCGAGCATGCATCT AGGGC

GGCCAATTCCGCCCCTCTCCCCCCCACCCCTCTCCCTCCCCCCCCCCTAACGTTACTG

GCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATA

TT GCCGTCTTTTGGCAAT GT GAGGGCCCGGAAACCTGGCCCT GTCTTCTT GACGAGCAT

TCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGG

AAGCAGTT CCT CTGGAAGCTT CTT G AAGACAAACAACGTCT GT AGCG ACCCTTT GT AGA

CAGCGGAACCCCCCACCTGGCGATAGATGCCTCTGCGGCCAAAAGCCACGTGTATAAG AT ACACCT GCAAAGGCGGCACAACCCCAGTGCCACGTT GT GAGTT GGAT AGTT GT GGA AAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAG GT ACCCCATT GT ATGGGAT CT GATCT GGGGCCT CGGT GCA CAT GCTTT ACAT GT GTTTA GTCGAGGTT AAAAAACGTCT AGGCCCCCCGAACCACGGGGACGTGGTTTT CCTTT GAAA AACACGAT GAT AAT AT GAT GCACAGCTCAGCACTGCT CT GTTGCCTGGT CCTCCT GACT GGGGTGAGGGCCAGCCCAGGCCAGGGCACCCAGTCTGAGAACAGCTGCACCCACTTC CCAGGCAACCTGCCT AACAT GCTTCGAGATCTCCGAGATGCCTT CAGCAGAGT GAAGA CTTT CTTT CAAAT G AAGG AT CAGCTGGACAACTT GTTGTT AAAGGAGTCCTT GCTGG AG GACTTT AAGGGTT ACCTGGGTT GCCAAGCCTT GTCT G AGAT G ATCCAGTTTT ACCT GGA GGAGGTGATGCCCCAAGCTGAGAACCAAGACCCAGACATCAAGGCGCATGTGAACTCC CTGGGGGAGAACCTGAAGACCCTCAGGCTGAGGCTACGGCGCTGTCATCGATTTCTTC CCT GT GAAAACAAGAGCAAGGCCGT GGAGCAGGT GAAGAAT GCCTTTAAT AAGCTCCAA GAG AAAGGC AT CT ACAAAGCCAT G AGT G AGTTT GACAT CTT CAT CAACT ACAT AG AAGC CT ACAT GACAAT GAAGAT ACGAAACGGGAGCGGCGCT ACT AACTT CAGCCTGCT GAAG CAGGCT GGAGACGTGGAGGAGAACCCTGGACCT AT GT ACAGAATGCAGCTGCT GAGCT GCATCGCCCTGAGCCTGGCCCTGGTGACCAACAGCGCACCCACGTCCTCTAGCACCAA GAAGACCCAGTT ACAGTTGGAGCATCT ACTTTT AGACCTGCAAAT GATTTT GAACGGCAT CAA CAACT ACAAGAAT CCT AAACTT ACTCGCAT GCTT ACCTT CAAATTTT ACATGCCCAA GAAGGCCACCGAACTGAAGCACTTGCAATGTCTGGAGGAAGAACTCAAGCCGCTGGAG GAAGTTCTCAACCTCGCGCAGTCCAAGAATTTCCACCTCCGGCCAAGAGACCTGATCAG T AACATT AAT GT GAT AGTGCTGGAGCT GAAGGGAAGCGAGACT ACATTT AT GTGCGAGT ACGCCGAT GAAACCGCT ACAAT CGTCGAGTTCCT GAAT AGATGGATCACATTTTGCCAG T CAATT AT CT CT ACTCT GACAT GAT AACTCGAGGT CT AG AGGGCCCGTTT AAACCCGCT GATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTG CCTTCCTT GACCCTGGAAGGTGCCACT CCCACT GTCCTTTCCTAAT AAAAT GAGGAAATT GCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACA GCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTA TGGCTTCTACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCCAGCTGGGGCG CCCTCTGGT AAGGTT GGGAAGCCCT GCAAAGT AAACTGGATGGCTTTCTCGCCGCCAA GGATCTGATGGCGCAGGGGATCAAGCTCTGATCAAGAGACAGGATGAGGATCGTTTCG CATGGCCAAAGAAGACAATATT GAAAT GCAAGGT ACCGTTCTT GAAACGTTGCCT AAT AC CATGTTCCGCGTAGAGTTAGAAAACGGTCACGTGGTTACTGCACACATCTCCGGTAAAA T GCGCAAAAACT ACATCCGCATCCT GACGGGCGACAAAGT GACT GTT GAACT GACCCC GT ACGACCT GAGCAAAGGCCGCATT GT CTTCCGT AGTCGCT GAT AAATT ATT AACGCTT ACAATTT CCT GATGCGGT ATTTT CTCCTT ACGCAT CTGTGCGGT ATTT CACACCGCAT AC AGGT GGCACTTTTCGGGGAAAT GT GCGCGGAACCCCT ATTT GTTT ATTTTTCT AAAT ACA TT CAAAT ATGTATCCGCT CAT GAGACAAT AACCCT GAT AAATGCTT CAAT AAT AGC ACGT GCT AAAACTT CATTTTT AATTT AAAAGG AT CT AGGT GAAG ATCCTTTTT GAT AAT CT CAT G ACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGAT CAAAGG AT CTT CTT GAGATCCTTTTTTT CTGCGCGT AATCT GCT GCTTGCAAACAAAAAA ACCACCGCT ACCAGCGGTGGTTT GTTT GCCGGATCAAGAGCT ACCAACTCTTTTTCCGA AGGT AACT GGCTTCAGCAGAGCGCAGATACCAAAT ACT GTTCTTCT AGT GT AGCCGT AG TTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCT GTT ACCAGTGGCT GCTGCCAGTGGCGAT AAGTCGT GTCTT ACCGGGTTGGACTCAAGA CGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAG CCCAGCTTGGAGCGAACGACCT ACACCGAACT GAGAT ACCT ACAGCGT GAGCT AT GAG AAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGG TCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAG TCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGG GGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGGCTTTTG CT GGCCTTTTGCT CACAT GTT CTT

Pharmaceutical compositions herein are preferably aqueous formulations comprising at least 50% water, more preferably at least 60% water, more preferably at least 75% water, more preferably at least 90% water, more preferably at least 95% water, and most preferably at least 99% water. The pharmaceutical compositions herein may alternatively be dry formulations, such as lyophilized formulations, intended for reconstitution, inhalation, intranasal instillation, intradermal administration, etc.

Pharmaceutical formulations herein are preferably administered without the use of methods for enhancing transformation, such as electroporation. In one embodiment, pharmaceutical formulations are intended for parenteral administration, e.g. subcutaneous administration, intradermal administration, intravenous administration, intra-muscular administration, etc. In another embodiment, pharmaceutical compositions herein may furthermore be administered topically, orally, rectally, or by inhalation.

Pharmaceutical compositions herein are preferably without addition of any condensation agents or other excipients that may induce local reactions. Formulations herein preferably contain free-radical scavengers (e.g. 1% ethanol) and/or chelators such as e.g. divalent cation scavengers (e.g. EDTA [CAS #60-00-4], EGTA [CAS #67-42-5], or DPTA [CAS #67-43-6]) in order to enhance stability of aqueous plasmid DNA. Pharmaceutical compositions herein may furthermore be in the form of a saline solution and/or a buffer solution or comprise a saline solution and/or comprise a buffer solution (e.g. PBS - phosphate buffered saline, TRIS buffer, or equivalent pharmaceutically acceptable buffers). Pharmaceutical formulations herein are preferably free from any adjuvants as well other typical vaccine ingredients such as e.g. aluminium hydroxide, phenol, sorbitol, silicone, etc.

Administration: The DNA immuno-therapy vaccine herein may be administered to an AA patient, or a patient in risk of developing AA. The vaccine may be administered e.g. on a daily basis, every second day, twice a week, once a week, twice monthly, once a month, every second month, four times a year, or once a year - frequency may be adjusted according to general or individual needs. The immuno-therapy herein may be chronic. The duration of therapy may be e.g. one month, two months, three months, 6 months, one year, two years, three years, five years, six years, seven years, eight years, nine years, or 10 years.

EMBODIMENTS

The following embodiments illustrate the invention and are not to be understood in any limiting way. It is understood that all embodiments can be combined in all possible ways.

1. A plasmid which encodes, preferably from a single operon: i. an alopecia associated antigen; ii. TGF-b; iii. IL-10, and iv. IL-2.

2. The plasmid according to embodiment 1, which said alopecia associated antigen is selected from gp100 and MART 1 , combinations thereof and/or functional fragments thereof.

3. The plasmid according to any one of the preceding embodiments, wherein the alopecia associated antigen is a combination of gp100 and MART 1 or functional fragments thereof.

4. The plasmid according to any one of the preceding embodiments, wherein the alopecia associated antigen comprises one or more functional fragments of gp100 and/or MART 1. 5. The plasmid according to any one of the preceding embodiments, wherein the alopecia associated antigen comprises one or more functional fragments of each of gp100 and MARTI

6. The plasmid according to any one of the preceding embodiments, wherein the alopecia associated antigen comprises gp100(149-167), gp100(204-222), gp100(275-293) and/or MARTI (22-40), or combinations thereof.

7. The plasmid according to any one of the preceding embodiments, wherein the alopecia associated antigen comprises gp 100(149- 167), gp100(204-222), gp100(275-293) and MARTI (22-40), or combinations thereof.

8. The plasmid according to any one of the preceding embodiments, wherein the alopecia associated antigen consists of gp100(149-167), gp100(204-222), gp100(275-293) and MARTI (22-40) (SEQ ID NO: 7).

9. A plasmid according to any one of the preceding embodiments, which co expresses/encodes (preferably from a single operon): (i) an alopecia associated antigen; (ii) TQR-b/TQR-b1 (such as in a constitutively active form); (iii) IL-10, and (iv) IL-2.

10. The plasmid according to any one of the preceding embodiments, wherein said plasmid comprises: (i) an F2A element separating the alopecia associated antigen encoding sequence and the TGF-b encoding sequence, (ii) an EMCV IRES element separating the TGF-b encoding sequence and the IL-10 encoding sequence, and (iii) a P2A element separating the IL-10 encoding sequence and the IL-2 encoding sequence.

11. The plasmid according to any of the preceding embodiments, wherein said plasmid comprises: (i) 2A element (such as an F2A or a P2A element) separating the alopecia associated antigen encoding sequence and the TGF-b encoding sequence, (ii) an EMCV IRES element (alternatively a bi-directional promoter) separating the TGF-b encoding sequence and the IL-10 encoding sequence (preferably, three alanine amino acids are encoded immediately N- terminal to the IL-10 gene), and (iii) a 2A element (such as a P2A element) separating the IL-10 encoding sequence and the IL-2 encoding sequence.

12. The plasmid according to any of the preceding embodiments, wherein the TGF-b encoding sequence encodes constitutively active TGF-b, preferably constitutively active human TGF-bI

13. The plasmid according to any of the preceding embodiments, wherein said plasmid comprises: (i) an encoding sequence, (ii) an F2A element, (iii) a TGF-b encoding sequence, (iv) an EMCV IRES element, (v) an IL-10 encoding sequence, (vi) a P2A element, (vii) an IL-2 encoding sequence, (viii) a polyadenylation/termination element, (ix) a selection gene, (x) an origin of replication, (xi) a eukaryotic promoter element, (xii) a eukaryotic translational start sequence, (xiii) an endosomal sorting sequence, and (xiv) optionally an intron.

14. The plasmid according to any of the preceding embodiments, wherein said plasmid comprises the following elements:

(i) a promoter (such as a CMV promoter),

(ii) an intron (located within the noncoding leader sequence), and

(iii) a eukaryotic translational start sequence (such as a Kozak element),

(iv) an encoding sequence (such as an alopecia associated antigen is selected from gp100 and/or MART 1 , functional fragments thereof and/or combinations thereof),

(v) an F2A element preferably separating the antigen encoding sequence and the TGF-b encoding sequence,

(vi) a TGF-b encoding sequence (such as a constitutively active human TGF-b encoding sequence, preferably a constitutively active human TGF-bI encoding sequence),

(vii) an EMCV IRES element (or alternatively a bi-directional eukaryotic promoter), wherein said EMCV IRES element separates the TGF-b encoding sequence and the IL-10 encoding sequences,

(viii) an IL-10 encoding sequence (such as a human IL-10 encoding sequence with a three alanine amino acid N-terminal addition), (ix) a 2A element, such as a P2A element, wherein said 2A element separates the IL-10 encoding sequence and the IL-2 encoding sequence,

(x) an IL-2 encoding sequence (such as a human IL-2 encoding sequence),

(xi) a termination element (such as a bGH_PA termination element),

(xii) a selection gene (such as a kanamycin encoding sequence or a wt infA encoding sequence),

(xiii) an origin of replication (such as a prokaryotic origin of replication, such as e.g. pUC ori).

15. The plasmid according to embodiment 14, wherein the elements (i)-(xiii) are arranged by order of expression.

16. The plasmid according to any one of embodiments 13-15, wherein the encoding sequence is SEQ ID NO: 7.

17. The plasmid according to any of the preceding embodiments, wherein the DNA sequence of the plasmid is as set forth SEQ ID NO: 9, or essentially as set forth in SEQ ID NO: 9.

18. The plasmid according to any one of the preceding embodiments, wherein a few minor modifications, resulting in e.g. one, two, three, or four amino acid substitution in the translated product of one or more of the alopecia associated antigen and/or the cytokines made from SEQ ID NO: 9 herein.

19. The plasmid according to any one of the preceding embodiments, wherein the DNA sequence of the plasmid is as set forth in SEQ ID NO: 9 or a modification of SEQ ID NO: 9 resulting in e.g. one, two, three or four amino acid substitutions in one or more of the alopecia associated antigen and/or cytokines, or a modification of SEQ ID NO: 9 which results in expression of the same polypeptide sequences as from SEQ ID NO: 9.

20. The plasmid according to any one of the preceding embodiments, wherein the DNA sequence of the plasmid is as set forth in SEQ ID NO: 9 or a modification of SEQ ID NO: 9 having less than 100 bases which are different than SEQ ID NO: 9. 21. The plasmid according to any of embodiments 1-16, wherein the DNA sequence of the plasmid is as set forth SEQ ID NO: 8, or essentially as set forth in SEQ ID NO: 8.

22. The plasmid according to embodiment 21, wherein a few minor modifications, resulting in e.g. one, two, three, or four amino acid substitution in the translated product of one or more of the alopecia associated antigen and/or the cytokines made from SEQ ID NO: 8 herein.

23. The plasmid according to any of embodiments 21-22, wherein the DNA sequence of the plasmid is as set forth in SEQ ID NO: 8 or a modification of SEQ ID NO: 8 resulting in e.g. one, two, three or four amino acid substitutions in one or more of the alopecia associated antigen and/or cytokines, or a modification of SEQ ID NO: 8 which results in expression of the same polypeptide sequences as from SEQ ID NO: 8.

24. The plasmid according to any of embodiments 21-23, wherein the DNA sequence of the plasmid is as set forth in SEQ ID NO: 8 or a modification of SEQ ID NO: 8 having less than 100 bases which are different than SEQ ID NO: 8.

25. The plasmid according to any one of the preceding embodiments, wherein said plasmid comprises a TGF-b gene coding for SEQ ID NO: 29 or SEQ ID NO: 29 having less than 10 amino acid substitutions.

26. The plasmid according to any of the preceding embodiments for use in delaying, preventing or treating Alopecia Areata, Alopecia Totalis, or Alopecia Universalis.

27. The plasmid according to any of the preceding embodiments for use in delaying or preventing Alopecia Areata, Alopecia Totalis, or Alopecia Universalis.

28. The plasmid according to any of the preceding embodiments for intra-muscular, intradermal, intranasal, or subcutaneous administration. 29. The plasmid according to embodiment 28 for subcutaneous administration.

30. The plasmid according to embodiment 28 for intra-muscular injection.

31. The plasmid according to any of the preceding embodiments for use in treating a medical condition in a subject, such as e.g. Alopecia Areata.

32. Use of a plasmid according to any one of embodiments 1-25 for the manufacture of a medicament for delaying, preventing or treating alopecia, such as Alopecia Areata.

33. Use of a plasmid according to any one of embodiments 1-25 for the manufacture of a medicament for delaying or preventing alopecia, such as Alopecia Areata.

34. A DNA immuno-therapy vaccine comprising the plasmid according to any one of embodiments 1-31.

35. The DNA immuno-therapy vaccine according to embodiment 34 for use in delaying or preventing or treating Alopecia Areata.

36. The DNA immuno-therapy vaccine according to any of embodiments 34-35 for intra-muscular, intradermal, intranasal, or subcutaneous administration.

37. The DNA immuno-therapy vaccine according to embodiment 34-36 for subcutaneous administration.

38. The DNA immuno-therapy vaccine according to embodiment 34-36 for intra muscular administration.

39. The DNA immuno-therapy vaccine according to any of embodiments 34-38 used in association with, or in parallel with other types of medical treatments such as e.g. JAK inhibitors, cortico-steroids, hair follicle epithelial/stem cell therapy, hair follicle epithelial/stem cell grafting, etc. to prolong the survival and efficacy of engrafted cells. 40. A pharmaceutical composition comprising the DNA immuno-therapy vaccine according to any of embodiments 34-39, or a plasmid according to any of embodiments 1-31, wherein said pharmaceutical composition comprises a saline solution and/or a buffer and/or a chelator.

41. A pharmaceutical composition comprising the DNA immuno-therapy vaccine according to any of embodiments 34-39, or a plasmid according to any of embodiments 1-31, wherein said pharmaceutical composition comprises a saline solution and/or a buffer and/or a chelator and/or ethanol.

42. The pharmaceutical composition according to any of embodiments 40-41, wherein the volume/volume percentage of ethanol is less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.

43. The pharmaceutical composition according to any of embodiments 40-42, wherein said composition does not comprise any virus, lipid co-packing agent, or condensation agent.

44. A method of producing a plasmid according to any of embodiments 1-31, wherein said method comprises (i) incubating a host cell, such as a host cell of bacterial origin (such as e.g. E. coli) transfected with said plasmid under suitable conditions and (ii) recovering/purifying said plasmid.

45. The method according to embodiment 44, wherein said host cell is a E. coli infA thermosensitive strain.

46. A method of delaying the onset of Alopecia Areata (AA) or symptoms thereof in a patient at risk of developing AA, or recently diagnosed with AA, said method comprising administering a DNA immuno-therapy vaccine comprising the plasmid according to any of embodiments 1-31.

47. A method of preserving hair follicle function in an individual, said method comprising administering a DNA immuno-therapy vaccine comprising the plasmid according to any of embodiments 1-31. 48. A method of treating a subject suffering from Alopecia Areata comprising administering a vaccine comprising the plasmid according to any of embodiments 1-31.

49. A vaccine for preventing or delaying the onset of Alopecia Areata (AA) symptoms in a patient at risk of developing, or recently diagnosed with AA, said vaccine comprising the plasmid according to any of embodiments 1-31.

Examples

General method and in vivo model:

C3H/HeJ mice, obtained from The Jackson laboratory (https://www.jax.org/; catalog # 000659) constitute a mouse model of Alopecia Areata: Immune function in autoimmunity relies on a complex network of cellular interactions that cannot be adequately evaluated in vitro. Disease suppression and/or treatment evaluations herein were carried out in the C3H/HeJ mouse model, which is an animal model of AA.

In vivo studies were performed as described by Wang et al., Journal of Investigative Dermatology 2015;135:2530, i.e. a lymph node cell transfer model.

Female C3H/HeJ mice, 10-12 weeks of age, were adoptively transferred twice one week apart with 10 million in vitro expanded skin-draining lymph node cells from mice with AA. Lymph node cells (LNCs) for expansion were prepared from excised inguinal, auxiliary and cervical skin draining lymph nodes. Pooled single cell suspensions of these LNCs were stimulated with anti-CD3 and anti-CD28 coated Dynabeads in complete advanced RPMI (cRPMI) supplemented with IL-2, IL-7 and IL-15 cytokines. To obtain sufficient numbers of cells for adoptive transfers, activated cells were expanded for one week by multiple passages in cytokine supplemented cRPMI. Post expansion, 10 million LNCs were injected intradermally on the lower dorsal side of the 10-12 weeks old Female C3H/HeJ mice. One week later, another injection of 10 million LNC was performed and this day marked the initiation for monitoring AA induction and sub-cutaneous dosing.

Dosing consisted of 3 sub-cutaneous doses per week till 13 weeks after LNC transfer with PBS, empty plasmid, or AA plasmid. The empty plasmid consists of a DNA backbone, but does not include AA antigen sequences, TGF-b, IL-10, IL-2, HTLV-1R and B- globin intron. AA plasmid is SEQ ID NO: 8 including AA associated antigen sequence of functional fragments of gp100 and MARTI SEQ ID NO: 8: full (non-annotated) plasmid sequence (6,270 base pairs). To determine the role of the encoded AA antigen, two studies were performed (Example 1 and Example 2 below). Both examples were conducted as described above.

Example 1 - Disease suppression efficacy for plasmids with and without antigen plus cytokines and during dosing: To determine the efficacy of the encoded antigen plus cytokines, the C3H/HeJ mice were dosed with 3 sub-cutaneous doses per week till 13 weeks after LNC transfer as described above. A total of 10 mice were initiated in each treatment arm, but 4 in each arm were sacrificed at 7 weeks for further analyses (data not included). The mice were examined for hair loss each week. The results are shown in Table 1 below and Figure 3:

Table 1: Mice with no hair loss at 5, 9 and 13 weeks.

It is seen from Table 1 and Figure 3 that when mice are dosed with AA plasmid, they are less likely to develop AA compared with mice dosed with PBS or an empty plasmid. Data in Figure 3 have been generated by Kaplan-Meier survival curves and analyzed by Log-rank tests (*p < 0.05; **p < 0.01).

Example 2 - Disease suppression efficacy for plasmids with and without antigen plus cytokines during dosing, and durability of tolerance following withdrawal: To determine the efficacy of the encoded antigen and the duration of the effect following withdrawal of dosing, the C3H/HeJ mice were first dosed with 3 sub-cutaneous doses per week till 13 weeks after LNC transfer as described above, and following the mice were monitored until 22 weeks after LNC transfer. Six mice in each treatment arm completed dosing and withdrawal period. The mice were examined visually for hair loss and results shown in Table 2 and Figure 4.

Table 2: Mice with no hair loss at 5, 9, 15 and 22 weeks.

It is seen from Table 2 and Figure 4 that AA is prevented in animals when dosed with AA plasmid as compared to the empty plasmid or PBS. In addition, it is seen that the effect is maintained even after dosing has stopped at 13 weeks. Data in Figure 4 have been generated by Kaplan-Meier survival curves and analyzed by Log-rank tests (*p < 0.05).

At 22 weeks the mice were sacrificed, and spleen and skin-draining lymph nodes were collected. After tissue processing, isolated cells were stained with antibodies allowing detection of NKG2D+ CD8+ T cells by flow cytometry. The results are shown in Table 3 and Figure 5.

Table 3: Frequencies of NKG2D+ CD8+ T-cells in spleen and skin-draining lymph nodes of mice presenting either hair loss or no hair loss. * Adjusted P Value (2way ANOVA with Sidak’s multiple comparisons test) p = 0.9322 (not significant); ** Adjusted P Value (2way ANOVA with Sidak’s multiple comparisons test) p < 0.0001 (****) The frequency of NKG2D+ CD8+ T cells within the CD8+ T cell population is decreased in skin-draining lymph nodes, but not spleen, of animals dosed with AA plasmid as compared to the empty plasmid or PBS (not shown). Moreover, it is seen from Table 3 and Figure 5 that the frequency of NKG2D+ CD8+ T cells within the CD8+ T cell population is increased in skin-draining lymph nodes of mice with hair loss in general, across the different treatment groups. However, no difference is seen in spleen cells. This is consistent with the hypothesis that NKG2D+ CD8+ T cells are disease-driving for AA, similar to what is seen for JAK inhibitors (Xing et al., Nat Med. 2014; 20(9), 1043-1049). While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims

1. A plasmid which encodes: i. an alopecia associated antigen comprising gp100 and/or MARTI, functional fragments thereof, and/or combinations thereof; ii. TGF-b; iii. IL-10, and iv. IL-2.

2. The plasmid according to claim 1 , wherein the alopecia associated antigen comprises functional fragments of gp100 and/or MART 1.

3. The plasmid according to any one of claims 1-2, wherein the alopecia associated antigen comprises gp100(149-167), gp100(204-222), gp100(275-293) and/or MARTI (22-40).

4. The plasmid according to any one of the preceding claims, wherein the alopecia associated antigen consists of gp100(149-167), gp100(204-222), gp100(275-293) and MARTI (22-40) (SEQ ID NO: 7).

5. The plasmid according to any one of the preceding claims, wherein the plasmid is SEQ ID NO: 9 or essentially as set forth in SEQ ID NO: 9.

6. The plasmid according to any one of the preceding claims, wherein said plasmid comprises: (i) an F2A element separating the alopecia associated antigen encoding sequence and the TGF-b encoding sequence, (ii) an EMCV IRES element separating the TGF-b encoding sequence and the IL-10 encoding sequence, and (iii) a P2A element separating the IL-10 encoding sequence and the IL-2 encoding sequence.

7. The plasmid according to any one of the preceding claims, wherein the TGF-b encoding sequence encodes constitutively active TGF-b.

8. The plasmid according to any one of the preceding claims, wherein said plasmid comprises a TGF-b gene coding for SEQ ID NO: 29 or SEQ ID NO: 29 having less than 10 amino acid substitutions.

9. A DNA immuno-therapy vaccine comprising a plasmid according to any one of the preceding claims.

10. The DNA immuno-therapy vaccine according to claim 9, or the plasmid according to any one of claims 1-8, for use in delaying, preventing or treating alopecia, such as Alopecia Areata.

11. The DNA immuno-therapy vaccine according to claim 9, or the plasmid according to any one of claims 1-8, for use in delaying or preventing alopecia, such as Alopecia Areata.

12. The DNA immuno-therapy vaccine according to claim 9-11, or a plasmid according to any one of claims 1-8, for subcutaneous or intra-muscular administration.

13. A pharmaceutical composition comprising the DNA immuno-therapy vaccine according to claim 9, or a plasmid according to any one of claims 1-8, wherein said pharmaceutical composition comprises a saline solution and/or a buffer and/or a chelator.

14. Use of the plasmid according to any one of claims 1-8 or the DNA immuno-therapy vaccine according to claims 9-12 for the manufacture of a medicament for delaying, preventing or treating alopecia, such as Alopecia Areata.

15. A method of delaying the onset of Alopecia Areata (AA) or symptoms thereof in a patient at risk of developing AA, or recently diagnosed with AA, said method comprising administering a DNA immuno-therapy vaccine comprising the plasmid according to any of embodiments 1-8.