Plasmid encoding insulin antigens and cytokines, DNA immunotherapy vaccine comprising said plasmid, pharmaceutical composition comprising said vaccine or said plasmid and use of said plasmid in DNA immunotherapy and / or in the delay or prevention of type 1 diabetes

Abstract

The present invention relates to plasmids encoding insulin antigens and cytokines for use in immunization tolerance, in particular for the prevention and / or delay of, for example, type 1 diabetes.

Description

1 / 84 PLASMID ENCODING INSULIN ANTIGENS AND CYTOKINES, DNA IMMUNOTHERAPY VACCINE COMPRISING SAID PLASMID, PHARMACEUTICAL COMPOSITION COMPRISING SAID VACCINE OR SAID PLASMID AND USE OF SAID PLASMID IN DNA IMMUNOTHERAPY AND / OR IN THE DELAY OR PREVENTION OF TYPE 1 DIABETES TECHNICAL FIELD

[001] The present invention relates to tolerogenic DNA immunotherapy vaccines for reducing antigen-specific T cell reactivity. BACKGROUND

[002] According to traditional vaccine approaches, the purified protein / antigen is injected into a person / patient / animal in order to stimulate immune responses specifically to that protein / antigen. This vaccine approach tends to primarily impact antibody production, while T cells tend not to be significantly affected, in addition to generating T cell memory of the antigen. Therefore, traditional vaccine approaches are not considered adequate for the treatment and / or prevention of T cell-driven diseases, such as, for example, type 1 diabetes (T1D), as T cell activation, especially CD8+ T cells, are considered the causative agent of this disease. Experimental approaches with tolerogenic protein-based vaccines have primarily targeted antibody-producing B cells rather than disease-relevant T cells.

[003] Generally, DNA-based vaccines, in contrast to protein-based vaccines, are plasmids that encode specific antigens - these plasmids are absorbed by cells in the host's body (transfected). These transfected host cells produce Petition 870220118140, dated 12 / 15 / 2022, page 19 / 107 2 / 84 then the antigen is processed into small fragments (T cell epitopes) for presentation to the immune system, in particular, to circulating T cells. Since 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 CD8+ T cells (or cytotoxic T cells), the main drivers, for example, of T1D pathology. Thus, DNA vaccines, rather than protein vaccines, are suitable for inducing T cell responses. Although there are currently no DNA vaccines available for human use, there are three licensed plasmid-mediated DNA vaccines for veterinary use that induce immunity to Equine Infectious Anemia Virus, West Nile Virus, and certain canine cancers.

[004] In contrast to stimulatory DNA vaccines, tolerogenic DNA immunotherapy vaccines are intended to suppress immune reactivity toward an antigen, rather than activating immune responses against it. These vaccines do not stimulate immunity against the encoded antigen nor alter the type of stimulus (as, for example, antigen desensitization vaccination approaches for allergies do); instead, they cause depletion and / or lack of function and / or death of autoreactive T cells. To achieve this, the antigen must be presented to the immune system without co-stimulation or inflammatory effects that would otherwise activate primary stimulatory immune responses. This approach of presenting an antigen to be ignored by the immune system, or tolerated, could be valuable in the treatment of autoimmune diseases, since the specific disease mechanism would thus be targeted, rather than systematically suppressing the entire immune response.A tolerogenic DNA immunotherapy vaccine is, therefore, a mild method of modulating unwanted immune responses.

[005] The ultimate goal of a vaccine for DNA immunotherapy Petition 870220118140, dated 12 / 15 / 2022, page 20 / 107 3 / 84 specific tolerogenic therapy for T1D is to preserve beta cell function and endogenous insulin production. This can be achieved through disease prevention or delay (especially valuable in pediatric and young adult cohorts where monitoring is difficult and normalcy of life is a major patient motivator) or by extending the honeymoon phase of minimal monitoring and insulin use that often occurs in the first six months after T1D diagnosis.

[006] Although DNA-based vaccines are known to be safe, no DNA vaccine (stimulatory or tolerogenic) that has been tested in clinical studies has sufficient potency as a stand-alone approach for the treatment of, for example, T1D. Tolerogenic DNA vaccines known in the art have shown little efficacy and typically required highly artificial systems to induce the desired effects. There is, therefore, a need in the art for tolerogenic DNA immunotherapy vaccines with significantly increased potency, without compromising the safety profile and, preferably, without requiring an inconvenient administration regimen. SUMMARY

[007] The present invention relates to a multicistronic vector / plasmid that co-expresses / encodes a cell-retained antigen, such as insulin, as well as secreted immunomodulators, such as TGF-β, IL-10 and, optionally, IL-2. The present invention further relates to DNA immunotherapy vaccines comprising such plasmids, as well as their pharmaceutical formulations and kits. Finally, the present invention relates to the medicinal use of such products, as well as methods for producing such plasmids.

[008] The DNA immunotherapy plasmids / vaccines in this document have therapeutic potential in the treatment of autoimmune diseases that Petition 870220118140, dated 12 / 15 / 2022, page 21 / 107 4 / 84 are primarily driven by T cells, such as, for example, type 1 diabetes (T1D).

[009] In one aspect, the present invention provides a plasmid that encodes: i. an insulin antigen; ii. TGF-β; and iii. IL-10. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1. Circular plasmid map.

[0011] Figure 2. mRNA and translated protein map for the vector products of the plasmid from Figure 1.

[0012] Figure 3. Shear stability of the plasmid in three injection passes via G30 needle.

[0013] Figure 4. Confirmation of the plasmid retention phenotype by growth at 30°C (1-50 passages using 17 hours of incubation and 51100 passages using 22 hours of incubation). DESCRIPTION

[0014] The inventor of the present invention has provided, in this document, a unique vector that drives the expression of multiple secreted cytokines, as well as a cell-retained antigen, from a single promoter / multicistronic mRNA.

[0015] DNA immunotherapy vaccination with a single vector encoding all components of the therapy in a single cell is highly preferable over immunotherapy vaccination with a mixture of separate vectors / plasmids, each driving the expression of individual components, since random transfection of cells with different vectors does not guarantee the expression of all components, or even any specific ratio of components, in a transfected cell. Petition 870220118140, dated 12 / 15 / 2022, page 22 / 107 5 / 84 specific provided.

[0016] Transfection of a single multicistronic plasmid / vector results in a specifically manipulated local environment / microenvironment around the transfected cell. In this way, combinations of immunomodulators can be added to the antigen so that they potentiate the desired immunological effect of single T cells without the need for high doses of systemic immunomodulator that could otherwise cause adverse events and widespread immunosuppression.

[0017] This local restriction of immunomodulator production in host cells transfected with the DNA immunotherapy vaccine allows the safe use of highly potent cytokine hormones, which are synergistic for modifying T cell responses, but cannot be dosed frequently enough for effect and / or titrated to give the desired response without unacceptable adverse events.

[0018] For example, Interleukin-10 (IL-10) and Transforming Growth Factor beta1 (TGF-31) are known to be able to induce regulatory T cells (Tregs) from unexposed CD4+ T cells. However, the combination of IL-10 / TGF-31 provides a synergistic effect (15 to 20 times more effective) in inducing Tregs than either cytokine alone (US6083919 A) and, moreover, this combination results in immune tolerance in a wider population of target cells than either cytokine alone (Zeller JC, Panoskaltsis-Mortari A, Murphy WJ, et al. 1999 J Immunol. 163(7):3684-91).

[0019] Furthermore, Interleukin-2 (IL-2) is known to expand and stabilize Tregs but, on the other hand, also contributes to inflammatory responses. The combination of IL-2 and IL-10, however, results in suppressor Tregs, rather than inflammatory stimulation. As circulating T cells encounter cells that are transfected with the vaccine to Petition 870220118140, dated 12 / 15 / 2022, page 23 / 107 6 / 84 DNA immunotherapy in this document, they are temporarily exposed to sub-ideal concentrations of IL-10 and IL-2. Circulating T cells are slightly tolerant, and if they are also reactive to the co-expressed antigen (e.g., insulin), they will bind to the transfected cell and thus receive a longer duration of exposure to the immunomodulator and, in addition, will also receive another signal that programs / reinstructs them for suppressive effects. In this way, T cells that are responsive to the encoded antigen are selectively re-instructed to a suppressive phenotype when they encounter the transfected cell.

[0020] The DNA immunotherapy plasmids / vectors / vaccines in this document are thus designed to induce antigen-specific Tregs that accumulate at sites of autoimmunity to restrain disease (e.g., the pancreas in T1D), rather than directly impacting the disease through expressed cytokine hormones.

[0021] In addition to an antigen (insulin in the T1D example), the vector / operon / plasmid in this document encodes at least two cytokines (e.g., TGF-β1 and IL-10) that, together, synergistically suppress antigen-presenting cells as well as T cell function, and lead to the induction of Tregs. This effect is enhanced if it also occurs in combination with effective exposure to the antigen.

[0022] In one embodiment, TGF-β1 is in a constitutively active form that does not require processing or an inflammatory environment to function. Although Tregs can be produced from unexposed T cells through exposure to antigen and TGF-β1, Tregs are nevertheless plastic, meaning they can dedifferentiate and convert into Th17 effector cells and then cause more, not less, autoimmune destruction. The combination of IL-10 with TGF-β1, in addition to being a more potent immunomodulator, suppresses the environment that would produce Th17 cells. Petition 870220118140, dated 12 / 15 / 2022, page 24 / 107 7 / 84 pathogenic, instead of Tregs.

[0023] In one embodiment, the multicistronic vector in this document also encodes IL-2 in addition to the antigen, TGF-31, IL-10. IL-2 expands Treg numbers and stabilizes their phenotype (prevents Treg cells from dedifferentiating into effector T cells) and thus increases their functional lifespan in inflamed target tissues.

[0024] These three cytokines (TGF-β1, IL-10, and IL-2), in combination with the antigen, thus have well-known synergistic effects to induce tolerance through the following mechanisms: (i) significantly increased generation of antigen-specific suppressor Tregs, (ii) longer Treg lifespan, and (iii) greater efficacy per individual Treg cell in suppressing inflammation / autoreactivity. However, the necessary concentrations of purified cytokine systemically infused would have several serious, or even lethal, side effects, such as: (i) lethal fibrosis from excess TGF-β1, (ii) flu-like symptoms, (iii) capillary leak syndrome from excess IL-2, (iv) widespread immunosuppression leading to chronic infections, (v) potentiated tumor development, as well as (vi) anemia from excess IL-10.

[0025] By co-expressing these cytokines from the same vector / plasmid and therefore by the same cell that presents the antigen to the immune system, the vector reaches the desired local environment for inducing tolerance without systemic action and corresponding side effects that would otherwise result from administering purified cytokines at high doses.

[0026] Injection of naked / unexposed plasmid / vector DNA (vector and buffer alone) has a very low uptake and transfection rate – less than one in about 100,000 plasmid molecules transfects a cell, while the rest is degraded and thus has no biological effect. This extremely low transfection inefficiency provides a mechanism Petition 870220118140, dated 12 / 15 / 2022, page 25 / 107 8 / 84 safety margin for distribution and limitation of transfected cells.

[0027] Administering systemically active amounts of any of these cytokines, whether by administering mature proteins or by transducing a highly efficient viral vector, would be difficult, if not impossible, to titrate to a safe and effective dose. Limiting total exposure to a very small systemic dose distributed across a few high-expression microenvironments leads to a highly advantageous safety and efficacy profile.

[0028] The combination of antigen and these three cytokines in this document produces efficient protection against the development of T1D and even appears to be able to stably reverse disease progression. Due to the low transfection efficiency of the unexposed plasmid / DNA vector injection, very few cells produce these recombinant proteins and therefore there is no detectable change in serum cytokine levels of the plasmid / vector-encoded cytokines – and therefore there is no detectable immune stimulation or immunosuppression for any antigens other than the plasmid / vector-encoded antigen (preproinsulin). This results in a desirable safety profile.

[0029] Typically, DNA vaccines perform poorly with subcutaneous (SC) injection and are therefore usually administered using intramuscular injection (often with electroporation) or, alternatively, using intradermal jet injection requiring a complicated device as well as significant maintenance and calibration. Since most side effect problems with intramuscular injection are related to the adjuvant (injection site irritation), they are therefore not a concern for the DNA immunotherapy vaccine format not disclosed in this document. Additionally, the injected volumes are generally relatively small and therefore do not cause significant muscle distension and pain. In one embodiment, the injected volumes are 1 ml or Petition 870220118140, dated 12 / 15 / 2022, page 26 / 107 9 / 84 less. In another modality, the injected volumes are approximately 0.6 or 0.5 ml. Regardless, the multi-cytokine plasmid / vector presented in this document appears to unexpectedly provide protection against T1D, even when administered subcutaneously, thus allowing for several potential dosing formats for patients.

[0030] 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 further simplified by providing the multicistronic plasmid in this document.

[0031] In contrast, if each of the protein products is produced from a separate plasmid, then the synergistic value of co-expression in the same transfected cell would 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 on the local environment of the transfected cell would potentially be lost; furthermore, multiple individual clinical trials would therefore be required (one for each plasmid and each combination). Producing all proteins from a single plasmid / vector and single mRNA alleviates the need to test multiple individual molecules and determine the ideal co-packaging ratios inherent in a multiple plasmid / vector format.

[0032] Any vector formats suitable for the present invention may be used in this document, such as plasmids (replicating or passive), minicircles, linear vectors (MiLVs), viral vectors (integrating [e.g., lentivirals] and non-integrating [e.g., adenovirals]), cosmids, bacterial artificial chromosomes (BACs), chromosomes Petition 870220118140, dated 12 / 15 / 2022, page 27 / 107 10 / 84 artificial human cells (HACs), etc.

[0033] In addition, any permitted transfection enhancement method may be used in this document: for example, electroporation, sonoporation (ultrasonic enhancement, with or without microbubble contrast enhancement), lipid / polymer aggregates, hydrodynamics (pressure through high volume injection), biolistics / gene-gun (deposition through the skin via compressed gas), etc.

[0034] In one embodiment, non-replicating episomal plasmid DNA is used in this document due to: i) multiple mRNA copies derived from a transfection from a single plasmid, and ii) prolonged stability and function of plasmid nucleic acids over mRNA and other DNA vector formats. Thus, while mRNA- and DNA-based expression systems can provide intracellular distribution and colocalization, plasmid-based systems provide greater dosage control and persistence.

[0035] In one embodiment, the plasmids / vectors encode four proteins: (i) an antigen, (ii) TGF beta 1 (TGF-βΙ), (iii) Interleukin-10 and (iv) Interleukin-2.

[0036] In one embodiment, the antigen is a T1D-relevant antigen targeted endosomally, such as insulin or GAD65. Endosomal targeting can be done by means of, for example, Ii / CD74 fusion, a LIMPII / SCARB fusion, or a transferrin receptor fusion.

[0037] In one embodiment, TGF-e1 is in an activated form.

[0038] The expression of four proteins from a Petition 870220118140, dated 12 / 15 / 2022, page 28 / 107 11 / 84 plasmid / vector is possible, for example, if the desired sequences are separated with A) separate promoters, B) IRES (Internal Entry Site of Ribosomes) sequences that recruit a new ribosome to translate each segment, or C) viral 2A sequences (e.g., FMDV 2A or TaV 2A sequences) that are translated and induce a ribosomal pause / skip resulting in the production of separate polypeptides from a single open reading frame. However, in practice, each of these strategies is complex and difficult to implement.

[0039] The 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 because A) it results in a very large, unstable plasmid that is difficult to produce due to the excessive length of multiple promoters, B) it results in unpredictable behavior of the translated proteins relative to each other (they are no longer produced in fixed ratios to each other), C) each promoter can be independently silenced, leading to the selective expression of some genes but not others necessary for full effectiveness, and D) lack of regulatory simplicity. In contrast, IRES elements and 2A sequences operate at the mRNA and translation levels and co-express, in a reproducible manner, fixed ratios of each protein from a single promoter.

[0040] Each of the four classes of IRES elements has different cofactor requirements to function, as well as different sequence requirements for the downstream gene to be translated. For example, the EMCV (Endomyocarditis Virus) IRES is a 630 base pair type 1 IRES that 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 cofactor but utilizes a non-eukaryotic initiation codon. Petition 870220118140, dated 12 / 15 / 2022, page 29 / 107 12 / 84 standard.

[0041] When IRES elements of different classes are used, they interfere with each other so that each type of IRES element can only be used once in each plasmid, and when they are used together, different types of IRES elements attenuate each other (decrease in effectiveness) in ways that are difficult to predict.

[0042] Furthermore, gene shuffling / IRES combinations result in unpredictable ratios of translated products, since gene interactions with IRES elements are not static but context-dependent on flanking nucleotide sequences. Additionally, IRES elements impose restrictions on early amino acid positions during or immediately after initiation. For example, CrPv IRES requires the first amino acid to be an alanine instead of the standard methionine, and EMCV IRES may not tolerate the amino acids P, W, C, R, or K within the first three codons. In one embodiment, to accommodate the N-terminal amino acid restrictions imposed by EMCV IRES, the DNA vaccine contains an extension of three alanines to the N-terminus of the IL-10 gene.

[0043] Furthermore, 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 produce industrially due to deletions and spontaneous recombinations. In addition, due to the high degree of secondary structure that IRES elements confer to the transcribed mRNAs containing them, they increase the likelihood of activating pathogen recognition receptors (Dabo S, Meurs EF. 2012 Viruses 4(11):2598-635.) in the transfected cell and producing stimulating effects contrary to the induction of tolerance that is Petition 870220118140, dated 12 / 15 / 2022, p. 30 / 107 13 / 84 intended.

[0044] Unlike IRES elements, 2A sequences do not interact with each other and therefore provide stable and consistent performance. However, they are translated into 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) at the 5' end of the sequences to be separated and also initiate the 3' sequence with a proline. Some proteins are permissive of these modifications and some are not, leading to practical restrictions on the use of 2A sequences. For example, the product of Interleukin-10 is permissive of the 2A tail, but both Interleukin-2 and TGF-β1 misfold and lose function if expressed upstream of a 2A marker.Therefore, although it is possible to express several independent proteins separated by 2A sequences, two of the four proteins in this document cannot terminate in 2A markers and, therefore, other strategies must be used.

[0045] Because each type of 2A amino acid sequence modifies ribosomal function during protein translation, it will have different efficiencies in the two central properties of the 2A family, namely (i) the separation of juxtaposed gene products and (ii) processivity (restart) in the second gene product. Different 2A sequences have different efficiencies in generating the ribosomal pause that breaks the main peptide structure (resulting in two separate proteins), as well as different efficiencies in restarting peptide synthesis of the second gene product.

[0046] The ability of 2A sequences to separate protein products and restart protein translation is 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 the 2A amino acid sequences Petition 870220118140, dated 12 / 15 / 2022, p. 31 / 107 14 / 84 result in significantly different mixtures of separate or fused flanking gene products, ranging from less than 5% (>95% fused) to completely separate (0% fused or 100% separate).

[0047] Furthermore, the inventor discovered in this document that the adjacent amino acid sequences encoding the two flanking protein products also affect the efficiency of restarting and separating the 2A sequences, leading to significant deviations from the reported results. Thus, the restart efficiency 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 protein separation will be determined by the 2A amino acid sequence used and its context.

[0048] In one embodiment, FMDV 2A is inserted between the antigen coding sequence and the TGF-β1 coding sequence in this document; resulting in 100% separation as well as a 1:1 ratio of the protein products.

[0049] In another embodiment, TaV 2A can be inserted between the IL-10 coding sequence and the IL-2 coding sequence in this document, resulting in approximately 50% separate products, as well as a 10 to 6 ratio of protein products. Thus, each transfected cell delivers a relatively low dose of Interleukin-2, which is unable to stimulate effector T cells, and a higher dose of Interleukin-10 to influence T cells toward the Treg phenotype. Since the production of fused IL-10 / IL-2 is disadvantageous, attempts have been made to design greater cleavage efficiency of the TaV 2A segment. An attempt to precede the 2A segment with an insulator segment, which is an element that extends the translated region upstream of TaV 2A to reduce the impact of the upstream sequence on the 2A element, did not improve the Petition 870220118140, dated 12 / 15 / 2022, p. 32 / 107 15 / 84 separation. In a different attempt to solve the fusion problem, an upstream uncoupling segment with a GSG-translated protein sequence was added; however, this approach resulted only in an incremental improvement in cleavage efficiency.

[0050] Thus, cytokine fusions resulting from the separation of genes encoding IL-10 and IL-2 by a TAV 2A will possibly be immunogenic.

[0051] In another embodiment, the vector / plasmid in this document has a 2A segment of P. Separation of the genes encoding IL-10 and IL-2 by a P2A results in complete or near-complete separation of the protein products, as well as a ratio of at least two times (or perhaps even four or five times) of IL-10 compared to IL-2.

[0052] In order to address the shortcomings of the IRES-only and 2A-only systems described above, the four cDNA sequences in this document (antigen, TGF-β1, 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 jumping and the production of independent proteins from each sequence in the polyprotein pair. Since TGF-β1 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 must terminate at the translated portion of the mRNA sequence.

[0053] The chronology / sequence of expressed proteins and IRES / 2A elements in this document can therefore be as follows: (i) Antigen, (ii) FMDV 2A, (iii) TGF beta 1, (iv) IRES, (v) IL-10, (vi) P 2A and (vii) IL-2. As a consequence, all four proteins can be independently expressed from a single gene operon / segment in a stable and predictable manner. Since each of these proteins is expressed from a single mRNA, the ratios of each product are fixed - it is not possible Petition 870220118140, dated 12 / 15 / 2022, page 33 / 107 16 / 84 can generate an excess of IL-2 over IL-10, for example.

[0054] In addition to using a combination of IRES and 2A elements for the separation of encoded genes, an alternative solution in this document could be the use of a bidirectional promoter to generate 2 mRNAs – these mRNAs would encode a pair of proteins, instead of all four in one mRNA molecule. Equivalent arrangements can therefore be constructed using pairs of expression cassettes arranged appropriately around a mammalian bidirectional promoter and using 2A sequence separation and / or IRES elements. However, this approach is associated with disadvantages, mainly due to the large size of bidirectional promoters, but also to a potential regulatory burden with separated mRNA elements included in a medicinal product. Therefore, the preferred embodiments in this document use a single promoter and a combination of IRES and 2A elements, instead of a bidirectional promoter.

[0055] In theory, some 2A sequences could be replaced by sequences sensitive to endogenous intracellular proteases. However, the inventor has discovered, in this document, that such proteases are associated with significant disadvantages (e.g., lack of reported function, resulting in the secretion of fused protein products).

[0056] For the antigen to be processed and presented to the immune system within the local environment of the plasmid-encoded cytokine hormones, the antigen must be retained within the transfected cell. In the case of type 1 diabetes, the production of active insulin would potentially lead to an undesirable reduction in blood glucose if it were secreted or released from the transfected cell.

[0057] In order to avoid antigen secretion, any signs of secretion can be removed from the antigen coding sequence, by Petition 870220118140, dated 12 / 15 / 2022, page 34 / 107 17 / 84 For example, removing the secretion signal coding sequence from the nucleic acid sequence encoding preproinsulin would generate proinsulin instead of preproinsulin, thus allowing the antigen to accumulate within the transfected cell. Although this translated antigen product (e.g., insulin) would not be actively secreted, it could be released during lysis due to necrosis resulting from attack by CD8+ T cells. Additionally, the insulin signal sequence is a region known to contain disease-relevant epitopes (potentially inducing autoimmunity), and inclusion of the signal sequence therefore ensures a broader induction of tolerance and a greater likelihood of reducing the disease.

[0058] Furthermore, cytoplasmic retention of the antigen only allows processing via the proteasome and presentation via the MHC class I pathway, which detects intracellular pathogens via CD8+ T cells. Since CD4+ T cells contribute significantly to pro-inflammatory cytokines and most, if not all, autoimmune-suppressing Tregs are CD4+, expanding antigen presentation to include MHC class II, which is recognized by CD4+ T cells, may be advantageous.

[0059] MHC class II processing and CD4+ T cell stimulation do not normally include intracellular antigen, as access to this pathway is via endocytosis of extracellular antigen. Normally, protein products produced within a transfected cell are presented only through the standard intracellular / proteasomal processing pathway and MHC class I, resulting in CD8+ T cell effects, but not CD4+ T cell effects. In order to target CD4+ and CD8+ T cells for immunomodulation, the preferred modality also includes factors that lead to MHC class II presentation.

[0060] In principle, to induce the presentation of the MHC class Petition 870220118140, dated 12 / 15 / 2022, page 35 / 107 18 / 84 In class II, 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. The transferrin receptor, also known as the iron transport protein receptor, fuses the extracellular space / plasma membrane cycle to the endosome and therefore can expose other immune cells to the whole antigen, such as B cells, macrophages, etc. LimpII / SCARB fusions target the endosome directly, but preferentially the early endosome, and sometimes result in overprocessing and total destruction of the antigen. Class II (CD74) fusions, which utilize 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 probability of effectively presenting the antigen in the context of MHC class II.Additionally, even with endosomal separation from Ii fusions, the pre-proinsulin secretion sequence must be inactivated, or the antigen would also be secreted and lost before processing.

[0061] Blocking insulin antigen secretion was alternatively achieved in this document by mutating two amino acids required for removal of the secretion marker by SRP (Signal Recognition Particle) in the Rough Endoplasmic Reticulum. Mutations from Ala (A) to Glu (E) completely abolish preproinsulin maturation and secretion, while maintaining the necessary antigen epitope structure for optimal tolerance induction.

[0062] In one embodiment, a plasmid DNA vaccine is used in this document. The plasmid is cultured / replicated, for example, in E. coli and isolated / purified from media, and subsequently formulated into liquid formulations, for example, water, saline, liquid PBS formulations, or as a lyophilized powder for intradermal jet injection, administration Petition 870220118140, dated 12 / 15 / 2022, page 36 / 107 19 / 84 intranasal, or inhalation. In one embodiment, the plasmid in this document is formulated in an aqueous pharmaceutical formulation comprising, optionally, stabilizers. Any suitable microbial system may be used for plasmid production.

[0063] Stabilizers in the formulation include, but are not limited to, chelating agents such as EDTA, EGTA, or DPTA for the sequestration of Mg++ and Fe+++, which may be involved, on the other hand, in DNA degradation, and / or citrate, which protects the plasmid from non-specific degradation effects. In one embodiment, the plasmid in this document may be formulated in isotonic PBS or, alternatively, in TRIS + citrate + EDTA. These plasmids have the advantages of being stable, easy to produce, and safe and convenient during use.

[0064] In another embodiment, delivery agents, such as viruses, lipids, liposomes, copackaging, etc., would be added in connection with the present invention. However, the use of delivery agents in this document may have potential problems with immunity, viral integration, etc. Definitions

[0065] Antigen: The DNA immunotherapy vaccine in this document encodes an antigen. The antigen in this document may be any type of immunogenic disease-associated protein or fragment thereof that can be recognized by the T-cell component of the immune system. For example, in the case of treatment or prevention of type 1 diabetes, an insulin antigen may be used. In one example, the insulin antigen is the immunodominant peptide InsB 9-23. For DNA immunotherapy vaccines for multiple sclerosis in this document, a myelin basic protein (MBP), myelin oligodendrocyte protein (MOG), and / or proteolipid protein antigen (PLP) may be used as the antigen. Sequences Petition 870220118140, dated 12 / 15 / 2022, page 37 / 107 20 / 84 similar protein antigen coding antigens for representative antigens of alopecia, polymyositis / dermatomyositis, celiac psilocystin, and protein allergens (e.g., peanut protein ara h 2) are also examples of suitable antigens for use in DNA immunotherapy vaccines in this document.

[0066] Antigen targeting: In one embodiment, the antigen in this document is targeted via the endosomal route. Antigens in this document include the whole protein, secretion-deficient pre-proteins, or a functional or immunodominant peptide fragment thereof.

[0067] For example, the insulin antigen in this document is an antigen for use in immune modulating therapy and not a glucose-lowering agent. Therefore, it must not be fully processed / matured or secreted in order to ensure that it is presented on MHC molecules to circulating T cells. The DNA immunotherapy vaccine in this document therefore does not result in increased blood insulin levels, but instead results in increased antigen presentation to the immune system, in particular to T cells.

[0068] Therefore, the insulin antigen in this document may be a small immunodominant peptide encoding fragments (e.g., insulin B chain 9-23 peptide, including swapped registry peptides displaying equivalent T cell epitopes), whole proinsulin, which lacks the necessary secretion sequence but is otherwise intact, or preproinsulin muteins that contain the secretion sequence but are modified to prevent secretory function.

[0069] Examples of insulin antigens in this document include:

[0070] Mouse proinsulin (SEQ ID NO 1): FVNQHLCG SHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQ PLALE GSLQKRGIVEQCCTSICSLYQLENYCN Petition 870220118140, dated 12 / 15 / 2022, page 38 / 107 21 / 84

[0071] Human proinsulin (SEQ ID NO 2): FVNQHLCGSHLVE ALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALE GSLQKRGIVEQCCTSICSLYQLENYCN

[0072] Modified mouse preproinsulin that is not secreted (substitutions relative to wt preproinsulin shown in bold and underlined (SEQ ID NO 3)): MALWMRRLLPLLALLALWG PDPEQEFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVG QVELGGGPGAGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCN

[0073] Modified human preproinsulin that is not secreted (substitutions with respect to wt preproinsulin shown in bold and underlined (SEQ ID NO 4)):

[0074] MALWMRLLPLLALLALWGPDPEQEFVNQHLCGSHLVEA LYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEG SLQKRGIVEQCCTSICSLYQLENYCN

[0075] Mouse wt preproinsulin (SEQ ID NO 5): ALW MRLLPLLALLALWGPDPAQAFVNQHLCGSHLVEALYLVCGERGFFY TPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKRGIVEQCC TSICSLYQLENYCN

[0076] Human wt pre-proinsulin (SEQ ID NO 6): MALWMRLL PLLALLALWGPDPAQAFVNQHLCGSHLVEALYLVCGERGFFYTPKTR REAEDLQVGQVELGGGPGAGSLQPLALEGSLQKRGIVEQCCTSICSL YQLENYCN

[0077] Mouse and human identical insulin peptide InsB 9-23: SHLVEALYLVCGERG (SEQ ID NO 7)

[0078] Modified InsB 9-23 (substitutions with respect to InsB 9-23 wt shown in bold and underlined (SEQ ID NO 8) and (SEQ ID NO 27)): SHLVEALYLVCGEEG and SHLVEALYLVCGGEG

[0079] The insulin antigens in this document may, therefore, Petition 870220118140, dated 12 / 15 / 2022, page 39 / 107 22 / 84 accumulate in the cytosol of the transfected host cell and can thus be presented via MHC class I, or released by cytolysis.

[0080] The endosomal targeting that results in MHC class II presentation can be achieved in this document by fusing the antigen sequence with leader sequences that form transmembrane segments with cytoplasmic sequences YXX0, where Y is tyrosine, X is any amino acid, and O 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 endosomal / lysosomal separation signals DXXLL, which are underlined in the following example sequences. The protein domains that include these signals, therefore, target or cycle to the endosome / lysosome and include: transferrin receptor, LimpII or CD74, also known as the invariant chain, MHC II chaperone, or Ii, or any similar domain.

[0081] Examples of endosomal targeting domains in this document include, but are not limited to:

[0082] Mouse CD74 endosomal targeting domain / invariant chain (Ii) (SEQ ID NO 9): MDDQRDLISNHEQL PILGNRPREPERCSRGALYTGVSVLVALLLAGQATTAYFLYQQQGRL DKLTITSQNLQLESLRMKLP

[0083] Human CD74 endosomal targeting domain / invariant chain (Ii) (SEQ ID NO 10): MHRRRSRSCREDQKP VMDDQRDLISNNEQLPMLGRRPGAPESKCSRGALYTGFSILVTLLLA GQATTAYFLYQQQGRLDKLTITSQNLQLESLRMKLP

[0084] Type 1 Diabetes: Type 1 diabetes (T1D) is considered a chronic autoimmune disease where self-destructive T cells infiltrate the islets of Langerhans in the pancreas and play a significant role in Petition 870220118140, dated 12 / 15 / 2022, p. 40 / 107 23 / 84 specifically destroys the population of insulin-producing beta cells. Once a significant number of islet cells are destroyed, reduced amounts of insulin, or no insulin at all, will result in insulin deficiency and hyperglycemia in the patient. Therefore, patients with T1D are unable to produce sufficient insulin and require regular insulin injections throughout their lives. Some patients with type 1 diabetes are diagnosed with type 1.5 diabetes, latent autoimmune diabetes / LADA, double diabetes, etc., which are diabetes disorders that carry symptoms of both type 1 and type 2 diabetes – all diabetes disorders that carry a succession of type 1 and type 2 diabetes are also thus included under the term type 1 diabetes in this document.

[0085] Tolerogenic DNA Vaccine The DNA-based immunotherapy vaccines / vectors / plasmids in this document are designed to switch off or downregulate the part of the immune system responsible for destroying normal healthy self cells and thus prevent or enhance T-cell-based autoimmunity.

[0086] The term DNA immunotherapy vaccine, as used in this document, is intended to mean a compound or composition comprising a DNA molecule that is administered to a subject in order to reduce the risk of that subject developing one or more diseases.

[0087] In some modalities, DNA-based immunotherapy vaccines are plasmids / vectors that encode specific antigens. After vaccination, these plasmids are assimilated by, in other words, transfected into antigen-presenting cells in the host body. The transfected host cells then produce the antigen and present small fragments of the antigen to the immune system, in particular, to T cells. This approach leads to a modification of antigen-specific T cell responses, as well as a modification Petition 870220118140, dated 12 / 15 / 2022, page 41 / 107 24 / 84 minimum immune responses to other (non-encoded or irrelevant) antigens. Only a few host cells are typically transformed with the DNA vaccine plasmid / vector in this document, meaning that probably less than one in a hundred thousand, one in five hundred thousand, or even less than one in a million plasmid / vector molecules eventually enter a host cell. The DNA vaccines in this document thus represent a very mild and specific approach to modulating immune responses to antigens such as insulin in patients with T1D or in patients at risk of developing T1D.

[0088] Plasmid: A plasmid is a small DNA molecule 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 DNA vaccines for immunotherapy. Plasmids are considered replicons, a unit of DNA capable of autonomous replication within a suitable host. Plasmids can be transmitted from one bacterium to another bacterium, which can be bacteria of the same species or different species, through three main mechanisms: transformation, transduction, and conjugation. DNA vaccine plasmids can be assimilated by a host cell through passive transformation – usually at a relatively low rate.The plasmids in this document replicate efficiently—but do not drive protein expression—in bacteria. Furthermore, the plasmids in this document drive protein expression—but not plasmid replication—in humans and other mammals, such as mice. In one embodiment, a pVAXI vector (Invitrogen / LifeTechnologies) is used as a scaffold in this. Petition 870220118140, dated 12 / 15 / 2022, page 42 / 107 25 / 84 document to incorporate the elements that are part of the present invention. Other suitable vector scaffolds in this document include any main vector structure containing a eukaryotic promoter element, a prokaryotic origin of replication with high copy production, and a selection system for plasmid maintenance.

[0089] Selection gene and selection system: In one aspect, DNA immunotherapy vaccines in this document comprise a selection gene / selection marker for manufacturing purposes. The selectable marker in this document is, for example, a gene that confers resistance to a cellular toxin – for example, an antibiotic such as ampicillin, kanamycin, chloramphenicol, streptomycin, etc.

[0090] Other types of selection systems suitable in this document include, for example, conditional lethal silencing systems (e.g., CcdA / CcdB or ParD / ParE Hok / Sok type systems), or sequences that complement a genomic defect in the production cell strain and thus allow the growth of an otherwise non-viable host (e.g., auxotrophic complementation by dapD- or pyrF-, translation initiation complementation by infA-, etc.)

[0091] Production cells harboring the plasmid / DNA vaccine, which includes the selection marker, will survive when exposed to the toxin / antibiotic / condition, while those that failed to assimilate the plasmid sequences will die. As such, in one embodiment, the DNA vaccines in this document comprise the nucleic acid sequence encoding a selection marker in order to provide higher yield / purity and more efficient yield / replication in production cells such as E. coli.

[0092] Although antibiotic selection is a common laboratory strategy, there may be advantages associated with antibiotic-free selection systems. Petition 870220118140, dated 12 / 15 / 2022, page 43 / 107 26 / 84 antibiotics - for example, in relation to more efficient regulatory processes. Although vectors that do not contain a selection mechanism, such as minicircles, synthetic linear vectors, etc., can also be used in this document, these implementations are associated with certain disadvantages in production, in particular, due to higher production and quality control costs.

[0093] Examples of complementation (rescue) strategies are known in the state of the art, however, these strategies suffer from several disadvantages.

[0094] Metabolic complementation systems, such as dapD [lysine biosynthesis] or pyrF [uridine biosynthesis] systems, generally result in cross-feeding during the production of high-density E. coli, where a plasmid-containing bacterium will produce and secrete an excess of the required compound and thus relax the selection pressure for neighboring bacteria without the plasmid.

[0095] Another example of a suitable selection system in this document are plasmids encoding essential proteins, such as infA, which encode IF1 / Initiation 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 since there is no way to exogenously supplement the necessary protein / infA (J Bacteriol. 1994 Jan; 176(1):198-205 and J Biotechnol. 2004 Jul 1;111(1):17-30).

[0096] In order to circumvent the disadvantages associated with the infA selection system, an alternative selection system has been provided in this document with a temperature-sensitive (or thermosensory) translational shift from the L. monocytogenes prfA invasion protein gene. Petition 870220118140, dated 12 / 15 / 2022, page 44 / 107 27 / 84 (Cell. 2002, September 6;110(5):551-61). By placing the hairpin-forming portion of a thermosensitive RNA sequence upstream of E. coli genomic copies of infA via standard recombination technology, its expression becomes regulated through fermentation temperature control, allowing slow growth of plasmid-free cells at 37°C, and rapid cell death at temperatures <30°C. Transformation of the thermosensitive E. coli production strain manipulated with infA wt-expressing plasmids thus allows normal full growth rates at all temperatures, permitting plasmid-free expansion at 37°C as well as rigorous plasmid selection at 30°C. Additionally, this system does not generate selective pressure for E. coli wt to retain the plasmid, and it is thus lost within 8 hours in culture – ensuring no environmental permanence of the therapeutic plasmid.

[0097] Nucleotide sequence of infA from E. coli wt (SEQ ID NO 11): ATGGCCAAAGAAGACAATATTGAAATGCAAGGTACCGTTCTT GAAACGTTGCCTAATACCATGTTCCGCGTAGAGTTAGAAAACGGT CACGTGGTTACTGCACACATCTCCGGTAAAATGCGCAAAAACTAC ATCCGCATCCTGACGGGCGACAAAGTGACTGTTGAACTGACCCC GTACGACCTGAGCAAAGGCCGCATTGTCTTCCGTAG TCGCTGA

[0098] IF1 protein sequence of E. coli wt resulting from translation of the infA gene (initial methionine / M not included in the prfA fusion - (SEQ ID NO 12)): MAKEDNIEMQGTVLETLPNTMFRVELENGHVVTAHISGKMRKNYIRILTGDKVTVELTPYDLSKGRIVFRSR

[0099] E. coli production cell lines used in this document for the production of vaccine plasmids for DNA immunotherapy may thus harbor the following thermosensitive prfA nucleotide sequence:

[00100] nucleotide sequence of prfA from L. monocytogenes wt Petition 870220118140, dated 12 / 15 / 2022, p. 45 / 107 28 / 84 (thermosensor hairpin) (Shine Dalgarno underlined, ATG initiation in bold - (SEQ ID NO 13)): TGTAAAAAACATCATTTAGCGT GACTTTCTTTCAACAGCTAACAATTGTTGTTACTGCCTAATGTTTTT AGGGTATTTTAAAAAAGGGCGATAAAAAACGATTGGGGGATGAGAAATGAACGCTCAA

[00101] prfA protein sequence of L. monocytogenes wt (the fused upstream of E. coli IF1 - resulting from the translation of SEQ ID NO 13): MNAQ

[00102] Origin of Replication (Ori): The origin of replication, also called the origin of replication, is a specific sequence in a genome where DNA strand replication is initiated. In one embodiment, the origin of replication sites in this document include the pUC Ori which allows replication in the bacterial E. coli production cell line – but not in mammalian host cells, i.e., cells from the body of the vaccinated subject / person / patient. Other suitable bacterial origins of replication in this document include, but are not limited to: R6K, pBR322, ColE1, pMB1, 15A, pSC101, etc. In one aspect, the origin of replication in this document is a high copy number version that produces a high plasmid / biomass ratio for more efficient production. Vectors that do not contain an origin of replication, such as minicircles, synthetic linear vectors, etc., may also be used in this document.

[00103] Promoter: A promoter is a region of DNA that initiates the transcription of a specific gene. Promoters are located near the transcription initiation sites of genes, on the same strand and upstream in the DNA, towards the 5' end of the sense strand. For transcription to occur, RNA polymerase must bind to DNA near a gene. Promoters contain specific DNA sequences, such as response elements, that provide a safe initial binding site for RNA polymerase. Petition 870220118140, dated 12 / 15 / 2022, p. 46 / 107 29 / 84 and for transcription factors that recruit RNA polymerase. Transcription factors have specific activator or repressor sequences that bind to specific promoters and regulate gene expression. Promoters thus represent critical elements that can work in combination with other regulatory regions, such as enhancers, silencers, limiting / isolating elements, to direct the transcription level of a given gene. A classic promoter leads to the production of a single messenger RNA (mRNA), while bidirectional promoters in this document lead to the production of two mRNAs immediately adjacent to the promoter, both upstream and downstream of the promoter.

[00104] In one embodiment, eukaryotic promoters are used in this document. Eukaryotic promoters do not necessarily obey the one gene / one promoter rule, such as multiple viral promoters, as well as promoters that exhibit broad expression (i.e., do not have restricted cell type specificities, such as expression only in neurons). Examples of promoters in this document that are capable of driving broad transcription of large mRNA molecules from multiple genes include: the immediate-early (IE) viral promoters of CMV and SV40; promoters of endogenous EF1a, PGK1, Ubc, and beta-actin; and synthetic promoters, such as the hybrid CAG promoter. Many other suitable mammalian promoters exist, and more are being designed through synthetic biology efforts. Any promoter that results in the desired expression characteristics in human cells can be used in the DNA immunotherapy vaccine plasmids in this document.

[00105] Enhancers: Enhancers are DNA elements that increase the efficiency of promoters in producing mRNA transcripts. The enhancers in this document may be matched (e.g., SV40 enhancer / CMV promoter) or unmatched. Any combination Petition 870220118140, dated 12 / 15 / 2022, p. 47 / 107 30 / 84 of a suitable enhancer / promoter for eukaryotic function can be used in this document.

[00106] Eukaryotic translation initiation: The eukaryotic translation initiation sequence is generally referred to as the Kozak consensus sequence. The Kozak sequence in an mRNA molecule is recognized by the ribosome as the translation initiation 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 correspond to consensus sequences. In fact, Kozak consensus sequences are typically less efficient than wild-type variants isolated from mammalian mRNAs. Although weak Kozak sequences are regularly isolated from native mRNAs and likely play a role in controlling the translation of low-abundance proteins, the DNA immunotherapy vaccines in this document preferentially encode a medium- or high-efficiency Kozak sequence.Examples of Kozak sequences useful in this document include the following nucleotide sequence: gccRccATGG (SEQ ID NO 14), where the lowercase bases are the most common nucleotides, but which may vary, while the uppercase nucleotides are fixed (R is the IUPAC uncertainty code for the bases A and G), and ATG indicates the translation initiation site of the methionine codon at position +1.

[00107] Endosome separation signal: An endosome is a membrane-bound compartment within eukaryotic cells. Some proteins can be transported into endosomes and there they are 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, Petition 870220118140, dated 12 / 15 / 2022, page 48 / 107 31 / 84 CD4+ T cells. Protein separation into endosomes is mediated by signals present in the cytosolic domains of the proteins. Endosomal signals are generally short linear amino acid sequences. The antigens in this document are preferentially targeted to endosomes using an endosome separation signal, such as, for example, endosomal / lysosomal separation signals YXX0, [DE]XXXL[LI], or DXXLL. Endosome separation signals include various synthetic or naturally occurring endosomal separation signals. Examples in this document include the endosome separation signals present in Cd74 / invariant chain / Ii, LimpII / SCARB, or transferrin receptor. Any endosomal targeting domain that is pharmaceutically acceptable and provides the desired function may be used. Fusion of these endosomal targeting domains to the antigens directs them to the endosomal compartment after translation for greater efficacy.Endosomal antigen segregation provides processing and presentation to the immune system in MHC class II complexes, in addition to constitutive presentation in MHC class I complexes, for a more complete and robust induction of tolerance and possible expansion of Tregs (which cannot be achieved through MHC class I / antigen complexes). In one embodiment, the tolerogenic DNA vaccines in this document encode a fusion of the antigen with the CD74 / invariant / Ii chain to drive endosomal targeting and antigen presentation via MHC class II.

[00108] Introns: Introns are non-coding sequences within an mRNA. Some introns are known to significantly enhance mRNA translation and function. In this sense, the inclusion of intron sequences can also be used in this document. Standard introns, such as beta-globin, or any intron that adheres to mammalian splicing conventions, such as MCM7, can be used. In one embodiment, vectors Petition 870220118140, dated 12 / 15 / 2022, page 49 / 107 32 / 84 of DNA immunotherapy vaccine vectors in this document comprise sequences that encode one or more introns. In another embodiment, DNA immunotherapy vaccine vectors in this document do not have sequences that encode introns.

[00109] Ribosomal pause marker: In connection with the present invention, it may be advantageous to include one or more ribosomal pause marker sequences between the protein-coding sequences in the DNA immunotherapy vaccine vector / plasmid in this document in order to separate the protein products.

[00110] One example is the FMDV viral 2A marker (foot-and-mouth disease virus 2A marker). The translated amino acid sequence of FMDV 2A is APVKQTLNFDLLKLAGDVESNPGP - (SEQ ID NO 15). The FMDV 2A marker is capable of pausing and restarting the ribosome. The ratio of the translated product before and after the FMDV 2A marker is close to 1:1, and the resulting protein products are normally completely separated. These types of ribosome markers have been used previously in connection with the co-expression of two different domains, for example, heavy chain and light chain in recombinant antibody production. However, the inventor of the present invention made the surprising discovery that they are useful in connection with multicistronic DNA vaccines for the separation of flanking products and for the control of expressed protein ratios due to the inherent efficiency of ribosomal restart.Sequence markers that favor a 1:1 ratio of translated products are, in this document, preferably inserted between protein-coding sequences that would preferentially be produced at a 1:1 ratio (or close to it), such as, for example, an insulin antigen and a potent cytokine, such as, for example, TGF-β.

[00111] Another example of a pause marker sequence Petition 870220118140, dated 12 / 15 / 2022, pp. 50 / 107 33 / 84 ribosomal marker in this document is the viral sequence marker TaV 2A (Thiesa virus 2A - TaV 2A amino acid sequence: RAEGRGSLLTCGDVEENPGP (SEQ ID NO 16)). The ratio of translated product before / upstream and after / downstream of this marker is reported to be 50:1 (or close to it). The inventor of the present invention made the surprising discovery that, although this type of marker can be used to control expression levels in cases where it is vital that one translated product completely dominates the other, the separation of flanking cytokine products is less than 50% compared to sequences disclosed in the literature and the expression ratio is about 10:6. In connection with the present invention, a 2A type of ribosomal pause marker sequence should preferably result in different expression levels of two proteins encoded by the same vector / plasmid.The expression of small amounts of a pleiotropic cytokine (such as IL-2) relative to an anti-inflammatory cytokine, such as IL-10, is desirable in this document, and fused products are not desirable.

[00112] Another example of a ribosomal pause marker amino acid sequence in this document is the viral sequence 2A of P (2A of porcine teschovirus-1, ATNFSLLKQAGDVEENPGP - (SEQ ID NO 17)). Sequence 2A of P functions appropriately when inserted between IL-10 and IL-2 in this document, resulting in almost complete separation with an expression ratio of >5:1 between IL-10 and IL-2.

[00113] Alternatively, proteinase-sensitive sequences, which allow endogenous cleavage between expressed plasmid polyproteins, can be used in this document. A furin-sensitive sequence (which recognizes RAKR motifs) or a carboxypeptidase-sensitive sequence (which recognizes RRRR, RKRR, or RRKR motifs) can be used in this document for the separation of protein products. However, the Petition 870220118140, dated 12 / 15 / 2022, p. 51 / 107 34 / 84 The inventor of the present invention made the surprising discovery that neither the furin nor the carboxypeptidase sequences result in separate products as described in this document - thus leading to the secretion of unwanted IL10 / IL-2 fusion proteins.

[00114] TGF-β / ε / ε1 (transforming growth factor beta / ε1): TGF-β is a secreted protein that controls proliferation, cell differentiation, and other functions in most cells. TGF-β is a very potent cytokine with significant effects on cell fate and phenotype in a context-dependent manner, for example, depending on other concomitantly received cytokine signals. Endogenous TGF-β 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, the TGF-β in this document is a modified form that is constitutively active. This is achieved by replacing cysteines at positions 223 and 225 with amino acids incapable of forming disulfide bridges. For example, serine and 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.

[00115] Human endogenous TGF-e1 sequence - SEQ ID NO 18: MPPSGLRLLLLLLPLLWLLVLTPGRPAAGLSTCKTIDMELVKRKRIEAI RGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPE PEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREA VPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPS DSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDI NGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTN YCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGCPPYIW SLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVE Petition 870220118140, dated 12 / 15 / 2022, page 52 / 107 35 / 84 QLSNMIVRSCKCS. Sequence of modified human TGF-βI that is constitutively active and secreted (substitutions relative to TGF-β1 wt shown in bold and underlined) - SEQ ID NO 19: MPPSGLRLLLLLLPLLWLLV LTPGRPAAGLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVP PGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVET HNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVE QHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSR GGEIegfrlsahvsvdsrdntlqvdingfttgrrgdlatihgmnrpf LLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRK DLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGA SAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS. Another modified human TGF-β1 sequence that can be used is SEQ ID NO 25: MPPSGLRLLLLLLPLLWLLVLTPGRPAAGLSTCKT IDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYN ST RDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSI YMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNS WRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHSS SDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHL QSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKG YHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALE PLPIVYYVGRKPKVEQLSNMIVRSCKCS.

[00116] Terminator sequence: A transcription terminator is a section of a nucleic acid sequence that marks the end of a gene during transcription. The release of the transcriptional complex releases RNA polymerase and related transcriptional machinery to initiate the transcription of new mRNAs. Additionally, the same cellular factors add a template-free poly-A tail that significantly increases lifespan. Petition 870220118140, dated 12 / 15 / 2022, page 53 / 107 36 / 84 and mRNA functionality. An example of a suitable transcription terminator in this document includes the bGH_PA terminator, CGACTGTGCCTTCTAGT TGCCAGCCATCTGTTGTTTGCCCCTCCCCGTGCCTTCCTTGACC CTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAA ATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGT GGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATA GCAGGCATGCTGGGGATGCGGTGGGCTCTATGG (SEQ ID NO 20).

[00117] Any acceptable terminator sequence may be used in this document. Variations include the use of two different flanking terminator sequences in the case of bidirectional promoters producing two oppositely oriented mRNAs.

[00118] In one embodiment, the plasmid of the invention has the sequence as shown in SEQ ID NO 24.

[00119] In a second embodiment, the plasmid of the invention has the sequence SEQ ID NO 26: complete plasmid sequence (not annotated) GA CTCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCAT AGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGC CCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAAT AATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGA CGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTA CATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAAT GACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTT ATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGC TATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTG GATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATT GACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTT Petição 870220118140, de 15 / 12 / 2022, pág. 54 / 107 37 / 84 TCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGC GGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCT GGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAAT ACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTT AAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGA ATTCTGCACTGCAGCTCGCATCTCTCCTTCACGCGCCCGCCGCCC TACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGC CTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCC CTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCT GACCCTGCTTGCTCAACTCTAGGTAAGTTAATGAGACAGATAGAA ACTGGTCTTGTAGAAACAGAGTAGTCGCCTGCTTTTCTGCCAGGT GCTGACTTCTCTCCCCTGGGCTTTTTTCTTTTTCTCAGGTTGAAA AGAAGAAGACGAAGAAGACGAAGAAGACAAACCGTCGTCGACTG CCATGCGCCGCTGATTAACGCCGCCACCATGGCCCACCGACGCA GATCCAGAAGCTGCCGTGAGGACCAGAAGCCCGTGATGGATGAT CAGAGGGACCTTATCTCTAACAATGAACAACTGCCAATGCTCGGC AGACGGCCTGGGGCCCCGGAGAGCAAGTGCAGCAGAGGAGCC TTGTACACGGGGTTCTCCATTTTAGTGACTCTCCTTCTCGCCGGC CAAGCTACCACCGCCTACTTTCTGTACCAACAGCAAGGCAGACTA GACAAACTGACAATCACAAGCCAGAACCTTCAGCTGGAGTCTCTG CGGATGAAGCTGCCCGCTTTGTGGATGAGATTGCTTCCTCTACTT GCTCTCCTGGCGCTCTGGGGACCTGACCCCGAGCAAGAGTTTGT TAATCAGCACCTGTGTGGGAGTCATCTGGTGGAGGCACTCTATT TAGTGTGCGGAGAGAGGGGCTTCTTCTACACTCCAAAGACCAGA CGGGAGGCCGAAGACCTTCAAGTGGGGCAAGTAGAACTGGGTG GCGGACCCGGTGCCGGGAGCCTTCAGCCGCTCGCCCTGGAGGG CTCTCTTCAGAAACGCGGCATCGTGGAGCAGTGTTGCACATCCA TTTGCTCACTCTACCAGCTGGAGAACTACTGCAACGGAAGCGGA Petição 870220118140, de 15 / 12 / 2022, pág. 55 / 107 38 / 84 GTGAAGCAGACGTTGAATTTTGATTTGTTGAAGTTGGCGGGGGAT GTGGAGAGCAATCCGGGGCCGATGCCCCCTAGTGGCCTCAGAC TTTTGTTATTGTTATTACCGCTTTTATGGCTCTTGGTGCTGACACC GGGCCGTCCGGCTGCTGGCTTGTCGACTTGTAAGACAATTGATAT GGAATTGGTGAAACGAAAACGGATTGAGGCCATCCGAGGACAGA TTTTGAGCAAGCTGCGGCTTGCCTCGCCACCCTCGCAAGGGGA AGTCCCACCCGGACCTCTACCAGAAGCAGTCCTAGCGCTGTACAA CAGTACAAGAGATAGAGTGGCCGGGGAATCCGCAGAACCAGAGC CTGAGCCTGAAGCCGATTATTATGCAAAGGAAGTGACTAGGGTCC TGATGGTCGAGACCCATAACGAAATCTACGACAAATTCAAACAAA GTACCCACTCTATCTACATGTTCTTCAACACCAGTGAGCTAAGAGA AGCCGTGCCCGAACCTGTGCTTCTTTCCCGCGCAGAACTCCGCC TCTTGAGACTCAAATTGAAAGTTGAACAACACGTAGAGCTTTACC AGAAATACTCTAATAATTCATGGCGATATCTTTCTAATCGTCTCCT CGCCCCATCTGACAGCCCTGAATGGCTCTCCTTCGACGTTACGGGAGTTGTGCGCCAGTGGCTCAGCAGAGGCGGAGAGATAGAGG GCTTTCGGCTGAGCGCACATAGCTCTAGCGACTCAAGGGACA ACACATTGCAAGTGGATATTAACGGTTTTACAACTGGACGGAGAG GGGACCTGGCGACCATCCACGGCATGAATAGACCTTTCCTGCTG CTGATGGCTACTCCCCTGGAGAGGGCACAGCACTTACAGTCTTCC AGACACCGGCGCGCCCTGGATACAAACTACTGCTTCAGCTCCACC GAAAAGAACTGTTGCGTGCGGCAGCTGTACATTGACTTCAGAAAGGATCTGGGCTGGAAGTGGATTCATGAGCCCAAGGGGTATCAT GCCAACTTCTGTCTTGGGCCATGCCCATACATCTGGTCACTGGAT ACCCAGTACTCCAAAGTTCTGGCCTTGTACAATCAACACAACCCT GGAGCTTCCGCCGCTCCTTGCTGTGTGCCCCAAGCCCTAGAGC CCCTGCCCATCGTTTATTATGTCGGACGCAAGCCCAAAGTAGAA CAGCTATCAAATATGATCGTGAGAAGCTGCAAGTGTAGCTGATA AACGCGTCGAGCATGCATCTAGGGCGGCCAATTCCGCCCCTCT Petição 870220118140, de 15 / 12 / 2022, pág. 56 / 107 39 / 84 CCCCCCCACCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCC GAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTA TTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAA CCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCC CTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAA GCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAG CGACCCTTTGTAGACAGCGGAACCCCCCACCTGGCGATAGATG CCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAG GCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGG AAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCT GAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTG GGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAA AAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTT GAAAAACACGATGATAATATGATGCACAGCTCAGCACTGCTCT GTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCAGCCCAGGC CAGGGCACCCAGTCTGAGAACAGCTGCACCCACTTCCCAGGC AACCTGCCTAACATGCTTCGAGATCTCCGAGATGCCTTCAGCAG AGTGAAGACTTTCTTTCAAATGAAGGATCAGCTGGACAACTTGTT GTTAAAGGAGTCCTTGCTGGAGGACTTTAAGGGTTACCTGGGTT GAGGTGATGCCCCAAGCTGAGAACCAAGACCCAGACATCAAGGC GCATGTGAACTCCCTGGGGGAGAACCTGAAGACCCTCAGGCTG AGGCTACGGCGCTGTCATCGATTTCTTCCCTGTGAAAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAATGCCTTTAATAAGCTCCAA GAGAAAGGCATCTACAAAGCCATGAGTGAGTTTGACATCTTC ATCAACTACATAGAAGCCTACATGACAATGAAGATACGAAACGG GAGCGGCGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGAC GTGGAGGAGAACCCTGGACCTATGTACAGAATGCAGCTGCTGA GCTGCATCGCCCTGAGCCTGGCCCTGGTGACCAACAGCGCACC CACGTCCTCTAGCACCAAGAAGACCCAGTTACAGTTGGAGCATC Petição 870220118140, de 15 / 12 / 2022, pág. 57 / 107 40 / 84 TACTTTTAGACCTGCAAATGATTTTGAACGGCATCAACAACTACA AGAATCCTAAACTTACTCGCATGCTTACCTTCAAATTTTACATGC CCAAGAAGGCCACCGAACTGAAGCACTTGCAATGTCTGGAGGA AGAACTCAAGCCGCTGGAGGAAGTTCTCAACCTCGCGCAGTCC AAGAATTTCCACCTCCGGCCAAGAGACCTGATCAGTAACATT AATGTGATAGTGCTGGAGCTGAAGGGAAGCGAGACTACATTTAT GTGCGAGTACGCCGATGAAACCGCTACAATCGTCGAGTTCCTGA ATAGATGGATCACATTTTGCCAGTCAATTATCTCTACTCTGACAT GATAACTCGAGGTCTAGAGGGCCCGTTTAAACCCGCTGATCAG CCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCC CCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCAC TGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAG TAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAG CAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGAT GCGGTGGGCTCTATGGCTTCTACTGGGCGGTTTTATGGACAGCA AGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTG GGAAGCCCTGCAAAGTAAACTGGATGGCTTTCTCGCCGCCAAGG ATCTGATGGCGCAGGGGATCAAGCTCTGATCAAGAGACAGGATG AGGATCGTTTCGCATGGCCAAAGAAGACAATATTGAAATGCAA GGTACCGTTCTTGAAACGTTGCCTAATACCATGTTCCGCGTAG AGTTAGAAAACGGTCACGTGGTTACTGCACACATCTCCGGTAAA ATGCGCAAAAACTACATCCGCATCCTGACGGGCGACAAAGTGA CTGTTGAACTGACCCCGTACGACCTGAGCAAAGGCCGCATTGTCTTCCGTAGTCGCTGATAAATTATTAACGCTTACAATTTCC TGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCC TATTTGTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGA GACAATAACCCTGATAAATGCTTCAATAATAGCACGTGCTAAAA CTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGAT Petition 870220118140, dated 12 / 15 / 2022, pp. 58 / 107 41 / 84 AATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTG AGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATC CTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCAC CGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAAC TCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCA AATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAA GAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGT TACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCG GGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCG GTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGA GCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTAT GAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTA TCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGA GCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGT TTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAG GGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTT TACGGTTCCTGGGCTTTTGCTGGCCTTTTGCTCACATGTTCTT.

[00120] In a third embodiment, the plasmid of the invention has the sequence SEQ ID NO 28: complete plasmid sequence (not annotated) GACTCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATT ATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCA TAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGG CCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAA TAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTG ACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAG TACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAA TGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTT ATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCT ATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGA Petição 870220118140, de 15 / 12 / 2022, pág. 59 / 107 42 / 84 TAGCGGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGAC GTCAATGGGAGTTTGTTTTGGCACCAAATCAACGGGACTTTCC AAAATGTCGTAACAACTCCGCCCCATTGACCAAATGGGCGGT AGGCGTGTACGGTGGGAGGTCTATAAGCAGAGCTCTCTGGC TAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGA CTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCT TGGTACCGAGCTCGGATCCACTAGTCCAGTGGTGGAATTCTG CAGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGG CCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGC CTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGC CTACCTAGACTCAGCCGCTTCCACGCTTTGCCTGACCCTGCT TGCTCAACTCTTAGTAAGTAATGAGACAGATAGAAACTGGTCTT GTAGAAACAGAGTAGTCGCCTGCTTTTTCTGCCAGGTGCTGACT TCTCTCCCTGGGCTTTTTTCTTTTTCTCAGGTTGAAAAGAAGAA GACGAAGAAGACGAAGAACAAACCGTCGTCGACTGCCA TGCGCCGCTGATTAACGCCGCACCATGGCCCACCGACGCAGAT CCAGAAGCTGCCGTGAGGACCCAGAAGCCGTGATGGATCAG AGGGACTTATCTCTTAACAATGAACAACTGCCAATGCTGGCAG ACGGCCTGGGCCCCGGAGAGCAAGTGCAGCAGAGGAGCCCTTG TACACGGGGTTTCCCATTTTAGTGACTTCCTTCTCGCCGGCC AAGCTACCACCCGGCCTACTTTCTGTACCAACAGCAAGGCAGACTAGACAAACTGACAATCACAAGCCAGAACCTTCAGCTGGAGTCT CTGCGGATGAAGCTGCCCGCTTTGTGGATGAGATTGCTTCCTC TACTTGCTCTCCTGGCGCTCTGGGGACCTGACCCCGAGCAAGAG TTTGTTAATCAGCACCTGTGTGGGAGTCATCTGGTGGAGGCAC TCTATTTAGTGTGCGGAGAGAGGGGCTTCTTCTACACTCCAAAGA CCAGACGGGAGGCCGAAGACCTTCAAGTGGGGCAAGTAGAACT GGGTGGCGGACCCGGTGCCGGGAGCCTTCAGCCGCTCGC Petição 870220118140, de 15 / 12 / 2022, pág. 60 / 107 43 / 84 CCTGGAGGGCTCTCTTCAGAAACGCGGCATCGTGGAGCAG TGTTGCACATCCATTTGCTCACTCTACCAGCTGGAGAACTACTG CAACGGAAGCGGAGTGAAGCAGACGTTGAATTTTGATTTGTTG AAGTTGGCGGGGGATGTGGAGAGCAATCCGGGGCCGATGCC CCCTAGTGGCCTCAGACTTTTGTTATTGTTATTACCGCTTTTAT GGCTCTTGGTGCTGACACCGGGCCGTCCGGCTGCTGGCTTGT CGACTTGTAAGACAATTGATATGGAATTGGTGAAACGAAAACGG ATTGAGGCCATCCGAGGACAGATTTTGAGCAAGCTGCGGCTTG CCTCGCCACCCTCGCAAGGGGAAGTCCCACCCGGACCTCTAC CAGAAGCAGTCCTAGCGCTGTACAACAGTACAAGAGATAGAG TGGCCGGGGAATCCGCAGAACCAGAGCCTGAGCCTGAAGCC GATTATTATGCAAAGGAAGTGACTAGGGTCCTGATGGTCGAG ACCCATAACGAAATCTACGACAAATTCAAACAAAGTACCCACTC TATCTACATGTTCTTCAACACCAGTGAGCTAAGAGAAGCCGT GCCCGAACCTGTGCTTCTTTCCCGCGCAGAACTCCGCCTCTTGA GACTCAAATTGAAAGTTGAACAACACGTAGAGCTTTACCAGAAA TACTCTAATAATTCATGGCGATATCTTTCTAATCGTCTCCTCG CCCCATCTGACAGCCCTGAATGGCTCTCCTTCGACGTTACGGG AGTTGTGCGCCAGTGGCTCAGCAGAGGCGGAGAGATAGAGGGCT TTCGGCTGAGCGCACATAGCTCTAGCGACTCAAGGGACAACAC ATTGCAAGTGGATATTAACGGTTTTACAACTGGACGGAGAGGGG ACCTGGCGACCATCCACGGCATGAATAGACCTTTCCTGCTGCTGATGGCTACTCCCCTGGAGAGGGCACAGCACTTACAGTCTTC CAGACACCGGCGCGCCCTGGATACAAACTACTGCTTCAGCTCCA CCGAAAAGAACTGTTGCGTGCGGCAGCTGTACATTGACTTCAG AAAGGATCTGGGCTGGAAGTGGATTCATGAGCCCAAGGGGTAT CATGCCAACTTCTGTCTTGGGCCATGCCCATACATCTGGTCACT GGATACCCAGTACTCCAAAGTTCTGGCCTTGTACAATCAACACA ACCCTGGAGCTTCCGCCGCTCCTTGCTGTGTGCCCCAAGCCCTA GAGCCCCTGCCCATCGTTTATTATGTCGGACGCAAGCCCAAAGTA Petição 870220118140, de 15 / 12 / 2022, pág. 61 / 107 44 / 84 GAACAGCTATCAAATATGATCGTGAGAAGCTGCAAGTGTAGCTGA TAAACGCGTCGAGCATGCATCTAGGGCGGCCAATTCCGCCCCTC TCCCCCCCACCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCC GAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTA TTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAA ACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCC CCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAG GAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGTAGACAGCGGAACCCCCCACCTGGCGATAGATGCCTCTGCGGCCAAAAGCCACG TGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAA GGATGCCCAGAAGGTACCCCATTG TATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGT GTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGG GGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGATGC ACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGT GAGGGCCAGCCCAGGCCAGGGCACCCAGTCTGAGAACAGCT GCACCCACTTCCCAGGCAACCTGCCTAACATGCTTCGAGATCTCC GAGATGCCTTCAGCAGAGTGAAGACTTTCTTTCAAATGAAGGA TCAGCTGGACAACTTGTTGTTAAAGGAGTCCTTGCTGGAGGACT TTAAGGGTTACCTGGGTTGCCAAGCCTTGTCTGAGATGATCCAGT TTTACCTGGAGGAGGTGATGCCCCAAGCTGAGAACCAAGACCCA GACATCAAGGCGCATGTGAACTCCCTGGGGGAGAACCTGAAGA CCCTCAGGCTGAGGCTACGGCGCTGTCATCGATTTCTTCCCTGTGAAAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAATGCCTTTAAT AAGCTCCAAGAGAAAGGCATCTACAAAGCCATGAGTGAGTTTGA CATCTTCATCAACTACATAGAAGCCTACATGACAATGAAGATACG Petition 870220118140, dated 12 / 15 / 2022, pp. 62 / 107 45 / 84 AAACGGGAGCGGCGCTACTAACTTCAGCCTGCTGAAGCAGGCT GGAGACGTGGAGGAGAACCCTGGACCTATGTACAGAATGCAGCT GCTGAGCTGCATCGCCCTGAGCCTGGCCCTGGTGACCAACAGCG CACCCACGTCCTCTAGCACCAAGAAGACCCAGTTACAGTTGGAG CATCTACTTTTAGACCTGCAAATGATTTTGAACGGCATCAACAAC TACAAGAATCCTAAACTTACTCGCATGCTTACCTTCAAATTTTA CATGCCCAAGAAGGCCACCGAACTGAAGCACTTGCAATGTCTG GAGGAAGAACTCAAGCCGCTGGAGGAAGTTCTCAACCTCGCGCAGTCCAAGAATTTCCACCTCCGGCCAAGAGACCTGATCAGTA ACATTAATGTGATAGTGCTGGAGCTGAAGGGAAGCGAGACTACAT TTATGTGCGAGTACGCCGATGAAACCGCTACAATCGTCGAGTTCC TGAATAGATGGATCACATTTTGCCAGTCAATTATCTCTACTCTGA CATGATAACTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCA GCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCC CCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCAC TGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAG TAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAG CAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGAT GCGGTGGGCTCTATGGCTTCTACTGGGCGGTTTTATGGACAGCA AGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTG GGAAGCCCTGCAAAGTAAACTGGATGGCTTTCTCGCCGCCAAG GATCTGATGGCGCAGGGGATCAAGCTCTGATCAAGAGACAGG ATGAGGATCGTTTCGCATGGCCAAAGAAGACAATATTGAAAT GCAAGGTACCGTTCTTGAAACGTTGCCTAATACCATGTTCCGCG TAGAGTTAGAAAACGGTCACGTGGTTACTGCACACATCTCCGG TAAAATGCGCAAAAACTACATCCGCATCCTGACGGGCGACAAAG TGACTGTTGAACTGACCCCGTACGACCTGAGCAAAGGCCGCATTGTCTTCCGTAGTCGCTGATAAATTATTAACGCTTACAATTTCCTGA TGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCA Petição 870220118140, de 15 / 12 / 2022, pág. 63 / 107 46 / 84 TACAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTG TTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATAGCACGTGCTAAAACTTCA TTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATC TCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGT CAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTT TTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTA CCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTT TTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACT GTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAAC TCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACC AGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTG GACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGG CTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACG ACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAG CGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTA AGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCA GGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCA CCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGC GGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTT CCTGGGCTTTTGCTGGCCTTTTGCTCACATGTTCTT.

[00121] In a fourth embodiment, the plasmid of the invention has the sequence SEQ ID NO 29: complete plasmid sequence (not annotated) GACTCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATT ATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCAT AGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGC CCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAA TAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATT GACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCA Petition 870220118140, dated 12 / 15 / 2022, pp. 64 / 107 47 / 84 GTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTC AATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGAC CTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCA TCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGG GCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCAC CCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAAC GGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGC AAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGC AGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTT ATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGC TAGCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCACTAG TCCAGTGTGGTGGAATTCTGCAGCTCGCATCTCTCCTTCACGC GCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAG TCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCT TTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTC TCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTAGGTAAGTTA ATGAGACAGATAGAAACTGGTCTTGTAGAAACAGAGTAGTCGCC TGCTTTTCTGCCAGGTGCTGACTTCTCTCCCCTGGGCTTTTTTCTTTTTCTCAGGTTGAAAAGAAGAAGACGAAGAAGACGAAGAAGACA AACCGTCGTCGACTGCCATGCGCCGCTGATTAACGCCGCCACCA TGGCCCACCGACGCAGATCCAGAAGCTGCCGTGAGGACCAGAAGCCCGTGATGGATGATCAGAGGGACCTTATCTCTAACAATGAA CAACTGCCAATGCTCGGCAGACGGCCTGGGGCCCCGGAGAGCAAGTGCAGCAGAGGAGCCTTGTACACGGGGTTCTCCATTTTAGT GACTCTCCTTCTCGCCGGCCAAGCTACCACCGCCTACTTTCTGTACCAACAGCAAGGCAGACTAGACAAACTGACAATCACAAGCC AGAACCTTCAGCTGGAGTCTCTGCGGATGAAGCTGCCCGCTTTG TGGATGAGATTGCTTCCTCTACTTGCTCTCCTGGCGCTCTGGGGA Petição 870220118140, de 15 / 12 / 2022, pág. 65 / 107 48 / 84 CCTGACCCCGAGCAAGAGTTTGTTAATCAGCACCTGTGTGGGAG TCATCTGGTGGAGGCACTCTATTTAGTGTGCGGAGAGAGGGG CTTCTTCTACACTCCAAAGACCAGACGGGAGGCCGAAGACCTT CAAGTGGGGCAAGTAGAACTGGGTGGCGGACCCGGTGCCGGGAGCCTTCAGCCGCTCGCCCTGGAGGGCTCTCTTCAGAAACG CGGCATCGTGGAGCAGTGTTGCACATCCATTTGCTCACTCTACC AGCTGGAGAACTACTGCAACGGAAGCGGAGTGAAGCAGACGTT GAATTTTGATTTGTTGAAGTTGGCGGGGGATGTGGAGAGCAAT CCGGGGCCGATGCCCCCTAGTGGCCTCAGACTTTTGTTATTGT TATTACCGCTTTTATGGCTCTTGGTGCTGACACCGGGCCGTC CGGCTGCTGGCTTGTCGACTTGTAAGACAATTGATATGGAATTGGTGAAACGAAAACGGATTGAGGCCATCCGAGGACAGATTTTGAGCAAGCTGCGGCTTGCCTCGC CACCCTCGCAAGGGGAAG TCCCACCCGGACCTCTACCAGAAGCAGTCCTAGCGCTGTACA ACAGTACAAGAGATAGAGTGGCCGGGGAATCCGCAGAACCAG AGCCTGAGCCTGAAGCCGATTATTATGCAAAGGAAGTGACTAG GGTCCTGATGGTCGAGACCCATAACGAAATCTACGACAAAT TCAAACAAAGTACCCACTCTATCTACATGTTCTTCAACACCAG TGAGCTAAGAGAAGCCGTGCCCGAACCTGTGCTTCTTTCCCGCG CAGAACTCCGCCTCTTGAGACTCAAATTGAAAGTTGAACAACAC GTAGAGCTTTACCAGAAATACTCTAATAATTCATGGCGATATCT TTCTAATCGTCTCCTCGCCCCATCTGACAGCCCTGAATGGCTCTCCTTCGACGTTACGGGAGTTGTGCGCCAGTGGCTCAGCAGAGGCGGAGAGATAGAGGGCTTTCGGCTGAGCGCACATAGCTCTAGCG ACTCAAGGGACAACACATTGCAAGTGGATATTAACGGTTTTACAA CTGGACGGAGAGGGGACCTGGCGACCATCCACGGCATGAATAG ACCTTTCCTGCTGCTGATGGCTACTCCCCTGGAGAGGGCACAGCACTTACAGTCTTCCAGACACCGGCGCGCCCTGGATACAAAC TACTGCTTCAGCTCCACCGAAAAGAACTGTTGCGTGCGGCAGCT Petição 870220118140, de 15 / 12 / 2022, pág. 66 / 107 49 / 84 GTACATTGACTTCAGAAAGGATCTGGGCTGGAAGTGGATTCATGA GCCCAAGGGGTATCATGCCAACTTCTGTCTTGGGCCATGCCCAT ACATCTGGTCACTGGATACCCAGTACTCCAAAGTTCTGGCCTTG TACAATCAACACAACCCTGGAGCTTCCGCCGCTCCTTGCTGTGT GCCCCAAGCCCTAGAGCCCCTGCCCATCGTTTATTATGTCGGACGCAAGCCCAAAGTAGAACAGCTATCAAATATGATCGTGAGAAGCT GCAAGTGTAGCTGATAAACGCGTCGAGCATGCATCTAGGGCGG CCAATTCCGCCCCTCTCCCCCCCACCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTG CGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCT CGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAA GACAAACAACGTCTGTAGCGACCCTTTGTAGACAGCGGAACC CCCCACCTGGCGATAGATGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGG AAAGAGTCAAATGGCTCTCCTCAAG CGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATT GTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATG TGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGG GACGTGGTTTTCCTTTGAAAAACACGATGATAATATGATGCACA GCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAG GGCCAGCCCAGGCCAGGGCACCCAGTCTGAGAACAGCTGCA CCCACTTCCCAGGCAACCTGCCTAACATGCTTCGAGATCTCCGA GATGCCTTCAGCAGAGTGAAGACTTTCTTTCAAATGAAGGAT CAGCTGGACAACTTGTTGTTAAAGGAGTCCTTGCTGGAGGACT TTAAGGGTTACCTGGGTTGCCAAGCCTTGTCTGAGATGATCCAG TTTTACCTGGAGGAGGTGATGCCCCAAGCTGAGAACCAAGACC CAGACATCAAGGCGCATGTGAACTCCCTGGGGGAGAACCTGAA Petição 870220118140, de 15 / 12 / 2022, pág. 67 / 107 50 / 84 GACCCTCAGGCTGAGGCTACGGCGCTGTCATCGATTTCTTCC CTGTGAAAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAATGCC TTTAATAAGCTCCAAGAGAAAGGCATCTACAAAGCCATGAGTG AGTTTGACATCTTCATCAACTACATAGAAGCCTACATGACA ATGAAGATACGAAACGGGAGCGGCGCTACTAACTTCAGC CTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTA TGTACAGAATGCAGCTGCTGAGCTGCATCGCCCTGAGCCTGG CCCTGGTGACCAACAGCGCACCCACGTCCTCTAGCACCAAGAA GACCCAGTTACAGTTGGAGCATCTACTTTTAGACCTGCA AATGATTTTGAACGGCATCAACAACTACAAGAATCCTAAAC TTACTCGCATGCTTACCTTCAAATTTTACATGCCCAAGAAGG CCACCGAACTGAAGCACTTGCAATGTCTGGAGGAAGAACTCAA GCCGCTGGAGGAAGTTCTCAACCTCGCGCAGTCCAAGAATT TCCACCTCCGGCCAAGAGACCTGATCAGTAACATTAATGTGA TAGTGCTGGAGCTGAAGGGAAGCGAGACTACATTTATGTGCGAG TACGCCGATGAAACCGCTACAATCGTCGAGTTCCTGAATAGATG GATCACATTTTGCCAGTCAATTATCTCTACTCTGACATGATAAC TCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTG TGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCC GTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTT CCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGT GTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAG GGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGA TGGCTTTCTCGCCGCCAAGGA TCTGATGGCGCAGGGGATCAAGCTCTGATCAAGAGACAGGATGA GGATCGTTTCGCATGGCCAAAGAAGACAATATTGAAATG Petição 870220118140, de 15 / 12 / 2022, pág. 68 / 107 51 / 84 CAAGGTACCGTTCTTGAAACGTTGCCTAATACCATGTTCCGCGTAGAGTTAGAAAACGGTCACGTGGTTACTGCACACATCTCCGGTAAAATGCGCAAAAACTACATCC GCATCCTGACGGGCGAC AAAGTGACTGTTGAACTGACCCCGTACGACCTGAGCAAAGGC CGCATTGTCTTCCGTAGTCGCTGATAAATTATTAACGCTTAC AATTTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTAT TTCACACCGCATACAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCC GCTCATGAGACAATAACCCTGATAAATGCTTCAATAATAGCACG TGCTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCC TTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTT CCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAA AAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGC TACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCA GATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACC ACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCT GCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTC GTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGC GCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCC AGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGT GAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCG GACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCG CACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGT CCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGG CTTTTGCTGGCCTTTTGCTCACATGTTCTT

[00122] termo GLP-1 / peptídeo GLP-1 / peptídeo agonista de GLP1R, conforme usado neste documento, refere-se a moléculas / peptí Petition 870220118140, dated 12 / 15 / 2022, pp. 69 / 107 52 / 84 GLP-1 proteins / variants / agonists, in this document, are molecules having GLP-1R agonist function, meaning they are agonists of the GLP-1 receptor. This class of drugs is commonly used to treat diabetes, particularly type 2 diabetes. The amino acid sequence of mature human GLP-1 is: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 21).

[00123] The term GLP-1 analog, as used in this document, refers to a peptide or compound that is a variant of GLP-1 (SEQ ID NO: 15). The terms GLP-1 analog and analog may be used interchangeably in this document.

[00124] GLP-1 analogues can be described by reference to i) the number of amino acid residues in human GLP-1 (SEQ ID NO: 15), which corresponds to the amino acid residues that are modified (i.e., the corresponding position in GLP-1 (SEQ ID NO: 15)), and ii) the actual modification.

[00125] The term GLP-1 derivatives refers to derivatives of GLP-1 analogs. The term derivative, as used in this document in the context of a GLP-1 analog, means a chemically modified GLP-1 analog in which one or more substituents have been covalently attached to the GLP-1 analog. The term substituent, as used in this document, means a chemical moiety or side group conjugated to the GLP-1 protein / agonist / analog. The derivative may comprise one or more modifications selected from among amides, carbohydrates, alkyl groups, acyl groups, esters, and the like.

[00126] In some embodiments, the substituent is covalently linked through an amino acid residue in said polypeptide, for example, at one of the selected amino acid positions of the group consisting of positions 22, 23, 27, 34, 35 and 36.

[00127] In some embodiments, the LPG-1 derivative comprises Petition 870220118140, dated 12 / 15 / 2022, pp. 70-107 53 / 84 a substituent comprising a lipophilic fraction. The term lipophilic fraction, as used in this document, means an aliphatic or cyclic hydrocarbon fraction with more than 6 and less than 30 carbon atoms, wherein said hydrocarbon fraction may comprise additional substituents.

[00128] Examples of GLP-1 agonists include (but are not limited to) exenatide, liraglutide, lixisentide, albiglutide, dulaglutide, taspoglutide, and semaglutide. DNA immunotherapy vaccines using the plasmids in this document may be initially combined with parallel GLP-1 agonist treatment in the treatment of, for example, patients with newly onset T1D. Co-administration of GLP-1 may be chronic or temporary and may include oral routes in addition to parenteral routes.

[00129] Liraglutide: (SEQ ID NO 22): the WtxrHAEGTFTSDVSSYLEGQAAN^Í—ef awlvrg rg-==-

[00130] Semaglutide (SEQ ID NO 23): O o -HH^^ACGTF TSO VSSY LEGQAAKv^EFIAWLVRGR G—=-

[00131] The pharmaceutical compositions in this document 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 in this document may alternatively be dry formulations, such as Petition 870220118140, dated 12 / 15 / 2022, pp. 71 / 107 54 / 84 lyophilized formulations, intended for reconstitution, inhalation, intranasal instillation, intradermal administration, etc.

[00132] The pharmaceutical formulations in this document are preferably administered without the use of methods to enhance transformation, such as electroporation. In one embodiment, the pharmaceutical formulations are intended for parenteral administration, for example, subcutaneous administration, intradermal administration, intravenous administration, intramuscular administration, etc. In another embodiment, the pharmaceutical compositions in this document may also be administered topically, orally, rectally, or by inhalation.

[00133] The pharmaceutical compositions in this document preferably do not have the addition of any condensing agent or other excipients that may induce local reactions. The formulations in this document contain free radical scavengers (e.g., 1% ethanol) and / or chelating agents, such as divalent cation scavengers (e.g., EDTA [CAS #60-00-4], EGTA [CAS #67-42-5], or DPTA [CAS #67-43-6]) in order to improve the stability of aqueous plasmid DNA. The pharmaceutical compositions in this document may, in addition, 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).The pharmaceutical formulations in this document are preferably free of any adjuvants, as well as other typical vaccine ingredients, such as, for example, aluminum hydroxide, phenol, sorbitol, silicone, etc.

[00134] Administration: The DNA immunotherapy vaccine in this document can be administered to a patient with T1D, or to a patient at risk of developing T1D. The vaccine can be administered, for example, Petition 870220118140, dated 12 / 15 / 2022, pp. 72 / 107 55 / 84 daily, every other day, twice a week, once a week, twice a month, once a month, every two months, four times a year, or once a year – the frequency may be adjusted according to general or individual needs. Immunotherapy in this document may be chronic. The duration of therapy may be, for example, one month, two months, three months, six months, one year, two years, three years, five years, six years, seven years, eight years, nine years, or ten years. Modalities

[00135] The following embodiments illustrate the invention and should not be understood as limiting. It is understood that all embodiments can be combined in all possible ways.

[00136] A plasmid that codes for: (i) an antigen; (ii) TGF-β; and (iii) IL-10.

[00137] The plasmid, according to embodiment 1, in which the said antigen is an insulin antigen.

[00138] 3. A plasmid that co-expresses / encodes (preferably from a single operon): (i) an antigen, such as, for example, an insulin antigen; (ii) TGF-β / TGF-β1 (such as in a constitutively active form); and (iii) IL-10.

[00139] 4. The plasmid, according to any of the preceding embodiments, in which the said insulin antigen is selected from the group consisting of: proinsulin, pre-proinsulin incapable of secretion, or a functional or immunodominant peptide fragment thereof.

[00140] 5. The plasmid, according to any of the preceding embodiments, in which the said insulin antigen is selected from the group consisting of: proinsulin, preproinsulin and a peptide fragment Petition 870220118140, dated 12 / 15 / 2022, pp. 73 / 107 56 / 84 functional or immunodominant of the same.

[00141] 6. The plasmid, according to any of the previous embodiments, in which the said insulin antigen is the endosomal target insulin.

[00142] 7. The plasmid, according to any of the preceding embodiments, in which said plasmid expresses the insulin antigen and TGF-β in a ratio of approximately 1:1.

[00143] 8. The plasmid, according to any of the previous embodiments, in which said plasmid expresses the insulin antigen and TGF-β in an amount at least 200 times lower than IL-10.

[00144] 9. The plasmid, according to any of the previous embodiments, in which said plasmid expresses the insulin antigen and TGF-β in an amount at least 2 times lower than IL-10.

[00145] 10. The plasmid, according to any of the previous embodiments, in which said plasmid also co-expresses Interleukin-2 (IL-2).

[00146] 11. The plasmid, according to any of the previous embodiments, in which said plasmid expresses an excess of IL-10 and IL-2 on the antigen (e.g., insulin) and TGF-β.

[00147] 12. The plasmid, according to any of the preceding embodiments, in which said plasmid expresses IL-10 and IL-2 at least about once, twice, five times or at least about one hundred times over TGF-β and insulin antigen (IL-10+IL2 to insulin+TGF-β ratio may be at least 1:1, or 2:1, or 5:1 or 100:1).

[00148] 13. The plasmid, according to any of the preceding embodiments, in which said plasmid expresses IL-10 and IL-2 at least about one hundred times, two hundred times, five hundred times, or at least one thousand times over TGF-β and insulin antigen (ratio Petition 870220118140, dated 12 / 15 / 2022, pp. 74 / 107 The ratio between IL-10+IL-2 and insulin+TGF-β (57 / 84) can be at least 100:1, or 200:1, or 500:1, or 1000:1.

[00149] 14. The plasmid, according to any of the preceding embodiments, in which said plasmid expresses IL-10 and IL-2 in a ratio of about 1:1 - 100:1, such as, for example, 1:1 - 50:1, such as, for example, 1:1 - 25:1, such as, for example, 1:1 - 10:1, alternatively 1:1 - 5:1, alternatively 1:1 - 3:1, alternatively 1:1 - 2:1. Alternatively, the ratio between expressed IL-10 and expressed IL-2 may be about 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4; 1:0.3, 1:0.2, or 1:0.1.

[00150] 15. The plasmid, according to any of the foregoing embodiments, wherein said plasmid comprises: (i) an FMDV 2A element that separates the insulin antigen coding sequence and the TGF-β coding sequence, (ii) an EMCV IRES element that separates the TGF-β coding sequence and the IL-10 coding sequence, and (iii) a 2A element that separates the IL-10 coding sequence and the IL-2 coding sequence.

[00151] 16. The plasmid, according to any of the preceding embodiments, in which said plasmid comprises:

[00152] a 2A element (such as an FMDV 2A or P 2A element) that separates the insulin antigen coding sequence and the TGF-β coding sequence,

[00153] an EMCV IRES element (alternatively a bidirectional promoter) that separates the TGF-β coding sequence and the IL-10 coding sequence (preferably, three alanine amino acids are encoded immediately from the N-terminal to the IL-10 gene), and

[00154] a 2A element (like a 2A element of P) that separates the IL-10 coding sequence and the IL-2 coding sequence.

[00155] 17. The plasmid, according to any of the Petition 870220118140, dated 12 / 15 / 2022, pp. 75 / 107 58 / 84 previous modalities, in which the TGF-β coding sequence encodes constitutively active TGF-β, preferentially constitutively active human TGF-β1.

[00156] 18. The plasmid, according to any of the preceding embodiments, wherein said plasmid comprises: (i) an endosomal target preproinsulin coding sequence, (ii) an FMDV 2A element, (iii) a TGF-β coding sequence, (iv) an EMCV IRES element, (v) an IL-10 coding sequence, (vi) a P 2A element, (vii) an IL-2 coding sequence, (viii) a polyadenylation / termination element, (ix) a selection gene, (x) an origin of replication, (xi) a eukaryotic promoter element, (xii) a eukaryotic translation initiation sequence, (xiii) an endosome severing sequence, and (xiv) optionally, an intron

[00157] 19. The plasmid, according to any of the previous embodiments, in which said plasmid comprises the following elements: a promoter (such as a CMV IE promoter), an intron (located within the non-coding leader sequence), and a eukaryotic translation initiation sequence (such as a Kozak element), an endosomally targeted antigen coding sequence (such as an endosomally targeted human secretion-deficient preproinsulin coding sequence), an FMDV 2A element that preferentially separates the antigen coding sequence and the TGF-β coding sequence, a TGF-β coding sequence (such as a constitutively active human TGF-β coding sequence, preferably a constitutively active human TGF^1 coding sequence), Petition 870220118140, dated 12 / 15 / 2022, pp. 76 / 107 59 / 84 an EMCV IRES element (or, alternatively, a bidirectional eukaryotic promoter), wherein said EMCV IRES element separates the TGF-β coding sequence and the IL-10 coding sequences, an IL-10 coding sequence (such as a human IL-10 coding sequence with an N-terminal addition of three alanine amino acids), a 2A element, such as a P 2A element, wherein said 2A element separates the IL-10 coding sequence and the IL-2 coding sequence. an IL-2 coding sequence (such as a human IL-2 coding sequence), a termination element (such as a bGH_PA termination element), a selection gene (such as a kanamycin coding sequence or an infA wt coding sequence), an origin of replication (such as a prokaryotic origin of replication, for example, pUC ori).

[00158] 20. The plasmid, according to embodiment 18, in which elements (i)-(xiii) are arranged in order of expression.

[00159] 21. The plasmid, according to any of the preceding embodiments, wherein the plasmid DNA sequence is as shown in SEQ ID NO 24, or essentially as shown in SEQ ID NO 24.

[00160] 22. The plasmid, according to embodiment 21, in which some minor modifications, resulting in, for example, one, two, three or four amino acid substitutions in one or more of the antigen and / or cytokines, are made in SEQ ID NO 24 in this document.

[00161] 23. The plasmid, according to any of the Petition 870220118140, dated 12 / 15 / 2022, pp. 77 / 107 60 / 84 embodiments 1-20, wherein the plasmid DNA sequence is as shown in SEQ ID NO:26 or a modification of SEQ ID NO:26, resulting in, for example, one, two, three or four amino acid substitutions in one or more of the antigen and / or cytokines, or a modification of SEQ ID NO:26, which results in the expression of the same polypeptide sequences as SEQ ID NO:26.

[00162] 24. The plasmid, according to any of embodiments 1-20, wherein the plasmid DNA sequence is as shown in SEQ ID NO:26 or a modification of SEQ ID NO:26 having fewer than 100 bases that are different from SEQ ID NO:26.

[00163] 25. The plasmid, according to any of embodiments 1-20, wherein the plasmid DNA sequence is as shown in SEQ ID NO:28 or a modification of SEQ ID NO:28 resulting in, for example, one, two, three or four amino acid substitutions in one or more of the antigen and / or cytokines, or a modification of SEQ ID NO:28 resulting in the expression of the same polypeptide sequences as SEQ ID NO:28.

[00164] 26. The plasmid, according to any of embodiments 1-20, wherein the plasmid DNA sequence is as shown in SEQ ID NO:28 or a modification of SEQ ID NO:28 having fewer than 100 bases that are different from SEQ ID NO:28.

[00165] 27. The plasmid, according to any of embodiments 1-20, wherein the plasmid DNA sequence is as shown in SEQ ID NO:29 or a modification of SEQ ID NO:29 resulting in, for example, one, two, three or four amino acid substitutions in one or more of the antigen and / or cytokines, or a modification of SEQ ID NO:29 resulting in the expression of the same polypeptide sequences as SEQ ID NO:29. Petition 870220118140, dated 12 / 15 / 2022, pp. 78 / 107 61 / 84

[00166] 28. The plasmid, according to any of embodiments 1-20, wherein the plasmid DNA sequence is as shown in SEQ ID NO:29 or a modification of SEQ ID NO:29 having fewer than 100 bases that are different from SEQ ID NO:29.

[00167] 29. The plasmid, according to any of embodiments 1-20, wherein said plasmid comprises a TGF-β gene comprising SEQ ID NO:25 or SEQ ID NO:25 having less than 10 base substitutions.

[00168] 30. The plasmid, according to any of the above embodiments, for use in delaying or preventing type I diabetes.

[00169] 31. The plasmid, according to any of the above modalities, for intramuscular, intradermal, intranasal or subcutaneous administration.

[00170] 32. The plasmid, according to embodiment 31, for subcutaneous administration.

[00171] 33. The plasmid, according to embodiment 31, for intramuscular injection.

[00172] 34. The plasmid, according to any of the foregoing embodiments, for use in the treatment of a medical condition in a subject, such as, for example, type I diabetes, early-onset type I diabetes, or increased risk of developing type I diabetes (including type 1.5 diabetes conditions).

[00173] 35. A DNA immunotherapy vaccine comprising a plasmid, according to any of the previous embodiments.

[00174] 36. The DNA immunotherapy vaccine, according to modality 35, for use in delaying or preventing type I diabetes.

[00175] 37. The DNA immunotherapy vaccine, according to any of the modalities 35-36, for intramuscular administration, Petition 870220118140, dated 12 / 15 / 2022, pp. 79 / 107 62 / 84 intradermal, intranasal or subcutaneous.

[00176] 38. The DNA immunotherapy vaccine, according to modality 37, for subcutaneous administration.

[00177] 39. The DNA immunotherapy vaccine, according to modality 37, for intramuscular administration.

[00178] 40. The DNA immunotherapy vaccine, according to any of the modalities 35-39, used in association with, or in parallel with, other types of medical treatments, such as, for example, beta cell / beta stem cell therapy, beta cell / beta stem cell grafting, etc., to prolong the survival and effectiveness of grafted cells.

[00179] 41. A pharmaceutical composition comprising a DNA immunotherapy vaccine, according to any of the embodiments 34-39, or a plasmid, according to any of the embodiments 134, wherein said pharmaceutical composition comprises a saline solution and / or a buffer and / or a chelating agent.

[00180] 42. A pharmaceutical composition comprising a DNA immunotherapy vaccine, according to any of the embodiments 35-40, or a plasmid, according to any of the embodiments 134, wherein said pharmaceutical composition comprises a saline solution and / or a buffer and / or a chelating agent and / or ethanol.

[00181] 43. The pharmaceutical composition, according to any of the embodiments 41-42, in which the volume / volume percentage of ethanol is less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.

[00182] 44. The pharmaceutical composition, according to any of the embodiments 41-43, in which said composition does not comprise any virus, lipid co-packing agent, or condensing agent.

[00183] 45. The pharmaceutical composition, according to any of the embodiments 41-44, in which the said composition further comprises a Petition 870220118140, dated 12 / 15 / 2022, pp. 80-107 63 / 84 GLP-1R agonist.

[00184] 46. The pharmaceutical composition, according to any of the embodiments 41-44, in which said composition further comprises a GLP-1 analogue / GLP-1R agonist.

[00185] 47. The pharmaceutical composition, according to any of the embodiments 45-46, in which said GLP-1 analogue or said GLP-1R agonist is selected from liraglutide, semaglutide or a mixture thereof.

[00186] 48. A kit comprising a pharmaceutical composition, according to any of the embodiments 41-47, and a pharmaceutical composition comprising a GLP-1 analogue / GLP-1R agonist (for example, liraglutide and / or semaglutide).

[00187] 49. A method for producing a plasmid, according to any of the embodiments 1-34, wherein said method comprises (i) incubating a host cell, such as a host cell of bacterial origin, such as, for example, E. coli) transfected with said plasmid under suitable conditions and (ii) recovering / purifying said plasmid.

[00188] 50. The method, according to embodiment 49, in which the host cell in question is a thermosensitive infA strain of E. coli.

[00189] 51. A method of delaying the onset of type-1 diabetes (T1D) or its symptoms in a patient at risk of developing T1D, or recently diagnosed with T1D, with said method comprising administering a DNA immunotherapy vaccine comprising the plasmid according to any of the embodiments 1-31, optionally in combination with a GLP-1 analogue / GLP1R agonist.

[00190] 52. A method of preserving beta cell function and / or endogenous insulin production in an individual, with the aforementioned Petition 870220118140, dated 12 / 15 / 2022, pp. 81 / 107 64 / 84 method comprising the administration of a DNA immunotherapy vaccine comprising the plasmid according to any of the embodiments 1-34, optionally in combination with a GLP-1 analogue / GLP-1R agonist.

[00191] 53. A method of treating a diabetic individual comprising administering a vaccine comprising the plasmid according to any of the embodiments 1-34, optionally in combination with a GLP-1 analogue / GLP-1R agonist (e.g., liraglutide and / or semaglutide).

[00192] 54. A vaccine to prevent or delay the onset of symptoms of type-1 diabetes (T1D) in a patient at risk of developing, or recently diagnosed with, T1D, with said vaccine comprising the plasmid according to any of the embodiments 1-34.

[00193] 55. A method of reducing insulin dosage in an individual having type-1 diabetes (T1D), or a person at risk of developing T1D, with said method comprising administering a DNA immunotherapy vaccine comprising a plasmid according to any of the embodiments 1-33, optionally in combination with a GLP-1 analogue / GLP-1R agonist (e.g., liraglutide and / or semaglutide). EXAMPLES

[00194] Non-obese diabetic mice (NOD mouse model of type 1 diabetes): Immune function in autoimmunity depends on a complex network of cellular interactions that cannot be adequately assessed in vitro.

[00195] The disease suppression and / or treatment assessments in this document were performed in the NOD mouse model, which is a polygenic spontaneous onset model, in which most of the Petition 870220118140, dated 12 / 15 / 2022, pp. 82 / 107 65 / 84 mice develop elevated blood glucose concentrations (BGV, blood glucose value, determined from needle and portable meter in the tail vein) between 12 and 30 weeks of age. The incidence and progression of the disease are unpredictable, with an overall incidence ranging from 60% to 95% at 30 weeks of age (WoA) and progression from diagnosis (two sequential BGV readings >250) to the end (two sequential BGV readings of 600 or higher) ranging from 2 days to 4 weeks. Elevated BGV replication in sequential readings is necessary since mice are allowed ad libitum food and water, resulting in moderate BGV variability beyond that caused by immunopathology.

[00196] An example of a plasmid nucleotide sequence in this document:

[00197] SEQ ID NO 24: complete plasmid sequence (unannotated) (6,401 base pairs) GACTCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATT ATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCAT AGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGC CCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAA TAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATT GACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCA GTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTC AATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGAC CTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATC GCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGT GGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCAT TGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACT TTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGC Petição 870220118140, de 15 / 12 / 2022, pág. 83 / 107 66 / 84 GGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCT GGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATA CGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTT AAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAA TTCTGCACTGCAGCTCGCATCTCTCCTTCACGCGCCCGCCGCCC TACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCG CCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTC CCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCT GACCCTGCTTGCTCAACTCTAGGTAAGTTAATGAGACAGATAGA AACTGGTCTTGTAGAAACAGAGTAGTCGCCTGCTTTTCTGCCAG GTGCTGACTTCTCTCCCCTGGGCTTTTTTCTTTTTCTCAGGTTGA AAAGAAGAAGACGAAGAAGACGAAGAAGACAAACCGTCGTCGA CTGCCATGCGCCGCTGATTAACGCCGCCACCATGGCCCACCGA CGCAGATCCAGAAGCTGCCGTGAGGACCAGAAGCCCGTGATGGA TGATCAGAGGGACCTTATCTCTAACAATGAACAACTGCCAATGC TCGGCAGACGGCCTGGGGCCCCGGAGAGCAAGTGCAGCAGAG GAGCCTTGTACACGGGGTTCTCCATTTTAGTGACTCTCCTTCTCG CCGGCCAAGCTACCACCGCCTACTTTCTGTACCAACAGCAAGGC AGACTAGACAAACTGACAATCACAAGCCAGAACCTTCAGCTGGA GTCTCTGCGGATGAAGCTGCCCGCTTTGTGGATGAGATTGCTTCCTCTACTTGCTCTCCTGGCGCTCTGGGGACCTGACCCCGAGCAAGAGTTTGTTAATCAGCACCTGTGTGGGAGTCATCTGGTGG AGGCACTCTATTTAGTGCGGAGAGAGGGGCTTCTTCTACAC TCCAAAGACCAGACGGGAGGCCGAAGACCTTCAAGTGGGGCA AGTAGAACTGGGTGGCGGACCCGGTGCCGGGAGCCTTCAGGC CGCTCGCCCTGGAGGGCTCTCTTCAGAAACGCGGCATCGTGG AGCAGTGTTGCACATCCATTTGCTCACTCTACCAGCTGGAGAAC TACTGCAACGGAAGCGGAGTGAAGCAGACGTTGAATTTTGATTT Petition 870220118140, of 15 / 12 / 2022, p. 84 / 107 67 / 84 GTTGAAGTTGGCGGGGGATGTGGAGAGCAATCCGGGGCCGA TGCCCCCTAGTGGCCTCAGACTTTGTTATTGTTATTACCGCTTTT ATGGCTCTTGGTGCTGACACCGGGCCGTCCGGCTGCTGGCTTG TCGACTTGTAAGACAATTGATATGGAATTGGTGAAACGAAAACG GATTGAGGCCATCCGAGGACAGATTTTGAGCAAGCTGCGGCTT GCCTCGCCACCCTCGCAAGGGGAAGTCCCACCCGGACCTCTAC CAGAAGCAGTCCTAGCGCTGTACAACAGTACAAGAGATAGAGTG GCCGGGGAATCCGCAGAACCAGAGCCTGAGCCTGAAGCCGAT TATTATGCAAAGGAAGTGACTAGGGTCCTGATGGTCGAGACCCA TAACGAAATCTACGACAAATTCAAACAAAGTACCCACTCTATCT ACATGTTCTTCAACACCAGTGAGCTAAGAGAAGCCGTGCCC GAACCTGTGCTTCTTTCCCGCGCAGAACTCCGCCTCTTGAGACT CAAATTGAAAGTTGAACAACACGTAGAGCTTTACCAGAAATACTC TAATAATTCATGGCGATATCTTTCTAATCGTCTCCTCGCCCCAT CTGACAGCCCTGAATGGCTCTCCTTCGACGTTACGGGAGTTGT GCGCCAGTGGCTCAGCAGAGGCGGAGAGATAGAGGGCTTTC GGCTGAGCGCACATGTATCTGTGGACTCAAGGGACAACACATT GCAAGTGGATATTAACGGTTTTACAACTGGACGGAGAGGGGAC CTGGCGACCATCCACGGCATGAATAGACCTTTCCTGCTGCTGATG GCTACTCCCCTGGAGAGGGCACAGCACTTACAGTCTTCCAGAC ACCGGCGCGCCCTGGATACAAACTACTGCTTCAGCTCCACCGAA AAGAACTGTTGCGTGCGGCAGCTGTACATTGACTTCAGAAAGGA TCTGGGCTGGAAGTGGATTCATGAGCCCAAGGGGTATCATGCCA ACTTCTGTCTTGGGCCATGCCCATACATCTGGTCACTGGATACCCAGTACTCCAAAGTTCTGGCCTTGTACAATCAACACAACCCTGGA GCTTCCGCCGCTCCTTGCTGTGTGCCCCAAGCCCTAGAGCCC CTGCCCATCGTTTATTATGTCGGACGCAAGCCCAAAGTAGAA CAGCTATCAAATATGATCGTGAGAAGCTGCAAGTGTAGCTGAT AAACGCGTCGAGCATGCATCTAGGGCGGCCAATTCCGCCCCT Petition 870220118140, dated 12 / 15 / 2022, pp. 85 / 107 68 / 84 CTCCCCCCCACCCCTCTCCCTCCCCCCCCCCTAACGTTACT GGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTAT ATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGG GCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGG GGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAA TGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAA ACAACGTCTGTAGCGACCCTTTGTAGACAGCGGAACCCCCCAC CTGGCGATAGATGCCTCTGCGGCCAAAAGCCACGTGTATAAGAT ACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTT GGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATT CAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATG GGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTT TAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGG GACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCTG CCGCTCATTCTAGTGCCCTTCTTTGCTGCCTGGTCCTGCTCAC CGGGGTGCGAGCTAGCCCTGGACAAGGGACACAATCCGAAA ACTCGTGCACCCACTTCCCGGGCAACCTCCCTAACATGCT GAGGGACCTCCGTGATGCCTTCAGTAGAGTGAAGACGTTCTT CCAAATGAAAGATCAGTTAGATAACCTGCTCCTGAAGGAGTCACTCTTAGAAGACTTCAAAGGATACCTCGGCTGCCAAGCACTTAGC GAGATGATTCAATTCTACTTAGAAGAAGTCATGCCTCAAGCTGA GAATCAAGACCCCGACATCAAAGCTCATGTGAATTCTTTGGGAG AAAATTTGAAGACTTTGCGGCTGCGGCTGCGGAGATGTCACC GCTTTCTGCCCTGTGAGAACAAATCAAAAGCGGTCGAGCAAGTTAAGAATGCCTTCAATAAGCTACAAGAGAAGGGCATCTACAAAG CAATGAGCGAGTTTGATATCTTTATCAATTACATTGAAGCCTAC ATGACAATGAAGATTAGGAATGCCGCGGGGAGCGGCGCTACTA ACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAAC CCTGGACCTATGTACAGAATGCAGCTGCTGAGCTGCATCGCCC TGAGCCTGGCCTGGTGACCAACAGCGCCACCACGTCCTCTAG Petition 870220118140, de 15 / 12 / 2022, pág. 86 / 107 69 / 84 CACCAAGAAGACCCAGTTACAGTTGGAGCATCTACTTTTAGACC TGCAAATGATTTTGAACGGCATCAACAACTACAAGAATCCTAAA CTTACTCGCATGCTTACCTTCAAATTTTACATGCCCAAGAAGGCCA CCGAACTGAAGCACTTGCAATGTCTGGAGGAAGAACTCAAGCC GCTGGAGGAAGTTCTCAACCTCGCGCAGTCCAAGAATTTCCACC TCCGGCCAAGAGACCTGATCAGTAACATTAATGTGATAGTGCTG GAGCTGAAGGGAAGCGAGACTACATTTATGTGCGAGTACGCCG ATGAAACCGCTACAATCGTCGAGTTCCTGAATAGATGGATCACAT TTTGCCAGTCAATTATCTCTACTCTGACATGATAACTCGAGG TCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTG CCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCT TCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATA AAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTA TTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTG GGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTAT GGCTTCTACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAA AGTAAACTGGATGGCTTTCTCGCCGCCAAGGATCTGATGGCGCA GGGGATCAAGCTCTGATCAAGAGACAGGATGAGGATCGTTTCG CATGGCCAAAGAAGACAATATTGAAATGCAAGGTACCGTTCTTG AAACGTTGCCTAATACCATGTTCCGCGTAGAGTTAGAAAACGG TCACGTGGTTACTGCACACATCTCCGGTAAAATGCGCAAAAACTACATCCGCATCCTGACGGGCGACAAAGTGACTGTTGAACTGAC CCCGTACGACCTGAGCAAAGGCCGCATTGTCTTCCGTAGTCG CTGATAAATTATTAACGCTTACAATTTCCTGATGCGGTATTTTC TCCTTACGCATCTGTGCGGTATTTCACACCGCATACAGGTGG CACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTA TTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAA TAACCCTGATAAATGCTTCAATAATAGCACGTGCTAAAAC TTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGA Petição 870220118140, de 15 / 12 / 2022, pág. 87 / 107 70 / 84 TAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCAC TGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAA CCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATA CCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTT CAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCT GTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCG GGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCG GGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAA CGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAA GCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTA AGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAG GGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACC TCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGG AGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCC TGGGCTTTTGCTGGCCTTTTGCTCACATGTTCTT Exemplo 1 - Plasmídeos codificadores de antígeno em comparação com plasmídeos codificadores de antígeno + IL-10:

[00198] It has been suggested in the state of the art that depletion of immunostimulatory CpG sequences in the main plasmid structure would be necessary for effective DNA immunotherapy treatment of T1D. This experiment was thus shaped after previously published experiments (2008 J Immunol. 181(12):8298-307).

[00199] Eight plasmid doses were given once weekly to NOD mice, starting at week 9 (age): either an empty vector (pVAX1, 50 µg), or a pVAX1-proinsulin Ag (not targeted endosomally, not pre-proinsulin), or a CpG-depleted pVAX1-proinsulin Ag, or a pVAX1-IL10-IRES-proinsulin construct Ag was administered. Petition 870220118140, dated 12 / 15 / 2022, pp. 88 / 107 71 / 84 bicistronic in equimolar ratios.

[00200] All administrations were intramuscular in the left quadriceps under isoflurane anesthesia and contained only plasmid in PBS + EDTA. BGVs were assessed in all mice weekly, and the incidence of type 1 diabetes was scored based on two BGV readings above 250 mg / dL. Mice were evaluated until 30 weeks of age or until a BGV of 600 was achieved, followed by sacrifice.

[00201] The results of this experiment (table 1) demonstrate that A) CpG depletion is neither necessary nor beneficial for efficacy, B) the inclusion of immunomodulatory cytokines significantly increases efficacy, and C) the main plasmid structure (empty vector) is equivalent to the untreated groups. Table 1: Incidence of T1D in NOD mice at 30 weeks of age. Plasmid Incidence of T1D disease at 30 weeks of age History of untreated colony incidence 77.8% pVAX1 (negative control of empty vector) 23 / 29 = 79.3% pVAX1-proinsulin antigen depleted for CpG (antigen+modified vector) 24 / 29 = 82.7% pVAX1-proinsulin antigen (antigen) 18 / 30 = 60% pVAX1-IL10-IRES-proinsulin antigen (antigen+IL-10) 10 / 26 = 38.5% Example 2 - Expressed protein products resulting from plasmids encoding antigen, IL-10, IL-2, and TGF-β

[00202] Multicistronic plasmids were created to co-express TGF-β, IL-10, and optionally IL-2. Freestyle293 cells were transiently transfected and cultured in serum-free media. Supernatants were collected and quantified by ELISA after 72 hours. Petition 870220118140, dated 12 / 15 / 2022, pp. 89 / 107 72 / 84

[00203] The results in Table 2 below show that: A) the expression of multiple independent cytokines is obtained from a single vector, B) significant amounts of each cytokine are produced and at the expected ratios, C) minor sequence alterations significantly improve IL-10 expression from the first-generation IL10 / proinsulin plasmid, and D) neither the main plasmid structure (empty vector) nor endosomal antigen targeting (IIAg) induces cytokine production or dysregulation. Table 2: ELISA quantification of expressed protein products. Plasmid TGF-β1 active (ng / ml) Interleukin-10 (ng / ml) Interleukin-2 (ng / ml) pVAX1 (empty vector) <0.0035 <0.0027 <0.0009 pVAX1-IL10 / Proinsulin (antigen+IL-10) <0.0035 85.3 <0.0009 pVAX1-IIAg / TGFp / IL10 / (antigen+TGFe+IL-10) 7.35 1238.8 <0.0009 pVAX1-IIAg / TGFp / IL10 / IL2 (antigen+TGFe+IL-10+IL2) 2.39 1259.5 777.0 Example 3 - Impact of TGF-β and IL-2 on disease suppression

[00204] Multicistronic plasmids were evaluated for disease prevention in NOD mice, as in Example 1, except that dosing was continued once weekly until sacrifice (onset of diabetes) or week 30. One mouse from each group (initial n=24) was sent for full necropsy after 10 weeks of dosing – including pathology in 10 standard highly perfused tissues, complete blood count, and clinical chemistry. Apart from minor muscle rupture and growth due to mechanical trauma at the injection site, there were no deviations from undosed animals.

[00205] The results in Table 3 below show that: A) the addition of TGFp significantly increases efficacy, B) the inclusion of Interleukin Petition 870220118140, dated 12 / 15 / 2022, pp. 90 / 107 73 / 84 can increase efficacy and does not induce pathology, C) chronic dosing with plasmids expressing IL-10 and the antigen increases efficacy in disease prevention, and D) chronic dosing with plasmids expressing TGF3, IL-10, and IL-2 increases efficacy without resulting in any safety signal. Table 3: Incidence of T1D in NOD mice. Plasmid Incidence of disease at 30 weeks of age History of incidence of untreated colony 77.8% Untreated (negative control) 18 / 21 = 85.7% pVAX1-Ag / IL10 (antigen+IL-10) 5 / 23 = 21.7% pVAX1-IIAg / TGFp / IL10 (antigen+TGFe+IL-10) 2 / 23 = 8.7% pVAX1-IIAg / TGFp / IL10 / IL2 (antigen+TGFe+IL-10+IL-2) 1 / 23 = 4.3% Example 4: Evaluation of IRES elements, introns, as well as subcutaneous administration.

[00206] Multicistronic plasmids were evaluated for disease prevention in NOD mice, as in Example 3, except for the dosing which started earlier (at week 5) in order to better mimic chronic pediatric administration. In addition to validating the pVAX1IIAg / TGFp / ILW and pVAX1-IIAg / TGFp / IL10 / IL2 intron-containing plasmids, other control groups were examined. Specifically, a different IRES segment (CrPV [Cricket Paralysis Virus], as opposed to EMCV [Encephalomyocarditis Virus]) was evaluated for expected increases in efficacy, as well as for intron segment deletion to assess its necessity. Due to the obvious lack of efficacy compared to the parental plasmid (pVAX1-IIAg / TGFp / IL10 / IL2), the CrPV and intron-free (ni = intron-free) groups were terminated early. Furthermore, the mouse cohort used in this experiment experienced more severe progression. Petition 870220118140, dated 12 / 15 / 2022, pp. 91 / 107 74 / 84 showed a faster progression of the disease than previous cohorts, with the time from diagnosis to sacrifice averaging 1.25 weeks instead of 2.75 in previous experiments. Finally, a subcutaneous administration group was added. This group was treated with a triple cytokine plasmid (pVAX1-IIAg / TGFp / IL10 / IL2) injected once weekly into the subcutaneous space in the nape of the neck without anesthesia.

[00207] The results in Table 4 show that: A) EMCV IRES elements provide significantly better efficacy than CrPV IRES, B) the inclusion of an intron (in this plasmid located within the CD74 endosomal targeting region) significantly increases efficacy, C) although the inclusion of IL-2 provides minimal benefit in moderate disease scenarios, its presence significantly increases the efficacy and robustness of treatment in aggressive disease scenarios, and D) subcutaneous dosing, which is ineffective in most DNA vaccine applications, here shows modest efficacy and a significant delay in disease progression even without optimization. Table 4: Incidence of T1D in NOD mice. Type of treatment Diabetics / total % diabetics Historical control 80% at 30 weeks Untreated 15 / 21 71.4% at 30 weeks Control with empty vector im 13 / 21 61.9% at 30 weeks pVAX1-IIAg / TGFp / IL10 / IL2 (without intron) im 10 / 24 41.6% at 22 weeks pVAX1-IIAg / TGFp / IL10 / IL2 (CrPv IRES, instead of EMCV IRES) im 7 / 22 31.8% at 22 weeks pVAX1-IIAg / TGFp / IL10 im (without IL-2) 12 / 42 28.6% at 30 weeks pVAX1-IIAg / TGFp / IL10 / IL2 im 1 / 42 2.4% at 30 weeks pVAX1-IIAg / TGFp / IL10 / IL2 sc 12 / 42 28.6% at 30 weeks Petition 870220118140, dated 12 / 15 / 2022, pp. 92 / 107 75 / 84 Example 5 Comparison of commercial antibiotic-free selection with antibiotic selection systems

[00208] An alternative plasmid core structure was evaluated with the aim of removing kanamycin resistance to meet European Medicines Agency guidelines. The same insert (IIAg / TGFp / IL10 / IL2, including intron) was cloned into the Nature Technology NTC9385R nanoplasmid core structure. The resulting plasmid was evaluated in NOD mice as in Example 3, except that treatment started at week 11 (late onset) and ended prematurely due to failure of the NTC9385R-based plasmid.

[00209] The results in Table 5 below show that: A) alterations in the plasmid major structure selection system surprisingly induced significant changes in plasmid effectiveness, and B) a late start to treatment results in early conversions. Data from other related experiments indicate that dosing with these tolerogenic DNA vaccine plasmids requires two to four weeks to become effective, so a late start to treatment results in several early cases of diabetes before the treatment becomes effective. Table 5: Incidence of T1D disease in NOD mice. Plasmid Incidence of disease at 30 weeks of age History of incidence of untreated colony 77.8% Untreated (negative control) 16 / 21 = 76.2% pVAX1-IIAg / TGFp / IL10 / IL2 with intron (kanamycin resistant) 5 / 21 = 23.8% pNTC9385R-IIAg / TGFp / IL10 / IL2 with intron (commercial antibiotic-free screening system) 13 / 21 = 61.9% Example 6: Disease suppression efficacy with antigen- and antigen-containing plasmids. Petition 870220118140, dated 12 / 15 / 2022, pp. 93 / 107 76 / 84

[00210] To determine the role of the encoded antigen in plasmid function, two experiments were performed (Examples 6 and 7). An alternative plasmid was evaluated with the aim of removing the antigen-coding region (pre-proinsulin) while retaining the CD74 targeting domain and all three secreted cytokines. The resulting plasmid was evaluated in NOD mice as in Example 3, except that treatment began at week 11 (late onset).

[00211] This experiment demonstrates that the antigen portion is necessary for full efficacy and that it is not merely cytokine production that drives plasmid function. This is one of the two criteria necessary to demonstrate the antigen specificity of the treatment. Table 6: Incidence of T1D in NOD mice. Plasmid Incidence of disease at 30 weeks of age History of incidence of untreated colony 77.8% pVAX1-II / TGFp / IL10 / IL2 (antigen+cytokines) 2 / 22 = 9.1% pVAX1-II / TGFp / IL10 / IL2 (no antigen+cytokines) 15 / 28 = 53.5%

[00212] Example 7 Impact of antigen immunotherapy in this document on the effectiveness of unrelated antigen vaccines

[00213] To determine the role of the encoded antigen in plasmid function, two experiments were performed (Examples 6 and 7). NOD mice were treated with sham injection of PBS or treated with the pVAX1-IIAg / TGFp / IL10 / IL2 plasmid as in Example 3. After four doses (i.e., at 13 weeks of age), each mouse was immunized ipsilaterally with 50 pg of an irrelevant antigen (Chicken Ovalbumin, OVA) in 100 Petition 870220118140, dated 12 / 15 / 2022, pp. 94 / 107 77 / 84 μL of a 1:1 alum suspension. Sham or plasmid treatments were continued once weekly until sacrifice three weeks (21 days) after immunization, at which time serum was collected. Switched-class antibodies (total IgG and IgG2a) against ovalbumin antigen were determined using commercial ELISA kits. No significant differences were observed between the plasmid-treated and sham-treated groups in their total anti-OVA IgG levels, nor did either group produce anti-OVA IgG2a.

[00214] The results in Table 7 below show that although the plasmid suppresses immune responses related to the target disease, it does not suppress immune reactivity toward unrelated antigens (i.e., any antigens not encoded by the plasmid). This is the second of two criteria needed to demonstrate the antigen specificity of the treatment. Since treatment of pediatric patients will involve the concomitant administration of standard childhood vaccination, this is a significant advantage over systemic / generic immunosuppression via agents such as methotrexate or cyclosporine A. Table 7: Response to irrelevant antigen in NOD mice that received DNA immunotherapy vaccination against T1D. Sample Treatment # Average mcg of anti-OVA IgG / mL Error Treated with plasmid 8 7.517 + / - 0.967 Treated with PBS (simulation) 5 8.954 + / - 1.227

[00215] These values ​​result in a non-significant p-value of 0.377 and a confidence interval of -1.99 to 4.87. These results indicate that treatment with the immunomodulatory plasmid does not affect the response. Petition 870220118140, dated 12 / 15 / 2022, pp. 95 / 107 78 / 84 immune to other antigens not encoded by the plasmid and therefore does not result in widespread or systemic immunosuppression. Example 8: Individual protein products expressed from the plasmid.

[00216] The TaV 2A element resulted in unexpected IL10+IL-2 fusion products in this document (data not shown) and other separation strategies were therefore evaluated. Initial separation technologies included upstream extensions of the TaV 2A sequence (leading to rapid degradation and absence of secreted IL-10) and also a carboxypeptidase cleavage site (which induced death of transfected cell lines). Other separation strategies evaluated were GSGTaV 2A, a furin cleavage site, a furin site followed by TaV 2A, P 2A and E 2A (equine rhinitis virus A).

[00217] Freestyle293 cells were transiently transfected and cultured in serum-free media. Cell pellets and supernatants were collected and subjected to semi-quantitative multicolor Western blotting after 72 hours.

[00218] The results in Table 8 below show that: A) unexpectedly, proteolytic cleavage sites fail to function between IL-10 and IL-2 genes, B) GSG markers (uncoupling sequences) between IL-10 and IL-2 are preferential for extended insulator sequences, C) 2A of P is preferential for 2A of TaV or 2A of E, and D) 2A sequences may have significant and unexpected effects on the degradation and secretion of upstream expressed proteins, such as IL-10. Table 8: Separation of protein products of expressed IL-10 and IL-2. Cellular Plasmid Interleukin-10 Secreted Cellular Interleukin-10 Interleukin-2 2A of GSG-TaV ++++ ++ - Furin cleavage site + - - Furin / 2A of TaV +++ ++ - Petition 870220118140, dated 12 / 15 / 2022, pp. 96 / 107 79 / 84 2A of P++++++ - 2A of E++++++ - Continuation Cellular Interleukin-2 Secreted Molten Product Secreted Molten Product +++ ++ - - ++++ ++ ++ ++ + +++ + - ++ ++ - Example 9 Comparison of a commercial selection system with a heat-sensitive selection system provided in this document, as well as a comparison between plasmids encoding IL-2 and plasmids not encoding IL-2 (subcutaneous administration)

[00219] The main plasmid structures were created and evaluated with the aim of removing kanamycin resistance to meet European Medicines Agency guidelines. The corrected insert (IIAg / GSGFMDV 2A / TGFp / EMCV IRES / IL10 / GSG-P 2A / IL2, including an intron in the upstream non-coding region) was cloned into a retrofitted / minimally modified pVAX1 vector containing the Nature Technology RNA-OUT selection marker or an equivalent minimally modified pVAX1 vector encoding wt infA (pNN) as main structures. In addition, plasmids containing an additional SV40 enhancer element or deficient in IL-2 were produced. The resulting plasmids were evaluated in NOD mice as in Example 3, except that administration was sc either once weekly or three times weekly (preferred).

[00220] The results shown in Table 9+10 below show that: A) the exchange of commercially available RNA-OUT for kanamycin antibiotic resistance in the pVAX1 main structure still underperforms, B) the antibiotic-free selection system of Petition 870220118140, dated 12 / 15 / 2022, pp. 97 / 107 A) 80 / 84 infA supplementation has equivalent performance to the parental pVAXI vector, B) Interleukin-2 is required for optimal efficacy, C) the addition of the SV40 enhancer element does not increase efficacy, and D) the corrected triple cytokine insert retains full functionality. Table 9: Incidence of T1D in NOD mice. Plasmid, administered 3 times a week (ideal) Disease incidence at 30 weeks of age History of untreated colony incidence 78.9% Untreated (negative control) 12 / 15 = 80% Empty pNN vector (negative control with heat-sensitive selection, but without protein-coding sequences) 12 / 16 = 75% pVAX1-IIAg / FMDV / TGFp / IL10 / TaV2A / IL2 (kanamycin selection and protein-coding sequences) 1 / 16 = 6.3% pNN-IIAg / FMDV / TGFp / IL10 / P2A / IL2 (temperature-selective system and protein-coding sequences) 1 / 23 = 4.3% pVAX1-RNA-OUT-IIAg / FMDV / TGFp / IL10 / P2A / IL2 (commercial selective system and protein-coding sequences) 9 / 23 = 39.1% Table 10: Incidence of T1D in NOD mice. Plasmid, administered once a week (subideal) Disease incidence at 27 weeks of age History of untreated colony incidence 78.9% Untreated (negative control) 12 / 15 = 80% pNN-IIAg / FMDV / TGFp / IL10 / P2A / IL2 (selective temperature and protein coding sequences) 16 / 37 = 43.2% pNN-SV40e-IIAg / FMDV / TGFp / IL10 / P2A / IL2 (selective temperature and coding sequences, as well as an enhancer) 20 / 37 = 54% pNN-IIAg / FMDV / TGFp / IL10 (IL-2 deficient) (selective temperature and protein coding sequences - except IL-2) 25 / 40 = 62.5% pVAX1-RNA-OUT- 27 / 38 = 71% Petition 870220118140, dated 12 / 15 / 2022, pp. 98 / 107 81 / 84 IIAg / FMDV / TGFp / IL10 / P2A / IL2 (commercial selective system and protein coding sequences) Example 10: Evaluation of the durability of the tolerance effect after plasmid withdrawal.

[00221] In the previous experiment (represented in Table 9), the pNN-IIAg / FMDV / TGFp / IL10 / P2A / IL2 group was not sacrificed at 30 weeks of age, but discontinued plasmid dosing. Blood glucose values ​​were followed for an additional ten (10) weeks, for a total of 40 weeks of age, to assess whether the plasmid had induced a durable state of tolerance or whether continued dosing was necessary for efficacy.

[00222] The results shown in Table 11 below indicate that continuous dosing is necessary for durability of tolerance, as a stable disease-free state for 30 weeks of age rapidly deteriorates after discontinuation of dosing. This indicates a beneficial safety profile, since any adverse event that may be encountered with plasmid dosing should also cease with dosing. Table 11: Incidence of T1D in NOD mice after discontinuation of plasmid dosing. Disease incidence up to 30 weeks of age: 1 / 23 = 4.3% 9 / 23 = 39.1% Example 11 Evaluation of plasmid stability and durability in injection

[00223] A major problem with plasmid administration is degradation during administration. In the case of injection, shear forces Petition 870220118140, dated 12 / 15 / 2022, pp. 99 / 107 The 82 / 84 characteristics found in large, viscous plasmid molecules, when passed through a fine needle under pressure, lead to the disruption of the plasmid's covalently closed circular structure – rendering it linear and subject to reduced transfection capacity and rapid destruction. Most plasmids experience 5–15% degradation to linear forms upon injection through needles of clinically acceptable sizes, resulting in reduced efficacy or a need for higher initial doses to compensate for the loss. Several types of sequence structures that can lead to plasmid unwinding and susceptibility to shear degradation have been intentionally minimized in the disclosed plasmids, with the intention of increasing robustness and reliability with injection protocols.In order to evaluate the shear degradation of the plasmid, which can vary with viscosity and therefore with concentration, the initial human plasmid was resuspended in Tris EDTA buffer at concentrations of 5, 7 and 9 mg / ml and passed three times through a G30 needle (expelled, withdrawn back into the syringe, then expelled again) and one (1) microgram samples were passed on an agarose gel against reference samples that were not passed through the injection process.

[00224] The results shown in Figure 3 surprisingly indicate that the plasmid is not visibly degraded by the three injection passes at any concentration or viscosity tested. Plasmid degradation would be visualized as a scattering of smaller bands (between the main supercoiled band at 6 Kb and the small process impurity band at the bottom of the gel or approximately 600 bp). These linearization / degradation spots are not seen for any sample passed through the injection process. This robust physical stability at the dosage is highly desirable and greater than predicted or previously reported in the literature. Petition 870220118140, dated 12 / 15 / 2022, pp. 100 / 107 83 / 84 Example 12: Verification of plasmid retention with infA complementation system.

[00225] In order to verify that the infA-based plasmid retention selection system functioned as desired, plasmid-transformed bacteria were cultured through 100 passages (approximately 36 duplications / generations per passage, for a total of 3,600 generations of potential plasmid carryover or loss examined). Passages 1-100 were generated at 11 per week, 2 passages per weekday at 37°C and one every weekend at 30°C. All were performed in animal component-free liquid LB media (Teknova soy-tone) supplemented with 15 micrograms / ml of naladexic acid (selecting for DH5a base strain, not for plasmid presence). Glycerol reserves were generated from each passage and maintained until all 100 passages were obtained for simultaneous processing.

[00226] Scrapings from glycerol reserves were used to inoculate 5 ml of cultures overnight, which were processed according to the manufacturer's instructions in Qiagen miniprep kits using a vacuum dispenser (both 16 and 32 cultures per run, due to gel size restrictions). No attempt was made to collect OD600 readings for normalization of cell intake, and all preparations were made based on standard volumes. One microliter of each miniprep was subjected to PstI / XhoI digestion to dissolve the main structure (approximately 2.4 Kbp) of the insert (approximately 4 Kbp), without correction for the resulting plasmid concentration of each miniprep. Each gel was run with Tridye 2-Log flanking ladders (NEB https: / / www.neb.com / products / n3200-2-log-dnaladder-01-100-kb), a first strip of undigested plasmid sample, and visualized with SybrSafe stain.In the gel images, despite the lack of control over the amount of nucleic acid, all digestion ranges are visible. Petition 870220118140, dated 12 / 15 / 2022, pp. 101 / 107 Figures 84 / 84 show the presence and expected digestion pattern for the plasmid (seen in the images for passages 1-16, 17-48, 49-80, and 81-100).

[00227] As further confirmation, glycerol pools for passages 1-100 were also streaked onto animal component-free, antibiotic-free LB agar plates from sector 50 and incubated overnight at 30°C. No attempt to control the streak inoculum was made. As shown in Figure 4, all streaks representing glycerol pools resulted in notable growth and thus plasmid retention. Example 13: Dose augmentation suitability with an infA supplementation system.

[00228] In order to verify that the infA-based plasmid retention selection system functioned as desired at the production scale, plasmid-transformed bacteria were used in a pilot 50 L fed-batch fermenter run with a specific yield increasing at the temperature change step. Minimal medium with the addition of yeast extract was used, reducing the doubling rate to 0.88 / hour. The fed-batch process was initiated at 17:00 after inoculation and dissolved oxygen regulation to 30% was achieved by successively increasing the pO2 cascade parameters (stirring at 32:15, pressure at 40:30, then airflow at 45:40). The biomass increase rate decreased immediately after the change to 42°C, as predicted. The amount of plasmid DNA produced was estimated at 1.03 ± 0.17 g / L, using a small-scale plasmid extraction procedure mimicking the immediate post-lysis yield. Petition 870220118140, dated 12 / 15 / 2022, pp. 102 / 107

Claims

1 / 3 CLAIMS 1. Expression plasmid CHARACTERIZED in that it comprises: i. a nucleotide sequence encoding an insulin antigen; ii. a nucleotide sequence encoding TGF-β; iii. a nucleotide sequence encoding IL-10; and iv. a nucleotide sequence encoding Interleukin-2 (IL-2), wherein said plasmid comprises: (i) an FMDV 2A element separating the insulin antigen coding sequence and the TGF-β coding sequence, (ii) an EMCV IRES element separating the TGF-β coding sequence and the IL-10 coding sequence, and (iii) a 2A element separating the IL-10 coding sequence and the IL-2 coding sequence; wherein the DNA sequence of the plasmid is as presented in the SEQ ID NOs. 24, 26, 28 or 29.

2. Plasmid, according to claim 1, CHARACTERIZED in that said insulin antigen is selected from the group consisting of: proinsulin, preproinsulin and a functional or immunodominant peptide fragment thereof.

3. Plasmid, according to claim 1 or 2, CHARACTERIZED in that said insulin antigen is an endosomal insulin target.

4. Plasmid, according to any one of claims 1 to 3, CHARACTERIZED in that said plasmid conducts expression of insulin antigen and TGF-β in an amount at least 2 times lower than IL-10.

5. Plasmid, according to any one of claims 1 to 4, CHARACTERIZED in that the coding sequence of TGF-β Petition 870260044556, dated 12 / 05 / 2026, page 9 / 15 2 / 3 encodes constitutively active TGF-β.

6. Plasmid, according to any one of claims 1 to 6, CHARACTERIZED in that it is for use in delaying or preventing type I diabetes.

7. Plasmid, according to any one of claims 1 to 6, CHARACTERIZED in that it is formulated for subcutaneous administration.

8. Plasmid, according to any one of claims 1 to 6, CHARACTERIZED in that it is formulated for intramuscular administration.

9. DNA immunotherapy vaccine CHARACTERIZED in that it comprises a plasmid as defined in any one of claims 1 to 5.

10. DNA immunotherapy vaccine, according to claim 9, CHARACTERIZED in that it is for use in delaying or preventing type I diabetes.

11. DNA immunotherapy vaccine, according to claim 9, CHARACTERIZED in that it is formulated for subcutaneous administration.

12. DNA immunotherapy vaccine, according to claim 9, CHARACTERIZED in that it is formulated for intramuscular administration.

13. Pharmaceutical composition CHARACTERIZED in that it comprises a DNA immunotherapy vaccine, as defined in claim 9, or a plasmid, as defined in any one of claims 1 to 5, wherein said pharmaceutical composition comprises a saline solution and / or a buffer and / or a chelating agent.

14. Pharmaceutical composition, according to claim 13, Petition 870260044556, dated 12 / 05 / 2026, page 10 / 15 3 / 3 CHARACTERIZED by the fact that said buffer does not comprise any virus, lipid copacking agent, or condensing agent.

15. Pharmaceutical composition, according to claim 13 or 14, CHARACTERIZED in that said composition further comprises a GLP-1R agonist. Petition 870260044556, dated 12 / 05 / 2026, page 11 / 15

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