virus

Abstract

The present invention is in the field of transgene delivery to target cells using viral vectors, particularly in the field of gene therapy. We have identified compositions that allow for oral administration of viral particles, particularly adenoviral particles. [Selection diagram] None

Description

[Technical Field]

[0001] The present invention relates to oral administration of viral particles, particularly adenoviral particles carrying a transgene, such as a therapeutic protein for gene therapy. The present invention particularly relates to methods for delivering a transgene to target cells by oral administration of a product comprising viral particles carrying the transgene. The present invention also provides capsules and tablets suitable for oral delivery to a patient, which package pharmaceutical compositions comprising the viral particles. The present invention further provides methods for preparing viral particles for oral administration to a patient. [Background technology]

[0002] Viral vectors are widely used in gene therapy and to express foreign antigens as vaccines. Adenoviral vectors have several advantages over other types of viral vectors. For example, adenoviruses are well-studied, can be propagated to high-titer stable stocks, can efficiently transduce both dividing and non-dividing cells, and can carry large segments of DNA. Currently, numerous gene therapy trials using adenoviral vectors are underway, many of which involve cancer treatment. Other studies have used adenoviruses to express therapeutic proteins to correct genetic defects. Nearly all of these clinical trials have demonstrated that adenoviral vectors are safe and well-tolerated.

[0003] Many adenoviral vectors are modified versions of Ad5 (serotype 5), and vectors can be either replication-competent or replication-defective. Replication-defective vectors generally lack the essential E1A and E1B genes, which are replaced by expression cassettes with high promoter activity to drive the expression of foreign transgenes. Many replication-defective vectors also lack the E3 and E4 regions. Another type of replication-defective adenoviral vector is a "helper-dependent" vector, which lacks much of the genome but retains the origin of replication and approximately 500 base pairs required for packaging into virions. These vectors are constructed and propagated in the presence of a replication-competent helper adenovirus, which provides the required early and late proteins. The helper adenovirus generally contains loxP sites flanking the packaging signal of the genome. The cell line used to produce the vector conditionally expresses Cre recombinase, which excises the loxP-flanked packaging signal from the helper adenoviral genome, allowing for the preferred packaging of the helper-dependent adenoviral genome (see, e.g., McConnell and Imperiale (2004) Human Gene Therapy, 1022-1033 and Vorburger and Hunt (2002) The Oncologist, 7, 46-59).

[0004] Replication-incompetent adenoviral vectors have been administered, for example, intranasally or via intrabronchial catheter when expressing cystic fibrosis transmembrane conductance regulator, by intramyocardial or skeletal muscle injection when expressing vascular endothelial growth factor, and by intratumoral injection when expressing cytosine deaminase. Summary of the Invention

[0005] The present inventors have surprisingly discovered that adenoviral particles can be administered orally. In particular, the present inventors have shown that adenoviral particles carrying insulin under the control of the glucose-dependent insulinotropic polypeptide promoter can reduce glucose levels when orally administered to diabetic rats. Not only were the adenoviral particles successfully delivered to the intestine, but they were also surprisingly able to enter cells and express therapeutic proteins. The therapeutic proteins were found to be detectable in the peripheral circulation.

[0006] Thus, the present invention provides a method for delivering a transgene to target cells in a patient, comprising oral administration of a product comprising viral particles carrying the transgene.

[0007] The present invention also provides a tablet or capsule for oral administration to a patient, which contains a pharmaceutical composition comprising a viral particle carrying a transgene.

[0008] The present invention further provides a method for preparing a viral particle carrying a transgene for oral administration to a patient, said method comprising: (a) culturing and purifying viral particles carrying the transgene; (b) incorporating the viral particles into a pharmaceutical composition; (c) optionally drying the composition; and (d) packaging the composition into a tablet or capsule for oral administration to a patient. [Brief explanation of the drawings]

[0009] [Figure 1]This figure shows the results of an in vivo evaluation of oral administration of adenoviral constructs containing a glucose-dependent insulinotropic polypeptide promoter driving either proinsulin or GFP expression. Results presented are for glucose assessment of tail vein plasma samples before (day -3) or after administration (days 3, 5, 8, 10, and 12, with the corresponding dose for day 1). Data are back-transformed adjusted means (n ​​= 5-6). SEM was calculated from the residuals of the statistical model. Data were analyzed by a general linear model with treatment as a factor and day 1 body weight and log (day -3) plasma glucose as covariates, followed by a Dunnett's test. Significant differences compared to vehicle are indicated by **p<0.01. [Figure 2] We demonstrated that we detected in vitro positive butyrylcholinesterase enzyme activity with butyrylthiocholine (BTC) iodide substrate in the supernatant of cell cultures (HEK293) after Ad5-CMV-BChE vector infection, essentially in the absence of null Ad5-CMV-GFP vector (both unencapsulated). [Figure 3] Tail vein blood (plasma samples) were sampled (on the day of dosing (pre-dose), 1 day, and 3 days post-dose) to demonstrate the in vivo evaluation of adenoviral excipient-loaded capsules administered orally to Brown Norway rats. Each adenoviral excipient loaded dose level was 2x10^10 vp / capsule, as measured spectrophotometrically (OD 260 nm). BCHe levels in individual plasma samples from n=8 subjects were measured using the DetectX® Butyrylcholinesterase Fluorescent Activity Kit (K016-F1), and elevated enzyme activity levels (P<0.01) were detected 3 days post-dose. [Figure 4] Figure 1 shows the weight loss rate of IFNαβ− / − mice when challenged with Zika virus up to 3 days post-infection. Vaccinated mice showed less weight loss than placebo-treated mice (p=0.0003). [Figure 5]Intraocular pressure in IFNαβ− / − mice challenged with Zika virus. Vaccinated mice were more protected than placebo (p=0.016). [Figure 6] Figure 1 shows viremia in mice after challenge with Zika virus. A reduction in viremia was observed after three doses of vaccine (VAC). Results for placebo are indicated as "PLA." [Figure 7-1] Response of immunocompetent mice (control for antibody production) to the vaccine is shown. [Figure 7-2] This is a continuation of Figure 7-1. [Figure 8] Figure 1 shows the reduction of viremia in black-eared marmosets (Callithrix penicillata) after infectious Zika virus challenge, followed by two doses of oral capsule vector. [Figure 9] The Zika virus genome is shown. DETAILED DESCRIPTION OF THE INVENTION

[0010] It is understood that different applications of the disclosed products and methods can be adapted to the particular needs of the art. The terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting.

[0011] Furthermore, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. So, for example, "an amino acid sequence" includes two or more such sequences, and so forth.

[0012] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0013] Transgene The present invention provides methods for delivering a transgene to a patient. In particular, the present invention provides methods for delivering a transgene to target cells in a patient. The methods involve oral administration of viral particles, particularly adenoviral particles, carrying the transgene.

[0014] Transgene (or heterologous gene), as used herein, is intended to refer to a polynucleotide that has been introduced into a cell by genetic engineering. A transgene can be a gene that is not naturally expressed by the cell. A transgene can also be a gene that is naturally expressed by the cell. For example, a transgene can complement the natural expression of a protein or correct a defect in the expression of a protein.

[0015] The transgene generally encodes a therapeutic protein. The methods of the present invention are generally methods of gene therapy, in which a nucleic acid is therapeutically delivered to a patient's cells, which then express the therapeutic protein to treat the disease. The therapeutic protein may be, for example, a hormone or an enzyme.

[0016] Protein and peptide therapeutics can be classified based on function and therapeutic application (see, e.g., Leader et al. (2008) Nature Reviews Drug Discovery, 7, 21-39). For example, therapeutic proteins can have enzymatic or regulatory activity (Group 1). Group 1 therapeutics can replace abnormal or defective proteins (often involved in endocrine or metabolic disorders), augment existing pathways (e.g., hematological and endocrine pathways and immune responses), or provide new functions or activities. Examples of proteins in this category include albumin, insulin, Factor VIII, Factor IX, and antithrombin III, enzymes such as pancreatic enzymes, lactase, β-glucocerebrosidase, alglucosidase-α laronidase, idursulfase, galsulfase, adenosine deaminase, and other proteins such as erythropoietin, darbepoietin-α, G-CSF, GM-CSF, interleukin-11, follicle-stimulating hormone, human chorionic gonadotropin, and various interferons. Examples of therapeutic agents that increase existing pathways include keratinocyte growth factor, platelet-derived growth factor, and trypsin. Examples of proteins that provide new functions or activities include botulinum toxin, collagenase, hyaluronidase, papain, and streptokinase.

[0017] Group 2 therapeutic proteins / peptides have specific targeting activity and may interfere with molecules or organisms or deliver other compounds or proteins. For example, Group 2 proteins may bind to molecules, block their function, target them for destruction, or stimulate signaling pathways. Examples include antibody-based drugs and Fc fusion proteins.

[0018] In the methods of the present invention, a therapeutic protein can be located in Group 1 or Group 2. In other words, in the present invention, a therapeutic protein can replace an abnormal or defective protein, augment an existing pathway, or provide a new function or activity. A therapeutic protein can also have specific targeting activity, such as an antibody or Fc fusion protein.

[0019] Group 3 molecules protect against harmful foreign agents, treat autoimmune diseases, or treat cancer (protein vaccines). Generally, Group 3 molecules include antigenic components derived from microorganisms such as Zika virus or herpes simplex virus (HSV) (preferably HSV2) or tumors. In the methods of the invention, the therapeutic protein may be a Group 3 therapeutic. In other words, the therapeutic protein may be an antigen.

[0020] In some cases, the transgene may also encode a diagnostic agent, such as a fluorescent protein or a protein that can be detected by an external scanning device.

[0021] The viral vector, however, is preferably a gene therapy vector. The viral vector may also be a vaccine vector that expresses an antigen.

[0022] Therapeutic agents are generally full-length proteins, e.g., when used in gene therapy. Therapeutic agents may also be functional variants, modified forms, or sequence variants that retain one or more activities (preferably all activities) of the full-length counterpart.

[0023] In the present invention, therapeutic proteins are not particularly limited as long as they benefit from oral administration of nucleic acids encoding the therapeutic proteins to patients and include hormones and enzymes. Such proteins are well known to those skilled in the art, and include, for example, insulin, glucagon antagonists, glucagon-like peptide-1, resistin, leptin, Acrp30, cholecystokinin, coagulation factors (e.g., factors VIII, IX, and X), growth factors (e.g., growth hormone, insulin-like growth factor 1, platelet-derived growth factor, epidermal growth factor, acidic and basic fibroblast growth factor, transforming growth factor β, etc.), and antibodies (e.g., human or humanized).

[0024] Additional transgenes encoding therapeutic proteins include cytokines, interferons (e.g., interferon (INF), INF-α 2b and 2a, INF-α N1, INF-β 1b, INF-γ), interleukins (e.g., IL-1 to IL-10), tumor necrosis factors (TNF-α, TNF-β), chemokines, granulocyte-macrophage colony-stimulating factor (GM-CSF), polypeptide hormones, antimicrobial polypeptides (e.g., antibacterial, antifungal, antiviral, and / or antiparasitic polypeptides), enzymes (e.g., adenosine deaminase), gonadotropins, chemotactins, lipid-binding proteins, filgrastim, hemoglobin, erythropoietin, insulinotropin, imiglucerase, sargramostim, tissue plasminogen activator (tPA), urokinase, streptokinase, neurite growth factor (NGF), phenylalanine ammonia-lyase, brain-derived neurite factor (BNF). These include BDNF, neurite growth factor (NGF), phenylalanine ammonia-lyase, thrombopoietin (TPO), superoxide dismutase (SOD), adenosine deaminase, catalase, calcitonin, endothelian, L-asparaginase, pepsin, uricase, trypsin, chymotrypsin, elastase, carboxypeptidase, lactase, sucrase, intrinsic factor, calcitonin, parathyroid hormone (PTH)-like hormone, soluble CD4, and antibodies and / or antigen-binding fragments (e.g., FAbs) thereof (e.g., orthoclone OKT-e (anti-CD3), GPIIb / IIa monoclonal antibody).

[0025] As stated above, the transgene is not intended to limit the present invention, and those skilled in the art can readily envision additional transgenes that encode therapeutic proteins.

[0026] The target cells are generally enterocytes. The intestine is the largest endocrine organ in the body, capable of producing large amounts of protein and containing rapidly regenerating tissue accessible to dividing cells. Target cells, such as K cells and stem cells, are primarily located in the upper intestine, where they are readily accessible to non-invasive gene therapy techniques, such as oral formulations. Thus, enterocytes, such as K cells, adjustably secrete proteins such as insulin, leptin, glucagon antagonists, GLP-1, GLP-2, ghrelin, cholecystokinin, growth hormone, clotting factors, and antibodies, among others, and are a means of treating, for example, diabetes, obesity, growth disorders, and other disorders treatable by producing proteins in mucosal tissue.

[0027] In the present invention, the therapeutic protein is preferably insulin. Accordingly, the present invention provides a method for treating diabetes, comprising orally administering to a patient in need thereof adenoviral particles encoding insulin. Other secondary disorders or conditions associated with diabetes can also be treated, including renal tubular calcification, liver degeneration, eye damage (diabetic retinopathy), diabetic foot, ulcers, excessive bleeding, delayed wound healing, slow clotting, increased risk of coronary heart disease, stroke, cardiovascular disease, dyslipidemia, hypertension, and obesity.

[0028] The methods of the invention can be used to treat and / or prevent a disease or condition (in other words, the methods of the invention can be therapeutic or prophylactic). A patient can already have a disease / condition or can be at risk of developing a disease / condition. Treatment typically reduces or prevents the severity or symptoms of a disease / condition in a subject. Treatment can prevent a disease / condition from worsening or can reverse a disease / condition. For example, delivery of insulin can lower blood glucose, improve glucose tolerance, provide normal glucose homeostasis, or prevent, ameliorate, or reverse histopathological changes such as those described above.

[0029] The insulin sequence can be a suitable therapeutic insulin sequence. For example, in the case of a human patient, the insulin can be a therapeutic human insulin. The transgene encodes a subsequence of full-length insulin, i.e., a functional subsequence that has one or more activities of its full-length counterpart, for example, maintaining glucose-lowering ability, providing normal glucose homeostasis, or reducing histopathological conditions associated with chronic or acute hyperglycemia. Such sequences are well known in the art.

[0030] Another example of a therapeutic protein suitable for administration using the methods of the present invention is butyrylcholinesterase. Again, the sequence of the butyrylcholinesterase is not limiting, provided it is suitable for therapeutic use (to treat butyrylcholinesterase deficiency) in a patient.

[0031] The present invention can also be used to deliver genetic payloads, such as gene editing payloads (CRISPR).

[0032] As mentioned above, in some instances, the transgene / therapeutic protein is an antigen (i.e., designed to stimulate an immune response in the host). Thus, the viral vector can be a vaccine vector that expresses the antigen and protects the host from challenge with the antigen (e.g., a microorganism). The antigen can generally be derived from a virus, but is not limited thereto. Antigens that can stimulate an immune response are known in the art.

[0033] The antigen may be derived from Zika virus. As shown in Figure 9, the Zika virus genome encodes various proteins. The vaccine vector of the present invention may express one or more of these proteins. Generally, the vaccine vector expresses the envelope protein E and / or NS1. The envelope protein E is involved in direct recognition of virus particles, and NS1 is thought to play an important role in pathogenesis. The vector may be designed to express either the full-length antigen sequence or an antigenic region of the full-length sequence (e.g., an antigenic region of the E protein or the complete E protein). Such antigens can be readily determined. As described in the Examples, expression of such antigens can be under the control of a CMV promoter. Those skilled in the art can readily design cassettes for antigen expression.

[0034] Antigens may also be derived from herpes simplex virus (HSV), preferably HSV2. The transgene may encode a suitable HSV antigen capable of stimulating an immune response. Various HSV antigens are known in the art.

[0035] For example, glycoprotein D (gD) is expressed on the viral surface and is known to be responsible for neutralizing antibody activity. Other HSV glycoproteins include glycoprotein C (gC) and glycoprotein E (gE). The transgene can encode antigens derived from any of gD, gC, and gE (or combinations thereof). Again, the transgene can encode (i.e., result in the expression of) the full-length protein or antigenic regions of the protein (with appropriate modifications as determined by one of skill in the art).

[0036] The transgene can specifically encode antigens derived from all of gD, gC, and gE (in some instances, the transgene can encode all of gD, gC, and gE). For example, the transgene can express a polyprotein.

[0037] Expression of such HSV antigens may occur under the control of a suitable promoter and may include other suitable regulatory sequences. The polyprotein expressed by the transgene may be cleavable (in some instances, the polyprotein may be self-cleavable). Those skilled in the art can readily design appropriate cassettes for expression of such HSV antigens.

[0038] virus particles As discussed further below, the present invention is generally practiced using adenovirus particles. However, other suitable viruses are adeno-associated viruses, lentiviruses, and oncolytic viruses such as HSV, and vaccinia viruses. Viruses with therapeutic applications are well known in the art.

[0039] Adenovirus particles The methods of the present invention generally involve orally administering adenovirus particles to a patient. Adenoviruses have been widely studied as infectious agents, the subject of basic research, and for potential use in gene therapy and vaccines. Numerous human adenovirus serotypes have been identified and classified into six subgenera (A through F) based on nucleic acid comparisons, fiber protein characteristics, and biological properties. For example, group A includes serotypes 12 and 31, group B includes serotypes 3 and 7, group C includes serotypes 2 and 5, group D includes serotypes 8 and 30, group E includes serotype 4, and group F includes serotypes 40 and 41.

[0040] In general structure, all adenoviruses investigated to date are non-enveloped, icosahedral, approximately 80 nanometers in diameter. Adenoviruses contain linear, double-stranded DNA complexed with core proteins and surrounded by an adenoviral capsid. Individual virions contain approximately 11 distinct proteins, designated by Roman numerals (II-XII) in order of decreasing size on an SDS gel.

[0041] The capsid is composed of seven structural proteins: II (hexon), III (penton), IIIa, IV (fiber), VI, VII, and IX. The capsid contains 252 capsomeres, of which 240 are hexon capsomeres and 12 are penton capsomeres. The hexon capsomeres are trimers of hexon proteins and comprise approximately 75% of the capsid's protein. The penton capsomeres are pentamers of penton proteins and are located at each of the 12 vertices of the virion. Each penton capsomere is connected to six adjacent hexon capsomeres and the fiber. The fiber, usually a trimer of fiber protein, protrudes from the penton capsomere. The hexon proteins, and to a lesser extent the fiber protein, contain the major antigenic determinants of adenoviruses and also determine serotype specificity.

[0042] Researchers have examined and compared the structures of capsid proteins, particularly the hexon protein, of different adenovirus serotypes to define the regions of the protein to which neutralizing antibodies are elicited. The predominant regions of the hexon protein against which neutralizing antibodies are directed appear to reside in loops 1 and 2 (i.e., LI or l1, and LII or l2, respectively), which protrude outward from the base of the hexon capsomere. Analysis of loops 1 and 2 from different adenovirus hexon proteins revealed the presence of seven distinct hypervariable regions (HVR1-HVR7) corresponding to positions in the hexon protein that vary considerably between serotypes.

[0043] The core of the adenovirus virion contains a linear, double-stranded DNA genome, associated proteins V, VII, X (mu), IVa2, and terminal protein (TP). The genome organization of different adenoviruses is conserved and is designed to function in a timing manner, with the ends of the genome being transcribed first (the immediate early genes E1 and E4 are located at opposite ends of the linear genome). Early transcription of E1 and E4 leads to the opening of the central region of the genome, allowing transcription of the central region.

[0044] The adenovirus genome generally contains eight RNA polymerase II transcription units: five early units, E1A, E1B, E2A-E2B, E3, and E4, two delayed early units, IX and IVa2, and a major late transcription unit. The major late transcription unit is further subdivided into L1-L5 regions based on the use of alternative splicing sites. Transcription units often express proteins with similar functions. For example, the E1A unit encodes two proteins responsible for activating S-phase transcription and induction during cell infection; the E1B transcription unit encodes two proteins that inhibit cell apoptosis; the E3 transcription unit is involved in evading immune responses; and the major late transcription unit encodes structural proteins required for capsid assembly.

[0045] For gene therapy and vaccination, recombinant adenoviral vectors have been designed to encode and express heterologous genes and antigens. The Ad2 and Ad5 serotypes have been most widely used in this context. Heterologous sequences have been inserted into the adenoviral genome, including in the early transcription unit and coding regions of various structural proteins, such as hexon, penton, and fiber. Deletions of the adenoviral genome (e.g., in the E1 region) are often used to generate replication-deficient adenoviral vectors, which are generally considered safer for administration to human subjects.

[0046] In the present invention, the adenovirus may be an adenovirus suitable for delivering a transgene to a target cell. For example, the adenovirus may be of any serotype, but is generally Ad5. The term "adenovirus vector" refers to wild-type, mutant, and / or recombinant adenovirus genomes and adenoviruses containing such genomes. The adenovirus vector may contain all or part of the genome of any adenovirus serotype, as well as a combination thereof (i.e., a hybrid genome).

[0047] Adenoviral vectors used in the present invention can be either self-replicating or non-replicating. Such vectors are well known. For example, in non-replicating vectors, the E1 region can be deleted and replaced with an expression cassette containing an exogenous promoter driving the expression of a heterologous transgene. Usually, the E3 region is also deleted. Deletion of E3 allows for larger insertions in the E1 region. Such vectors can be propagated in appropriate cell lines, such as HEK293 cells, which retain and express E1A and E1B proteins. Later generation vectors also lack the E4 region, and some vectors additionally lack the E2 region. E2 and E4 vectors must be propagated in cell lines that complement the deletions of E1, E4, and E2.

[0048] Vectors may also be helper-dependent vectors, which lack many or all of the adenoviral genes but retain cis-acting sequences, such as the inverted terminal repeats, as well as packaging sequences necessary to package and replicate the genome. These vectors grow in the presence of a helper adenovirus, which must be removed from the vector stock. Again, such systems are well known in the art.

[0049] In the present invention, the vector is generally a vector that is not capable of self-replication (such as the E1 / E3 deleted vector used in the Examples), but the vector may also be helper-dependent or self-replicating. Self-replicating vectors are well known. Those skilled in the art can easily select an appropriate vector system depending on the intended application.

[0050] Expression of the transgene can be driven via operably linking to an appropriate promoter. The vectors used in the present invention may also use other appropriate expression control elements, such as enhancers and regulatory elements. Enhancers are broadly defined as cis-acting elements that, when operably linked to a promoter / gene sequence, increase the transcription of that gene sequence. Enhancers can function from positions much farther away from the sequence of interest than other expression control elements (e.g., promoters) and can function when positioned in either direction relative to the sequence of interest. Enhancers have been identified from several viral sources, including adenovirus.

[0051] Adenoviral vectors may use highly active promoters such as the cytomegalovirus (CMV) promoter, the SV40 large T antigen promoter, the mouse mammary tumor virus LTR promoter, the adenovirus major late promoter (MLP), the mouse mammary tumor virus LTR promoter, or the SV40 early promoter.

[0052] The vector can also use an appropriate promoter to switch on expression of the transgene when required, for example, in the target cell (tissue-specific or inducible promoter). The promoter may be selected to be compatible with the target cell in which expression is designed.

[0053] Tissue-specific promoters are generally active in a particular cell type because they are recognized by a transcriptional activator protein or other transcriptional regulator that is specific to that cell type. For example, in the present invention, a promoter can target expression of a transgene to endocrine cells of the intestine (an "enteroendocrine cell-specific promoter").

[0054] The promoter can maintain transcriptional homeostasis of the transgene product in response to stimuli resulting from the activity of the transgene product.

[0055] Expression of the transgene is preferably controlled by operably linking it to the glucose-dependent insulinotropic polypeptide promoter (GIP), a specific example of an enteroendocrine cell promoter. When this construct is introduced into endocrine cells, the encoded protein is expressed and secreted in a controlled manner. The GIP promoter has previously been shown to target the expression and secretion of human insulin in K cells of the gastrointestinal tract. Most preferably, the GIP promoter is used to control insulin expression.

[0056] The examples of this application use the 1.2 kb rat GIP insert described in Biol. Res. (2011) 44, 301-305. Those skilled in the art can easily select an appropriate promoter depending on the application.

[0057] Other examples of tissue-specific promoters and enhancers for targeting protein expression to intestinal endocrine cells include glucokinase, chromogranin A and B, cholecystokinin, proglucagon, adenosine deaminase, secretin, gastrin, somatostatin, motilin, and ghrelin. Tissue-specific expression control elements may be active in other tissues, but to a much lesser extent than the target tissue. Promoters can also increase or decrease expression of operably linked nucleic acids in response to or upon nutrient withdrawal. Such nutrient-regulatable elements are also widely known. Other expression control elements may be constitutively active or may be expressed at specific stages of the cell cycle.

[0058] As used herein, the term "operably linked," or grammatical variations thereof, refers to the physical or functional juxtaposition of components as described that permits them to function as intended. In the example of an expression control element in operably linked relationship with a nucleic acid, the relationship is such that the control element regulates expression of the nucleic acid.

[0059] Preparations containing viral particles The viral particles are formulated with an excipient that provides heat stability.

[0060] WO2011 / 121306 discloses a formulation that was able to stabilize viral particles against damage caused by freezing, freeze-drying, and thawing, and also maintained viral activity during long-term stability testing.

[0061] In the present invention, viral particles may be formulated in a composition comprising a compound of formula (I) or a physiologically acceptable salt or ester thereof and / or a compound of formula (II) or a physiologically acceptable salt or ester thereof, and optionally one or more sugars. The viral particles are generally contacted in an aqueous solution with the compound of formula (I) or a physiologically acceptable salt or ester thereof and / or a compound of formula (II) or a physiologically acceptable salt or ester thereof, and optionally one or more sugars, and the resulting solution containing the viral particles is then dried to form a composition incorporating the viral particles.

[0062] Thus, viral particles can be mixed with an aqueous solution of a compound of formula (I) or a physiologically acceptable salt or ester thereof and / or a compound of formula (II) or a physiologically acceptable salt or ester thereof, and optionally one or more sugars. The resulting solution is then dried to form a composition incorporating the viral particles. The dried composition can be in the form of a cake or powder. If necessary, the cake can be crushed to a powder.

[0063] The viral particles are maintained in the aqueous solution prior to the drying step, which allows the aqueous solution to be stored after preparation for a period of time at which the drying step can be carried out without excessive loss of viral activity.

[0064] The compounds of formula (I) and formula (II) may exist as their physiologically acceptable salts or esters.

[0065] Salts are generally salts with physiologically acceptable acids, and thus include salts formed with inorganic acids such as hydrochloric acid or sulfuric acid, or organic acids such as citric acid, tartaric acid, malic acid, maleic acid, mandelic acid, fumaric acid, or methanesulfonic acid. Hydrochloride salts are preferred.

[0066] Esters are generally C 1-6 Alkyl esters, preferably C 1-4 Alkyl esters. The esters may therefore be methyl, ethyl, propyl, isopropyl, butyl, isobutyl or tert-butyl esters. Ethyl esters are preferred.

[0067] As used herein, C 1-6 The alkyl group is preferably C 1-4 Alkyl groups. Preferred alkyl groups are selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl and tert-butyl. Methyl and ethyl are particularly preferred.

[0068] For the avoidance of doubt, the definitions of compounds of formula (I) and formula (II) also include compounds wherein the carboxylate anion is protonated to -COOH and the ammonium or sulfonium cation is associated with a pharmaceutically acceptable anion. Further, for the avoidance of doubt, the above defined compounds may be used in their tautomeric or enantiomeric forms.

[0069] Compounds of formula (I) Generally, R1 is hydrogen or C 1-6 R represents alkyl and R represents hydrogen. Generally, R represents hydrogen or C 1-6 Preferably, R1 is hydrogen or C 1-6 R represents alkyl, R represents hydrogen, and R represents hydrogen or C 1-6 More preferably, R represents hydrogen or C 1-6 R4 represents hydrogen, and R2 represents C 1-6 Represents alkyl.

[0070] Preferably, the compound of formula (I) is NC 1-6 Alkyl-, N,N-di(C 1-6 alkyl)- or N,N,N-tri(C 1-6 alkyl)-glycine or a physiologically acceptable salt or ester thereof, more preferably N,N-di(C 1-6 alkyl)- or N,N,N-tri(C 1-6 The alkyl group is generally C 1-4 Alkyl groups. Preferred alkyl groups are selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl and tert-butyl. Methyl and ethyl are particularly preferred.

[0071] Preferred compounds of formula (I) are N-methylglycine, N,N-dimethylglycine, or N,N,N-trimethylglycine, or physiologically acceptable salts or esters thereof. N-methylglycine is also called sarcosine. N,N-dimethylglycine is also called dimethylglycine (DMG) or 2-(dimethylamino)-acetic acid. N,N,N-trimethylglycine is called trimethylglycine (TMG). The most preferred compound of formula (I) is DMG.

[0072] Alternatively, the compound of formula (I) is generally a glycine derivative of formula (IA) or a physiologically acceptable salt or ester thereof,

[0073] [ka] wherein R5 and R6 are independently C 1-6 Alkyl, e.g., methyl or ethyl, 1-4 R7 represents alkyl, and C 1-6 Alkyl, for example, methyl or ethyl 1-4 Alkyl or -(CH2) 2-5 NHC(O)(CH2) 5-15represents CH3. Preferred compounds of formula (IA) are trimethylglycine (TMG) and cocamidopropyl betaine (CAPB) or physiologically acceptable salts or esters thereof. Trimethylglycine is preferred.

[0074] Alternatively, the compound of formula (I) is generally a proline derivative of formula (IB) or a physiologically acceptable salt or ester thereof,

[0075] [ka] In the formula, R8 and R9 are independently C 1-6 Alkyl, e.g., methyl or ethyl, 1-4 Preferably, R8 and R9 both represent methyl, and the compound is known as proline betaine. S-proline betaine or a physiologically acceptable salt or ester thereof is particularly preferred.

[0076] [ka]

[0077] Compounds of formula (IA) or physiologically acceptable salts or esters thereof are preferred.

[0078] Preferably, the compound of formula (I) is N,N-dimethylglycine or N,N,N-trimethylglycine or a physiologically acceptable salt or ester thereof. Most preferably, the compound of formula (I) is N,N-dimethylglycine or a physiologically acceptable salt or ester thereof.

[0079] Compound of formula (II) Generally, carboxylic acids and R c The amine substituent in R c Attached to the same carbon atom of the alkyl moiety. c is C 2-4 or C2-3 It is the alkyl portion.

[0080] The compound of formula (II) is generally a sulfone compound of formula (IIA) or a physiologically acceptable salt or ester thereof:

[0081] [ka] In the formula, R c and R d independently C 1-6 Alkyl, e.g., C 1-4 represents alkyl. Preferred alkyl groups are selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and tert-butyl. Methyl and ethyl are particularly preferred. The most preferred sulfone compound is methylsulfonylmethane (MSM), also known as dimethylsulfone (DMSO).

[0082] The compound of formula (II) may be a compound of formula (IIB) or a physiologically acceptable salt or ester thereof:

[0083] [ka] In the formula, R e and R f independently, C 1-6 Alkyl, e.g., methyl or ethyl, 1-4 represents alkyl, and R g is a C substituted with a carboxylate anion and an amine (-NH) moiety. 1-6 Alkyl, e.g., methyl or ethyl, 1-4 represents alkyl. Preferably, the carboxylate and amine substituents are attached to the same carbon atom. A preferred compound of formula (IIB) is S-methyl-L-methionine (SMM) or a physiologically acceptable salt or ester thereof.

[0084] Most preferably, in the present invention, the compound of formula (I) is DMG or a physiologically acceptable salt or ester thereof, and the compound of formula (II) is MSM or a physiologically acceptable salt or ester thereof.

[0085] sugar Sugars suitable for use in the present invention include reducing sugars such as glucose, fructose, glyceraldehyde, lactose, arabinose, and maltose, and preferably non-reducing sugars such as sucrose and raffinose, more preferably sucrose. Sugars may be monosaccharides, disaccharides, trisaccharides, or other oligosaccharides. The term "sugar" includes sugar alcohols. Thus, in one embodiment, the use of non-reducing sugars or sugar alcohols is preferred.

[0086] Monosaccharides such as galactose and mannose, disaccharides such as sucrose, lactose and maltose, trisaccharides such as raffinose, and tetrasaccharides such as stachyose are contemplated. Trehalose, umbelliferose, verbascose, isomaltose, cellobiose, maltulose, turanose, melezitose, and melibiose are also suitable for use in the present invention. A suitable sugar alcohol is mannitol. When mannitol is used, cakes with improved appearance can be obtained by freeze-drying.

[0087] The presence of sugars can improve stability. The addition of sugars can also provide other benefits, such as improved solubility for a modified lyophilized cake and rapid reconstitution. Generally, when lyophilization is used, one or more sugars are present. When one sugar is used, the sugar is preferably sucrose or mannitol.

[0088] Preservation of viral activity is particularly effective when two or more sugars are used in the preservation mixture. Two, three, or four sugars may be used. Preferably, the aqueous solution is a solution of sucrose and raffinose. Sucrose is a disaccharide of glucose and fructose. Raffinose is a trisaccharide composed of galactose, fructose, and glucose.

[0089] In the present invention, the compound of formula (I) is preferably DMG or a physiologically acceptable salt or ester thereof, and the compound of formula (II) is preferably MSM or a physiologically acceptable salt or ester thereof. The composition also preferably includes sucrose.

[0090] Other components of the aqueous composition In the present invention, the aqueous solution containing virus particles, optionally one or more sugars, and a compound of formula (I) or a physiologically acceptable salt or ester thereof and / or a compound of formula (II) or a physiologically acceptable salt or ester thereof is generally dried. Any suitable aqueous solution may be used. The solution may be buffered. The solution may be HEPES, phosphate buffer, Tris buffer, or pure water.

[0091] The solution may have a pH of 2 to about 12 and may be buffered. The solution may be buffered with HEPES buffer, phosphate buffer, Tris buffer, sodium citrate buffer, bicine buffer (i.e., N,N-bis(2-hydroxyethyl)glycine buffer), or MOPS buffer (i.e., 3-(N-morpholino)propanesulfonic acid buffer). The solution may or may not contain NaCl. The solution may therefore be saline sodium citrate (SSC) buffer.

[0092] Typically, a preparation of viral particles is mixed with a preservation mixture, i.e., an aqueous solution of a compound of formula (I) or a physiologically acceptable salt or ester thereof and / or a compound of formula (II) or a physiologically acceptable salt or ester thereof, and optionally one or more sugars. The preservation mixture may itself be buffered. It may be HEPES, phosphate buffer, Tris buffer, or pure water.

[0093] Alternatively, the aqueous solution may consist essentially of or consist of virus particles, a compound of formula (I) or a physiologically acceptable salt or ester thereof and / or a compound of formula (II) or a physiologically acceptable salt or ester thereof, and optionally one or more sugars.

[0094] The concentrations of the compound of formula (I) or its physiologically acceptable salt or ester and / or the compound of formula (II) or its physiologically acceptable salt or ester, and any respective sugars can be determined by routine experimentation. Therefore, the optimized concentrations that provide the best stability can be selected. The compound of formula (I) or its physiologically acceptable salt or ester and / or the compound of formula (II) or its physiologically acceptable salt or ester may act synergistically to improve stability.

[0095] When present in aqueous solution for drying, the sugar concentration is at least 0.01M, typically up to saturation. Typically, the sugar concentration present is at least 0.1M, at least 0.2M, or at least 0.5M up to saturation, e.g., saturation or up to 3M, 2.5M, or 2M at room temperature. Thus, the sugar concentration may range, for example, from 0.1M to 3M or from 0.2M to 2M. Preferably, a sugar is present. Alternatively, the sugar concentration, or the total sugar concentration when more than one sugar is present, may range from 0.08M to 3M, 0.15M to 2M, or 0.2M to 1M. Suitable ranges are 0.05 to 1M.

[0096] When two or more sugars are present, preferably one of the sugars is sucrose, which may be present at a concentration of from 0.05M, 0.1M, 0.25M or 0.5M up to saturation, for example up to saturation or 3M, 2.5M or 2M at room temperature.

[0097] The ratio of the molar concentration of sucrose to the molar concentration of other sugars is generally 1:1 to 20:1, for example, 5:1 to 15:1. Thus, when two sugars are present, particularly when sucrose and raffinose are present, the ratio of the molar concentrations of sucrose is generally 1:1 to 20:1, for example, 5:1 to 15:1, preferably about 10:1.

[0098] The concentration of each compound of formula (I) or a physiologically acceptable salt or ester thereof, or compound of formula (II) or a physiologically acceptable salt or ester thereof in the aqueous solution upon drying is generally in the range of 0.001 M to 2.5 M, more particularly 0.01 M to 2.5 M. For example, the concentration range may be 0.1 M to 2.5 M.

[0099] Alternatively, for example, when the compound of formula (I) is DMG or a salt or ester thereof, the concentration of each compound of formula (I) or a physiologically acceptable salt or ester thereof, or the compound of formula (II) or a physiologically acceptable salt or ester thereof in the aqueous solution upon drying is generally in the range of 0.1 mM to 3 M, or 1 mM to 2 M. The concentration may be 1 mM to 1.5 M, 5 mM to 1 M, or 0.07 M to 0.7 M. A preferred concentration is 7 mM to 1.5 M or 0.07 M to 1.2 M. In particular, when the compound of formula (I) is an N-alkylated glycine derivative such as DMG, another preferred range is 0.5 to 1.5 M.

[0100] The particular concentration of the compound of formula (I) or a physiologically acceptable salt or ester thereof or the compound of formula (II) or a physiologically acceptable salt or ester thereof used will depend on several factors, including the type of virus particle being preserved, the particular compound used, whether one or more sugars are present and the identity of the sugar, and the drying procedure and conditions. Thus, The concentration of the compound of formula (II) where X represents -S(O)- or the compound of formula (IIA), such as MSM, or a physiologically acceptable salt or ester thereof, is preferably 0.2 mM to 1 M, for example, 0.35 mM to 1 M, 3.5 mM to 0.5 M, 0.035 M to 0.5 M, or 0.035 M to 0.25 M. The compound of formula (I), or the compound of formula (IA) or formula (IB), such as TMG, or a physiologically acceptable salt or ester thereof, is preferably used at a concentration of 0.01 M to 2 M, for example, 0.07 M to 2 M, 0.2 M to 1.5 M, 0.23 M to 1.5 M, or 0.07 M to 0.7 M. - X is -S + (R c The concentration of the compound of formula (II) representing S-methyl-L-methionine or the compound of formula (IIB) such as S-methyl-L-methionine, or a physiologically acceptable salt or ester thereof is preferably 0.005 M to 2 M, for example, 0.007 M to 2 M, 0.02 M to 2 M, 0.023 M to 1.5 M, or 0.07 M to 1 M. The concentration of the compound of formula (I), such as N,N-dimethylglycine (DMG), or a physiologically acceptable salt or ester thereof, in the absence of sugar is 5 mM to 1.5 M, or 70 mM to 1.5 M, or 1.2 M, or 7 mM to 1 M. More preferred concentrations are 0.023 M to 0.7 M or 1 M, or 0.07 M to 0.7 M or 1 M, for example about 0.7 M. The concentration of the compound of formula (I), such as N,N-dimethylglycine (DMG), or a physiologically acceptable salt or ester thereof, when one or more sugars are present, is generally low, in the range of 1 mM to 1 M or 1.5 M, or 5 mM to 1 M. More preferred concentrations are 0.007 M to 0.7 M or 1 M, for example about 0.007 M. An especially preferred range is 0.5 to 1.5 M.

[0101] When a compound of formula (I) or a physiologically acceptable salt or ester thereof and a compound of formula (II) or a physiologically acceptable salt or ester thereof are present, preferably when an N-alkylated glycine derivative or a salt or ester thereof and a sulfone compound of formula (IIA) or (IIC) are present, the compounds may be present in synergistic amounts, for example: The concentration of the N-alkylated glycine derivative or its salt or ester in the aqueous solution for drying is generally in the range of 0.1 mM to 3 M or 1 mM to 2 M. The concentration may be 1 mM to 1.5 M or 5 mM to 1 M. The preferred concentration is 0.1 M to 1.5 M or 0.5 M to 1.25 M. The concentration of the sulfone compound of formula (IIA) or (IIC) in the aqueous solution for drying is generally in the range of 0.1 mM to 3 M, 1 mM to 2 M or 0.2 mM to 1 M. The concentration may also be 0.1 M to 1.5 M or 0.5 M to 1.25 M.

[0102] The compositions may also contain other preservatives such as antioxidants, lubricants and binders that are well known in the art.

[0103] Tablets and capsules The pharmaceutical compositions described herein may be administered in the form of an aqueous suspension or solution or a lozenge, but are generally administered as tablets or capsules. The compositions may also be administered as gelatin wafers. Tablets may be coated or uncoated. Preferably, the compositions are incorporated into a capsule, such as a gelatin capsule.

[0104] Pharmaceutically compatible binders and / or adjuvant substances can be included in the oral formulations. Tablets, capsules, troches, etc. can contain any of the following ingredients: binders such as microcrystalline cellulose, gum tragacanth, or gelatin; excipients such as starch or lactose; disintegrants such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotes; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavorings such as peppermint, methyl salicylate, or flavorings, or compounds of a similar nature.

[0105] Capsules and tablets are generally enterically coated. Those skilled in the art can easily select and apply an appropriate enteric coating depending on the transgene to be delivered using methods known in the art. For example, the enteric coating may target delivery to the duodenum. An example of such a coating agent is poly(methacrylic acid-co-ethyl acrylate) 1:1 copolymer, as used in the examples. The enteric coating may have a threshold pH of 5.8 to 6.8.

[0106] Delivery of viral particles to patients The patient is generally a human, but can also be an animal, such as a livestock animal, a companion animal (such as a dog or cat) or a farm animal (sheep, pig, cow).

[0107] The administration of viral particles may be for either prophylactic or therapeutic purposes. A dose or "effective amount" for treating a subject is preferably sufficient to measurably or detectably improve one, some, or all of the symptoms of the condition, although preventing or inhibiting the progression or worsening of the disorder or condition or symptoms would be a satisfactory outcome. The dose and frequency of administration depend on the condition being treated and the desired outcome, and can be easily ascertained by one skilled in the art. The appropriate amount depends on the desired therapeutic effect and the individual subject (e.g., bioavailability of the subject, gender, age, etc.). For example, partial restoration of normal glucose homeostasis in a subject may reduce the frequency of insulin injections, although it may not result in complete freedom from insulin injections.

[0108] An effective amount can be determined by measuring the relevant physiological effect. For example, in the case of diabetes or other hyperglycemic conditions, a reduction in blood glucose or an improvement in glucose tolerance tests can be used to determine whether an amount of insulin is effective to treat the hyperglycemic condition. In the case of hemophilia, an effective amount is one that reduces clotting time or the frequency or duration of a subject's bleeding episodes.

[0109] The subject treatment methods of the present invention can also be used in conjunction with other forms of therapy. Adjunctive therapies include drug therapy, dietary changes (low sugar, low fat, etc.), surgical resection, transplantation, radiation therapy, etc. For example, the subject treatment methods for hyperglycemic conditions can be used in combination with drugs or other pharmaceutical preparations that increase insulin or lower glucose levels in a subject. Drugs for treating diabetes include, for example, biguanides and sulfonylureas (e.g., tolbutamide, chlorpropamide, acetohexamide, tolazamide, glibenclamide, and glipizide). Appetite suppressants are also well known and can be used in conjunction with the subject treatment methods. Adjunctive therapies can be administered before, simultaneously with, or after the subject treatment methods. Those skilled in the art can easily identify therapies that can be used in combination with the subject treatment methods in a regimen.

[0110] The present invention also provides a method for preventing and / or treating Zika virus infection using the viral particles of the present invention. In particular, the viral particles can be used as a vaccine for preventing and / or treating Zika virus infection. As mentioned above, the viral vector here can preferably express Zika virus antigens derived from envelope protein E and / or NS1.

[0111] The dosage and administration regimen of the Zika virus vaccine can be easily determined by one skilled in the art. For example, the prophylactic vaccine may be administered multiple times (e.g., two or three times) at appropriate intervals. Such intervals may be once per week or once every two weeks. For example, the prophylactic vaccine may be administered two or three times, approximately once every two weeks (at intervals of about 14 days).

[0112] Method for preparing viral particles The present invention also provides methods for preparing viral particles for oral administration to a patient. The methods generally include culturing and purifying the viral particles, formulating the particles into a composition, optionally drying the composition, and packaging the composition into a tablet or capsule for oral administration to a patient.

[0113] Viral particles carrying a transgene can be prepared using standard techniques well known to those skilled in the art, for example, standard molecular biology techniques can be used to generate genetic constructs for expressing the transgene.

[0114] The virus can be prepared by infecting cultured host cells with the virus strain to be used, allowing the infection to proceed so that the virus replicates in the cultured cells, and then releasing the virus by standard methods known in the art for virus recovery and purification. Suitable cells include human embryonic kidney (HEK) 293 cells or HEK293-derived cell clones, which are appropriate for the type of adenovirus used. The cells can be cultured under appropriate conditions to produce viral particles.

[0115] For example, the examples of this application show that the virus is grown in HEK293 cells, then purified by double cesium chloride density gradient centrifugation, and then subjected to desalting column treatment (e.g., PD-10) into an excipient formulation, which is described above.

[0116] Drying is generally achieved by freeze-drying, vacuum drying, fluidized bed drying, or spray drying. Freeze-drying is preferred. By reducing the water content of the material and sealing it in a vial, the material can be easily stored, shipped, and later reconstituted to its original form. Drying conditions can be optimized by routine experimentation.

[0117] Upon drying, a composition is formed that incorporates the viral particles. A matrix is ​​produced that incorporates the viral particles. The composition is generally an amorphous solid. Thus, a solid matrix, generally an amorphous solid matrix, is generally formed. "Amorphous" means unstructured, having no observable regular or repeating organization of molecules (i.e., non-crystalline).

[0118] When present, the sugar provides an amorphous matrix to the dried composition. The compound of formula (I) or its physiologically acceptable salt or ester and / or the compound of formula (II) or its physiologically acceptable salt or ester is dispersed in the sugar matrix. The compound of formula (I) or its physiologically acceptable salt or ester and / or the compound of formula (II) or its physiologically acceptable salt or ester is thus incorporated into the sugar matrix. The virus particles are also incorporated into the sugar matrix. A drying procedure can then be carried out, for example, by freeze-drying, to form an amorphous cake into which the virus particles are incorporated.

[0119] A drying step is generally carried out immediately or shortly after the aqueous solution is prepared. Alternatively, the aqueous solution is generally stored before the drying step. During storage, the virus particles in the aqueous solution are preserved by the compound of formula (I) or a physiologically acceptable salt or ester thereof and / or the compound of formula (II) or a physiologically acceptable salt or ester thereof, and optionally one or more sugars.

[0120] The aqueous solution or bulk intermediate solution is generally stored for up to 5 years, for example, up to 4, 3, 2, or 1 year. Preferably, the solution is stored for up to 6 months, more preferably up to 3 or 2 months, for example, 1 to 1 month or 1 to 1 week. Before drying, the solution is generally stored in a refrigerator or freezer. The temperature of the refrigerator is generally 2 to 8°C, preferably 4 to 6°C, or for example, about 4°C. The temperature of the freezer is generally -10 to -80°C, preferably -10 to -30°C, for example, about -20°C.

[0121] The solution is generally stored in a sealed container, preferably a sealed inert plastic container such as a bag or bottle. The solution is generally sterile. The volume of the bulk intermediate solution is 0.1 to 100 L, preferably 0.5 to 100 L, for example, 0.5 to 50 L, 1 to 20 L, or 5 to 10 L. The container generally has a volume of 0.1 to 100 L, preferably 0.5 to 100 L, for example, 0.5 to 50 L, 1 to 20 L, or 5 to 10 L.

[0122] If the stored bulk intermediate solution is to be lyophilized, it is generally poured into lyophilization trays prior to the drying step.

[0123] There are three main stages of freeze-drying: freezing, primary drying, and secondary drying. Freezing is generally performed using a freeze dryer. In this step, it is important to cool the biological material below the eutectic point (Teu) for simple crystalline products, or below the glass transition temperature (Tg') for amorphous products, i.e., below the lowest temperature at which the solid and liquid phases of the material can coexist. This ensures that sublimation rather than melting occurs in the following primary drying stage.

[0124] During primary drying, pressure is controlled by applying an appropriate level of vacuum and sufficient heat is provided to allow the water to sublimate. At least 50%, typically 60-70%, of the water in the material sublimes during this stage. Primary drying can be performed slowly, as excessive heat can disrupt or alter the structure of the biological material. The cold condenser and / or condenser plates provide a surface on which the water vapor can be trapped as it resolidifies.

[0125] In the secondary drying step, the water of hydration is removed by further application of heat. Generally, the pressure is also reduced to facilitate further drying. After the freeze-drying step is complete, the vacuum may be broken with an inert gas such as nitrogen before sealing, or the material may be sealed under vacuum.

[0126] In certain embodiments, vacuum drying is performed using a vacuum of about 1300 Pa. However, vacuum drying is not required for the present invention, and in other embodiments, the preservation mixture in contact with the viral particles is rotated (i.e., rotary dried) or freeze-dried. Advantageously, the method of the present invention further comprises subjecting the preservation mixture containing the viral particles to a vacuum. Conveniently, the vacuum is applied at a pressure of 20,000 Pa or less, preferably 10,000 Pa or less. Advantageously, the vacuum is applied for at least 10 hours, preferably 16 hours or more. As known to those skilled in the art, the duration of vacuum application depends on the size of the sample, the machine used, and other parameters.

[0127] In another embodiment, drying of the viral particles mixed with the preservation mixture of the present invention is achieved by spray drying or spray freeze drying. These techniques are well known to those skilled in the art and involve drying a liquid feed with a gas, such as air, an oxygen-free gas or nitrogen, or, in the case of spray freeze drying, liquid nitrogen. The liquid feed is atomized into a spray of droplets. The droplets are then dried by contact with gas or liquid nitrogen in a drying chamber.

[0128] In a further embodiment, drying of the virus particles mixed with the preservation mixture is achieved by fluidized bed drying. This technique is well known to those skilled in the art and generally involves passing a gas (e.g., air) through a product bed under controlled velocity conditions to create a fluidized state. This technique may involve stages of drying, cooling, agglomerating, granulating, and coating the particulate product material. Heat may be provided by fluid gas and / or other heated surfaces (e.g., panels or tubes) immersed in the fluid bed. Cooling can be achieved using cooled gas and / or cooled surfaces immersed in the fluid bed. The agglomeration and granulation steps are well known to those skilled in the art and can be carried out in a variety of ways depending on the product characteristics to be achieved. Coating of particulate products, such as powders, granules, or tablets, can be achieved by spraying a liquid onto the fluidized particles under controlled conditions.

[0129] Thus, a composition having a low residual moisture content can be obtained. A certain level of residual moisture content can be achieved during long-term storage at temperatures higher than refrigeration temperatures, for example, in the range of 40°C to 56°C or higher, or at temperatures lower than refrigeration temperatures, for example, in the range of 0 to -70°C or lower. The dry composition can therefore have a residual moisture content of 10% by weight or less, 5% by weight or less, 2% by weight or less, or 1% by weight or less. Preferably, the residual moisture content is 0.5% or more, or 1% or more. Generally, the dry composition has a residual moisture content of 0.5 to 10% by weight, preferably 1 to 5% by weight.

[0130] The composition can be obtained in dry powder form. For example, the cake obtained from freeze-drying can be pulverized into powder form. The solid composition according to the present invention can therefore be in the form of free-flowing particles.

[0131] In the present invention, the powder may be compressed into tablet form, tablets being discussed above. The powder may also be filled into capsules, again, capsules being discussed above.

[0132] The following examples illustrate the invention. [Example]

[0133] [Example 1] Two adenovirus (human adenovirus type 5 (dE1 / E3)) constructs were prepared using standard molecular biology techniques. The first adenovirus carried a GIP (glucose-dependent insulinotropic polypeptide) promoter (designated GIP-INS) driving INS (proinsulin) expression. The sequence used was a 1.2-kb rat GIP insert described in Biol Res 44:301-305, 2011 and the NCBI reference sequence (proinsulin) NM_019129.2 / 3. The second adenovirus carried a "null" eGFP (720 bp, Gene. 1996;173(1 Spec No):33-8) construct (designated GIP-GFP) expressing enhanced green fluorescent protein. GFP expression was controlled using the same promoter system as the insulin cassette.

[0134] High titer adenovirus stocks were prepared by viral culture in HEK293 cells, then purified by double cesium chloride density gradient centrifugation, and then desalting column (e.g., PD-10) into the following excipient formulations: - N,N-dimethylglycine (DMG) (0.2M), - Methylsulfonylmethane (MSM) (0.2M) and - Sucrose (0.4M).

[0135] The final excipient formulated adenovirus was freeze-dried, and the resulting cake was powdered with a spatula and then filled into porcine gelatin capsules size 9 (Torpac Inc. NJ, 07006). Finally, the capsules were coated with poly(methacrylic acid-co-ethyl acrylate) 1:1 copolymer by enteric coating methods known to those skilled in the art for targeted delivery to the duodenum.

[0136] In vivo evaluation of orally administered (by gavage) adenoviral excipient-loaded capsules was performed in STZ (35 mg / kg)-induced high-fat diet-fed diabetic rats (Cardiovascular Diabetology 2013 12:136). Glucose was assessed in tail vein blood (plasma samples) before (day -3) and after (days 3, 5, 8, 10, 12, and 15) administration, with the corresponding doses on day 1. The results are presented in Figure 1. The dose of each orally administered adenoviral excipient was titrated by TCID50 and CPE assays in HEK293 cells to quantify the orally administered live virus dose. These were -GIP-INS at a dose of 9.6E+07 TCID and GIP-GFP at a dose of 1.6E+07 TCID. In Figure 1, the diabetic state of all treated rats is indicated by pre-treatment (day -3) blood glucose levels that were approximately four times the normal level (approximately 7 mM).

[0137] Data were back-transformed adjusted means (n ​​= 5-6). SEM was calculated from the residuals of the statistical model. Data were analyzed by a general linear model with treatment as a factor and day 1 body weight and log (day -3) plasma glucose as covariates, followed by a Dunnett's test. Significant differences compared to vehicle were ** Provided with p<0.01.

[0138] [Example 2] An adenovirus (human adenovirus type 5 (dE1 / E3)) construct consisting of a CMV (cytomegalovirus) promoter driving BChE (butyrylcholinesterase) expression was prepared by standard molecular biology techniques and designated Ad5-CMV-BChE. The NCBI reference sequence: -butyrylcholinesterase NM_022942.1, rat species, was used.

[0139] Next, high-titer adenovirus stocks were prepared by viral culture in HEK293 cells, followed by adenovirus chromatography and final concentration in a DMG (0.2 M) / MSM (0.2 M) / sucrose (0.4 M) vehicle formulation. The final adenovirus-loaded vehicle was lyophilized, and the resulting cake was ground into a powder and then manually filled into size 9 porcine gelatin capsules (Torpac Inc. NJ, 07006). Finally, the capsules were enterically coated (poly(methacrylic acid-co-ethyl acrylate) 1:1 copolymer) for targeted delivery to the duodenum.

[0140] We detected positive in vitro butyrylcholinesterase enzyme activity with butyrylthiocholine (BTC) iodide substrate in the supernatant of cell cultures (HEK293) after infection with the Ad5-CMV-BChE vector and essentially in the absence of the null Ad5-CMV-GFP vector (both unencapsulated). The results are shown in Figure 2.

[0141] In vivo evaluation of orally administered adenovirus excipient-loaded capsules was performed by sampling tail vein blood (plasma samples) from Brown Norway rats on the day of administration (pre-dose), 1 day, and 3 days after administration. The loading dose level of each adenovirus excipient was determined spectrophotometrically (OD260nm) to be 2x10^10 vp / capsule.

[0142] BCHe levels in individual plasma samples from n=8 subjects were measured using the DetectX® Butyrylcholinesterase Fluorescent Activity Kit (K016-F1), and elevated enzyme activity levels (P<0.01) were detected 3 days after administration. The results are presented in Figure 3.

[0143] [Example 3] An adenovirus (serotype 5) construct (dE1 / E3) consisting of a CMV promoter driving expression of Zika antigens (E and NS1) was prepared and designated Ad5_FP(E / NS1)_GW. High-titer virus stocks were prepared by viral culture in HEK293 cells, then combined with excipients (0.4 M sucrose, 0.2 M DMG, 0.2 M MSM) and lyophilized. The resulting cake powder was filled into porcine gelatin capsules and then enteric-coated to enhance lower GI delivery.

[0144] This product was tested in IFNαβ - / - A mouse study was conducted (6 mice were treated with vaccine and 6 mice were treated with placebo). SV129 mice were also used as immunocompetent controls for antibody production.

[0145] Mice were administered 4x10 mAbs by gavage on days 0, 14, and 28, and then challenged with Zika virus (HS-2015-BA-01 isolate, 4x10 mAbs in the tail vein on day 42). 5 PFU was administered intravenously.) Body weight, intraocular pressure, and cerebral viremia were measured.

[0146] Figure 4 shows the weight loss rate of vaccine- and placebo-administered mice 3 days after virus challenge (IFNαβ - / - Mice lost weight upon Zika virus challenge.) The vaccine provided protection (p=0.0003).

[0147] Figure 5 shows the effect of viral challenge on intraocular pressure for vaccinated and placebo-treated mice. Again, vaccination provided protection (p=0.016).

[0148] Figure 6 then shows the brain, optic nerve and testicular viremia (recorded on day 49). A protective effect was observed with a reduction in the amount of viremia relative to the placebo control.

[0149] [Example 4] An oral Zika vaccine was tested in black-eared marmosets (2 pre-immune controls, 2 placebos, and 2 vaccinated animals). The vaccine formulation was prepared as previously described and filled into enteric-coated capsules (2.1 x 10 per dose). 8 TCID 50 of vaccines and 2.5x10 8 TCID 50 The capsules were stored at ambient temperature for approximately one month after manufacture. The capsules were administered twice, on days 0 and 13.

[0150] Animals were then challenged with the Zika virus HS-2015-BA-01 clinical isolate (5x10 5 PFU, administered subcutaneously on day 27 (14 days after the last dose of vaccine). Blood was collected on days 1, 3, 6, 9, and 12, and saliva and urine were collected on days 3, 6, 9, and 12 after challenge.

[0151] Figure 8 shows the relative viral load up to 5 days post-challenge. The vaccine provided immunity equivalent to pre-exposure to Zika virus. No Zika virus was detected in the vaccinated or pre-immune groups (LOD=4). Thus, the vaccine was effective even after 1 month of storage at ambient temperature. The present invention encompasses, for example, the following embodiments: [Embodiment 1] A method of delivering a transgene to target cells in a patient, comprising oral administration of a product comprising viral particles carrying the transgene. [Embodiment 2] The method of embodiment 1, wherein the viral particles are adenovirus particles. [Embodiment 3] The method of embodiment 1 or 2, wherein the product further comprises an enteric coating. [Embodiment 4] The method of any one of embodiments 1 to 3, wherein the introduced gene encodes a therapeutic protein. [Embodiment 5] The method of embodiment 4, wherein the therapeutic protein is insulin or butyrylcholinesterase. [Embodiment 6] The method of any one of embodiments 1 to 4, wherein the transgene encodes one or more antigens. [Embodiment 7] The method of embodiment 6, wherein the transgene encodes one or more Zika virus antigens. [Embodiment 8] The method of embodiment 7, wherein the Zika virus antigen is derived from the envelope protein (E) and / or NS1. [Embodiment 9] The method of embodiment 6, wherein the transgene encodes one or more herpes simplex virus (HSV), optionally HSV2, antigens. [Embodiment 10] The method of embodiment 9, wherein the HSV antigen is derived from glycoprotein C (gC), glycoprotein D (gD) and / or glycoprotein E (gE). [Embodiment 11] A method according to any one of embodiments 1 to 10, wherein expression of the introduced gene is controlled by a tissue-specific promoter. [Embodiment 12] The method of embodiment 11, wherein the promoter is a glucose-dependent insulinotropic polypeptide promoter. [Embodiment 13] The method of embodiment 12, wherein the therapeutic protein is insulin and the expression of insulin is controlled by a glucose-dependent insulinotropic polypeptide promoter. [Embodiment 14] Adenovirus particles (a) virus particles, (b) optionally one or more sugars; and (c) a compound of formula (I) or a physiologically acceptable salt or ester thereof [ka] (In the formula, R1 is hydrogen or C 1-6 represents alkyl, R4 represents hydrogen, or R1 and R4 together with the atoms to which they are attached form a pyrrolidine ring; R2 is hydrogen, C 1-6 Alkyl or -(CH2) 2-5 NHC(O)(CH2) 5-15 represents CH3, R3 is C 1-6 (representing alkyl) and / or a compound of formula (II) or a physiologically acceptable salt or ester thereof [ka] (In the formula, X is -S(O)2- or -S + (R c )-, R a and R b independently C 1-6 represents alkyl, R c is a C substituted with a carboxylate anion and an amine (-NH) moiety. 1-6 14. The method of any of embodiments 1 to 13, wherein the compound is formulated in a pharmaceutical composition comprising: [Embodiment 15] The method of embodiment 14, wherein the aqueous solution comprises a compound of formula (I) or a physiologically acceptable salt thereof, or a compound of formula (II) or a physiologically acceptable salt thereof. [Embodiment 16] The compound of formula (I) is N,N-di(C 1-6 alkyl)-, N,N,N-tri(C 1-6 alkyl)- or NC 1-6 16. The method of embodiment 14 or 15, wherein the alkyl-glycine is an alkyl-glycine or a physiologically acceptable salt or ester thereof. [Embodiment 17] The method of embodiment 16, wherein the compound of formula (I) is (a) N,N-dimethylglycine, N,N,N-trimethylglycine, or N-methylglycine or a physiologically acceptable salt or ester thereof, or (b) N-methylglycine, N,N-dimethylglycine, or N,N,N-trimethylglycine or a hydrochloride salt thereof. [Embodiment 18] The method of embodiment 17, wherein the compound of formula (I) is N,N-dimethylglycine or a physiologically acceptable salt or ester thereof. [Embodiment 19] The method according to embodiment 14 or 15, the compound of formula (I) is a compound of formula (IA) or a physiologically acceptable salt or ester thereof, [ka] (Wherein R5 and R6 are independently C 1-4 represents alkyl, and R7 is C 1-4 Alkyl or -(CH2) 2-5 NHC(O)(CH2) 5-15 (represents CH3) the compound of formula (I) is a compound of formula (IB) or a physiologically acceptable salt or ester thereof, [ka] (Wherein R8 and R9 are independently C 1-4 (representing alkyl) the compound of formula (II) is a compound of formula (IIA) or a physiologically acceptable salt or ester thereof, [ka] (In the formula, R c and R d independently C 1-4 (representing alkyl) or The compound of formula (II) is a compound of formula (IIB) or a physiologically acceptable salt or ester thereof, [ka] (In the formula, R e and R f independently C 1-4 represents R g is a C substituted with a carboxylate anion and an amine moiety. 1-4Representing alkyl), methods. [Embodiment 20] The method of any one of embodiments 14, 15, or 19, wherein the compound of formula (I) or (II) is dimethyl sulfone, trimethylglycine, cocamidopropyl betaine, proline betaine, or S-methyl-L-methionine, or a physiologically acceptable salt or ester thereof. [Embodiment 21] A method according to any one of embodiments 15 to 20, wherein (a) the aqueous solution contains one or more sugars, and the concentration of the compound of formula (I) or (II) or a physiologically acceptable salt or ester thereof is 0.1 mM to 2.5 M, and the sugar concentration, or if two or more sugars are present, the total sugar concentration, is at least 0.01 M; or (b) the concentration of the compound of formula (I) or (II) or a physiologically acceptable salt or ester thereof is 0.1 mM to 3 M. [Embodiment 22] The method of any one of embodiments 15 to 20, wherein the concentration of the compound of formula (I) or (II) or a physiologically acceptable salt or ester thereof is (a) 0.001 M to 2.5 M, 0.01 M to 2.5 M, or 0.1 M to 2 M, or (b) 7 mM to 1.5 M or 0.07 M to 0.7 M, or (c) 7 mM to 1.5 M or 0.07 M to 1 M, or (d) 0.05 M to 2 M, 0.02 M to 2 M, or 0.07 M to 1 M. [Embodiment 23] The method according to embodiment 14, wherein the aqueous solution comprises a compound of formula (I) or a physiologically acceptable salt thereof and a compound of formula (II) or a physiologically acceptable salt thereof. [Embodiment 24] An aqueous solution comprising a compound of formula (I) or a physiologically acceptable salt thereof as defined in any one of embodiments 13 to 15 and a sulfone compound of formula (IIC): [ka] (In the formula, R a and R b independently C 1-6 24. The method of embodiment 23, comprising a compound of formula (II), wherein R represents alkyl. [Embodiment 25] The method according to embodiment 24, wherein the concentration of the compound of formula (I) or a physiologically acceptable salt thereof in the aqueous solution is 0.1 to 1.5 M. [Embodiment 26] The method of embodiment 24 or 25, wherein the sulfone compound of formula (IIC) is methylsulfonylmethane. [Embodiment 27] The method according to any one of embodiments 24 to 26, wherein the concentration of the sulfone compound of formula (IIC) in the aqueous solution is 0.1 to 1.5 M. [Embodiment 28] A method according to any one of embodiments 14 to 27, wherein (a) the sugar concentration or total sugar concentration is 0.1M to 3M or 0.2M to 2M, (b) the solution contains one or more sugars at a concentration of at least 0.1M or, if two or more sugars are present, at a total sugar concentration, or (c) the sugar concentration of the aqueous solution is 0.05 to 1M. [Embodiment 29] The method of any of embodiments 14 to 28, wherein the composition comprises a non-reducing sugar or sugar alcohol. [Embodiment 30] A method according to any one of embodiments 14 to 29, wherein two or more sugars are used, one of the sugars being sucrose. [Embodiment 31] The method described in embodiment 30, wherein the ratio of the concentration of sucrose to the other sugars is 1:1 to 20:1. [Embodiment 32] The method of embodiment 30 or 31, wherein the other sugar is raffinose. [Embodiment 33] The method of any one of embodiments 14 to 31, wherein the composition comprises mannitol. [Embodiment 34] The method of any one of embodiments 14 to 29, wherein one sugar is present which is mannitol, or two sugars are present which are sucrose and raffinose. [Embodiment 35] The method of embodiment 29, wherein the aqueous solution contains sucrose or mannitol. [Embodiment 36] The method described in embodiment 14, wherein the composition comprises 0.2 M N,N-dimethylglycine, 0.2 M dimethyl sulfone, and 0.4 M sucrose. [Embodiment 37] The method of any one of embodiments 14 to 36, wherein the composition is a dry powder. [Embodiment 38] The method of any of embodiments 14 to 37, wherein the composition is administered to the patient in the form of a tablet or capsule. [Embodiment 39] The method of embodiment 37, wherein the tablet or capsule is enterically coated. [Embodiment 40] A tablet or capsule for oral administration to a patient, comprising a pharmaceutical composition comprising viral particles carrying a transgene. [Embodiment 41] A tablet or capsule according to embodiment 40, wherein the virus is an adenovirus. [Embodiment 42] A tablet or capsule according to embodiment 40 or 41, wherein the composition comprises a compound of formula (I) as defined in any of embodiments 14 or 16 to 25 or a physiologically acceptable salt or ester thereof, and / or a compound of formula (II) as defined in any of embodiments 14, 19 to 24, 26 or 27 or a physiologically acceptable salt or ester thereof, and optionally one or more sugars, preferably sucrose. [Embodiment 43] A tablet or capsule according to embodiment 42, wherein the sugar is as defined in any of embodiments 28 to 35. [Embodiment 44] A tablet or capsule described in embodiment 40 or 41, wherein the composition comprises 0.2 M N,N-dimethylglycine, 0.2 M dimethyl sulfone, and 0.4 M sucrose. [Embodiment 45] A tablet or capsule described in any of embodiments 40 to 44, wherein the transgene encodes a therapeutic protein. [Embodiment 46] The tablet or capsule of embodiment 45, wherein the therapeutic protein is insulin or butyrylcholinesterase. [Embodiment 47] A tablet or capsule described in any of embodiments 40 to 45, wherein the transgene encodes one or more antigens. [Embodiment 48] The tablet or capsule described in embodiment 47, wherein the transgene encodes one or more Zika virus antigens. [Embodiment 49] The tablet or capsule described in embodiment 48, wherein the transgene encodes one or more Zika virus antigens derived from the envelope protein (E) and / or NS1. [Embodiment 50] The tablet or capsule described in embodiment 47, wherein the transgene encodes one or more HSV, optionally HSV2, antigens. [Embodiment 51] The tablet or capsule described in embodiment 50, wherein the transgene encodes one or more HSV antigens derived from glycoprotein C (gC), glycoprotein D (gD) and / or glycoprotein E (gE). [Embodiment 52] A tablet or capsule described in any of embodiments 40 to 51, wherein expression of the introduced gene is controlled by a tissue-specific promoter. [Embodiment 53] A tablet or capsule according to embodiment 52, wherein the promoter is a glucose-dependent insulinotropic polypeptide promoter. [Embodiment 54] The tablet or capsule described in embodiment 53, wherein the therapeutic protein is insulin and the expression of insulin is controlled by the glucose-dependent insulinotropic polypeptide promoter. [Embodiment 55] A tablet or capsule according to any one of embodiments 40 to 54, wherein the composition is dried before packaging into the tablet or capsule. [Embodiment 56] A tablet or capsule described in any one of embodiments 40 to 55, which is coated with an enteric coating. [Embodiment 57] A method for preparing viral particles carrying a transgene for oral administration to a patient, comprising: (a) culturing and purifying viral particles carrying the transgene; (b) incorporating the viral particles into a pharmaceutical composition; (c) optionally drying the composition; and (d) packaging the composition into a tablet or capsule for oral administration to a patient. [Embodiment 58] The method of embodiment 57, wherein the viral particles are adenovirus particles. [Embodiment 59] The method described in embodiment 57 or 58, wherein the viral particles are incorporated into a composition described in any of embodiments 14 to 36. [Embodiment 60] The method described in embodiment 57, 58 or 59, wherein the introduced gene encodes a therapeutic protein. [Embodiment 61] The method of embodiment 60, wherein the therapeutic protein is insulin or butyrylcholinesterase. [Embodiment 62] A method described in any of embodiments 57 to 60, wherein the introduced gene encodes one or more antigens. [Embodiment 63] The method of embodiment 62, wherein the transgene encodes one or more Zika virus antigens. [Embodiment 64] The method described in embodiment 63, wherein the introduced gene encodes one or more Zika virus antigens derived from the envelope protein (E) and / or NS1. [Embodiment 65] The method of embodiment 62, wherein the transgene encodes one or more HSV, optionally HSV2, antigens. [Embodiment 66] The method described in embodiment 65, wherein the introduced gene encodes one or more HSV antigens derived from glycoprotein C (gC), glycoprotein D (gD) and / or glycoprotein E (gE). [Embodiment 67] A method according to any one of embodiments 57 to 66, wherein expression of the introduced gene is controlled by a tissue-specific promoter. [Embodiment 68] The method described in embodiment 67, wherein the promoter is a glucose-dependent insulinotropic polypeptide promoter. [Embodiment 69] The method described in embodiment 68, wherein the therapeutic protein is insulin and the expression of insulin is controlled by a glucose-dependent insulinotropic polypeptide promoter. [Embodiment 70] The method of any of embodiments 57 to 69, wherein the tablet or capsule is coated with an enteric coating. [Embodiment 71] A product comprising viral particles for use in a method for delivering a transgene to target cells in a patient, the method comprising oral administration of viral particles carrying the transgene. [Embodiment 72] The product for use described in embodiment 71, wherein the viral particles are adenoviral particles.

Claims

1. 1. A tablet or capsule comprising adenoviral particles carrying a transgene encoding one or more antigens for use in a method of transgene delivery to target cells in a patient comprising oral administration of said tablet or capsule comprising said adenoviral particles, Adenovirus particles are (a) sucrose at concentrations between 0.05 and 1 M; (b) N,N-dimethylglycine or a physiologically acceptable salt or ester thereof at a concentration of 0.07 M to 1 M, and (c) Dimethyl sulfone or a physiologically acceptable salt or ester thereof at a concentration of 0.07M to 1M and The composition is dried before being packaged into tablets or capsules, and the tablets or capsules are enteric coated. The tablet or capsule.

2. (i) the transgene encodes one or more Zika virus antigens, such as Zika virus antigens derived from the envelope protein (E) and / or NS1; or (ii) the transgene encodes one or more herpes simplex virus (HSV) antigens, e.g., HSV2 antigens, optionally wherein the HSV antigens are derived from glycoprotein C (gC), glycoprotein D (gD), and / or glycoprotein E (gE); 2. The tablet or capsule of claim 1.

3. 3. The tablet or capsule of claim 1, wherein expression of the transgene is controlled by a tissue-specific promoter.

4. A tablet or capsule described in any one of claims 1 to 3, wherein the physiologically acceptable salt of N,N-dimethylglycine is the hydrochloride salt.

5. (a) Using two or more sugars, optionally with a ratio of sucrose to other sugar concentrations of 1:1 to 20:1, and / or the other sugar is raffinose; or (b) the composition comprises 0.2 M N,N-dimethylglycine, 0.2 M dimethyl sulfone, and 0.4 M sucrose; 5. A tablet or capsule according to any one of claims 1 to 4.

6. 1. A tablet or capsule for oral administration to a patient comprising a pharmaceutical composition comprising adenoviral particles carrying a transgene encoding one or more antigens, Adenovirus particles (a) sucrose at concentrations between 0.05 and 1 M; and (b) N,N-dimethylglycine or a physiologically acceptable salt or ester thereof at a concentration of 0.07 M to 1 M, and (c) Dimethyl sulfone or a physiologically acceptable salt or ester thereof at a concentration of 0.07M to 1M and The composition is dried before being packaged into tablets or capsules, and the tablets or capsules are enteric coated. Tablets or capsules.

7. (a) The physiologically acceptable salt of N,N-dimethylglycine is hydrochloride. (b) using two or more sugars, optionally with a concentration ratio of sucrose to the other sugar of 1:1 to 20:1, and / or the other sugar is raffinose; or (c) the composition comprises 0.2 M N,N-dimethylglycine, 0.2 M dimethyl sulfone, and 0.4 M sucrose; 7. The tablet or capsule of claim 6.

8. (a)(i) the transgene encodes one or more Zika virus antigens, e.g., Zika virus antigens derived from the envelope protein (E) and / or NS1; or (ii) the transgene encodes one or more herpes simplex virus (HSV) antigens, e.g., HSV2 antigens, optionally HSV antigens derived from glycoprotein C (gC), glycoprotein D (gD), and / or glycoprotein E (gE); or (b) the expression of the transgene is controlled by a tissue-specific promoter; 8. The tablet or capsule according to claim 6 or 7.

9. 1. A method for preparing adenoviral particles carrying a transgene encoding one or more antigens for oral administration to a patient, comprising: (a) culturing and purifying adenoviral particles carrying the transgene; and (b) Virus particles (i) sucrose at a concentration of 0.05 to 1 M; (ii) N,N-dimethylglycine or a physiologically acceptable salt or ester thereof at a concentration of 0.07 M to 1 M, and (iii) dimethyl sulfone or a physiologically acceptable salt or ester thereof at a concentration of 0.07M to 1M and formulating the compound into a pharmaceutical composition comprising the compound. (c) drying the composition; and (d) packaging the composition into an enteric-coated tablet or capsule for oral administration to a patient; A method comprising:

10. (a) The physiologically acceptable salt of N,N-dimethylglycine is hydrochloride. (b) using two or more sugars, optionally with a concentration ratio of sucrose to the other sugar of 1:1 to 20:1, and / or the other sugar is raffinose; or (c) the composition comprises 0.2 M N,N-dimethylglycine, 0.2 M dimethyl sulfone, and 0.4 M sucrose; The method of claim 9.

11. (a)(i) the transgene encodes one or more Zika virus antigens, e.g., Zika virus antigens derived from the envelope protein (E) and / or NS1; or (ii) the transgene encodes one or more herpes simplex virus (HSV) antigens, e.g., HSV2 antigens, optionally HSV antigens derived from glycoprotein C (gC), glycoprotein D (gD), and / or glycoprotein E (gE); or (b) the expression of the transgene is controlled by a tissue-specific promoter; The method according to claim 9 or 10.

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