WO2020227465A2 - Methods for preparing stable dna compositions - Google Patents

Abstract

This disclosure relates to the preparation and use of stable plasmid DNA compositions useful for delivering therapeutic or immunogenic proteins to a patient. Disclosed herein are compositions of plasmid DNA, formulated through the addition and dissolution of solid calcium salt, that demonstrates improved stability relative to compositions of plasmid DNA formulated through the addition of aqueous calcium salt to solution. Also disclosed herein are methods of preparing, methods of administering to treat a patient in need thereof, and kits for treatment comprising the stable DNA compositions disclosed.

Description

METHODS FOR PREPARING STABLE DNA COMPOSITIONS

RELATED APPLICATION

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S provisional application, U.S.S.N. 62/845,668, filed May 9, 2019, which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[0002] This invention relates to the preparation and use of stable plasmid DNA

compositions useful for delivering therapeutic or immunogenic proteins to a patient.

BACKGROUND OF THE INVENTION

[0003] The use of plasmid DNA vectors to deliver therapeutic or immunogenic proteins to patients is well known. For example, DNA vectors have been used to deliver viral, bacterial and tumor antigens to induce immune responses for treatment and/or prevention of a number of diseases. Such vectors have also been used to deliver autoantigens associated with a variety of autoimmune diseases. In the case of treating autoimmune diseases, an antigen- specific plasmid vaccine approach has the advantage of decreasing the autoimmune response while leaving intact other important, desirable, physiologic roles of the immune system, such as immune surveillance against tumors, and immune responses against infectious agents. Use of vectors encoding autoantigens for treatment of multiple sclerosis, rheumatoid arthritis, Type 1 diabetes, neuromyelitis optica, and other autoimmune diseases is described, for example, in U.S. Patent Nos. 7,544,669, 7,811,813, and 8,748,404, as well as US Publication No. 2016/0317629.

[0004] In the case of Type 1 diabetes, a tolerizing plasmid DNA vector encoding human proinsulin (TOL-3021) has been shown to preserve beta islet cell function in Type 1 diabetes patients through reduction of insulin-specific CD8 T cells ( see Roep el al, Sci Transl Med , 2013, 5:191).

[0005] Although formulations of plasmid DNA vectors have proven useful in a variety of contexts, such formulations have been known to form particulates or precipitates during manufacture and storage. BRIEF SUMMARY OF THE INVENTION

[0006] The present disclosure provides methods of preparing stable DNA compositions comprising a plasmid DNA vector, calcium, and phosphate. The disclosed methods comprise providing an initial solution comprising a plasmid DNA vector and phosphate, adding an amount of solid calcium salt to the initial solution; and dissolving the amount of solid calcium salt in the initial composition thereby preparing the stable DNA composition, wherein the dissolved calcium concentration in the stable DNA composition is between 0.01- 3 mM. In certain embodiments, the calcium salt is calcium chloride dihydrate, and the initial solution comprises a phosphate buffer, such as phosphate buffered saline.

[0007] Preparation of existing formulations of plasmid DNA vehicles in which small amounts of a solid component, e.g., calcium chloride dihydrate, normally comprise first dissolving the solid component in liquid and then adding it to the formulation. This step has been performed because of a longstanding understanding that adding solid directly to the formulation typically results in non-uniformity of addition. Formulations prepared in this manner, however, have been known to readily form particulates or precipitates during storage. In particular, formulations of plasmid DNA vectors generated through the stepwise addition and dissolution of aqueous calcium chloride were known to induce precipitation of large calcium phosphate crystals within two months of storage, within one month of storage, within days or weeks of storage, and sometimes immediately.

[0008] Formation of large calcium phosphate precipitates disrupts formulation stability. It also leads to failure of compliance with United States Pharmacopoeia specifications for maximum particle size attributes of an injectable drug product. Specifically, a mandatory standard for an injectable drug solution is that it be free of particles over a certain size. Small volume injectables (volume <100 ml) comply with the standard if no more than 6000 particles per container are equal to or greater than 10 pm and no more than 600 particles per container are equal to greater than 25 pm. See Int. J. ofPharm. Compounding , 10(3): 202- 204 (2006), citing USP <788>.

[0009] In addition, calcium phosphate precipitates in existing formulations sometimes re dissolve and then re-precipitate as hydroxyapatite (Caio(P04)6(OH)2), an inert material.

Often, the precipitation of calcium phosphate crystals may be irreversible. This precipitation may have harmful effects on transfection efficiencies as well.

[0010] Further, existing standard protocols and kits for use of calcium salts for in vitro transfection require the use of very high concentrations of calcium salts, e.g., as high as 2M. [0011] Thus, there is a need in the art for improved formulations of plasmid DNA vectors that exhibit little to no precipitation of large calcium phosphate crystals during manufacture and storage. There is also a need in the art for transfection methods and kits that require lower concentrations of calcium salts, while concomitantly retaining transfection efficiency.

[0012] The compositions, methods, and kits of the present disclosure satisfy these and other needs. Disclosed herein are stable plasmid DNA compositions, formulated through the dissolution of solid calcium salt, which exhibit improved short- and long-term stability (or “shelf life”) relative to compositions of plasmid DNA formulated through the addition of aqueous calcium salt to solution. Also disclosed herein are compositions, methods, and kits that involve the use of about 0.9 mM concentrations of calcium salt, which is closer to physiological calcium concentrations.

[0013] The calcium in the disclosed stable DNA compositions may remain dissolved after several months of storage at about -20°C or less followed by several days or weeks of storage at about 2 °C to 8 °C. For example, the calcium in the disclosed stable DNA compositions may remain dissolved after five to six months of storage at about -20 °C or less followed by at least one to six weeks, or at least twenty-five weeks, of storage at about 2 °C to 8 °C. In addition, the calcium in the disclosed stable DNA compositions may remain dissolved following a single freeze-thaw cycle. The calcium in the disclosed stable DNA compositions may remain dissolved following at least two freeze-thaw cycles.

[0014] The plasmid DNA vector of the disclosed compositions may comprise an expression cassette that comprises a transgene of interest. The transgene of interest may encode a therapeutic peptide. In particular embodiments, the transgene of interest encodes an autoantigen associated with an autoimmune disease. In some embodiments, the autoantigen is proinsulin.

[0015] The disclosure further provides stable DNA compositions comprising a plasmid DNA vector, calcium, and phosphate, prepared by the disclosed methods. The stable DNA compositions may be administered to a patient in need thereof. The patient may be suffering from an autoimmune disease, such as Type 1 diabetes. In a typical embodiment, the stable DNA composition is administered by intramuscular injection.

DEFINITIONS

[0016] The term“plasmid DNA vector” refers to a DNA molecule capable of transferring nucleic acid sequences to target cells. For the purposes of the present disclosure,“vector construct,”“expression vector,” and“gene transfer vector,” generally refer to any nucleic acid construct capable of directing the expression of a gene of interest and which is useful in transferring the gene of interest into cells in a targeted tissue (e.g., skeletal muscle cells, central nervous system cells, optic nerve cells, hypodermal cells, or hypodermal adipose cells). Thus, the term includes cloning and expression vehicles, as well as integrating vectors, however, in some instances a vector may be configured to prevent, eliminate or inhibit the integration of the vector into a host cell genome. The term also includes tolerizing or tolerogenic plasmids. The term encompasses the use of one, two, three or more

autoantigens to be expressed, at varying concentrations, within a single vehicle (e.g., a tolerogenic plasmid that encodes two autoantigens).

[0017] The term“plasmid” refers to any genetic element that is capable of replication when present in a host cell by comprising proper control and regulatory elements. The term encompasses all forms of replicable genetic elements, including varying forms of

supercoiling, multimers, and open chain or“nicked” plasmids. Starting plasmids are commercially available, publicly available on an unrestricted basis, may be constructed from available plasmids in accord with published procedures, may be isolated from organisms harboring the plasmid (e.g., naturally occurring organisms or laboratory stocks (e.g., bacterial stocks, etc.)), or synthesized, in whole or in part, on a standard or custom basis. In addition, where equivalent plasmids to those described are known in the art, such plasmids will be readily apparent to the ordinarily skilled artisan, and the nucleic acid sequences of such plasmids may be readily available.

[0018] The term“expression cassette” refers to any recombinant expression system for the purpose of expressing a nucleic acid sequence of interest in vitro or in vivo, constitutively or inducibly, in any cell. The term includes linear and circular expression systems. The term includes all vectors. Specifically, the term includes circular plasmids, Cas protein/guide RNA mediated expression vectors (as used in CRISPR techniques, e.g., Cas9-gRNA vectors), and viral particles (e.g., recombinant adeno-associated viral particles (rAAV)). The cassettes can remain episomal or integrate into the host cell genome.

[0019] The term“calcium chloride,” as used herein, refers to all hydrate and solvate forms of the calcium chloride salt, including monohydrate and dihydrate forms.

[0020] By“freeze-thaw” cycle, it is meant to refer to a freezing and thawing of the composition, in other words a change of state between a frozen (solid) state and a fluid (liquid) state. The compositions of the invention may be subjected to at least one freeze-thaw cycle. The compositions may also be subjected to a series of alternating freezing and thawing events, such as at least two freeze-thaw cycles, at least three freeze-thaw cycles, at least four freeze-thaw cycles, at least five, at least six, at least seven, at least eight, at least nine, or at least ten freeze-thaw cycles. The time interval between freezing and thawing, or frozen and fluid states, can be any period of time, for example, hours, days, weeks or months. For example, once a composition has been frozen or is in a frozen state, it can be continually stored in the frozen state at sub-zero temperatures, for example, at -20°C or less (such as between about -20 °C and -80 °C), until it needs to be thawed for use again. For example, the composition could exist in a frozen state for up to a year, for more than a year, and even up to 7-10 years. Freezing of a composition can be performed rapidly, for example, in liquid nitrogen, or gradually, for example, at a freezing temperature, e.g., between about -20 °C and -80 °C. Thawing of a frozen composition can be performed at any temperature above 0 °C, either rapidly, such as at room temperature, or gradually, for example, in a refrigerator at 2-8 °C or on ice.

[0021] The terms“polypeptide” and“protein” are used interchangeably to refer to a polymer of amino acid residues linked by peptide bonds, and for the purposes of the instant disclosure, have a minimum length of at least 5 amino acids. Oligopeptides, oligomers, multimers, and the like, typically refer to longer chains of amino acids and are also composed of linearly arranged amino acids linked by peptide bonds, whether produced biologically, recombinantly, or synthetically and whether composed of naturally occurring or non- naturally occurring amino acids, are included within this definition. Both full-length proteins and fragments thereof greater than 5 amino acids are encompassed by the definition. The terms also include polypeptides that have co-translational (e.g., signal peptide cleavage) and post-translational modifications of the polypeptide, such as, for example, disulfide-bond formation, glycosylation, acetylation, phosphorylation, proteolytic cleavage (e.g., cleavage by furins or metalloproteases), and the like. Furthermore, as used herein, a“polypeptide” refers to a protein that includes modifications, such as deletions, additions, and substitutions (generally conservative in nature as would be known to a person in the art) to the native sequence, as long as the protein maintains the desired activity relevant to the purposes of the described methods. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts that produce the proteins, or errors due to PCR amplification or other recombinant DNA methods.

[0022] The terms“identical” or percent“identity,” in the context of two or more polynucleotide or polypeptide sequences (e.g., TOL-3021 and a related vector) refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, e.g., when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms that follow, or by visual inspection. Deviations in percent identity between two polypeptides or polynucleotides include chemical modifications, e.g., substitutions of different amino acid residues, as well as truncations. This term thus embraces sequences that are functionally comparable fragments of one another.

[0023] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0024] Algorithms that are suitable for determining percent sequence identity and sequence similarity include the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389- 3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov/).

[0025] The phrase“substantially identical,” in the context of two polynucleotides or polypeptides of the disclosure, refers to two or more sequences or subsequences that have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the above sequence comparison algorithms or by visual inspection. In the typical embodiment, the sequences are at least about 80% identical, usually at least about 90% identical, and often at least 95% identical. Substantial identity may be determined over a subsequence in a given polynucleotide or polypeptide or over the entire length of the molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIGURE 1 is a structural diagram of TOL-3021. TOL-3021 is a 3.3 Kb bacterial plasmid expression vector containing the coding sequences for human proinsulin (hINS) gene. Important functional and control features of TOL-3021 include the human

cytomegalovirus (CMV) immediate-early gene promoter/enhancer, the bovine growth hormone gene polyadenylation signal, the kanamycin resistance gene, and pUC origin of replication for propagation of the vector in E. coli. The backbone of TOL-3021 has been modified to decrease the number of immunostimulatory CpG sequences and substitute immunosuppressive sequences.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

[0027] This disclosure provides methods for preparing stable plasmid DNA compositions comprising calcium phosphate. The compositions disclosed herein comprise plasmid DNA in which no precipitation of large calcium phosphate crystals ( i.e ., greater than 1 pm in diameter) occurs, even after extended storage at i) about 2-8 °C, at ii) about -20 °C, or at iii) about -20 °C or less followed by extended storage at about 2-8 °C. The disclosed

compositions may further comprise plasmid DNA that does not exhibit breaking and maintains integrity of supercoiled structure, even after extended storage at i) about 2-8 °C, at ii) about -20 °C, or at iii) about -20 °C or less followed by extended storage at about 2-8 °C. The disclosed compositions may further exhibit high efficacy (or potency) of transfection into target cells, even after extended storage at i) about 2-8 °C, at ii) about -20 °C, or iii) about -20 °C or less followed by extended storage at about 2-8 °C.

[0028] In particular embodiments, compositions of plasmid DNA are provided wherein i) the plasmid maintains an 80%, a 90%, a 95%, or a greater extent of supercoiling, and ii) no precipitates of large calcium phosphate crystals form, after extended storage at about -20 °C followed by extended storage at about 2-8 °C. In particular embodiments, the compositions are provided wherein i) the plasmid maintains an 80%, a 90%, a 95%, or a greater extent of supercoiling, and ii) no large calcium phosphate crystal precipitates form, after storage for up to seven years at about -20 °C followed by storage for up to thirty- six months at about 4 °C.

In some embodiments, the disclosed compositions are subjected to at least one such freeze- thaw cycle. In other embodiments, the disclosed compositions are subjected to at least two or more such freeze-thaw cycles.

[0029] In some embodiments, the disclosed compositions are immediately stored at about 2-8 °C and are not stored at about -20 °C or less beforehand. Thus, compositions are provided wherein i) the plasmid maintains an 80%, a 90%, a 95%, or a greater extent of supercoiling, and ii) no large calcium phosphate crystal precipitates form, after storage for up to thirty- six months at about 4 °C.

[0030] In the disclosed compositions, the plasmid DNA vector forms a complex with calcium phosphate to form a single complexed particle, or nanoparticle. These nanoparticles may have a size of less than 1 pm, e.g., less than 200 nm or less than 100 nm. In certain embodiments, these nanoparticles have a size of about 85 nm. A. Plasmid DNA Vectors

[0031] The disclosed compositions are suitable for use with any plasmid DNA vector. For example, any vector useful to deliver a desired polypeptide, e.g., a therapeutic protein, a bacterial antigen, viral antigen, tumor antigen or autoantigen may be used. In some embodiments, the plasmid DNA vector comprises a transgene that encodes an autoantigen and is used to treat autoimmune disease. In some embodiments, the plasmid DNA vector comprises more than one transgene that each encodes a therapeutic protein or an autoantigen.

[0032] Techniques for construction of plasmid DNA vectors are well-known in the art, and the skilled artisan will be familiar with standard procedures for preparing plasmid DNA vectors. The vectors will typically include an expression cassette comprising a

polynucleotide sequence encoding the desired polypeptide, as well as a promoter and other control sequences suitable for use in a particular host cell. The vectors are typically purified free of bacterial endotoxin for delivery to mammals (e.g., humans) as a therapeutic agent.

[0033] Nucleotide sequences selected for use in the vectors may be derived from known sources, for example, by isolating the nucleic acid from cells containing a desired gene or nucleotide sequence using standard techniques. Similarly, the nucleotide sequences may be generated synthetically using standard modes of polynucleotide synthesis that are well known in the art. Synthetic oligonucleotides can also be prepared using commercially available automated oligonucleotide synthesizers. The nucleotide sequences can thus be designed with appropriate codons for a particular amino acid sequence. In general, one will select preferred codons for expression in the intended host. The complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence.

[0034] In the case of treating autoimmune diseases, the vectors may comprise one or more transgenes that encode autoantigens associated with a number of diseases. Examples include multiple sclerosis (MS), rheumatoid arthritis (RA), Type 1 diabetes, autoimmune uveitis (AU), primary biliary cirrhosis (PBC), myasthenia gravis (MG), neuromyelitis optica, Sjogren's syndrome, pemphigus vulgaris (PV), scleroderma, pernicious anemia, systemic lupus erythematosus (SLE) and Grave's disease. Table 1 provides a list of autoimmune diseases and associated autoantigens that may be expressed by vectors in the disclosed compositions.

[0035] The plasmid DNA vectors may also be modified in a variety of ways. For example, in the case of vectors used to deliver autoantigens, one or more immunostimulatory CpG dinucleotides of the formula 5'-purine-pyrimidine-C-G-pyrimidine-pyrimidine-3' present in the vector may be mutated by substituting the cytosine of the CpG dinucleotide with a non cytosine nucleotide, which renders the vector less immunostimulatory (see, US Patent No. 7,811,813, herein incorporated by reference).

[0036] In some embodiments, the plasmid DNA vector is TOL-3021 (SEQ ID NO:l). TOL-3021 comprises a CMV promoter, which drives the expression of human proinsulin; bovine growth hormone termination and polyA sequences; and a pUC origin of replication and a Kanamycin resistance gene (Kan^r), which accomplish vector propagation and selection, respectively (see FIG. 1). Preparation of TOL-3021 is described in detail in US Publication No. 2016/0068585, published on March 10, 2016, which is incorporated herein by reference. In certain embodiments, the plasmid DNA vector comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to the following sequence:

GCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGT

TATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCC

GCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCG

CCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTC

CATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATC

AAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCC

CGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTAC

ATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCA

ATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGA

CGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGT

AACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTC

TATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCG

AAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTAAGTATCAAG

GTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGA

AGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTT

TCTCTCCACAGGCTTAAGCTTATGGCCTTTGTGAACCAACACCTGTGCGGCTCAC

ACCTGGTGGAAGCTCTCTACCTAGTGTGCGGGGAACGAGGCTTCTTCTACACACC

CAAGACCCGCCGGGAGGCAGAGGACCTGCAGGTGGGGCAGGTGGAGCTGGGCG

GGGGCCCTGGTGCAGGCAGCCTGCAGCCCTTGGCCCTGGAGGGGTCCCTGCAGA

AGCGTGGCATTGTGGAACAATGCTGTACCAGCATCTGCTCCCTCTACCAGCTGGA

GAACTACTGCAACTAGCTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCC

TCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTC

CTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATT

GCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGG

ACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTG

GGCTCTATGGCTTCTACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCC

AGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGGC

TTTCTTGCGGCCAAGGATCTGATGGCGCAGGGGATCAAGCTCTGATCAAGAGAC AGGATGAGGATGGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCC

GGCAGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGG

CTGCTCTGATGCCGCCGTGTTCAGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTT

GTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGG

CTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCA

CTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCC

TGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCG

GCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACAT

CGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGAT

CTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAG

GCGAGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTG

CCGAATATCATGGTGGAAAATGGCAGGTTTTCTGGATTCATCGACTGTGGCCGGC

TGGGTGTGGCGGACAGGTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGA

AGAGCTTGGCGGCGAATGGGCTGACAGGTTCCTCGTGCTTTACGGTATTGCGGCT

CCCGATTCGCAGCGCATTGCCTTCTATAGGCTTCTTGACGAGTTCTTCTGAATTAT

TAACGCTTACAATTTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTT

CACACCGCATCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTT

TATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATA

AATGCTTCAATAATAGCACGTGCTAAAACTTCATTTTTAATTTAAAAGGATCTAG

GTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTT

CCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTT

TTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGG

TTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAG

CAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCAC

TTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGT

GGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAG

TTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCC

AGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGA

GAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGG

CAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGT

ATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGA

TGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTA

CGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTT (SEQ ID NO: 1) [0037] In other embodiments, the plasmid DNA vector comprises a transgene that encodes an autoantigen that is an aquaporin cDNA, e.g., as included in a DNA tolerizing vaccine, may be derived from human aquaporin-4 (AQP4) nucleotide sequence. Reference is made to US Publication No. 2016/0317629, herein incorporated by reference. In some embodiments, an aquaporin nucleotide sequence has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to the following sequence:

ATGAGTGACAGACCCACAGCAAGGCGGTGGGGTAAGTGTGGACCTTT

GTGTACCAGAGAGAACATCATGGTGGCTTTCAAAGGGGTCTGGACTC

AAGCTTTCTGGAAAGCAGTCACAGCGGAATTTCTGGCCATGCTTATT

TTTGTTCTCCTCAGCCTGGGATCCACCATCAACTGGGGTGGAACAGA

AAAGCCTTTACCGGTCGACATGGTTCTCATCTCCCTTTGCTTTGGAC

TCAGCATTGCAACCATGGTGCAGTGCTTTGGCCATATCAGCGGTGGC

CACATCAACCCTGCAGTGACTGTGGCCATGGTGTGCACCAGGAAGAT

CAGCATCGCCAAGTCTGTCTTCTACATCGCAGCCCAGTGCCTGGGGG

CCATCATTGGAGCAGGAATCCTCTATCTGGTCACACCTCCCAGTGTG

GTGGGAGGCCTGGGAGTCACCATGGTTCATGGAAATCTTACCGCTGG

TCATGGTCTCCTGGTTGAGTTGATAATCACATTTCAATTGGTGTTTA

CTATCTTTGCCAGCTGTGATTCCAAACGGACTGATGTCACTGGCTCA

ATAGCTTTAGCAATTGGATTTTCTGTTGCAATTGGACATTTATTTGC

AATCAATTATACTGGTGCCAGCATGAATCCCGCCCGATCCTTTGGAC

CT GC AGTT AT CAT GGG A A ATTGGG A A A ACC ATTGG AT AT ATT GGGTT

GGGCCCATCATAGGAGCTGTCCTCGCTGGTGGCCTTTATGAGTATGT

CTTCTGTCCAGATGTTGAATTCAAACGTCGTTTTAAAGAAGCCTTCA

GCAAAGCTGCCCAGCAAACAAAAGGAAGCTACATGGAGGTGGAGGAC

AACAGGAGTCAGGTAGAGACGGATGACCTGATTCTAAAACCTGGAGT

GGTGCATGTGATTGACGTTGACCGGGGAGAGGAGAAGAAGGGGAAAG

ACCAATCTGGAGAGGTATTGTCTTCAGTATGA (SEQ ID NO: 2)

[0038] In some embodiments, the aquaporin cDNA, e.g., as included in a DNA tolerizing vaccine, may be derived from mouse AQP4 nucleotide sequence. In such embodiments, an aquaporin nucleotide sequence has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to the sequence:

ATGAGTGACAGAGCTGCGGCAAGGCGGTGGGGTAAGTGTGGACATTC

CTGCAGTAGAGAGAGCATCATGGTGGCTTTCAAAGGAGTCTGGACTC

AGGCTTTCTGGAAGGCAGTCTCAGCAGAATTTCTGGCCACGCTTATC TTTGTTTTGCTCGGTGTGGGATCCACCATAAACTGGGGTGGCTCAGA

AAACCCCTTACCTGTGGACATGGTCCTCATCTCCCTTTGCTTTGGAC

TCAGCATTGCTACCATGGTGCAGTGCTTTGGCCACATCAGTGGTGGC

CACATCAATCCCGCTGTGACTGTAGCCATGGTGTGCACACGAAAGAT

CAGCATCGCTAAGTCCGTCTTCTACATCATTGCACAGTGCCTGGGGG

CCATCATTGGAGCCGGCATCCTCTACCTGGTCACACCTCCCAGTGTG

GTTGGAGGATTGGGAGTCACCACGGTTCATGGAAACCTCACCGCTGG

CCATGGGCTCCTGGTGGAGTTAATAATCACTTTCCAGTTGGTGTTCA

CTATTTTTGCCAGCTGTGATTCCAAACGAACTGATGTTACTGGTTCA

ATAGCTTTAGCAATTGGATTTTCCGTTGCAATTGGACATTTGTTTGC

AATCAATTATACTGGAGCCAGCATGAATCCAGCTCGATCTTTTGGAC

CCGCAGTTATCATGGGAAACTGGGCAAACCACTGGATATATTGGGTT

GGACCAATCATGGGCGCTGTGCTGGCAGGTGCCCTTTATGAGTATGT

CTTCTGTCCTGATGTGGAGCTCAAACGTCGCCTTAAGGAAGCCTTCA

GCAAAGCCGCGCAGCAGACAAAAGGGAGCTACATGGAGGTGGAGGAC

AACCGGAGCCAAGTGGAGACGGAAGACTTGATCCTGAAGCCCGGAGT

GGTGCATGTGATTGACATTGACCGTGGAGAAGAGAAGAAGGGGAAAG

ACT CTTC GGG AG AGGT ATT GTCTT CC GT ATG A (SEQ ID NO: 3)

[0039] One of skill will recognize that TOL-3021 and other vectors disclosed herein may be modified by adding, deleting or substituting nucleotides that do not change the function of the vector, e.g., for expressing proinsulin and inhibiting an autoimmune response.

Accordingly, the disclosure provides vectors that share at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleic acid sequence identity to an exemplified sequence (e.g., SEQ ID NOs: 1, 2 or 3), as measured using an algorithm known in the art, e.g., BLAST or ALIGN, set with standard parameters, as described above. Sequence identity may be determined with respect to, e.g., the full-length of the backbone, the full-length of the encoded protein (e.g., proinsulin autoantigen), or the full-length of the vector.

B. Pharmaceutical Compositions

[0040] The disclosed plasmid DNA vectors may be incorporated into a variety of compositions for therapeutic administration. The compositions may be formulated for any mode of administration, for example, oral, buccal, parenteral, intravenous, intradermal, subcutaneous, intramuscular, transdermal, intrarectal, intravaginal, and the like.

Pharmaceutically compatible binding agents, and/or adjuvant materials may be included as part of the compositions. In some embodiments, the vector is formulated for intramuscular administration. Compositions for injection may be presented in individual aliquots in unit dosage form, e.g., in ampules or in multi-dose containers, with or without an added preservative.

[0041] In some embodiments, the vector is formulated in an aqueous solution or buffer, for example, in physiologically compatible balanced salt buffers such as phosphate buffered saline (PBS), Dulbecco’s PBS, TRIS-buffered saline (TBS), Hank’s solution, Ringer’s solution, Alsever’s solution, Earle’s solution, Gey’s balanced salt solution (GBSS), Puck’s solution, Simm’s solution (SBSS), or Tyrode’s solution (TBSS). In particular embodiments, the vector is formulated in Dulbecco’s PBS. Dulbecco’s PBS typically comprises the following: NaCl (137 mM), KC1 (2.7 mM), Na₂HP0₄ (8.1 mM) and KH₂P0₄ (2 mM), at pH 7.4. In some embodiments, the pH of the buffer is adjusted to 7.1 ± 0.1. In particular embodiments, the buffer is isotonic. As used herein, the terms“phosphate buffered saline” and“PBS” embrace Dulbecco’s PBS.

[0042] The vector is also formulated with one or more calcium salts. The calcium salt may be, for example, calcium chloride, calcium phosphate, calcium acetate, calcium carbonate, calcium citrate, calcium gluconate, calcium lactate, calcium malate, calcium glubionate, calcium propionate, or calcium gluceptate, and/or any hydrates or solvates thereof. In particular embodiments, calcium chloride dihydrate is used. In particular embodiments, the one or more calcium salts are added to the formulation solution as a solid.

[0043] The compositions disclosed herein may comprise calcium at a final concentration from about 0.01 mM to about 3 mM. In particular embodiments, the concentration is about equal to physiological levels (e.g., about 0.9 mM). In other embodiments, the concentration of calcium is about 0.08 mM, about 0.1 mM, about 0.3 mM, about 0.5 mM, about 0.7 mM, about 0.8 mM, about 1.0 mM, about 1.1 mM, about 1.2 mM, about 1.3 mM, about 1.4 mM, or about 1.6 mM. In certain embodiments, the composition comprises calcium phosphate dihydrate in a concentration about 0.9 mM.

[0044] As noted above, the disclosed plasmid DNA vector compositions are stable, that is, the components of the compositions (and in particular the calcium phosphate in solution) do not form large particulates or precipitate even after extended periods of storage. The disclosed compositions are also stable in that they maintain integrity of the plasmid DNA vector. As is known in the art, plasmid integrity may be measured in terms of the extent of DNA breakage. Plasmid integrity may also be measured in terms of the relative content of the supercoiled circular (%SC) form of the plasmid. In certain embodiments, plasmid integrity is measured by evaluating and comparing the relative signals produced by the linear (L), open circular (OC) and supercoiled circular (SC) forms present in solution. Plasmid integrity (SC content) may be measured using capillary electrophoresis, anion exchange high- performance liquid chromatography, agarose gel electrophoresis, gel staining and subsequent photography, densitometric scanning, digital PCR, and various chromatographic and electrochemical techniques known in the art.

[0045] The supercoiled form of plasmid provides for enhanced transfection efficiency and improved biological activity in comparison to other plasmid forms. Thus, stability may also be measured in terms of the degree of efficacy or potency of transfection of the formulation into target cells. As such, the disclosed compositions are also stable in that they exhibit high efficacy or potency of transfection in target cells ( e.g ., skeletal muscle cells).

[0046] In some embodiments, the compositions comprise stable plasmid DNA in which minimal precipitation of calcium phosphate crystals of any size (large or small) occurs.

[0047] The degree of stability of a formulated composition can be a function of the composition’s preparation; accordingly, the high degree of stability of the disclosed compositions may result by following the steps of the disclosed methods. In particular, the disclosed methods require that solid calcium salt is added to an initial solution comprising the plasmid DNA vector. The final concentration of the calcium is typically between about 0.01 mM and about 3 mM. Usually the concentration is about 0.9 mM. In a typical embodiment, the initial solution comprises the plasmid DNA vector in PBS. The final concentration of the vector may be between 1 mg/ml and 3 mg/ml. In certain embodiments, the concentration is about 2 mg/ml. In other embodiments, the concentration is about 1.6 mg/ml, about 1.7 mg/ml, about 1.75 mg/ml, about 1.8 mg/ml, about 1.85 mg/ml, about 1.9 mg/ml, about 1.95 mg/ml, about 2.05 mg/ml, about 2.1 mg/ml, or about 2.15 mg/ml. The disclosed methods are typically carried out at room temperature (25 °C).

[0048] In some embodiments, the methods of preparing the plasmid DNA vector composition includes mixing the composition as the calcium salt is added. To minimize shearing of the plasmid DNA vector, shear rates during mixing are less than about 16500 s ¹, usually less than about 10,000 s ¹ and often less than about 5,000 s ¹. In a typical

embodiment, mixing is carried out using a stirring device (e.g., a magnetic stirrer). Shear rates can generally be correlated with RPM of a stirring device, as follows:

RPM SHEAR RATE

400 4700

500 5850 600 7000

700 8150

800 9350

900 10500

1000 11650

1100 12850

1200 14000

1300 15150

1400 16350

[0049] In the methods disclosed herein, the calcium salt may be added manually. In other embodiments, addition of calcium salt may be automatically time-triggered.

[0050] As noted above, the disclosed plasmid DNA vector compositions exhibit no precipitation of large particles of calcium phosphate ( e.g , particles larger than 10 pm) even after extended periods of storage at about -20 °C or less and/or about 2-8 °C. Precipitation may be evaluated according to standard methods known to those of skill in the art. Methods include visual inspection, dynamic light scattering, laser diffraction, dynamic image analysis, or nanoparticle tracking. In certain embodiments, particle size is detected by dynamic light scattering (where time-dependent fluctuations in scattered light intensity from particles are determined by their diffusion in solution by Brownian motion, which is dependent upon their hydrodynamic radii).

[0051] In certain embodiments, at least 95%, at least 96%, at least 97%, often at least 98%, at least 99%, and often 100% of individual aliquots of the disclosed compositions (e.g., unit doses) are stable after 8 months to 10 years of storage at about -20 °C. In certain

embodiments, at least 95%, at least 96%, at least 96%, often at least 98%, at least 99%, and often 100% of individual aliquots (e.g., unit doses) of the disclosed compositions are stable after 3 months of storage, and often after 6 months of storage at about -20°C. The disclosed compositions are considered to be stable at a particular time point if little to no precipitation is detectable by visual inspection, dynamic light scattering, laser diffraction, dynamic image analysis or nanoparticle tracking analysis. Thus, in certain embodiments, at least 95%, at least 96%, at least 97%, often at least 98%, at least 99%, and usually 100% of individual aliquots of the disclosed compositions exhibit little to no precipitation after 2 months, after 3 months, or after 6 months, of storage at about -20°C, as detected by dynamic light scattering.

[0052] In certain embodiments, at least 95%, at least 96%, at least 97%, often at least 98%, at least 99%, and often 100% of individual aliquots of the disclosed compositions are stable following a single freeze-thaw cycle. In certain embodiments, at least 95%, at least 96%, at least 97%, often at least 98%, at least 99%, and often 100% of individual aliquots of the disclosed compositions are stable following two freeze-thaw cycles. In certain embodiments, the compositions are stable following at least three freeze-thaw cycles, at least four freeze- thaw cycles, at least five freeze-thaw cycles, at least six freeze-thaw cycles, at least seven freeze-thaw cycles, at least eight freeze-thaw cycles, at least nine freeze-thaw cycles, or at least ten freeze-thaw cycles or more.

[0053] In specific embodiments, at least 95%, at least 96%, at least 97%, often at least 98%, at least 99%, and often 100% of individual aliquots of the disclosed compositions are stable after 3 months of storage, and often after 6 months of storage at about -20 °C, followed by a thaw to a temperature of about 2 °C to about 8 °C ( e.g ., about 4 or 4.5 °C), followed by storage at one to six weeks at about 2 °C to about 8 °C (e.g., about 4 or 4.5 °C). In certain embodiments, at least 95%, at least 96%, at least 97%, often at least 98%, at least 99%, and often 100% of individual aliquots of the disclosed compositions are stable after 3 months, 4 months, twenty weeks, 5 months, 6 months, twenty-five weeks, 7 months, or 8 months of storage at about -20 °C, followed by a thaw to and storage for about 3 days, 5 days, one week, 1.5 weeks, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, or longer at about 2 °C to about 8 °C (e.g., 4.0 °C or 4.5 °C). In certain embodiments, at least 95%, at least 96%, at least 97%, often at least 98%, at least 99%, and often 100% of individual aliquots of the disclosed compositions are stable after at least eight months to at least two years, at least four years, at least five years, at least six years, at least eight years, or at least ten years of storage at about - 20 °C. In certain embodiments, the individual aliquots of the disclosed compositions are frozen for a length of time between six months and ten years, and then thawed to and stored for at least one month, two months, three months, four months, six months, eight months, ten months, twelve months, fifteen months, eighteen months, twenty months, twenty-two months, twenty-four months, twenty-eight months, thirty-two months, or at least thirty-six months at about 2 °C to about 8 °C (e.g., 4.0 °C or 4.5 °C). In some embodiments, the compositions are stable after storage for longer than thirty-six months.

[0054] In certain embodiments, the solution is stored long-term at temperatures less than - 20 °C. For instance, the solution may be stored at temperatures of -30 °C, -40 °C, -50 °C, -60 °C, -70 °C, or -80 °C.

[0055] In some embodiments, the solution is not frozen before storage at about 2 °C to about 8 °C (e.g., 4.0 °C or 4.5 °C). In certain embodiments, at least 95%, at least 96%, at least 97%, often at least 98%, at least 99%, and often 100% of individual aliquots of the disclosed compositions are stable after storage for at least one month, two months, three months, four months, six months, eight months, ten months, twelve months, fifteen months, eighteen months, twenty months, twenty-two months, twenty-four months, twenty-eight months, thirty-two months, or at least thirty-six months at about 2 °C to about 8 °C, in the absence of any prior freezing. In some embodiments, the compositions are stable after storage for longer than thirty-six months at this temperature. For example, the compositions may be stable for at least forty, fifty, or sixty months.

[0056] In several embodiments, the plasmid vector remains stable throughout the manufacture, storage and freeze-thaw cycles described above. In certain embodiments, the plasmid vector remains stable such that at least 95% remains in supercoiled circular (SC) form. In certain embodiments, at least 90% remains in supercoiled circular form. In certain embodiments, at least 85% or at least 80% remains in supercoiled circular form. Regulations for pharmaceutical grade products for use in gene therapy may require at least 80% of the plasmid to be in the supercoiled circular form. See, e.g., CBER Draft Guidance of

Considerations for Plasmid DNA Vaccines for Infectious Disease Indications. Biotechnology Law Report 24, 304-311 (2005), herein incorporated by reference.

[0057] In certain embodiments, the size of the calcium phosphate-plasmid complexed particles in solution is less than 200 nm. In certain embodiments, the size of the calcium phosphate-plasmid complexed particles is about 100 nm, 90 nm, 85 nm, 80 nm, 75 nm or less. In other embodiments, the size of the calcium particle-plasmid complexed particles is about 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, or 1 pm.

[0058] The disclosed compositions may be filtered after preparation. In some

embodiments, the disclosed compositions are terminally sterilized. Terminal sterility filtration may provide minimal or no impact on plasmid integrity (%SC). In some embodiments, the disclosed compositions are filtered through a sterile cartridge (e.g., 0.22 pm cartridge) following their preparation. The plasmid-calcium phosphate particle will pass through the filter, while particles of sizes greater than 0.22 pm will not.

[0059] In some embodiments, an overlay of an inert gas is applied over the final formulation to reduce oxidation. In certain embodiment, the inert gas is nitrogen. In other embodiments, the inert gas is argon.

[0060] In certain embodiments, the final plasmid vector formulation (or composition) is prepared for transfection of the plasmid vector into a cell, or introduction into an animal in vivo. In particular embodiments, the animal is a mammal. In certain embodiments, the animal is a mouse, or a human. In certain embodiments, the cell is a mammalian skeletal muscle cell. In other embodiments, the cell is a mammalian central nervous cell or an optic nerve cell. In other embodiments, the cell is a hypodermal cell or a hypodermal adipose cell.

[0061] In certain embodiments, transfection of the vector is achieved by intramuscular injection, as described in more detail for example in US Publication No. 2016/0317629, published on November 3, 2016 and US Publication No. 2016/0068585, published on March 10, 2016, both of which are herein incorporated by reference.

C. Methods of Administration

[0062] Provided herein are methods for use or administration of the compositions comprising the plasmid DNA vectors (e.g., TOL-3021) for treating, reducing, preventing and inhibiting the symptoms of a disease (e.g., an autoimmune disease, such as Type 1 diabetes).

[0063] The parameters of different treatment and maintenance regimens, e.g., defined by a combination of dose amount, dosing frequency and dosing period, may be adjusted based on the ranges of dose, frequency and time period described herein. Therapeutic regimens will generally differ from maintenance regimens in delivering a higher level of the plasmid DNA vector (e.g., by delivering a higher dose more often or for a longer period) to the patient in order to improve and stabilize disease symptoms. Supplemental or maintenance regimens deliver a lower level of the DNA vector to the patient in order to maintain stabilizes symptoms and prevent relapse.

[0064] Therapeutically effective amounts of vector may be in the range of about 0.1 mg to about 10 mg per administration. An administration may comprise one to several injections. On a per injection basis, therapeutically effective amounts of vector may be in the range of 0.3 to 3.0 mg.

[0065] In the case of treating autoimmune disease or diabetes, therapeutically effective amounts of vector may be in the range of about 0.3 mg to about 6 mg. For example, a therapeutic amount of vector is in the range of about 1 mg to 3 mg, for example, in doses of about 1 mg, about 1.5 mg, about 2 mg, about 2.5 mg, or about 3 mg per administration. In other embodiments, a therapeutically effective amount of vector may be about 0.3 mg, about 0.4 mg or about 0.5 mg. In other embodiments, a therapeutically effective amount of vector may be about 4 mg, about 5 mg, about 6 mg, or about 7 mg. The dosing may be adjusted to higher or lower doses, as desired or necessary, over the course of treatment.

[0066] With respect to frequency of administration or dosing, the vector can administered, e.g., weekly, bi-weekly (i.e., every other week or twice monthly) or monthly to achieve a therapeutic effect. With respect to the period of dosing or administration of the plasmid DNA vector comprising one or more transgenes encoding autoantigens, the plasmid DNA vector may be administered for a period of weeks, months, years, or the life of the patient.

[0067] For administration to target tissues and cells, the vector may be formulated in the disclosed compositions at a concentration range of about 0.1 mg/mL to 10 mg/mL. In certain embodiments, the vector is formulated at a concentration of about 1.0 mg/ml to about 3 mg/ml, for example, about 2 mg/ml.

[0068] In some embodiments, the vector is delivered by intramuscular (“IM”) injection. In other embodiments, the vector is delivered by subcutaneous injection. The vector may be injected in a volume sufficient to deliver the vector without undesirable side effects, for example, a volume of about 2 ml or less is injected at a single site, for example, a volume of about 1.5 ml, about 1 ml, about 0.5 ml or less is injected at a single site. In some

embodiments the full dose of the vector is delivered at, i.e., divided between, two or more sites. In some embodiments, about 3.0 mg, about 2.5 mg, about 2.0 mg, about 1.5 mg, about 1.0 mg, about 0.75 mg, about 0.5 mg, about 0.4 mg, or about 0.3 mg of vector is delivered in each injection. In certain embodiments, about 1 mg of vector is delivered in each injection.

[0069] Also provided herein are kits for use in the disclosed methods. The disclosed kits include any combination of components and compositions for performing the disclosed methods. In some embodiments, a kit can include the following: a DNA tolerizing vaccine, a vaccine delivery device, a suitable buffer and any combination thereof.

[0070] In some embodiments, a disclosed kit includes lyophilized DNA tolerizing vaccine and a suitable diluent for resuspending the lyophilized DNA tolerizing vaccine before use where the DNA tolerizing vaccine and the diluent are present in separate containers. In some embodiments, a subject kit may include one or more pre-formulated doses of DNA tolerizing vaccine in“ready-to-use” format. In instances where a dosing regimen is desired that includes multiple administrations of one or more DNA tolerizing vaccines, a subject kit may include two or more doses of DNA tolerizing vaccine, in a pre-formulated or an unformulated configuration, and may, optionally, include instructions ( e.g ., instructions as to when each dose should be administered, instruction for preparing unformulated doses, instructions for dose delivery, etc.). In some instances, a subject kit may include one or more testing reagents or testing devices or combinations thereof for assaying a subject’s need for therapy (e.g., before or after therapy), assaying the effectiveness of therapy (e.g., during or after therapy), etc.

[0071] In addition to the above components, the disclosed kits may further include (in certain embodiments) instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, and the like. Yet another form of these instructions is a computer readable medium, e.g., diskette, compact disk (CD), flash drive, and the like, on which the information has been recorded. Yet another form of these instructions that may be present is a website address which may be used via the internet to access the information at a removed site.

[0072] By way of providing non-limiting examples, following are exemplary procedures for preparing formulations of the TOL-3021 vector according to the disclosure.

EXAMPLES

[0073] The following examples are offered to illustrate, but not to limit the claimed invention.

Example 1:

[0074] A plasmid DNA composition was prepared by executing the following steps:

[0075] Pooled bulk plasmid DNA drug (TOL-3021) was combined with Dulbecco’s PBS dilution buffer [1.44 g sodium phosphate, dihydrate, dibasic (NaiHPCkdHiOk), 8.02 g sodium chloride, 0.20 g potassium chloride, 0.2 g anhydrous potassium phosphate, monobasic (KH2PO4), and sufficient WFI quality water to bring the volume of solution to 1.0 L, pH 7.1 ± 0.1], until a concentration of TOL-3021 of 2.0 mg/ml was reached.

Concentration was measure by A260 absorbance. The solution was mixed at a speed of 100 to 150 rpm where a slight visible vortex was achieved while avoiding foaming, until the solution appeared homogenous, about 5 to 10 minutes. The solution appeared clear and colorless. Mixing occurred at room temperature.

[0076] To finalize the plasmid DNA formulation, 132 mg of solid calcium chloride dihydrate (CaCl₂*(H₂0)₂) was added slowly and manually to the 5 L bottle of solution. Calcium chloride was weighed in a weigh boat. The operator controlled the speed of addition by visual inspection. The solution was mixed at a speed of 100 to 150 rpm where a slight visible vortex was achieved while avoiding foaming, until the solution appeared homogenous, about 10 to 15 minutes. [0077] Mixing occurred in a class A environment at room temperature. The final concentration of calcium chloride in the stable formulation solution was 0.90 mM. The solution was filtered through a sterile 0.22 pm cartridge.

[0078] Little to no precipitation of large calcium phosphate crystals was detected (by dynamic light scattering) in the final formulation solution after the completion of calcium chloride addition. Most surprisingly, little to no precipitation was detected after six months of storage at about -20 °C, followed by a thaw, followed by twenty-five weeks of storage at about 4 °C.

[0079] Based on dynamic light scattering measurements, the formulation solution had a high degree of homogeneity. In view of the manner of addition of calcium chloride ( i.e adding a solid powder at a speed controlled manually), this uniformity of solution was an unexpected finding.

[0080] In addition, based on dynamic light scattering measurements, the particle size of TOL-3021 plasmid as complexed with calcium phosphate was substantially the same as that of prior TOL-3021 formulations generated through the stepwise addition of aqueous calcium chloride. That is, the size of the plasmid-calcium phosphate complexed particle was approximately 85 nm.

[0081] Based on capillary electrophoresis or AEX-HPLC measurements, the TOL-3021 plasmid in the final formulation had a relative supercoiled circular content of about 90%.

[0082] TOL-3021 plasmid DNA formulation was injected into CD-I mice in both (left and right; 50 pg each side) quadricep muscles. Transfection efficiencies may be improved relative to prior TOL-3021 formulations generated through the stepwise addition of aqueous calcium chloride.

Example 2:

[0083] Use of a machine for calcium chloride addition facilitates scale-up of the formulation of the TOL-3021 to the much larger volumes (e.g., 1000 L) required for large-scale manufacturing.

[0084] A plasmid DNA formulation solution was prepared by combining pooled bulk plasmid DNA drug (TOL-3021) with PBS dilution buffer as above, until a concentration of TOL-3021 of 1.9 mg/ml was reached.

[0085] Using an automatic time-triggered mechanism, 132 g of solid calcium chloride dihydrate (CaCl₂*(H₂0)₂) was added to the solution to make a 1000 L batch. The solution was mixed at a speed of 100 rpm about 15 minutes. The solution was filtered through a sterile 0.22 pm cartridge.

[0086] Little to no precipitation of calcium phosphate crystals was detected (as measured by dynamic light scattering) in the formulation after storage for about five to six months at about -20 °C, followed by a single thaw, followed by six weeks of storage at about 2-8 °C.

[0087] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

CLAIMS What is claimed is:

1. A method of preparing a stable DNA composition comprising a plasmid DNA vector, calcium, and phosphate, the method comprising:

providing an initial solution comprising the plasmid DNA vector and phosphate; adding an amount of solid calcium salt to the initial solution; and

dissolving the amount of solid calcium salt in the initial composition thereby preparing the stable DNA composition, wherein the dissolved calcium concentration in the stable DNA composition is between 0.01-3 mM.

2. The method of claim 1, wherein the calcium salt is calcium chloride.

3. The method of claim 1 or claim 2, wherein the calcium concentration in the stable DNA composition is about 0.9 mM.

4. The method of any one of claims 1-3, wherein the concentration of the plasmid DNA vector in the stable DNA composition is about 2 mg/ml.

5. The method of any one of claims 1-4, wherein the initial solution comprises a phosphate buffer.

6 . The method of claim 5, wherein the initial solution comprises phosphate buffered saline (PBS).

7. The method of any one of claims 1-6, wherein the plasmid DNA vector comprises one or more transgenes encoding a therapeutic protein and/or an autoantigen.

8. The method of any one of claims 1-7, wherein the plasmid DNA vector comprises one or more transgenes encoding an autoantigen.

9. The method of claim 7 or 8, wherein the autoantigen is proinsulin.

10. The method of claim 9, wherein the plasmid DNA vector comprises a nucleic acid sequence having at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:l (TOL- 3021).

11. The method of claim 7 or 8, wherein the autoantigen is acetylcholine receptor.

12. The method of claim 7 or 8, wherein the autoantigen is aquaporin.

13. The method any one of claims 1-12, wherein the stable DNA composition remains stable after at least one to six weeks of storage at about 2 °C to about 8 °C.

14. The method of any one of claims 1-13, wherein the DNA composition remains stable after at least twenty-five weeks of storage at about 2 °C to about 8 °C.

15. The method of any one of claims 1-14, wherein the DNA composition remains stable after at least about six months of storage at about -20 °C, followed by at least one to six weeks of storage at about 2 °C to about 8 °C.

16. The method of any one of claims 1-15, wherein the DNA composition remains stable after at least about six months of storage at about -20 °C, followed by at least one to six weeks of storage at about 4 °C.

17. The method any one of claims 1-16, wherein the DNA composition remains stable following at least one freeze-thaw cycle.

18. The method any one of claims 1-17, wherein the DNA composition remains stable following at least two freeze-thaw cycles.

19. The method of any one of claims 1-18, wherein the plasmid DNA vector has a supercoiled circular (SC) content of at least 90%.

20. The method of any one of claims 1-19, wherein the plasmid DNA vector has a supercoiled circular (SC) content of at least 80%.

21. The method of any one of claims 1-20, wherein the dissolving includes mixing at a shear rate of less than about 16500 s ¹.

22. The method of any one of claims 1-21 further comprising applying an overlay of nitrogen gas to the composition.

23. A stable DNA composition comprising a plasmid DNA vector, calcium, and phosphate, the stable DNA composition prepared by the method of any one of claims 1-22.

24. The stable DNA composition of claim 23, wherein the calcium is at a concentration of about 0.9 mM.

25. The stable DNA composition of claim 23 or 24, wherein the plasmid DNA vector is at a concentration of about 2 mg/ml.

26. The stable DNA composition of any one of claims 23-25, wherein the plasmid DNA vector comprises a nucleic acid sequence having at least 85%, at least 90%, or at least 95% identity with SEQ ID NO:l (TOL-3021).

27. The stable DNA composition of any one of claims 23-25, wherein the plasmid DNA vector comprises a nucleic acid sequence encoding an acetylcholine receptor autoantigen.

28. The stable DNA composition of any one of claims 23-25, wherein the plasmid DNA vector comprises a nucleic acid sequence encoding an autoantigen that is aquaporin.

29. The stable DNA composition of any one of claims 23-28 further comprising an adjuvant.

30. The stable DNA composition of any one of 23-29, wherein the plasmid DNA vector is incorporated into a Cas protein/guide RNA-mediated expression vector or a viral particle.

31. A stable DNA composition comprising:

(a) TOL-3021 plasmid; and

(b) calcium chloride dihydrate, wherein the composition has a pH of 7.1 ± 0.1, further wherein the plasmid DNA vector has a supercoiled circular (SC) content of at least 80%, and further wherein the plasmid DNA vector is complexed with calcium phosphate with a particle of a size of less than 200 nm.

32. The composition of claim 31, wherein the concentration of TOL-3021 plasmid is 2.0 mg/ml.

33. The composition of claim 31, wherein the concentration of calcium chloride dihydrate is about 0.9 mM.

34. The composition of claim 31, wherein plasmid DNA vector has a supercoiled circular (SC) content of at least 90%.

35. A method of use of the stable DNA composition of any one of claims 23-34, comprising storing the composition for at least at least six weeks at about 2 °C to about 8 °C.

36. The method of claim 35 further comprising storing the composition for at least thirty- six weeks at about 2 °C to about 8 °C.

37. A method of use of the stable DNA composition of any one of claims 23-34, comprising:

(a) storing the composition for at least about six months at about -20 °C;

(b) thawing the composition; and

(c) storing the composition for at least about six weeks at about 2 °C to about 8 °C.

38. The method of claim 37 further comprising storing the composition for at least thirty- six weeks at about 2 °C to about 8 °C.

39. A method of treating a disorder, the method comprising administering to a patient in need thereof, a therapeutically effective amount of the stable DNA composition of any one of claims 23-34.

40. The method of claim 39, wherein the disorder is Type 1 diabetes.

41. The method of claim 39 or 40, wherein the administering comprises intramuscular injection.

42. The method of any one of claims 39-41, wherein the stable DNA composition comprises a plasmid DNA vector comprising a nucleic acid sequence having at least 85%, at least 90%, or at least 95% identity with SEQ ID NO:l (TOL-3021).

43. A method of treating a disorder, the method comprising administering to a patient in need thereof, a therapeutically effective amount of the stable DNA composition of claim 27.

44. The method of claim 43, wherein the disorder is myasthenia gravis.

45. A method of treating a disorder, the method comprising administering to a patient in need thereof, a therapeutically effective amount of the stable DNA composition of claim 28.

46. The method of claim 45, wherein the disorder is neuromyelitis optica.

47. A kit for treating a patient suffering from Type 1 diabetes, comprising:

a) a lyophilized dose of the composition of claim 26; and

b) a suitable diluent for resuspending the lyophilized dose.

48. A kit for treating a patient suffering from myasthenia gravis, comprising:

a) a lyophilized dose of the composition of claim 27; and

b) a suitable diluent for resuspending the lyophilized dose.

49. A kit for treating a patient suffering from neuromyelitis optica, comprising:

a) a lyophilized dose of the composition of claim 28; and

b) a suitable diluent for resuspending the lyophilized dose.