Alphavirus replicon particles

Abstract

To provide an alphavirus replicon particle that can be used as a gene delivery system.SOLUTION: Provided is an alphavirus replicon particle (ARP), which comprises (i) alphavirus structural proteins comprising capsid and / or envelope, and (ii) an alphavirus replicon comprising a polynucleotide encoding alphavirus non-structural proteins nsp1, nsp2, nsp3 and nsp4 and at least one gene of interest wherein at least one of capsid, and E3 and E2 in the envelope comprises one or more amino acid alteration but E1 in the envelope comprises no amino acid alteration.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to alphavirus replicon particles that can be used as a gene delivery system.

Background Art

[0002] Gene therapy is designed to introduce genetic material into cells to correct abnormal genes or to produce beneficial proteins. When a mutant gene causes a defective or missing protein, gene therapy may be able to introduce a normal copy of the gene to restore the function of the protein.

[0003] Gene therapy is an emerging field in medicine and pharmacy because of its potential in treating chronic diseases such as cancer, viral infection, myocardial infarction, and genetic disorders.

[0004] Genes inserted directly into cells usually do not function. Instead, a carrier called a vector is genetically engineered to deliver the gene. Certain viruses are often used as vectors because they can deliver new genes by infecting cells. Viruses are modified so that they cannot cause disease when used by people. Some types of viruses, such as retroviruses, integrate their genetic material (including the new gene) into the chromosomes in human cells. Other viruses, such as adenoviruses, introduce their genes into the cell nucleus, but the genes are not integrated into the chromosomes.

[0005] The vector can be directly injected into a specific tissue that is taken up by individual cells or can be administered intravenously (by IV) into the body. Alternatively, a sample of the patient's cells can be removed and exposed to the vector in a laboratory setting. The cells containing the vector are then returned to the patient. If the treatment is successful, the new gene delivered by the vector makes a functional protein. (https: / / ghr.nlm.nih.gov / primer / therapy / procedures, Int J Pharm Investig. 2013 Jan-Mar; 3(1): 1-7.)

[0006] Alphaviruses include a set of mosquito-borne viruses of the Togaviridae family that are genetically, structurally, and serologically related. Alphaviruses include Eastern Equine Encephalitis Virus (EEEV), Venezuelan Equine Encephalitis Virus (VEEV), Everglades Virus, Mucambo Virus, Pixuna Virus, Western Equine Encephalitis Virus (WEEV), Sindbis Virus, Semliki Forest Virus, Middleburg Virus, Chikungunya Virus (CHIKV), O’nyong-nyong Virus, Ross River Virus, Barmah Forest Virus, Getah Virus, Sagiyama Virus, Bebaru Virus, Mayaro Virus, Una Virus, Aura Virus, Whataroa Virus, Babanki Virus, Kyzylagach Virus, Highlands J virus, Fort Morgan Virus, Ndumu Virus, and Buggy Creek Virus. The structural subunit containing the capsid, which is a single viral protein, associates with the RNA genome in an icosahedral nucleocapsid.In virions, the capsid is surrounded by a lipid envelope covered by a regular array of transmembrane protein spikes, each of which consists of a heterodimeric complex of two glycoproteins, E1 and E2.

[0007] Alphavirus replicon particles (ARPs) are produced in cells or cultures and incorporate a "replicon" that can express non-alphavirus genes within a virion shell containing alphavirus structural proteins and membrane lipids.

[0008] Alphavirus replicon particles are described in U.S. Patent No. 7,045,335, International Publication No. 2004 / 085660, and Virology 239, 389 - 401, 1997. Their production process is described in U.S. Patent No. 7,078,218, and the content of the documents cited in this paragraph is incorporated by reference.

[0009] Clinical applications of gene therapy are still limited due to the lack of suitable methods for the proper introduction of genes into cells, and thus this is an area of interest for many researchers. The development of an appropriate gene delivery system can be one of the most important factors in achieving successful gene therapy (Int. J Pharm Investig. 2013 Jan - Mar; 3(1): 1 - 7).

Prior Art Documents

Patent Documents

[0010]

Patent Document 1

Patent Document 2

Patent Document 3

Non - Patent Documents

[0011]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0012] The present invention relates to a gene delivery system comprising improved alphavirus replicon particles.

Means for Solving the Problems

[0013] In one aspect, the alphavirus replicon particle (ARP) comprises (i) an alphavirus structural protein comprising a capsid and / or an envelope, and (ii) an alphavirus replicon comprising a polynucleotide encoding alphavirus non-structural proteins nsp1, nsp2, nsp3 and nsp4 and at least one gene of interest. and at least one of E3 and E2 in the capsid and / or envelope contains one or more amino acid modifications, while E1 in the envelope does not contain amino acid modifications.

[0014] In one aspect, the present invention provides an ARP in which the viral structural protein has one or more modifications in the alphavirus capsid protein nuclear localization signal (NLS).

[0015] In one aspect, the alphavirus capsid protein is an EEEV, WEEV, VEEV, CHIKV, Ross River virus, or Barmah Forest virus capsid protein. In various embodiments of the above or any other aspect of the invention described herein, one or more modifications are in the NLS at amino acids 67-70 of the EEEV capsid protein; amino acids 67-70 of the WEEV capsid protein; amino acids 64-68 of the VEEV capsid protein; amino acids 62-69 of the CHIKV capsid protein; amino acids 71-74 of the Ross River virus capsid protein; or amino acids 64-68 of the Barmah Forest virus capsid protein.

[0016] In various embodiments of the above or any other aspect of the invention described herein, the modification is a substitution in a charged amino acid of the NLS or a basic charged amino acid of the NLS. In some embodiments, the charged amino acid or basic charged amino acid is lysine or arginine. In certain embodiments, lysine or arginine is substituted by a non-lysine or non-arginine amino acid. In specific embodiments, lysine or arginine is substituted by asparagine or alanine.

[0017] In various embodiments of the above or any other aspect of the invention described herein, the EEEV virus capsid protein NLS is modified at amino acid 67. In certain embodiments, the EEEV virus capsid protein NLS has the substitution K67N.

[0018] In various embodiments of the above or any other aspect of the invention described herein, the WEEV virus capsid protein NLS is modified at one or more of amino acids 67, 68, and 69. In certain embodiments, the WEEV capsid protein NLS includes K67N, K68N, and / or K69N.

[0019] In various embodiments of the above or any other aspect of the invention described herein, the VEEV capsid protein NLS is modified at one or more of amino acids 64, 65, and 67. In certain embodiments, the VEEV virus capsid protein NLS includes K64N, K65A or K65N, and / or K67A or K67N.

[0020] In various embodiments of the above or any other aspect of the invention described herein, the Ross River virus capsid protein NLS is modified at one or more of amino acids 71, 72, 73, and 74. In certain embodiments, the Ross River virus capsid protein NLS includes R71N, R72N, R73N, and / or R74N.

[0021] In various embodiments of the above or any other aspect of the invention described herein, the Barmah Forest virus capsid protein NLS is modified at one or more of amino acids 64, 65, 67, and 68. In certain embodiments, the Barmah Forest virus capsid protein NLS includes K64A, K65A or K65N, K67A, K67N, K68A and / or K68N.

[0022] Modifications in the capsid NLS are described in detail in U.S. Patent Publication Nos. 2014-170186 or 2017-073377. The contents of these publications are incorporated herein by reference.

[0023] In one aspect, the alphavirus E2 protein may have a non-lysine residue (e.g., asparagine) at the amino acid position corresponding to amino acid 234 in the CHIKV E2 protein and / or a modification at the amino acid position corresponding to amino acid 251 in the CHIKV E2 protein that destabilizes the E2 protein during virus budding.

[0024] In one aspect, the alphavirus E3 protein may contain modifications / mutations in its amino acid sequence at the Furin site (Arg-X-X-Arg).

[0025] The term "Arg-X-X-Arg" refers to the minimal cleavage site of Furin, and "X-X" includes any combination of two amino acids. Examples of modifications to the amino acid sequence at the Furin site include modifications to Ile-Glu / Asp-Gly-Arg, Asp-Asp-Asp-Asp-Lys, or Ser-Gly-Gly-Gly-Ser. Details regarding Furin site modifications are described in U.S. Patent Publications Nos. 2016-0040134 and 2016-0200775, which are incorporated herein by reference.

[0026] For example, the VEEV CT83 strain has a Furin site of RKRR at the end of its E3 region, and RKRR can be replaced with SGGGS.

[0027] According to the present invention, an alphavirus replicon contains nucleotides encoding alphavirus non-structural proteins nsp1, nsp2, nsp3, and nsp4, as well as at least one gene of interest. The alphavirus non-structural proteins may be derived from the same alphavirus from which the structural proteins are derived. The alphavirus non-structural proteins may be derived from an alphavirus different from the alphavirus from which the structural proteins are derived (chimeric alphavirus replicon particles).

[0028] For example, an alphavirus replicon containing an alphavirus structural protein derived from CHIKV, nucleotides encoding VEEV nsp1, nsp2, nsp3, and nsp4, and a gene of interest can be provided according to the present invention.

[0029] In one aspect, the present invention (i) CHIKV structural proteins including a capsid and / or an envelope, and (ii) a VEEV replicon comprising a polynucleotide encoding VEEV non-structural proteins nsp1, nsp2, nsp3 and nsp4 and at least one gene of interest to provide an alphavirus replicon particle (ARP).

[0030] The gene of interest can be selected from a wide variety of sequences derived from any desired source, such as viruses, prokaryotes, eukaryotes, archaea. Examples of categories of genes of interest include immunogens, including, for example, antigen proteins, cytokines, toxins, therapeutic proteins, enzymes, antisense sequences, and immune response modulators.

[0031] In another aspect, the present invention is a method for preparing an alphavirus replicon particle, comprising: i) a vector comprising a polynucleotide encoding alphavirus non-structural proteins nsp1, nsp2, nsp3 and nsp4 and at least one gene of interest, ii) a vector comprising a polynucleotide encoding an alphavirus capsid protein, and iii) a vector comprising a polynucleotide encoding alphavirus E3-E2-6K-E1 co-transfecting into a cell, wherein at least one of the capsid, E3 and E2 comprises one or more amino acid modifications, but E1 does not comprise an amino acid modification, culturing the transfected cells, and purifying the ARP from the cell culture to provide a method.

[0032] Generally, nucleotides encoding alphavirus structural proteins include nucleotides encoding E1, E2, 6k, and E3. During the expression of wild-type virus structural proteins, 6K and E3 are naturally cleaved and removed from the ARP during the assembly process. Mature wild-type ARP may include the capsid, E1, and E2 proteins. When one or more modifications to the amino acid sequence are introduced, for example, into the furin site of the E3 protein, E3 is not cleaved and may be included in the ARP. As used herein and in the claims, the term "virus structural protein" refers to not only those having 6k and / or E3, but also those not having 6K and / or E3.

[0033] Representative ARPs in which the alphavirus is CHIKV or VEEV are illustrated in FIG. 1.

[0034] In one aspect, the present invention provides (i) a CHIKV structural protein comprising a capsid and / or an envelope, and (ii) a VEEV replicon comprising a polynucleotide encoding VEEV non-structural proteins nsp1, nsp2, nsp3, and nsp4 and at least one gene of interest. The present invention provides chimeric alphavirus replicon particles comprising the same. BRIEF DESCRIPTION OF THE DRAWINGS

[0035]

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Mode for Carrying Out the Invention

[0036] Definition As used herein, "alphavirus" means an RNA-containing virus belonging to the Togaviridae family of viruses. Exemplary togaviruses include, but are not limited to, Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus, Mukambo virus, Pixuna virus, Western equine encephalitis virus (WEEV), Sindbis virus, Semliki Forest virus, Middelburg virus, Chikungunya virus (CHIKV), O'nyong'nyong virus, Ross River virus, Barmah Forest virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Wataroa virus, Babanki virus, Kyzylagach virus, Highlands J virus, Fort Morgan virus, Ndumu virus, Baggie Creek virus, and Ockelbo virus.

[0037] "Alphavirus structural protein" means a polypeptide or a fragment thereof having at least about 80% amino acid sequence identity with a naturally occurring viral capsid or envelope protein. In one embodiment, the alphavirus structural protein has at least about 85%, 90%, 95% or more amino acid sequence identity with Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus, Mukambo virus, Pixuna virus, Western equine encephalitis virus (WEEV), Sindbis virus, Semliki Forest virus, Middelburg virus, Chikungunya virus (CHIKV), O'nyong'nyong virus, Ross River virus, Barmah Forest virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Wataroa virus, Babanki virus, Kyzylagach virus, Highlands J virus, Fort Morgan virus, Ndumu virus, and Baggie Creek virus. The wild-type amino acid sequence of the alphavirus structural protein can be obtained from GenBank.

[0038] In a specific embodiment, the alphavirus is CHIKV, such as CHIKV strain 37997 or LR2006 OPY-1. In other embodiments, the alphavirus is VEEV, such as VEEV strain TC-83.

[0039] An "alphavirus replicon" means an RNA molecule that can induce its own amplification in vivo in a target cell. The replicon encodes polymerases (nspl, nsp2, nsp3, nsp4) that catalyze RNA amplification and contains cis RNA sequences required for replication that are recognized and utilized by the encoded polymerases. An alphavirus replicon typically contains elements in the following order: 5'UTR, a sequence encoding alphavirus nonstructural proteins (nsp1, nsp2, nsp3, nsp4), 3'UTR, and a polyA signal. The alphavirus replicon also contains one or more viral subgenomic promoters that induce the expression of the gene of interest. These sequences may have one or more mutations taught in the prior art.

[0040] An "alphavirus replicon particle" (ARP) means an alphavirus replicon packaged with alphavirus structural proteins. The ARP does not contain a polynucleotide encoding any of the alphavirus structural proteins.

[0041] A "drug" means any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or a fragment thereof.

[0042] As used herein, the term "adjuvant" means a compound that, when used in combination with a particular immunogen in a formulation, serves to increase, alter, or modify the resulting immune response. In certain embodiments, the adjuvant is used in combination with an ARP. Modification of the immune response includes enhancing or expanding the specificity of either or both of the antibody and cellular immune responses. Modification of the immune response may also mean a decrease or suppression of certain antigen-specific immune responses. In one embodiment, the adjuvant is a Ribi adjuvant.

[0043] "Improve" means to reduce, suppress, attenuate, mitigate, stop or stabilize the occurrence or progression of a disease or its symptoms.

[0044] "Modify" means a change in an amino acid or nucleotide at a specific position with respect to a polypeptide sequence or polynucleotide sequence. As used herein, a modification includes a substitution, deletion, or insertion of an amino acid or nucleotide at a specific position of a polypeptide or polynucleotide. In some embodiments, modification of the alphavirus capsid protein nuclear localization signal includes substitution of a charged amino acid (e.g., lysine or arginine) with an uncharged amino acid (e.g., alanine or asparagine, or any amino acid other than basic charged amino acids such as lysine or arginine).

[0045] "Modify" means a change (increase or decrease) in the expression level or activity of a gene or polypeptide that is detected by known methods of standard techniques, such as those described herein. As used herein, a modification includes a change of 10%, 25%, 50%, 75%, 100% or more in the expression level. A modification includes a change of 10, 20, 50, 70, 80, 90, 100, 200, 500, 1000-fold or more in the expression level.

[0046] "Analog" means a molecule that is not identical but has similar functional or structural characteristics. For example, a polypeptide analog retains the biological activity of the corresponding naturally occurring polypeptide but has certain biochemical modifications that enhance the function of the analog compared to the naturally occurring polypeptide. Such biochemical modifications can, for example, increase the protease resistance, membrane permeability, or half-life of the analog without modifying ligand binding. An analog may contain non-natural amino acids.

[0047] In the present disclosure, terms such as "comprises," "comprising," "contains," and "has" can have their meanings ascribed in United States patent law and can mean "includes," "including," etc. "Consisting essentially of" or "essentially consisting of" similarly have meanings ascribed in United States patent law. These terms are open-ended and allow for more than what is recited, as long as the basic or novel features of what is recited are not changed by the presence of more than what is recited, while excluding prior art embodiments.

[0048] "Detecting" refers to identifying the presence, absence, or amount of an analyte to be detected.

[0049] "Disease" means any condition or disorder that impairs or interferes with the normal function of cells, tissues, or organs.

[0050] "Effective amount" means the amount of an agent required to improve the symptoms of a disease as compared to an untreated patient. The effective amount of an active compound used to practice the present invention for the prevention or treatment of a disease will vary depending on the mode of administration, the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosing regimen. Such an amount is referred to as an "effective" amount.

[0051] "Fragment" means a portion of a polypeptide or nucleic acid molecule. This portion preferably contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the full length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0052] As used herein, "marker" means any protein or polynucleotide having an altered expression level or activity associated with a disease or disorder.

[0053] As used herein, "nuclear localization signal" or "NLS" is an amino acid sequence that targets a polypeptide to the cell nucleus when present on the surface of the polypeptide. NLS sequences are known in the art. See, for example, Goldfarb, D., and N. Michaud (1991) Trends Cell Biol. 1, 20-24; Gorlich, D., and I. W. Mattaj (1996) Science 271, 1513-1518. In one embodiment, the NLS comprises one or more short sequences of positively charged amino acids such as lysine or arginine. Consensus sequences for the NLS include K-K / R-X-K / R (Schneider, J. et al. (1988) Cell 54,117-125) and two clusters of basic amino acids separated by a spacer of about 10 amino acids, for example, KR[PAATKKAGQA]KKKK (Dingwall et al., / Cell Biol. 107 (3): 841-9). With respect to the alphavirus amino acid sequences of the present invention, the NLS is present at amino acids 67-70 (KRKK) of the EEEV capsid protein; amino acids 67-70 (KKKK) of the WEEV capsid protein; amino acids 64-68 (KKPKK) of the VEEV capsid protein; amino acids 62-69 (RRNRKNKK) of the CHIKV capsid protein; amino acids 71-74 (RKKK) of the Ross River virus capsid protein; and amino acids 64-68 (KKPKK) of the Barmah Forest virus capsid protein. For example, K64N of the VEEV TC83 capsid protein may be utilized.

[0054] As used herein, "obtaining", such as "obtaining an agent", includes synthesizing, purchasing, or otherwise acquiring the agent.

[0055] "Reduce" means a negative modification of at least 10%, 25%, 50%, 75%, or 100%.

[0056] "Reference" means a standard or control condition.

[0057] "Reference sequence" is a defined sequence used as a basis for sequence comparison. The reference sequence may be a subset or the entirety of a particular sequence; for example, a segment of a full-length cDNA or gene sequence, or a complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence is generally at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence is generally at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, even more preferably about 100 nucleotides or about 300 nucleotides or any integer around or between them.

[0058] Sequence identity is typically measured using sequence analysis software (e.g., the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or the PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach for determining the degree of identity, the BLAST program may be used, and probability scores between e<”3> and e<”100> indicate closely related sequences.

[0059] "Structural polyprotein" means a complex amino acid molecule comprising at least two separable polypeptides that contribute to a viral capsid or envelope. In one embodiment, the polypeptides are susceptible to cleavage by viral enzymes [e.g., a capsid autoproteinase and a signalase].

[0060] "Subject" means a mammal, including but not limited to human or non-human mammals such as bovine, equine, canine, ovine, or feline.

[0061] The ranges provided herein are to be understood as being inclusive of all values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0062] As used herein, the terms "treating", "being treated", "treatment", etc. refer to reducing or ameliorating a disorder and / or its associated symptoms. It is understood that treating a disorder or condition, without being limited thereto, does not require complete elimination of the disorder, condition, or their associated symptoms.

[0063] As used herein, unless specifically stated otherwise or not apparent from the context, the term "or" is understood to be inclusive.

[0064] As used herein, unless specifically stated otherwise or not apparent from the context, the terms "a", "an", and "the" are understood to be singular or plural.

[0065] As used herein, unless specifically stated otherwise or not apparent from the context, the term "about" is understood to be within the normal tolerance in the art, e.g., within 2 standard deviations of the mean value. About can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the recited value. Unless otherwise clearly indicated from the context, all numerical values provided herein are modified by the term "about".

[0066] Any composition or method provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0067] Alphavirus replicon When delivered to a eukaryotic cell, an alphavirus replicon can direct the production of multiple daughter RNAs by transcription from itself (via an antisense copy generated from itself). The alphavirus replicon can be translated directly after delivery to the cell, and this translation provides an RNA-dependent RNA polymerase, which then produces both antisense and sense transcripts from the delivered RNA. Thus, the delivered RNA directs the production of multiple daughter RNAs. These daughter RNAs can be translated themselves to provide in situ expression of the encoded protein, as can collinear subgenomic transcripts, or can be transcribed to provide additional transcripts having the same sense as the delivered RNA that is translated to provide in situ expression of the protein. The overall result of this sequence of transcription is a large amplification in the number of introduced replicon RNAs, such that the encoded protein becomes the major polypeptide product of the cell.

[0068] According to the present invention, an alphavirus replicon comprises a polynucleotide encoding non-structural proteins n1, n2, n3 and n4, and at least one gene of interest. The alphavirus replicon does not encode any of the alphavirus structural proteins.

[0069] The alphavirus non-structural protein may be a wild-type protein derived from one of the alphaviruses described above or may have one or more modifications in the wild-type amino acid sequence. Modifications of the alphavirus non-structural protein are disclosed in various prior art references, and those skilled in the art can select an appropriate alphavirus non-structural protein based on this known information.

[0070] Alphavirus replicons are well known in the art, and any of these previously disclosed replicons can be utilized (e.g., Virology. 1997 Dec 22;239(2):389-401., International Publication No. WO 2009 / 131604, International Publication No. WO 2011 / 005799, International Publication No. WO 2012 / 031043, International Publication No. WO 2014 / 1270493, and International Publication No. WO 2015 / 095167, the contents of which are incorporated herein by reference).

[0071] Alphavirus structural protein ARP has alphavirus structural proteins of capsid and envelope proteins. Preferably, the alphavirus structural protein includes a capsid protein and E2 and E1 proteins of the envelope, and may also have an E3 protein of the envelope. According to the present invention, at least one of the capsid and the envelope has at least one modification that enhances ARP expression in mammalian cells.

[0072] In one embodiment, the alphavirus structural protein is an alphavirus capsid protein having a non-lysine residue (e.g., alanine or asparagine) at the amino acid position corresponding to the lysine residue in the alphavirus capsid protein NLS, and / or at least includes a non-arginine residue (e.g., alanine or asparagine) at the amino acid position corresponding to the arginine residue in the alphavirus capsid protein NLS. In a specific embodiment, the alphavirus capsid protein is a WEEV CBA87 strain capsid protein having one or more of the modifications K67N, K68N, and K69N. In a certain specific embodiment, the alphavirus capsid protein is a VEEV TC83 strain capsid protein having one or more of the modifications K64N, K65A, K65N, K67A, and K67N. In some embodiments, the alphavirus capsid protein is an EEEV PE-6 strain capsid protein having the modification K67N. In a specific embodiment, the alphavirus capsid protein is a CHIKV strain 37997 capsid protein having one or more of the modifications R62A, R63A, R65A, K66A, K68A, and K69A; the alphavirus capsid protein is a Ross River virus capsid protein having one or more of the modifications R71N, K72N, K73N, and K74N; the alphavirus capsid protein is a Venezuelan equine encephalitis virus capsid protein having one or more of the modifications K64A, K64N, K65A, K65N, K67A, K67N, K68A and K68N. The wild-type capsid protein amino acid sequences of the alphaviruses described above are available in GenBank.

[0073] In one embodiment, the alphavirus E2 protein has a non-lysine residue (e.g., asparagine) at the amino acid position corresponding to amino acid 234 in the CHIKV E2 protein and / or a modification at the amino acid position corresponding to amino acid 251 in the CHIKV E2 protein that destabilizes the E2 protein during virus budding.

[0074] In one embodiment, the polynucleotide encoding alphavirus E3 can be modified to include a modification / mutation (Arg-X-X-Arg) to the amino acid sequence at the furin site.

[0075] The term “Arg-X-X-Arg” refers to the minimal cleavage site of furin, and “X-X” includes any combination of two amino acids. Examples of modifications to the amino acid sequence at the furin site include modifications to Ile-Glu / Asp-Gly-Arg, Asp-Asp-Asp-Asp-Lys, or Ser-Gly-Gly-Gly-Ser. Detailed descriptions are provided in U.S. Patent Publications Nos. 2016-0040134 and 2016-0200775, which are incorporated herein by reference.

[0076] For example, the VEEV CT83 strain has a furin site containing RKRR at the end of its E3 region, and the polynucleotide sequence encoding RKRR can be replaced with the polynucleotide sequence encoding SGGGS.

[0077] In some embodiments of the invention, the protein may contain mutations that result in silent substitutions, additions, or deletions, but that do not change the properties or activity of the encoded protein or the method by which the protein is made.

[0078] Method for preparing ARP ARP can be prepared by procedures known in the art. Exemplary procedures for producing ARP are disclosed in Virology 239, 389-401, 1997, the contents of which are incorporated herein by reference.

[0079] Generally, ARP can be produced by co - transfection of a suitable host cell with a vector encoding an alphavirus replicon, i.e., a polynucleotide encoding nsp1, nsp2, nsp3 and nsp4, and a vector containing the gene of interest; and at least one helper vector encoding an alphavirus structural protein. Preferably, the cells are co - transfected with a vector encoding an alphavirus replicon, a vector encoding a capsid protein and a vector encoding an envelope protein.

[0080] In particular, the present invention provides a method for preparing alphavirus replicon particles, comprising: i) a vector comprising a polynucleotide encoding alphavirus non - structural proteins nsp1, nsp2, nsp3 and nsp4 and at least one gene of interest, ii) a vector comprising a polynucleotide encoding an alphavirus capsid protein, and iii) a vector comprising a polynucleotide encoding alphavirus E3 - E2 - 6K - E1 co - transfecting the cells, wherein at least one of capsid, E3 and E2 contains one or more amino acid modifications, but E1 does not contain amino acid modifications, culturing the transfected cells, and purifying ARP from the cell culture The method is provided.

[0081] One skilled in the art in the field of molecular biology understands that any of a variety of expression systems can be used to produce ARP. The exact cell (host cell) to be co-transfected is not important for the present invention. ARP can be produced in a prokaryotic host [e.g., Escherichia coli (E. coli)], or in a eukaryotic host [e.g., Saccharomyces cerevisiae, insect cells, e.g., Sf21 cells, or mammalian cells, e.g., NIH 3T3, HeLa, COS cells]. Such cells are available from a wide range of sources (e.g., American Type Culture Collection, Rockland, Md.; see also, e.g., Ausubel et al., supra). Non-limiting examples of insect cells are Spodoptera frugiperda (Sf) cells, e.g., Sf9, Sf21, Trichoplusia ni cells, e.g., High Five cells, and Drosophila S2 cells. Examples of fungal (including yeast) host cells are Saccharomyces cerevisiae, Kluyveromyces lactis [K. lactis], Candida species including C. albicans and C. glabrata, Aspergillus nidulans, Schizosaccharomyces pombe [S. pombe], Pichia pastoris, and Yarrowia lipolytica. Examples of mammalian cells are COS cells, baby hamster kidney cells, mouse L cells, LNCaP cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, African green monkey cells, CV1 cells, HeLa cells, MDCK cells, Vero and Hep-2 cells.African clawed frog (Xenopus laevis) oocytes, or other cells of amphibian origin can also be used. Prokaryotic host cells include bacterial cells such as Escherichia coli, Bacillus subtilis, and mycobacteria.

[0082] Methods for obtaining the polynucleotide encoding the protein are known in the art. For example, genes encoding specific alphavirus proteins such as CHIKV, WEEV, EEEV, VEEV, Ross River virus, or Venezuelan equine encephalitis virus structural proteins can be isolated by RT-PCR from polyadenylated mRNA extracted from cells infected with the virus. The resulting gene product can be cloned as a polynucleotide insert into a vector.

[0083] The term "vector" refers to a means by which a nucleic acid sequence can be propagated and / or transferred between organisms, cells, or cell components. Vectors include plasmids, viruses, bacteriophages, proviruses, phagemids, transposons, artificial chromosomes, etc., which can replicate autonomously or be integrated into the chromosome of the host cell. Vectors can also be naked RNA polynucleotides, naked DNA polynucleotides, polynucleotides composed of both DNA and RNA within the same strand, poly-lysine conjugated DNA or RNA, peptide conjugated DNA or RNA, liposome conjugated DNA, etc., which do not replicate autonomously. In many, but not all, common embodiments, the vectors of the present invention are plasmids or bacmids.

[0084] Typically, the nucleic acid molecule to be expressed is "operably linked" to a promoter and / or enhancer and is subject to transcriptional regulatory control by the promoter and / or enhancer.

[0085] The method of transfection and the choice of expression medium depend on the host system selected. Methods of transfection are described, for example, in Ausubel et al. (supra); expression media can be selected from those provided, for example, in Cloning Vectors: A Laboratory Manual (P. H. Pouwels et al., 1985, Supp. 1987). The references cited in this paragraph are incorporated herein by reference.

[0086] There are various expression systems for the production of ARP of the present invention. Expression vectors useful for producing such ARP include, but are not limited to, vectors derived from chromosomes, episomes, and viruses, such as bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses such as baculovirus, papovaviruses such as SV40, vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus and retroviruses, and vectors derived from combinations thereof.

[0087] The constructs and / or vectors used herein include an alphavirus polynucleotide encoding a structural protein, including an envelope protein or capsid protein described herein. Also, the constructs and / or vectors used herein include an alphavirus polynucleotide encoding non-structural proteins nsp1, nsp2, nsp3 and nsp4, and a gene of interest.

[0088] The vector may be, for example, a phage, plasmid, virus, or retroviral vector. The construct and / or vector containing nucleotides is operably linked to a suitable promoter such as, by way of non-limiting example, the CMV promoter, phage lambda PL promoter, E. coli lac, phoA and tac promoters, SV40 early and late promoters, and the promoter of the retroviral LTR. Other suitable promoters are known to those skilled in the art depending on the host cell and / or the desired expression rate. The expression construct further contains sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcript expressed by the construct preferably contains a translation start codon at the beginning and a termination codon appropriately positioned at the end of the polypeptide to be translated.

[0089] The vector preferably contains at least one selectable marker. Such markers include dihydrofolate reductase for eukaryotic cell culture, G418 or neomycin resistance, and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Preferred among the vectors are viral vectors such as baculovirus, poxviruses (e.g., vaccinia virus, avipox virus, canarypox virus, fowlpox virus, squirrelpox virus, swinepox virus, etc.), adenoviruses (e.g., canine adenovirus), herpesviruses, and retroviruses. Other vectors that can be used in the present invention include vectors for use in bacteria, including pQE70, pQE60 and pQE-9, pBluescript vector, Phagescript vector, pNH8A, pNH16a, pNH18A, pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5. Preferred eukaryotic vectors include pFastBacl pWINEO, pSV2CAT, pOG44, pXTl and pSG, pSVK3, pBPV, pMSG, and pSVL. Other suitable vectors will be apparent to those skilled in the art.

[0090] Recombinant constructs can be prepared and used for transfection and can express viral proteins in eukaryotic and / or prokaryotic cells, including those described herein. Thus, the present invention provides one vector (or a plurality of vectors) containing nucleic acids encoding alphavirus structural proteins, including capsid, E3, E2, 6K, and El or portions thereof, and nucleic acids encoding alphavirus nsp1, nsp2, nsp3, and nsp4, and a host cell comprising one vector containing at least one gene of interest under conditions that allow for the formation of ARP.

[0091] In one embodiment, the vector is a recombinant baculovirus. In another embodiment, the recombinant baculovirus is transfected into insect cells. In a preferred embodiment, the cells are insect cells. In another embodiment, the insect cells are Sf9 cells.

[0092] One particular bacterial expression system for polypeptide production is the E. coli pET expression system (Novagen, Inc., Madison, Wis). According to this expression system, DNA encoding a polypeptide is inserted into a pET vector in a direction designed to allow expression. Since the gene encoding such a polypeptide is under the control of the T7 regulatory signal, expression of the polypeptide is achieved by inducing the expression of T7 RNA polymerase in the host cell. This is typically achieved using a host strain that expresses T7 RNA polymerase in response to IPTG induction. Once produced, the recombinant polypeptide is then isolated by standard methods known in the art, for example, according to the methods described herein.

[0093] Another bacterial expression system for polypeptide production is the pGEX expression system (Pharmacia). This system utilizes a GST gene fusion system designed for high-level expression of genes or gene fragments as fusion proteins, along with rapid purification and recovery of functional gene products. The protein of interest is fused to the carboxyl terminus of the glutathione S-transferase protein from Schistosoma japonicum and can be easily purified from bacterial lysates by affinity chromatography using glutathione sepharose 4B. The fusion protein can be recovered under mild conditions by elution with glutathione. Cleavage of the glutathione S-transferase domain from the fusion protein is facilitated by the presence of a recognition site for a site-specific protease upstream of this domain. For example, the protein expressed in the pGEX-2T plasmid can be cleaved by thrombin, and the protein expressed in pGEX-3X can be cleaved by factor Xa.

[0094] Depending on the vector and host cell selected, ARP is produced by growing host cells transfected with the vector under conditions where the recombinant protein is expressed, an alphavirus replicon is generated, and an ARP containing the alphavirus replicon packaged with particles of alphavirus structural proteins is formed. In one embodiment, the invention includes a method of producing ARP that includes co-transfecting into a suitable host cell a vector comprising polynucleotides encoding alphavirus nonstructural proteins nsp1, nsp2, nsp3, and nsp4 and at least one gene of interest, and at least one vector encoding at least one alphavirus protein each, and expressing the alphavirus protein under conditions that allow ARP formation. In another embodiment, the eukaryotic cell is selected from the group consisting of yeast, insect, amphibian, avian, or mammalian cells. Selection of appropriate growth conditions is within the skill of the art or within the purview of one of ordinary skill in the art.

[0095] Methods for growing cells that produce ARP of the present invention include, but are not limited to, batch, batch feed, continuous, and perfusion cell culture techniques. In one embodiment, cells co-transfected with a vector encoding an alphavirus replicon and a vector containing a polypeptide encoding a capsid, and a vector containing a polynucleotide encoding an envelope protein such as those derived from CHIKV or VEEV grow in a bioreactor or fermentation chamber where the cells proliferate and express a protein (e.g., a recombinant protein) for purification and isolation. Typically, cell culture is performed under aseptic, controlled temperature, and atmospheric conditions. A bioreactor is a chamber used for culturing cells that can monitor environmental conditions such as temperature, atmosphere, agitation, and / or pH. In one embodiment, the bioreactor is a stainless steel chamber. In another embodiment, the bioreactor is a pre-sterilized plastic bag (e.g., Cellbag™, Wave Biotech, Bridgewater, N.J.). In other embodiments, the pre-sterilized plastic bag is a bag of about 50 L to 1000 L.

[0096] ARP is isolated using methods that preserve its integrity, such as gradient centrifugation, e.g., cesium chloride, sucrose, and iodixanol, and standard purification techniques including, e.g., ion exchange and gel filtration chromatography.

[0097] The following are examples of how ARP of the present invention can be made, isolated, and purified. Those skilled in the art will understand that there are additional methods that can be used to make and purify ARP. Accordingly, the present invention is not limited to the methods described herein.

[0098] Generally, the production of the ARP of the present invention is achieved by seeding mammalian cells [e.g., human embryonic kidney (293T) cells] or Sf9 cells (non-infected) in a shaker flask, expanding the cells, and scaling up as the cells grow and proliferate (e.g., from a 125 ml flask to a 50 L Wave bag). The medium used to grow the cells is formulated for the appropriate cell line (preferably a serum-free medium, e.g., insect medium ExCell-420, JRH). Next, the appropriate vector (e.g., a mammalian expression vector or for SF) is transfected or infected with a cell recombinant baculovirus at the most effective multiplicity of infection (e.g., about 1 to about 3 plaque-forming units per cell). The polynucleotide or its portion is expressed in cells in which they self-assemble into ARP and secreted from the cells about 24 to 72 hours post-infection (hpi). Usually, transfection or infection is most effective when the cells are in the mid-log phase of growth (4 - 8 × 10<6> cells / ml) and at least about 90% are viable. Further, the transfected cells can be exposed to high pH conditions (pH > 7.2, e.g., pH 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, or higher) in the cell culture to increase ARP production.

[0099] The ARP of the present invention is recovered about 48 to 120 hours post-infection when the level of ARP in the cell culture medium is close to the maximum but before extensive cell lysis. The cell density and viability at the time of recovery are about 0.5 × 10 6 cells / ml to about 1.5 × 10 6It can be in cells / ml. Next, the medium is removed and clarified. To avoid ARP aggregation, NaCl can be added to the medium to a concentration of about 0.4 to about 1.0 M, preferably about 0.5 M. Removal of cells and cell debris from the cell culture medium containing ARP of the present invention can be achieved by tangential flow filtration (TFF) using a disposable pre-sterilized hollow fiber 0.5 or 1.00 μm filter cartridge or a similar device.

[0100] Furthermore, ARP can be exposed to high pH conditions (pH > 7.2, for example, pH 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, or higher) during purification to increase ARP production.

[0101] Next, the ARP in the clarified culture medium is concentrated by ultrafiltration using a disposable pre-sterilized 500,000 molecular weight cut-off hollow fiber cartridge. To remove residual medium components, the concentrated ARP can be diafiltered against 10 volumes of phosphate buffered saline (PBS) at pH 7.0 - 8.0 containing 0.5 M NaCl.

[0102] The concentrated and diafiltered ARP can be further purified in a 20% - 60% discontinuous sucrose gradient in PBS buffer at pH 7.2 with 0.5 M NaCl by centrifugation at 6,500 × g for 18 hours at about 4°C to about 10°C. Usually, ARP forms a visible band characteristic at sucrose or the interface (20% and 60% step gradients) between about 30% and about 40% that can be recovered from the gradient and stored. This product can be diluted to contain 200 mM NaCl for preparation for the next step in the purification process. This product contains ARP and can contain intact baculovirus particles.

[0103] Further purification of ARP can be achieved by anion-exchange chromatography or by 44% isopycnic sucrose cushion centrifugation. In anion-exchange chromatography, the sample from the sucrose gradient (see above) is loaded onto a column containing a medium with anions (e.g., Matrix Fractogel EMD TMAE), and eluted through a salt gradient (about 0.2 M to about 1.0 M NaCl) that can separate ARP from other contaminants (e.g., baculovirus and DNA / RNA). In the sucrose cushion method, the sample containing ARP is added to a 44% sucrose cushion and centrifuged at 30,000 g for about 18 hours. ARP forms a band at the top of the 44% sucrose, while the baculovirus precipitates at the bottom and other contaminating proteins remain in the 0% sucrose layer at the top. The ARP peak or band is collected.

[0104] If desired, intact baculovirus can be inactivated. Inactivation can be achieved by chemical methods, e.g., formalin or beta-propiolactone (BPL). Removal and / or inactivation of intact baculovirus can also be largely achieved by using selective precipitation and chromatography methods known in the art, as exemplified above. The inactivation method includes incubating the sample containing ARP in 0.2% BPL for 3 hours at about 25 °C to about 27 °C. Baculovirus can also be inactivated by incubating the sample containing ARP at 4 °C for 3 days, then at 37 °C for 1 hour with 0.05% BPL.

[0105] After the inactivation / removal step, the product containing ARP can be passed through another diafiltration step to remove all of the reagents from the inactivation step and / or all of the residual sucrose, and ARP can be placed in a desired buffer (e.g., PBS). The solution containing ARP can be sterilized by methods known in the art (e.g., sterile filtration) and stored in a refrigerator or freezer.

[0106] The above-described technology can be implemented over a variety of scales. For example, from T flasks, shaking flasks, spinner bottles to industrial-sized bioreactors. The bioreactor may include either a stainless steel tank or a pre-sterilized plastic bag (e.g., a system sold by Wave Biotech, Bridgewater, NJ). Those skilled in the art know what is most desirable for their purposes.

[0107] As described herein, upon administration to a desired host, the ARP of the present invention is taken up by cells normally infected by the alphavirus from which the structural protein is derived. The gene of interest contained in the replicon is internalized into the cell upon ARP entry. This property facilitates the use of the ARP described herein as a gene delivery vehicle, as it enables the delivery of the gene of interest to the desired cells.

[0108] While the natural alphavirus genome encodes viral structural proteins in addition to the non-structural replicase polyprotein, the alphavirus replicon does not encode alphavirus structural proteins. Thus, the alphavirus replicon can direct the production of its own genomic RNA copies within the cell, but cannot direct the production of virions containing the RNA. The inability to produce these virions means that, unlike wild-type alphaviruses, the replicon cannot perpetuate itself in an infectious form. The alphavirus structural proteins required for perpetuation in the wild-type virus are not present in the replicon, and their positions are determined by at least one gene of interest, such that the subgenomic transcript encodes the protein of interest rather than the structural alphavirus structural protein.

[0109] Thus, in certain embodiments, the ARP can be, or can contain a gene of interest that encodes, a therapeutic or diagnostic agent that needs to be delivered to a subject, such as, for example, a contrast agent, nucleic acid sequences (including siRNA and microRNA), radionuclides, hormones, peptides, antiviral agents, anti-tumor / chemotherapeutic agents, cell growth regulators, cell growth inhibitors, cytokines, antigens, adjuvants, and toxins. A replicon packaged into a particle of a viral structural protein should not adversely affect the stability of the ARP. This can be determined by producing an ARP containing a gene of interest and, if any, evaluating its effect on ARP stability.

[0110] Accordingly, the present invention provides a method for introducing a gene of interest into a cell. According to the present invention, the gene of interest is contained in an alphavirus replicon, and the alphavirus replicon is packaged with particles of alphavirus structural proteins. In related embodiments, the ARP contacts the cell. In related embodiments, the ARP is capable of entering the cell, thereby effecting delivery of the gene of interest into the cell.

[0111] As used herein, the terms "treating," "being treated," "treatment," etc. refer to reducing or ameliorating a disorder and / or its associated symptoms. It is understood that treating a disorder or condition, without being limited thereto, does not require completely eliminating the disorder, condition, or their associated symptoms.

[0112] As used herein, the terms "preventing," "being prevented," "prevention," "preventive treatment," etc. refer to reducing the likelihood of developing a disorder or condition in a subject that does not have the disorder or condition but is at risk of or prone to developing the disorder or condition.

[0113] The gene of interest may be a gene encoding an antigen. The ARP of the present invention can be prepared in an injectable form either as a liquid solution or a suspension. Solid forms suitable for injection can also be prepared as emulsions or in combination with ARP encapsulated in liposomes. The vaccine antigen is usually combined with a pharmaceutically acceptable carrier, including any carrier that does not induce the production of antibodies harmful to the subject receiving the carrier. Suitable carriers typically include large macromolecules that are slowly metabolized, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, lipid aggregates, and inactivated virus particles. Such carriers are well known to those skilled in the art. These carriers can also function as adjuvants.

[0114] The ARP described herein can be administered in combination with an adjuvant (e.g., Ribi). An adjuvant is an immunostimulatory agent that enhances the effectiveness of a vaccine. If desired, an ARP containing one or more alphavirus polypeptides or fragments or variants thereof is administered in combination with an adjuvant that enhances the effectiveness of the immune response generated against the antigen of interest. Effective adjuvants include, but are not limited to, aluminum salts such as aluminum hydroxide and aluminum phosphate, muramyl peptides, bacterial cell wall components, saponin adjuvants, and other substances that act as immunostimulatory agents to enhance the effectiveness of the composition.

[0115] The immunogenic composition, i.e., the ARP described herein, the pharmaceutically acceptable carrier, and the adjuvant typically also contain diluents such as water, saline, glycerol, ethanol, etc. Auxiliary substances such as wetting agents or emulsifying agents, pH buffering substances may also be present. Proteins can be formulated into the vaccine in either neutral or salt form. The immunogenic composition is typically administered parenterally by injection, and such injection can be either subcutaneous or intramuscular. Further formulations are suitable for other modes of administration such as suppositories or oral administration. Oral compositions can be administered as solutions, suspensions, tablets, pills, capsules, or sustained-release formulations.

[0116] The immunogenic composition is administered in a manner compatible with the dosage regimen. The immunogenic composition comprises an immunologically effective amount of the ARP described herein and the other aforementioned components. An immunologically effective amount means a composition administered in a single dose or multiple dose schedule that is effective for the treatment or prevention of infection. The dosage administered will vary depending on the subject being treated, the health and physical condition of the subject, the ability of the immune system of the subject to produce antibodies, the degree of protection desired, and other relevant factors. The exact amount of the active ingredient required depends on the judgment of the physician, but typically ranges from 5 μg to 250 μg of antigen per dose.

[0117] Pharmaceutical Compositions and Administration The present invention features a pharmaceutical composition comprising the ARP described herein. Pharmaceutical compositions useful herein include any pharmaceutical that does not itself induce the production of an immune response harmful to the vertebrate receiving the composition, and contain a pharmaceutically acceptable carrier, including any suitable diluent or excipient that can be administered without undue toxicity, and the ARP of the present invention. As used herein, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency for use in mammals, particularly humans, or listed in the U.S. Pharmacopeia, European Pharmacopeia or other generally recognized pharmacopeia. These compositions can be useful as vaccines and / or antigenic compositions for inducing a protective immune response in vertebrates.

[0118] Pharmaceutically acceptable carriers include, but are not limited to, physiological saline, buffered physiological saline, dextrose, water, glycerol, sterile isotonic aqueous buffers, and combinations thereof. A detailed description of pharmaceutically acceptable carriers, diluents, and other excipients is presented in Remington's Pharmaceutical Sciences (Mack Pub. Co. N.J. current edition). The formulation shall be adapted to the mode of administration. In a preferred embodiment, the formulation is suitable for administration to humans and is preferably sterile, non-particulate, and / or non-pyrogenic.

[0119] If desired, the composition may also contain small amounts of wetting or emulsifying agents, or pH buffering agents. The composition may be in solid form, such as lyophilized powder suitable for reconstitution, liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. Oral formulations may contain standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc.

[0120] In certain embodiments, the ARP composition is provided in liquid form, for example, in a sealed container indicating the amount and concentration of the ARP composition.

[0121] Preferably, the liquid form of the ARP composition is provided in a sealed container at at least about 50 μg / ml, more preferably at least about 100 μg / ml, at least about 200 μg / ml, at least 500 μg / ml, or at least 1 mg / ml.

[0122] Alternatively, the vaccine formulation is administered intranasally by any of intravenous drip, large particle aerosol (greater than about 10 microns) or spraying into the upper respiratory tract or small particle aerosol (less than 10 microns) or spraying into the lower respiratory tract. Any of the above delivery routes results in an immune response, but intranasal administration provides the additional benefit of inducing mucosal immunity at the site of entry of many viruses, including alphaviruses such as CHIKV or VEEV.

[0123] Accordingly, the present invention also includes a method of formulating a vaccine or antigenic composition that induces immunity against infection in a mammal or at least one symptom thereof, the method comprising the step of adding to the formulation an effective amount of ARP, such as an alphavirus (e.g., CHIKV or VEEV).

[0124] In certain cases, stimulation of immunity with a single dose is preferred, but additional dosages may also be administered by the same or different routes to achieve the desired effect. For example, in neonates and infants, multiple administrations may be required to elicit sufficient levels of immunity. Administration may be continued at intervals throughout childhood as necessary to maintain sufficient levels or protection.

[0125] Similarly, adults who are particularly susceptible to recurrent or severe infections, such as healthcare workers, daycare workers, families with young children, the elderly, and individuals with impaired cardiorespiratory function or immune system, may require multiple immunizations to establish and / or maintain a protective immune response. The level of induced immunity can be monitored, for example, by measuring the amount of neutralizing secreted antibodies and serum antibodies, and the dosage can be adjusted or vaccination repeated as necessary to induce and maintain the desired level of protection.

[0126] The dosage of the pharmaceutical preparation can be readily determined by one of ordinary skill in the art, for example, first, by identifying a dosage effective to induce a prophylactic or therapeutic immune response, for example, by measuring the serum titer of virus-specific immunoglobulins, or by measuring the inhibition ratio of antibodies in a serum sample, or a urine sample, or mucosal secretions. The dosage can be determined from animal experiments. A non-limiting list of animals used to study the effectiveness of vaccines includes guinea pigs, hamsters, ferrets, chinchillas, mice and cotton rats, as well as non-human primates. Most animals are not natural hosts for infectious agents, yet they can still be useful for studying various aspects of the disease. For example, a vaccine candidate, such as the ARP of the present invention, can be administered to any of the above animals to partially characterize the induced immune response and / or to determine whether any neutralizing antibodies are produced. For example, many studies have been conducted in mouse models because mice are small and their low cost allows researchers to scale up their studies.

[0127] Furthermore, human clinical studies can be conducted by one of ordinary skill in the art to determine the preferred effective dosage for humans. Such clinical studies are conventional and well known in the art. The exact dosage utilized also depends on the route of administration. The effective dosage can be estimated from a dose-response curve derived from in vitro or animal test systems.

[0128] Also, as is well known in the art, the immunogenicity of a particular composition can be enhanced by the use of a non-specific stimulator of the immune response, known as an adjuvant. Adjuvants have been used experimentally to promote a general increase in immunity to unknown antigens. Immunization protocols have used adjuvants for many years to stimulate responses, and thus adjuvants are well known to those of skill in the art. Some adjuvants affect the manner in which an antigen is presented. For example, the immune response increases when a protein antigen is precipitated by alum. Emulsification of an antigen also extends the period of antigen presentation. The inclusion of any adjuvant described in Vogel et al., 「A Compendium of Vaccine Adjuvants and Excipients (2nd Edition)」, which is hereby incorporated by reference in its entirety for all purposes, is contemplated within the scope of the present invention.

[0129] Exemplary adjuvants include complete Freund's adjuvant [a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis], incomplete Freund's adjuvant, and aluminum hydroxide adjuvant. Other adjuvants include GMCSP, BCG, aluminum hydroxide, MDP compounds such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). Also contemplated is RIBI, which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM), and cell wall skeleton (CWS), in a 2% squalene / Tween-80 emulsion. MF-59, Novasomes.RTM., MHC antigens may also be used.

[0130] The ARP of the present invention can also be formulated with "immunostimulatory factors". These are body-specific chemical messengers (cytokines) for increasing the response of the immune system. Immunostimulatory factors include, but are not limited to, various cytokines, lymphokines and chemokines with immunostimulatory activity, immunopotentiating activity, and inflammatory activity, such as interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-12, IL-13); growth factors [e.g., granulocyte macrophage (GM) colony-stimulating factor (CSF)]; and other immunostimulatory molecules such as macrophage inflammatory factor, Flt3 ligand, B7.1; B7.2. The immunostimulatory molecules can be administered in the same formulation as the ARP or separately. Either a protein or an expression vector encoding the protein can be administered to produce an immunostimulatory effect. Thus, in one embodiment, the present invention includes antigen formulations and vaccine formulations containing an adjuvant and / or immunostimulatory factors.

[0131] According to the present invention, the delivery system containing the ARP can deliver the gene of interest to the cytoplasm of eukaryotic cells, thereby treating cancer, viral infection, neuropathy, autoimmune diseases, graft rejection and monogenic or polygenic hereditary diseases.

[0132] The present invention will be described in detail with reference to the following examples, which are not intended to limit the scope of the present application.

Examples

[0133] Construction of VEEV replicon particles (VRP) The general procedure is shown in Figure 2. Establishment of a VEEV replicon plasmid expressing luciferase 1) Full-length VEEV TC83 non-structural protein (nsp) 1, nsp2, nsp3 and nsp4 fragments were synthesized (ThermoFisher). Similarly, gblocks corresponding to various fragments of the replicon construct were synthesized as follows (IDT).

[0134] 2) VEEV gblock1 - VEEV CA gene up to the ATG, ClaI - RSVp - 5’UTR - nsp1 (bp1 - 470) - RsrII - ApaI - 26Sp - sgRNA. This fragment had a 36 - bp overlap with the pBR322 backbone plasmid at the 5’ end. This is fragment #1.

[0135] 3) VEEV gblock2 - This fragment started at the ApaI site up to the ATG start codon and had a 64 - bp overlap with VEEV gblock1. This was followed by the Nano Luciferase ORF (Promega) and the first 72 bases of the VEEV 3’UTR.

[0136] 4) Cloning of fragment #2 - VEEV gblock2, full - length VEEV 3’UTR, and the VEEV polyA signal (A (n=55) ) were first assembled by overlap extension PCR using the oligomers shown in the following table:

[0137]

Table 1

[0138] VEEV_Oligo2 has a 15 - bp overlap with the pBR322 backbone plasmid.

[0139] 5) Next, VEEV gblock1 (fragment #1), fragment #2, and the pBR322 backbone (digested with ClaI and NruI - HF) were assembled using Gibson Assembly to obtain the pBR322 - RSVp - RsrII - ApaI - 26Sp - sgRNA - NanoLuc - A(n = 55) - SV40 pA backbone construct (BB). This backbone construct lacked the full - length TC83 nsp1 - 4 fragment.

[0140] 6) The nsp1-4 fragment (bp470-7461) was amplified from the synthesized Thermo plasmid obtained in 1) using primers containing RsrII and ApaI restriction sites - i) 5'-ccggccCGGACCGacaagtctctatcacc-3' (SEQ ID NO: 11, fwd primer) and ii) 5'-ggccggGGGCCCctctcaggtagctgaatg-3' (SEQ ID NO: 12, rev primer). The nsp fragment amplified by this PCR was cloned into the BB backbone plasmid using the RsrII and ApaI sites to obtain the full-length VEEV TC83 replicon construct. (Figure 3)

[0141] Helper plasmid Helper plasmid constructs encoding the VEEV TC83 capsid are shown in Figures 4 and 5. This construct expresses the wild-type VEEV capsid protein (SEQ ID NO: 1) and the VEEV capsid with a mutation in the NLS (K64N, SEQ ID NO: 2), respectively.

[0142] Helper plasmid constructs expressing the VEEV TC83 glycoprotein E3-E2-6K-E1 used herein are shown in Figures 6 and 7. This construct expresses the wild-type VEEV TC83 glycoprotein E3-E2-6K-E1 (SEQ ID NO: 3) and the E3-modified E3-E2-6K-E1 (where the furin site at the end of E3 RKRR was replaced with SGGGS, SEQ ID NO: 4), respectively.

[0143] Cell cultures and co-transfection 293T cells were seeded in 6-well plates containing complete DMEM supplemented with 10% FBS, penicillin, and streptomycin. Using PEI (1.7 μg of each plasmid), equal amounts of VEEV RSVp-NLuc constructs containing nsp1-4 (VR) (SEQ ID NO: 5) or lacking the nsp1-4 fragment (BB) were co-transfected into the cells together with a helper plasmid encoding the capsid and a helper plasmid encoding the glycoproteins E1-6K-E2-E3. The combinations of helper plasmids were (SEQ ID NO: 1 and 3), (SEQ ID NO: 1 and 4), (SEQ ID NO: 2 and 3), or (SEQ ID NO: 2 and 4). The cells were incubated with the transfection mixture at 37 °C for approximately 3 hours, after which the transfection mixture was removed, the cells were washed with 1× PBS, and fresh DMEM was added. The packaged virus replicon particles (VRPs) were harvested either at 48 hpt (hours post-transfection) or 72 hpt. To harvest the VRPs, the culture supernatant of the transfected cells was obtained by centrifuging the cell culture at 1200 rpm for 5 minutes at 4 °C to pellet all cell debris. The supernatant was filtered through a 0.45 μm filter. The harvested VRPs were stored either at 4 °C or -80 °C.

Example

[0144] Determination of the packaging ability of VEEV replicons into VEE virus replicon particles (VRPs) Infection and luciferase assay The general protocol is shown in Figure 8. 293T cells were seeded in complete DMEM in a 96-well plate at a density of approximately 10,000 cells per well. The recovered VRP, either undiluted or serially diluted 2-fold, was used to infect the cells at 37°C. At 14 hours post-infection (hpi), the VRP was removed, the cells were washed with PBS, and fresh DMEM was added to the wells. The cells were further incubated and harvested at 24, 48, and 72 hpi for luciferase assays. The luciferase assay was performed by adding equal amounts of infected cells and the Nano-Glo Luciferase Assay System (Promega) to a white-bottom opaque 96-well plate (Costar). Luciferase activity was immediately measured using a Bio-Tek Synergy HTX microplate reader.

[0145] Results The results are shown in Figure 9. Luciferase expression was confirmed in cells infected with VRP (VR) as early as 24 hpi. Cells infected with the backbone construct lacking nsp1-4(BB) showed little to no luciferase expression.

Example

[0146] New VEEV replicon construct A new VEEV TC83 replicon plasmid construct was prepared by introducing a multiple cloning site (MCS) to enable the introduction of various "genes of interest". The schematic protocol is shown in Figure 10. The construct is shown in Figure 11 and SEQ ID NO: 6. In the construct of Figure 11, nucleotides encoding luciferase are introduced as the gene of interest. The promoter in the plasmid can be selected considering the cells to be infected.

Example

[0147] Preparation of VRP with or without mutations in capsid and / or E3-E2-6K-E1 and having the gene of interest In this example, similar to Example 1, a VEEV replicon plasmid containing the target gene was co-transfected into 293T cells together with a VEEV capsid helper plasmid and a VEEV E3-E2-6K-E1 glycoprotein helper plasmid. As the target gene, a gene encoding luciferase, GFP, IKK, or JNK2 was used.

[0148] The following plasmids were used: i-1) A plasmid containing a polynucleotide encoding the VEEV CT83 wild-type capsid protein, or i-2) A plasmid containing a polynucleotide encoding the VEEV CT83 capsid protein (K64N) having a mutation in the NLS. ii-1) A plasmid containing a polynucleotide encoding wild-type VEEV CT83 E3-E2-6K-E1, or ii-2) A plasmid containing a polynucleotide encoding VEEV CT83 E3-E2-6K-E1 having a mutation at the furin site of E3 and iii) A plasmid containing polynucleotides encoding VEEV CT83 nsp1, nsp2, nsp3, and nsp4 and the target gene encoding luciferase, GFP, IKK, or JNK2.

[0149] Co-transfection A VEEV TC83 replicon plasmid containing the target gene, a helper plasmid encoding VEEV capsid (WT or mutant), and a helper plasmid encoding E3-E2-6K-E1 (WT or mutant) were co-transfected into 293T cells. Three plasmids (a VEEV replicon containing 1 μg of capsid, 1 μg of E3-E2-6K-E1, and 10 μg of the target gene) were transfected into 293T cells (by the PEI method), and the cells were incubated for 4 to 7 days. Then, VRP was recovered from the supernatant and purified by OptiPrep density sedimentation.

[0150] The purified VRP was confirmed by Western blotting using anti-VEEV antibody (ATCC) as the primary antibody (1:2000) and anti-mouse IgG (1:4000) as the secondary antibody. The results of VRP obtained by using the following plasmids are shown in Figure 12: i) A plasmid containing a polynucleotide encoding the VEEV CT83 capsid protein (K64N) having a mutation in the NLS. ii) A plasmid containing a polynucleotide encoding wild-type VEEV CT83 E3-E2-6K-E1, and iii) A plasmid containing polynucleotides encoding VEEV CT83 nsp1, nsp2, nsp3, and nsp4, and also a polynucleotide encoding GFP, IKK, or JNK2.

Example

[0151] The VRP obtained in Example 4 was used using the following vectors: i) A plasmid containing a polynucleotide encoding the VEEV CT83 capsid protein (K64N) having a mutation in the NLS. ii) A plasmid containing a polynucleotide encoding wild-type VEEV CT83 E3-E2-6K-E1, and iii) A plasmid containing polynucleotides encoding VEEV CT83 nsp1, nsp2, nsp3, and nsp4, and also a polynucleotide encoding IKK or JNK2.

[0152] 293T cells were infected with VRP containing the IKK or JNK2 gene. Infection of 293T cells with VRP was performed in the same manner as in Example 2.

[0153] The expression of IKK in VRP-infected 293T cells was confirmed by Western blotting. As a positive control, 293T cells transfected with an IKK expression vector plasmid were used. The cell lysates were subjected to Western blotting using an Rb anti-IKK antibody (Proteintech) as the primary antibody (1:500) and HRP-labeled anti-RbIgG as the secondary antibody (1:4000).

[0154] The results are shown in Figure 13. In Figure 13, lane 1 shows 293T cells infected with VRP. Lane 2 shows the positive control, i.e., cells transfected with the IKK expression vector plasmid. As shown in this figure, 293T cells infected with VRP expressed the IKK protein.

[0155] The expression of JNK2 from the replicon-inserted JNK2 gene in infected 293T cells was confirmed by Western blotting. As a control, uninfected 293T cells were used. A mouse anti-JNK2 antibody (Santa Cruz) was used as the primary antibody (1:500), and HRP-labeled anti-mouse IgG was used as the secondary antibody (1:4000). The results are shown in Figure 14. It was confirmed that 293T cells infected with VRP having JNK2 as the target gene expressed JNK2.

Example

[0156] Effect of mutations in the capsid NLS Two types of VRP obtained in Example 4 were used with the following plasmids: i-1) A plasmid containing a polynucleotide encoding the wild-type VEEV CT83 capsid protein, or i-2) A plasmid containing a polynucleotide encoding the VEEV CT83 capsid protein (K64N) having a mutation in the NLS. ii) A plasmid containing a polynucleotide encoding wild-type VEEV CT83 E3-E2-6K-E1, and iii) A plasmid comprising a polynucleotide encoding VEEV CT83 nsp1, nsp2, nsp3 and nsp4, and a polynucleotide encoding luciferase.

[0157] VRP was used to infect 293T cells in the same manner as in Example 2. A luciferase assay was performed in the same manner as in Example 2. The results are shown in Figure 15.

[0158] In this figure, the control corresponds to the background of luciferase activity. VRP with a capsid having a mutation in the NLS showed much higher luciferase activity (900556) compared to VRP with a WT capsid (118063).

[0159] This data showed that modification of the capsid led to higher yields and expression compared to alphavirus replicon particles without modification in the capsid.

Example

[0160] The VEE virus replicon particles (VRP) obtained in Example 4 were used by co - transfection of 293T cells with the following vectors: i) A plasmid comprising a polynucleotide encoding the VEEV CT83 capsid protein (K64N) having a mutation in the NLS. ii) A plasmid comprising a polynucleotide encoding wild - type VEEV CT83 E3 - E2 - 6K - E1. iii) A plasmid comprising a polynucleotide encoding VEEV CT83 nsp1, nsp2, nsp3 and nsp4, and a polynucleotide encoding GFP. VRP obtained in the same manner as in Example 2 was used to infect 293T cells. The control shows normal cells without infection. The expression of GFP in the cells was confirmed. The results are shown in Figure 16.

Example

[0161] The same VEEV replicon particles as those used in Example 7 were used. Macrophage J774.A1 cells and bone marrow-derived macrophage (BMDM) cells were infected with VRP. The gene of interest encoding GFP was expressed in the transfected macrophage cells. Both cell types were polarized by treatment with IL-4 for 48 hours and analyzed for GFP expression. The results are shown in Fig. 17.

Example

[0162] In vivo efficacy study regarding CT26 model The schematic protocol for this example is shown in Fig. 18. The VRP used in this example was prepared in Example 4 using the following plasmids: i) A plasmid containing a polynucleotide encoding the VEEV CT83 capsid protein (K64N) having a mutation in NLS, ii) A plasmid containing a polynucleotide encoding wild-type VEEV CT83 E3-E2-6K-E1, and iii) A plasmid containing polynucleotides encoding VEEV CT83 nsp1, nsp2, nsp3 and nsp4 and a polynucleotide encoding human IκB kinase (IKK).

[0163] This study consisted of a total of 32 animals in 4 treatment groups, n = 8. The animals were randomized on day 0 when the tumor volume reached 60 - 100 mm 3 and administration was started on day 0 at 10 mg / kg (BIW×3 and IP) with G1: vehicle, G2: anti-PD-1 mAb, G3: VRP and G4: combination of VRP and anti-PD-1 mAb. Tumor growth was monitored until 30 days after the start of treatment.

[0164] For groups 1, 3, and 4, mice were injected intratumorally with 50 μl of medium or VRP dispersed in medium on days 0, 2, 4, 6, 8, and 10. The VRP or medium was administered intratumorally in the right flank using a 0.3 ml insulin syringe. The aim was to use one entry point, but to distribute the substance through the tumor by inserting and removing the needle.

[0165] Tumor size was measured twice a week. The humane endpoint for this study was a tumor burden of 3000 mm 3 and / or a weight loss of 20% or more. The results for all 8 animals in each group are shown in Figure 19.

[0166] The data showed that VRP containing the gene encoding IKK and the combination of VRP and anti-PD-1 antibody demonstrated superior antitumor effects compared to control and anti-PD-1 monotherapy.

Example

[0167] Chimeric alphavirus replicon particles were prepared as in Example 4 using the following set of vectors. Chimeric ARP1 i) A plasmid containing a polynucleotide encoding the wild-type CHIKV 37997 strain capsid protein, ii) A plasmid containing a polynucleotide encoding the wild-type CHIKV 37997 strain E3-E2-6K-E1, and iii) A plasmid containing polynucleotides encoding VEEV CT83 nsp1, nsp2, nsp3, and nsp4 and a polynucleotide encoding GFP.

[0168] Chimeric ARP2 i) A plasmid containing a polynucleotide encoding the wild-type CHIKV OPY-1 strain capsid protein, ii) A plasmid containing a polynucleotide encoding the wild-type CHIKV OPY-1 strain E3-E2-6K-E1, and iii) A plasmid comprising a polynucleotide encoding VEEV CT83 nsp1, nsp2, nsp3 and nsp4, and a polynucleotide encoding GFP.

[0169] The obtained ARP was purified in the same manner as in Example 1. The purified chimeric ARP was confirmed by Western blotting using anti-CHIKV rabbit serum (1:2000) as the primary antibody and goat anti-rabbit IgG-HRP (1:4000) as the secondary antibody. The results are shown in Figure 20. The production of both chimeric ARPs was confirmed.

[0170] 293T cells were infected with the chimeric ARP encoding GFP. After infection, the cells were incubated for 48 hours, and the expression of GFP was confirmed by FACS analysis. Uninfected cells were used as a control. The results are shown in Figure 21. 5.21% and 4.07% of the cells infected with ARPS expressed GFP, suggesting that the chimeric ARP successfully expressed the GFP protein (the gene of interest is the nucleotide encoding GFP) intracellularly.

Claims

**Claim 1** (i) a structural protein of Chikungunya virus (CHIKV) comprising a capsid and / or an envelope, and (ii) a Venezuelan equine encephalitis virus (VEEV) replicon comprising a polynucleotide encoding VEEV non-structural proteins nsp1, nsp2, nsp3 and nsp4, and at least one gene of interest Alpha virus replicon particles (ARPs) comprising the same. **Claim 2** The alpha virus replicon particles according to claim 1, wherein the E2 protein in the envelope further comprises one or more modifications. **Claim 3** The alpha virus replicon particles according to claim 1 or 2, wherein the furin site of the E3 protein in the envelope further comprises one or more modifications. **Claim 4** The alpha virus replicon particles according to any one of claims 1 to 3, wherein the CHIKV is CHIKV strain 37997 or strain OPY-1. **Claim 5** The alpha virus replicon particles according to any one of claims 1 to 4, wherein the VEEV is VEEV strain TC-83. **Claim 6** The alpha virus replicon particles according to claim 5, wherein the CHIKV is CHIKV strain 37997 or strain OPY-1, and the VEEV is VEEV strain TC-83. **Claim 7** The alpha virus replicon particles according to any one of claims 1 to 6, wherein the gene of interest encodes an antigen.

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