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  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
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  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
  • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
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  • C07K2319/00—Fusion polypeptide
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  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/16011—Orthomyxoviridae
  • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
  • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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  • C12N2760/00011—Details
  • C12N2760/16011—Orthomyxoviridae
  • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
  • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/20011—Rhabdoviridae
  • C12N2760/20111—Lyssavirus, e.g. rabies virus
  • C12N2760/20122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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  • C12N2760/00011—Details
  • C12N2760/20011—Rhabdoviridae
  • C12N2760/20111—Lyssavirus, e.g. rabies virus
  • C12N2760/20134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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  • C12N2770/00011—Details
  • C12N2770/36011—Togaviridae
  • C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
  • C12N2770/36122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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  • C12N2770/00011—Details
  • C12N2770/36011—Togaviridae
  • C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
  • C12N2770/36141—Use of virus, viral particle or viral elements as a vector
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  • C12N2770/00011—Details
  • C12N2770/36011—Togaviridae
  • C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
  • C12N2770/36141—Use of virus, viral particle or viral elements as a vector
  • C12N2770/36143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

• the present disclosure relates to the field of molecular virology and immunology, and particularly relates to nucleic acid molecules encoding modified viral genomes and replicons (e.g., self-replicating RNAs), pharmaceutical compositions containing the same, and the use of such nucleic acid molecules and compositions for production of desired products in cell cultures or in a living body. Also provided are methods for inducing pharmacodynamic effects, e.g., eliciting an immune response in a subject in need thereof, as well as methods for preventing and/or treating various health conditions.
• viral -based expression vectors have been deployed for expression of heterologous proteins in cultured recombinant cells.
• modified viral vectors for gene expression in host cells continues to expand.
• Recent advances in this regard include further development of techniques and systems for production of multi- subunit protein complexes, and co-expression of protein-modifying enzymes to improve heterologous protein production.
• Other recent progresses regarding viral expression vector technologies include many advanced genome engineering applications for controlling gene expression, preparation of viral vectors, in vivo gene therapy applications, and creation of vaccine delivery vectors.
• the present disclosure relates generally to the development of immunotherapeutics, such as recombinant nucleic acids constructs and pharmaceutical compositions including the same for use in the prevention and management of various health conditions such as proliferative disorders and microbial infection.
• immunotherapeutics such as recombinant nucleic acids constructs and pharmaceutical compositions including the same for use in the prevention and management of various health conditions such as proliferative disorders and microbial infection.
• some embodiments of the disclosure provide, inter alia, nucleic acid constructs encoding recombinant alphavirus with a coding sequence for at least one heterologous one nonstructural protein (nsP) or a portion thereof.
• nsP nonstructural protein
• nucleic acid constructs including a modified genome or RNA replicon (e.g., self-repli eating RNA) of an alphavirus species, wherein at least one nonstructural protein (nsP), or a portion thereof, of the modified alphavirus genome or RNA replicon is heterologous relative to the remainder of the modified alphavirus genome or RNA replicon.
• nsP nonstructural protein
• Non-limiting embodiments of the nucleic acid constructs of the disclosure can include one or more of the following features.
• the at least one heterologous nsP or portion thereof is nsPl, nsP2, nsP3, nsP4, or a portion of any thereof, or a combination of any of the foregoing. In some embodiments, the at least one heterologous nsP or portion thereof is derived from another strain of the same alphavirus species. In some embodiments, the at least one heterologous nsP or portion thereof is derived from another alphavirus species.
• the modified alphavirus genome or RNA replicon (e.g., self-replicating RNA) is devoid of at least a portion of the nucleic acid sequence encoding one or more viral structural proteins. In some embodiments, the modified viral genome or RNA replicon is devoid of a substantial portion of the nucleic acid sequence encoding one or more viral structural proteins. In some embodiments, the modified viral genome or RNA replicon includes no nucleic acid sequence encoding viral structural proteins.
• the modified alphavirus genome or RNA replicon (e.g., self-replicating RNA) of the disclosure further includes one or more expression cassettes, wherein each of the expression cassettes includes a promoter operably linked to a heterologous nucleic acid sequence.
• at least one of the expression cassettes includes a subgenomic (. g) promoter operably linked to a heterologous nucleic acid sequence.
• the sg promoter is a 26S subgenomic promoter.
• the modified alphavirus genome or RNA replicon of the disclosure further includes one or more untranslated regions (UTRs). In some embodiments, at least one of the UTRs is a heterologous UTR.
• At least one of expression cassettes includes a coding sequence for a gene of interest (GO I).
• the GOI encodes a polypeptide selected from the group consisting of a therapeutic polypeptide, a prophylactic polypeptide, a diagnostic polypeptide, a nutraceutical polypeptide, an industrial enzyme, and a reporter polypeptide.
• the GOI encodes a polypeptide selected from the group consisting of an antibody, an antigen, an immune modulator, an enzyme, a signaling protein, and a cytokine.
• the coding sequence of the GOI is optimized for expression at a level higher than the expression level of a reference coding sequence. In some embodiments, the coding sequence of the GOI is optimized for enhanced RNA stability.
• the modified alphavirus genome or RNA replicon (e.g., self-replicating RNA) of the disclosure is of an alphavirus species selected from the group consisting of Aura virus (AURAV), Babanki virus (BABV), Barmah Forest virus (BFV), Bebaru virus (BEBV), Buggy Creek virus, Caaingua virus, Cabassou virus, Chikungunya virus (CHIKV), Eastern equine encephalitis virus (EEEV), Eilat virus, Everglades virus (EVEV), Fort Morgan virus (FMV), Getah virus (GETV), Highlands J virus (HJV), Kyzylagach virus (KYZV), Madariaga virus (MADV), Mayaro virus (MAYV), Middelburg virus (MIDV), Mosso das Pedras virus, Mucambo virus (MUCV), Ndumu virus (NDUV), O'nyong'nyong virus (ONNV), Pixuna virus (PIXV),
• AURAV Aura virus
• the modified alphavirus genome or RNA replicon (e.g., self-replicating RNA) of the disclosure is of a Sindbis virus (SINV).
• the modified alphavirus genome or RNA replicon of the disclosure is of a SINV strain Girdwood.
• At least one heterologous nsP or portion thereof of the modified genome or RNA replicon is derived from a SINV strain AR86. In some embodiments, at least one heterologous nsP or portion thereof of the modified genome or RNA replicon is derived from a SINV strain Girdwood.
• the at least one heterologous nsP or portion thereof is nsPl, nsP3, nsP4, or a portion of any thereof, or a combination of any of the foregoing.
• the modified genome or RNA replicon e.g., self-replicating RNA
• the at least one heterologous nsP or portion thereof of the modified SINV-AR86 genome or RNA replicon is derived from a SINV strain Girdwood.
• the at least one heterologous nsP or portion thereof of the modified SINV- AR86 genome or RNA replicon is derived from nsP2 of a SINV strain Girdwood.
• the nucleic acid construct of the disclosure is incorporated into a vector.
• the vector is a self-replicating RNA (srRNA) vector.
• the nucleic acid construct including a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1-4.
• recombinant cells including a nucleic acid construct as disclosed herein.
• the recombinant cell is a eukaryotic cell.
• the recombinant cell is an animal cell.
• the animal cell is a vertebrate animal cell or an invertebrate animal cell.
• the animal cell is an insect cell.
• the insect cell is a mosquito cell.
• the recombinant cell is a mammalian cell.
• the recombinant cell is selected from the group consisting of a monkey kidney CV1 cell transformed by SV40 (COS-7), a human embryonic kidney cell (e.g., HEK 293 or HEK 293 cell), a baby hamster kidney cell (BHK), a mouse sertoli cell (e.g., TM4 cells), a monkey kidney cell (CV1), a human cervical carcinoma cell (HeLa), canine kidney cell (MDCK), buffalo rat liver cell (BRL 3 A), human lung cell (W138), human liver cell (Hep G2), mouse mammary tumor (MMT 060562), TRI cell, , FS4 cell, a Chinese hamster ovary cell (CHO cell), an African green monkey kidney cell (Vero cell), a human A549 cell, a human cervix cell, a human CHME5 cell, a human PER.C6 cell, a NS0 murine myeloma cell, a human epidermoid laryn
• transgenic animals including a nucleic acid construct as described herein.
• the animal is a vertebrate animal or an invertebrate animal.
• the animal is an insect.
• the animal is a mammal.
• the mammal is a non-human mammal.
• methods for producing a polypeptide of interest include (i) rearing a transgenic animal as disclosed herein; or (ii) culturing a recombinant cell including a nucleic acid construct as disclosed herein under conditions wherein the transgenic animal or recombinant cell produces the polypeptide encoded by the GOI.
• kits for producing a polypeptide of interest in a subject include administering to the subject a nucleic acid construct as disclosed herein.
• the subject is vertebrate animal or an invertebrate animal.
• the subject is an insect.
• the insect is a mosquito.
• the subject is a mammalian subject.
• the mammalian subject is a human subject.
• provided herein are recombinant polypeptides produced by a method of the disclosure.
• compositions including a pharmaceutically acceptable excipient and: a) a nucleic acid construct of the disclosure; b) a recombinant cell of the disclosure; and/or c) a recombinant polypeptide of the disclosure.
• compositions including a nucleic acid construct as disclosed herein and a pharmaceutically acceptable excipient are provided herein.
• the compositions include a recombinant polypeptide of as disclosed herein and a pharmaceutically acceptable excipient.
• the compositions are immunogenic compositions.
• the immunogenic compositions are formulated as a vaccine. In some embodiments, the immunogenic compositions are substantially non-immunogenic to a subject. In some embodiments, the pharmaceutical compositions are formulated as an adjuvant. In some embodiments, the pharmaceutical compositions are formulated for one or more of intranasal administration, intranodal administration, transdermal administration, intraperitoneal administration, intramuscular administration, intratumoral administration, intraarticular administration, intravenous administration, subcutaneous administration, intravaginal administration, intraocular administration, oral administration, and rectal administration.
• RNA replicon e.g., self-replicating RNA
• methods for functionalizing/engineering an alphavirus genome or RNA replicon including: (a) providing a non-functional alphavirus genome or RNA replicon; (b) replacing a nonstructural protein (nsP), or a portion thereof, of the non-functional alphavirus genome or RNA replicon with a heterologous coding sequence for the corresponding nsP or portion thereof derived from a different alphavirus strain to generate a modified alphavirus genome or RNA replicon; (c) assessing functionality of the modified alphavirus genome or RNA replicon; and (d) identifying the modified alphavirus genome or RNA replicon as being functional if the modified alphavirus genome or RNA replicon is capable of RNA replication and/or expression.
• nsP nonstructural protein
• Non-limiting exemplary embodiments of the p methods for functionalizing and/or engineering an alphavirus genome or RNA replicon (e.g., self-replicating RNA) of the disclosure can include one or more of the following features.
• the heterologous nsP or portion thereof is derived from another strain of the same alphavirus species.
• the heterologous nsP or portion thereof is derived from another alphavirus species.
• the heterologous nsP or portion thereof is nsPl, nsP2, nsP3, nsP4, or a portion of any thereof.
• the non-functionality of the alphavirus genome or RNA replicon is determined by a deficiency in self-replication within a host cell.
• the assessing functionality of the modified alphavirus genome or RNA replicon includes an assay selected from the group consisting of: detection of RNA replication, detection of viral protein expression, detection of cytopathic effect (CPE), and detection of heterologous transgene expression.
• kits for inducing a pharmacodynamic effect in a subject and, in particular, methods for eliciting an immune response in a subject in need thereof include administering to the subject a composition including: a) a nucleic acid construct of the disclosure; b) a recombinant cell of the disclosure; c) a recombinant polypeptide of the disclosure; and/or d) a pharmaceutical composition of the disclosure.
• kits for preventing and/or treating a health condition in a subject in need thereof include prophylactically or therapeutically administering to the subject a composition including: a) a nucleic acid construct of the disclosure; b) a recombinant cell of the disclosure; c) a recombinant polypeptide of the disclosure; and/or d) a pharmaceutical composition of any one of the disclosure.
• Non-limiting exemplary embodiments of the methods of the disclosure can include one or more of the following features.
• the condition is a proliferative disorder or a microbial infection.
• the subject has or is suspected of having a condition associated with proliferative disorder or a microbial infection.
• the administered composition results in an increased production of interferon in the subject.
• the composition is administered to the subject individually as a single therapy (monotherapy) or as a first therapy in combination with at least one additional therapies.
• the at least one additional therapies is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, targeted therapy, and surgery.
• kits for inducing a pharmacodynamic effect, for eliciting an immune response, for the prevention, and/or for the treatment of a health condition or a microbial infection including: a) a nucleic acid construct of the disclosure; b) a recombinant cell of the disclosure; c) a recombinant polypeptide of the disclosure; and/or d) a pharmaceutical composition of the disclosure.
• FIG. 1 is a schematic representation of four non-limiting examples of alphavirus genome designs in accordance with some embodiments of the disclosure.
• Non- structural proteins nsPl, nsP2, nsP3, and nsP4 are shown. These alphavirus designs each contain (i) a heterologous gene of interest (GO I) placed under control of a 26S subgenomic promoter; and (ii) native 5’ UTR and 3’ UTR sequences derived from the SINV strain AR86.
• GO I heterologous gene of interest
• native 5’ UTR and 3’ UTR sequences derived from the SINV strain AR86 In AR86-Girdwood Hybrid 1, the structural proteins nsPl, nsP3, and nsP4 are from SINV strain AR86, while nsP2 is from SINV strain Girdwood.
• nsP4 is from AR86 strain, while nsPl, nsP2, and nsP3 are from Girdwood strain.
• nsP3 is from AR86 strain, whereas nsPl, nsP2, and nsP4 are from Girdwood strain.
• nsPl are from AR86 strain, and nsP2, nsP3, whereas nsP4 is from Girdwood.
• FIG. 2A is a schematic structure of the base Sindbis AR86-Girdwood Hybrid 1 vector described in FIG. 1 without coding sequence for a gene of interest (GO I).
• FIG. 2B is a schematic structure of the Sindbis AR86-Girdwood Hybrid 1 vector described in FIG. 1 with coding sequence for an exemplary GO I, e.g., hemagglutinin precursor (HA) of the influenza A virus H5N1 (H5N1 HA), which is placed under control of a 26S subgenomic promoter.
• HA hemagglutinin precursor
• FIG. 3A is a schematic structures of the base Sindbis AR86-Girdwood Hybrid 2 vector described in FIG. 1 without coding sequence for a GOI.
• FIG. 3B is a schematic structures of the Sindbis AR86-Girdwood Hybrid 2 vector described in FIG. 1 with coding sequence for an exemplary GOI, e.g., H5N1 HA placed under control of a 26S subgenomic promoter.
• an exemplary GOI e.g., H5N1 HA placed under control of a 26S subgenomic promoter.
• FIG. 4A is a schematic structure of the base Sindbis AR86-Girdwood Hybrid 3 vector described in FIG. 1 without coding sequence for a GOI.
• FIG. 4B is a schematic structure of Sindbis AR86-Girdwood Hybrid 3 vectors described in FIG. 1 with coding sequence for an exemplary GOI, e.g., H5N1 HA placed under control of a 26S subgenomic promoter.
• an exemplary GOI e.g., H5N1 HA placed under control of a 26S subgenomic promoter.
• FIG. 5A is a schematic structure of Sindbis AR86-Girdwood Hybrid 4 vectors described in FIG. 1 without coding sequence for a GOI.
• FIG. 5B is a schematic structure of Sindbis AR86-Girdwood Hybrid 4 vectors described in FIG. 1, with coding sequence for an exemplary GOI, e.g., H5N1 HA placed under control of a 26S subgenomic promoter.
• an exemplary GOI e.g., H5N1 HA placed under control of a 26S subgenomic promoter.
• FIG. 6 graphically summarizes the results of experiments performed to demonstrate that a non-functional alphavirus genome or RNA replicon (e.g., self-replicating RNA) can be functionalized by replacing a defective nsP sequence with a corresponding functional nsP derived from a heterologous alphavirus genome or RNA replicon.
• FIG. 6 depicts contour plots of BHK-21 cells which have been transformed with exemplary alphavirus genome designs in accordance with some embodiments of the disclosure.
• the alphavirus genome designs were each introduced into BHK-21 cells by electroporation, and 20 hours following transformation, the cells were fixed and permeabilized and stained using a PE- conjugated anti-double stranded RNA (dsRNA) mouse monoclonal antibody (J2, Scicons) to quantify the frequency of dsRNA+ cells by fluorescence flow cytometry.
• dsRNA PE- conjugated anti-double stranded RNA
• J2, Scicons PE- conjugated anti-double stranded RNA
• FIG. 7 graphically summarizes the results of experiments performed to demonstrate that expression of a GOI can be detected from srRNA vectors with heterologous nonstructural protein genes.
• FIG. 7 is a bar chart illustrating the quantification of relative expression of HA polypeptide of avian influenza A H5N1 in cells which have been transformed by srRNA vector designs in accordance with some embodiments of the disclosure.
• the alphavirus srRNA designs were each introduced into BHK-21 cells by electroporation, and 20 hours following transformation, the cells were fixed and permeabilized and stained using an APC-conjugated anti-H5Nl mouse monoclonal antibody (2B7, Abeam; APC: allophycocyanin) to quantify mean fluorescence intensity (MFI) of H5N1+ cells by fluorescence flow cytometry.
• APC-conjugated anti-H5Nl mouse monoclonal antibody (2B7, Abeam; APC: allophycocyanin
• FIGS. 8A-8B schematically summarize the results of experiments demonstrating that modified srRNA vectors containing heterologous nonstructural protein genes designed in accordance with some embodiments of the disclosure can be used to express two exemplary bioactive proteins: (i) interleukin-1 receptor antagonist protein (IL-IRA) and (ii) interleukin- 12 (IL- 12)
• FIGS. 8A-8B are bar charts illustrating the quantification of secreted protein bioactivity from BHK-21 cells which were transformed with the srRNAs. The srRNAs shown in FIGS.
• FIG. 8A- 8B are SINV AR86-Girdwood Hybrid 1 srRNAs (RBI307, RBI308) each encoding two proteins IL-IRA and IL-12 in two different configurations. Also included in these experiments were four control VEEV-based srRNAs as follows: VEEV srRNAs encoding both IL-IRA and IL-12 in two configurations (RBI299, RBI300) and VEEV srRNAs with control transgenes (RBI296, RBI298). Also included in these experiments were two control SINV Girdwood-based srRNAs (RBI309, RBI310) each encoding two proteins IL-IRA and IL-12 in two different configurations.
• FIG. 8A shows the quantification of bioactive IL- IRA in the cell culture media at 24 and 48 hours following srRNA transformation.
• FIG. 8B shows the quantification of bioactive IL- 12 in the cell culture media at 24 and 48 hours following srRNA transformation.
• FIGS. 9A-9B are bar charts illustrating in vivo immunogenicity of a panel of srRNAs encoding an exemplary viral antigen, which is an envelope glycoprotein G of a rabies virus (RABV-G).
• the panel included srRNAs derived from Venezuelan equine encephalitis virus (VEE.TC83), Chikungunya virus strains S27 (CHIK.S27) and DRDE-06 (CHIK.DRDE), Sindbis virus strains Girdwood (SIN.GW) and AR86-Girdwood Hybrid 1 (SIN.AR86), and Eastern equine encephalitis virus (EEE.FL93).
• FIG. 9A shows the quantification of antigen-specific splenic T cell responses evaluated by ELISpot after two immunizations.
• FIG. 9B shows antirabies neutralizing antibody titers from sera after two immunizations.
• FIGS. 10A-10C are bar charts showing in vivo immunogenicity of a panel of srRNAs encoding exemplary tumor-associated antigens for use as vaccine, e.g., for eliciting an immune response in a subject.
• the panel included srRNAs derived from Sindbis AR86- Girdwood Hybrid 1 (SIN.AR86) and five other alphaviruses: Venezuelan equine encephalitis virus (VEE.TC83), Chikungunya virus strains S27 (CHIK.S27) and DRDE-06 (CHIK.DRDE), Sindbis virus strain Girdwood (SIN.GW) and Eastern equine encephalitis virus (EEE.FL93).
• Sindbis AR86- Girdwood Hybrid 1 SIN.AR86
• VEE.TC83 Venezuelan equine encephalitis virus
• CHIK.S27 Chikungunya virus strains S27
• DRDE-06 DRDE-06
• Each srRNA includes sequences encoding for three polypeptides: sequence for estrogen receptor 1 (ESRI), human epidermal growth factor 2 HER2), and human epidermal growth factor 2 (HER3).
• FIGS. 10A-10C show splenic T cell responses to these three antigens determined using ELISpot analysis in mice having received two immunizations, with statistical comparisons between each antigen tested.
• nucleic acid constructs such as, e.g., expression constructs and vectors, containing a modified genome or replicon RNA (e.g., self-replicating RNA) of an alphavirus species, wherein at least one nonstructural protein (nsP), or a portion thereof, of the modified alphavirus genome or RNA replicon is heterologous relative to the remainder of the modified alphavirus genome or RNA replicon.
• nsP nonstructural protein
• viral-based expression vectors including one or more expression cassettes encoding a polypeptide of interest encoded by a gene of interest (GO I).
• recombinant cells that are genetically engineered to include one or more of the nucleic acid constructs disclosed herein. Biomaterials and recombinant products derived from such recombinant cells are also within the scope of the application.
• compositions and methods useful inducing pharmacodynamic effects e.g., for eliciting an immune response, in a subject in need thereof, as well as methods for preventing and/or treating various health conditions.
• RNA viruses e.g., alphaviruses
• RNA viruses e.g., alphaviruses
• an advantage of using alphaviruses such as SINV as viral expression vectors is that they can direct the synthesis of large amounts of recombinant proteins in recombinant host cells.
• polypeptides such as therapeutic single chain antibodies can be most effective if expressed at high levels in vivo.
• high protein expression from a replicon RNA can increase overall yields of the antibody product.
• high level expression can induce the most robust immune response in vivo.
• Alphaviruses utilize motifs contained in their UTRs, structural regions, and non- structural regions to impact their replication in host cells. These regions also contain mechanism to evade host cell innate immunity. However, significant differences among alphavirus species have been reported, for example in mechanisms of immune evasion, tissue tropism, xenotropic hosts, as well as disease symptoms and severity.
• the publicly available alphavirus genomic data does not always provide nucleotide sequences that are capable of direct replacement of the nucleic acid sequences encoding the structural proteins with a gene of interest (GOI) to result in self-replicating RNA and transgene-expressing replicons.
• GOI gene of interest
• a large number of publicly available alphavirus genomes were found non-functional, e.g., incapable of undergoing replication and/or expressing a transgene.
• RNA replicon e.g., self-replicating RNA
• nsP nonstructural protein
• full-length viruses and synthetic replicons do not have the same capacity for replication.
• many full-length viruses and replicons from publicly available resources are functionally defective.
• a modification approach to functionalize (e.g., to render functional) a defective alphavirus genome or RNA replicon is to revert one or more key point mutations related to virulence that diverged between strains, for example mutations that diverse between a functional strain (e.g., Girdwood) and a non-functional strain (e.g., AR86).
• each of the nsP subunits e.g., nsPl, nsP2, nsP3, nsP4
• nsP subunits e.g., nsPl, nsP2, nsP3, nsP4
• nsP polyprotein complex that performs genomic and subgenomic transcription function.
• each nsP itself is reasonably biologically self-contained in terms of contributing to the overall function of the replicon (e.g., self-replicating RNA) and should be treated as a discrete modular unit.
• some embodiments of the present disclosure relate to a method of functionalizing a non-functional alphavirus genome or RNA replicon, wherein each of the nsP subunits is treated as a discrete modular unit and can be replaced (swapped) by a corresponding modular unit from another virus (e.g., another species or another strain of the same species), resulting in a chimeric virus with new characteristics.
• the presently disclosed method allows for a new combinatorial look at the effect of swapping out nsPs in a following minimal set: (1) a replicon that does not launch in vitro, and (2) a replicon that does launch in vitro.
• This approach rapidly gives information on which nsP is creating issues for any given strain without the need to create a large number of new constructs.
• the presently described new procedure is a rapid, technically feasible method that is significantly improved than any of the currently known methods.
• some embodiments of the disclosure relate to self-replicating RNA (srRNAs) vectors containing heterologous nonstructural protein genes that have been engineered to express one or more heterologous genes of interest (GOI).
• srRNAs self-replicating RNA
• GOI heterologous genes of interest
• a Sindbis srRNA vector containing heterologous nonstructural protein genes (SINV AR86-Girdwood Hybrid 1) has been engineered to replace the structural polyprotein gene with a synthetic human IL-IRA gene or IL-12 gene cassette to produce self-replicating vectors capable of RNA replication and transgene expression in transfected BHK-21 cells (see e.g., FIG. 8).
• SINV AR86-Girdwood Hybrid 1 -based srRNA constructs as described herein can be employed for expression of an antigenic molecule of interest and formulated as a vaccine with a measurable pharmacodynamic effect in vivo (see, e.g., FIG.
• FIGS. 7A-7B demonstrate that SINV AR86-Girdwood Hybrid 1 -based srRNA vectors can be useful for expression of multiple proteins whose coding sequences are operably linked to one another within a single open reading frame (e.g., in a polycistronic ORF) and have bioactivity as measured by pharmacodynamic effect in vivo (see, e.g., FIG. 10).
• these studies further demonstrate the use of srRNA vectors with heterologous nonstructural protein genes and SINV AR86-Girdwood Hybrid 1 -based srRNA vectors in therapeutic and vaccine applications.
• a cell includes one or more cells, comprising mixtures thereof.
• a and/or B is used herein to include all of the following alternatives: “A”, “B”, “A or B”, and “A and B”.
• aspects and embodiments of the disclosure described herein include “comprising”, “consisting”, and “consisting essentially of’ aspects and embodiments.
• “comprising” is synonymous with “including”, “containing”, or “characterized by”, and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.
• “consisting” excludes any elements, steps, or ingredients not specified in the claimed composition or method.
• “consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claimed composition or method.
• administration refers to the delivery of a bioactive composition or formulation by an administration route comprising, but not limited to, intranasal, transdermal, intravenous, intra-arterial, intramuscular, intranodal, intraperitoneal, subcutaneous, intramuscular, oral, intravaginal, and topical administration, or combinations thereof.
• administration route comprising, but is not limited to, intranasal, transdermal, intravenous, intra-arterial, intramuscular, intranodal, intraperitoneal, subcutaneous, intramuscular, oral, intravaginal, and topical administration, or combinations thereof.
• administration route comprising, but not limited to, intranasal, transdermal, intravenous, intra-arterial, intramuscular, intranodal, intraperitoneal, subcutaneous, intramuscular, oral, intravaginal, and topical administration, or combinations thereof.
• the term includes, but is not limited to, administering by a medical professional and self-administering.
• cell refers not only to the particular subject cell, cell culture, or cell line but also to the progeny or potential progeny of such a cell, cell culture, or cell line, without regard to the number of transfers or passages in culture. It should be understood that not all progeny are exactly identical to the parental cell.
• progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein, so long as the progeny retain the same functionality as that of the original cell, cell culture, or cell line.
• a composition of the disclosure e.g., nucleic acid constructs, recombinant cells, recombinant polypeptides, and/or pharmaceutical compositions
• a composition of the disclosure generally refers to an amount sufficient for the composition to accomplish a stated purpose relative to the absence of the composition (e.g., achieve the effect for which it is administered, stimulate an immune response, prevent or treat a disease, or reduce one or more symptoms of a disease, disorder, infection, or health condition).
• an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.”
• a “reduction” of a symptom means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s).
• the exact amount of a composition including a “therapeutically effective amount” will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols.
• nucleic acid construct refers to a recombinant nucleic acid molecule including one or more isolated nucleic acid sequences from heterologous sources.
• nucleic acid constructs of the disclosure can be chimeric nucleic acid molecules in which two or more nucleic acid sequences of different origin are assembled into a single nucleic acid molecule.
• nucleic acid constructs include any constructs that contain (1) nucleic acid sequences, including regulatory and coding sequences that are not found adjoined to one another in nature (e.g, at least one of the nucleotide sequences is heterologous with respect to at least one of its other nucleotide sequences), or (2) sequences encoding parts of functional RNA molecules or proteins not naturally adjoined, or (3) parts of promoters that are not naturally adjoined.
• nucleic acid constructs can include any recombinant nucleic acid molecules, linear or circular, single-stranded or double-stranded DNA or RNA nucleic acid molecules, derived from any source, such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid sequences have been operably linked.
• Constructs of the present disclosure can include the necessary elements to direct expression of a nucleic acid sequence of interest that is also contained in the construct. Such elements can include control elements such as a promoter that is operably linked to (so as to direct transcription of) the nucleic acid sequence of interest, and optionally includes a polyadenylation sequence.
• the nucleic acid construct can be incorporated within a vector.
• vector is used herein to refer to a nucleic acid molecule or sequence capable of transferring or transporting another nucleic acid molecule.
• vector encompasses both DNA-based vectors and RNA-based vectors.
• vector includes cloning vectors and expression vectors, as well as viral vectors and integrating vectors.
• An “expression vector” is a vector that includes a regulatory region, thereby capable of expressing DNA sequences and fragments in vitro, ex vivo, and/or in vivo.
• a vector may include sequences that direct autonomous replication in a cell such as, for example a plasmid (DNA-based vector) or a self-replicating RNA vector.
• a vector may include sequences sufficient to allow integration into host cell DNA.
• a vector may include DNA sequences that can be transcribed into RNA in vitro and/or in vivo.
• Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors.
• the vector of the disclosure can be single-stranded vector (e.g., ssDNA or ssRNA). In some embodiments, the vector of the disclosure can be double-stranded vector (e.g., dsDNA or dsRNA). In some embodiments, a vector is a gene delivery vector. In some embodiments, a vector is used as a gene delivery vehicle to transfer a gene into a cell.
• the vector can include, for example, one or more selectable markers, one or more origins of replication, such as prokaryotic and eukaryotic origins, at least one multiple cloning site, and/or elements to facilitate stable integration of the construct into the genome of a cell.
• Two or more constructs can be incorporated within a single nucleic acid molecule, such as a single vector, or can be containing within two or more separate nucleic acid molecules, such as two or more separate vectors.
• An “expression construct” generally includes at least a control sequence operably linked to a nucleotide sequence of interest.
• promoters in operable connection with the nucleotide sequences to be expressed are provided in expression constructs for expression in a cell.
• compositions and methods for preparing and using constructs and cells are known to one skilled in the art.
• a composition of the disclosure e.g, nucleic acid constructs, recombinant cells, recombinant polypeptides, and/or pharmaceutical compositions
• a composition of the disclosure generally refers to an amount sufficient for the composition to accomplish a stated purpose relative to the absence of the composition (e.g., achieve the effect for which it is administered, stimulate an immune response, prevent or treat a disease, or reduce one or more symptoms of a disease, disorder, infection, or health condition).
• an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.”
• a “reduction” of a symptom means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s).
• the exact amount of a composition including a “therapeutically effective amount” will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols.
• operably linked denotes a physical or functional linkage between two or more elements, e.g., polypeptide sequences or polynucleotide sequences, which permits them to operate in their intended fashion.
• operably linked when used in context of the nucleic acid molecules described herein or the coding sequences and promoter sequences in a nucleic acid molecule means that the coding sequences and promoter sequences are in-frame and in proper spatial and distance away to permit the effects of the respective binding by transcription factors or RNA polymerase on transcription. It should be understood that operably linked elements can be contiguous or non-contiguous (e.g, linked to one another through a linker).
• operably linked refers to a physical linkage (e.g., directly or indirectly linked) between amino acid sequences (e.g., different segments, portions, regions, or domains) to provide for a described activity of the constructs.
• Operably linked segments, portions, regions, and domains of the polypeptides or nucleic acid molecules disclosed herein can be contiguous or non-contiguous (e.g., linked to one another through a linker).
• percent identity refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acids that are the same (e.g., about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher sequence identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection.
• sequences are then said to be “substantially identical.”
• This definition also refers to, or can be applied to, the complement of a sequence.
• This definition also includes sequences that have deletions and/or additions, as well as those that have substitutions.
• Sequence identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux et al, Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, FASTA (Atschul et al., J Mol Biol 215:403, 1990). Sequence identity can be measured using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), with the default parameters thereof.
• portion can refer to a fraction.
• a portion of an amino acid sequence comprises at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, and at least 90% of the amino acids of said amino acid sequence.
• said discontinuous fraction is composed of 2, 3, 4, 5, 6, 7, 8, or more parts of a structure, each part being a continuous element of the structure.
• a discontinuous fraction of an amino acid sequence can be composed of 2, 3, 4, 5, 6, 7, 8, or more, for example not more than 4 parts of said amino acid sequence, wherein each part comprises at least 2, 3, 4, 5 continuous amino acids, at least 10 continuous amino acids, at least 20 continuous amino acids, at least 30 continuous amino acids of the amino acid sequence.
• pharmaceutically acceptable excipient refers to any suitable substance that provides a pharmaceutically acceptable carrier, additive, or diluent for administration of a compound(s) of interest to a subject.
• pharmaceutically acceptable excipient can encompass substances referred to as pharmaceutically acceptable diluents, pharmaceutically acceptable additives, and pharmaceutically acceptable carriers.
• pharmaceutically acceptable carrier includes, but is not limited to, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds (e.g., antibiotics and additional therapeutic agents) can also be incorporated into the compositions.
• recombinant when used with reference to a cell, a nucleic acid, a protein, or a vector, indicates that the cell, nucleic acid, protein or vector has been altered or produced through human intervention such as, for example, has been modified by or is the result of laboratory methods.
• recombinant proteins and nucleic acids include proteins and nucleic acids produced by laboratory methods.
• Recombinant proteins can include amino acid residues not found within the native (non-recombinant or wild-type) form of the protein or can be include amino acid residues that have been modified, e.g., labeled.
• the term can include any modifications to the peptide, protein, or nucleic acid sequence.
• Such modifications may include the following: any chemical modifications of the peptide, protein or nucleic acid sequence, including of one or more amino acids, deoxyribonucleotides, or ribonucleotides; addition, deletion, and/or substitution of one or more of amino acids in the peptide or protein; creation of a fusion protein, e.g., a fusion protein comprising an antibody fragment; and addition, deletion, and/or substitution of one or more of nucleic acids in the nucleic acid sequence.
• ’’recombinant when used in reference to a cell is not intended to include naturally-occurring cells but encompass cells that have been engineered/modified to include or express a polypeptide or nucleic acid that would not be present in the cell if it was not engineered/modified.
• replicon RNA refers to RNA which contains all of the genetic information required for directing its own amplification or selfreplication within a permissive cell. Therefore, replicon RNA is sometimes also referred to as “self-amplifying RNA” (saRNA) or “self-replicating RNA” (srRNA).
• saRNA self-amplifying RNA
• srRNA self-replicating RNA
• the RNA molecule 1) encodes polymerase, replicase, or other proteins which may interact with viral or host cell-derived proteins, nucleic acids or ribonucleoproteins to catalyze the RNA amplification process; and 2) contain c/.s-acting RNA sequences required for replication and transcription of the subgenomic replicon-encoded RNA. These sequences may be bound during the process of replication to its self-encoded proteins, or non-self-encoded cell-derived proteins, nucleic acids or ribonucleoproteins, or complexes between any of these components.
• an alphavirus replicon RNA molecule (e.g., srRNA or saRNA molecule) generally contains the following ordered elements: 5' viral RNA sequence(s) required in cis for replication, sequences coding for biologically active alphavirus non-structural proteins (e.g., nsPl, nsP2, nsP3, and nsP4), promoter for the subgenomic RNA (sgRNA), 3' viral sequences required in cis for replication, and a polyadenylate tract (poly(A)).
• replicon RNA e.g., srRNA or saRNA molecule
• the replicon RNA may be of length different from that of any known, naturally-occurring alphavirus.
• the replicon RNA does not contain the sequences of at least one of structural viral protein; and/or sequences encoding structural genes can be substituted with heterologous sequences.
• the replicon RNA can contain one or more sequences, so-called packaging signals, which serve to initiate interactions with alphavirus structural proteins that lead to particle formation.
• a “subject” or an “individual” includes animals, such as human (e.g., human individuals) and non-human animals.
• a “subject” or “individual” is a patient under the care of a physician.
• the subject can be a human patient or an individual who has, is at risk of having, or is suspected of having a health condition of interest (e.g., cancer or infection) and/or one or more symptoms of the health condition.
• the subject can also be an individual who is diagnosed with a risk of the health condition and/or disease of interest at the time of diagnosis or later.
• non-human animals includes all vertebrates, e.g., mammals, e.g., rodents, e.g., mice, non-human primates, and other mammals, such as e.g., sheep, dogs, cows, chickens, and non-mammals, such as amphibians, reptiles, etc.
• mammals e.g., rodents, e.g., mice, non-human primates, and other mammals, such as e.g., sheep, dogs, cows, chickens, and non-mammals, such as amphibians, reptiles, etc.
• a range includes each individual member.
• a group having 1- 3 articles refers to groups having 1, 2, or 3 articles.
• a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.
• Alphaviruses are small, enveloped RNA viruses with a single-stranded, positivesense RNA genome.
• the alphavirus genus includes, inter alia, the Sindbis virus (SINV), the Semliki Forest virus (SFV), the Ross River virus (RRV), Venezuelan equine encephalitis virus (VEEV), and Eastern equine encephalitis virus (EEEV), which are all closely related and are able to infect various vertebrates such as mammalians, rodents, fish, avian species, and larger mammals such as humans and horses as well as invertebrates such as insects.
• Sindbis virus SINV
• SFV Semliki Forest virus
• RRV Ross River virus
• VEEV Venezuelan equine encephalitis virus
• EEEV Eastern equine encephalitis virus
• Non-limiting exemplary alphavirus species include Aura virus (AURAV), Babanki virus (BABV), Barmah Forest virus (BFV), Bebaru virus (BEBV), Buggy Creek virus, Caaingua virus, Cabassou virus, Chikungunya virus (CHIKV), Eastern equine encephalitis virus (EEEV), Eilat virus, Everglades virus (EVEV), Fort Morgan virus (FMV), Getah virus (GETV), Highlands J virus (HJV), Kyzylagach virus (KYZV), Madariaga virus (MADV), Mayaro virus (MAYV), Middelburg virus (MIDV), Mosso das Pedras virus, Mucambo virus (MUCV), Ndumu virus (NDUV), O'nyong'nyong virus (ONNV), Pixuna virus (PIXV), Rio Negro virus (AURAV), Babanki virus (BABV), Barmah Forest virus (BFV), Bebaru virus (BEBV), Buggy Creek virus, Caaing
• the alphavirus genome is approximately 12 kb long, and it consists of two open reading frames (ORFs): a 7 kb frame encoding the nonstructural proteins (nsPs) and a 4 kb frame encoding the structural polyprotein.
• the non-structural polyprotein (nsP) is cleaved into four different proteins (nsPl, nsP2, nsP3, and nsP4) which are necessary for the transcription and translation of viral mRNA inside the cytoplasm of host cells.
• the nsPl protein is an mRNA capping enzyme that possesses both guanine-7- methyltransf erase (MTase) and guanylyltransferase (GTase) activities, where they direct the methylation and capping of newly synthesized viral genomic and subgenomic RNAs.
• MTase guanine-7- methyltransf erase
• GTase guanylyltransferase
• the MTase motif in the N-terminal domain of nsPl catalyzes the transfer of the methyl group from S- adenosylmethionine (AdoMet) to the N7 position of a GTP molecule (m7Gppp).
• GTase then binds the m7Gppp, forming a covalent link with a catalytic histidine (m7Gp-GTase) and releasing PPi.
• the GTase then transfers the m7Gp molecule to the 5 ’-diphosphate RNA to create m7GpppNp-RNA.
• the resulting cap structure is essential for viral mRNA translation and prevents the mRNA from being degraded by cellular 5’ exonucleases.
• Following the N-terminal domain are features that allow the association of the nsPl protein to cellular membranes.
• nsPl protein and nsPl- containing replication complex anchor onto the plasma membrane, possibly through nsPl interaction with the membrane’s anionic phospholipids.
• the nsP2 protein possesses numerous enzymatic activities and functional roles.
• the N-terminal region contains a helicase domain that has seven signature motif of Superfamily 1 (SF1) helicases. It functions as an RNA triphosphatase that performs the first of the viral RNA capping reactions. It also functions as a nucleotide triphosphatase (NTPase), fueling the RNA helicase activity.
• the C-terminal region of nsP2 contains a papain-like cysteine protease, which is responsible for processing the viral non-structural polyprotein. The protease recognizes conserved motifs within the polyprotein. This proteolytic function is highly regulated and is modulated by other domains of nsP2.
• the alphavirus nsP2 protein has also been described as a virulence factor responsible for the transcriptional and translational shutoff in infected host cells and the inhibition of interferon (IFN) mediated antiviral responses contributing to the controlling of translational machinery by viral factors.
• IFN interferon
• nsP3 protein has three recognized domains: the N-terminal macrodomain with phosphatase activity and nucleic acid binding ability, the alphavirus unique domain (AUD) and the C-terminal hypervariable domain. It has been demonstrated that the deletion of this domain in SFV nsP3 resulted in low viral pathogenicity, suggesting its importance in viral RNA transcription regulation.
• the nsP4 polymerase is the most highly conserved protein in alphaviruses, with the most divergent being >50% identical in amino acid sequence when compared with other alphaviral nsP4s.
• the nsP4 contains the core RNA-dependent RNA polymerase (RdRp) domain at the C-terminal end, determined to be solely responsible for the RNA synthetic properties of the viral replication complex.
• the RdRp participates in replicating the genomic RNA via a negative strand RNA and transcribing the 26S subgenomic RNA.
• the N-terminal domain is alphavirus-specific and can be partially disordered structurally.
• nSPs nonstructural proteins
• nsP3 creates a ring structure that encircles nsP2. These two proteins have an extensive interface. Mutations in nsP2 that produce noncytopathic viruses or a temperature sensitive phenotypes cluster at the P2/P3 interface region. P3 mutations opposite the location of the nsP2 noncytopathic mutations prevent efficient cleavage of P2/3. This in turn can affect RNA infectivity altering viral RNA production levels.
• the 3’ one-third of the genome comprises subgenomic RNA which serves as a template for translation of all the structural proteins required for forming viral particles: the core nucleocapsid protein C, and the envelope proteins P62 and El that associate as a heterodimer.
• the viral membrane-anchored surface glycoproteins are responsible for receptor recognition and entry into target cells through membrane fusion.
• the subgenomic RNA is transcribed from the p26S subgenomic promoter present at the 3’ end of the RNA sequence encoding the nsP4 protein.
• the proteolytic maturation of P62 into E2 and E3 causes a change in the viral surface.
• glycoprotein “spikes” form an E1/E2 dimer or an E1ZE2/E3 trimer, where E2 extends from the center to the vertices, El fills the space between the vertices, and E3, if present, is at the distal end of the spike.
• El Upon exposure of the virus to the acidity of the endosome, El dissociates from E2 to form an El homotrimer, which is necessary for the fusion step to drive the cellular and viral membranes together.
• the alphaviral glycoprotein El is a class II viral fusion protein, which is structurally different from the class I fusion proteins found in influenza virus and HIV.
• the E2 glycoprotein functions to interact with the nucleocapsid through its cytoplasmic domain, while its ectodomain is responsible for binding a cellular receptor. Most alphaviruses lose the peripheral protein E3, while in Semliki viruses it remains associated with the viral surface.
• RNA polyprotein nsPl-4
• RNA polymerase activity that produces a negative strand complementary to the genomic RNA.
• the negative strand is used as a template for the production of two RNAs, respectively: (1) a positive genomic RNA corresponding to the genome of the secondary viruses producing, by translation, other nsP and acting as a genome for the virus; and (2) subgenomic RNA encoding the structural proteins of the virus forming the infectious particles.
• the positive genomic RNA/subgenomic RNA ratio is regulated by proteolytic autocleavage of the polyprotein to nsPl, nsP2, nsP3 and nsP4.
• the viral gene expression takes place in two phases. In a first phase, there is main synthesis of positive genomic strands and of negative strands. During the second phase, the synthesis of subgenomic RNA is virtually exclusive, thus resulting in the production of large amount of structural protein.
• nucleic acid constructs a nucleic acid sequence encoding a modified viral genome or replicon RNA (e.g., self-replicating RNA) of an alphavirus species, a recombinant cell comprising the nucleic acid construct, a transgenic animal comprising the nucleic acid construct, and a recombinant polypeptide produced by the methods of the present disclosure.
• a modified viral genome or replicon RNA e.g., self-replicating RNA
• Some embodiments of the disclosure provide a modified alphavirus genome or replicon RNA (e.g., self-replicating RNA) in which at least one structural protein (nsP), or a portion thereof, of the modified alphavirus genome or RNA replicon is heterologous relative to the remainder of the modified alphavirus genome or RNA replicon, wherein the at least one heterologous nsP or portion thereof is nsPl, nsP2, nsP3, nsP4, or a portion of any thereof, or a combination of any of the foregoing.
• nsPl or a portion thereof is heterologous relative to the remainder of the modified alphavirus genome or RNA replicon.
• nsP2 or a portion thereof is heterologous relative to the remainder of the modified alphavirus genome or RNA replicon.
• nsP3 or a portion thereof is heterologous relative to the remainder of the modified alphavirus genome or RNA replicon.
• nsP4 or a portion thereof is heterologous relative to the remainder of the modified alphavirus genome or RNA replicon.
• two nsP proteins are heterologous relative to the remainder of the modified alphavirus genome or RNA replicon.
• three nsP proteins are heterologous relative to the remainder of the modified alphavirus genome or RNA replicon.
• nsPl, nsP2, and nsP3 or a portion thereof are heterologous relative to the remainder of the modified alphavirus genome or RNA replicon. In some embodiments, nsPl, nsP2, and nsP4 or a portion thereof are heterologous relative to the remainder of the modified alphavirus genome or RNA replicon. In some embodiments, nsP2, nsP3, and nsP4 or a portion thereof are heterologous relative to the remainder of the modified alphavirus genome or RNA replicon.
• nucleic acid constructs including a nucleic acid sequence encoding a modified genome or replicon RNA (e.g., self-replicating RNA) of an alphavirus, such as Sindbis virus (SINV), wherein at least one nonstructural protein (nsP), or a portion thereof, of the modified alphavirus genome or RNA replicon is heterologous relative to the remainder of the modified alphavirus genome or RNA replicon.
• a modified genome or replicon RNA e.g., self-replicating RNA
• an alphavirus such as Sindbis virus (SINV)
• nsP nonstructural protein
• the at least one heterologous nsP or portion thereof is nsPl, nsP2, nsP3, nsP4, or a portion of any thereof, or a combination of any of the foregoing.
• a portion of a nucleic acid sequence encoding a nonstructural polypeptide can include enough of the nucleic acid sequence encoding the nonstructural polypeptide to afford putative identification of that polypeptide, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (see, for example, in “Basic Local Alignment Search Tool”; Altschul SF et al.. J. Mol. Biol.
• a portion of a nucleotide sequence comprises enough of the sequence to afford specific identification and/or isolation of a nucleic acid fragment comprising the sequence.
• a portion of a nucleic acid sequence can include at least about 20%, for example, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% of the full-length nucleic acid sequence.
• Non-limiting exemplary alphavirus species suitable for the compositions and methods of the present disclosure include Aura virus (AURAV), Babanki virus (BABV), Barmah Forest virus (BFV), Bebaru virus (BEBV), Buggy Creek virus, Caaingua virus, Cabassou virus, Chikungunya virus (CHIKV), Eastern equine encephalitis virus (EEEV), Eilat virus, Everglades virus (EVEV), Fort Morgan virus (FMV), Getah virus (GETV), Highlands J virus (HJV), Kyzylagach virus (KYZV), Madariaga virus (MADV), Mayaro virus (MAYV), Middelburg virus (MIDV), Mosso das Pedras virus, Mucambo virus (MUCV), Ndumu virus (NDUV), O'nyong'nyong virus (ONNV), Pixuna virus (PIXV), Rio Negro virus (RNV), Ross River virus (RRV), Salmon pancreas disease virus (SPDV), Sem
• the alphavirus is Venezuelan equine encephalitis virus (VEEV). In some embodiments, the alphavirus is Eastern Equine Encephalitis virus (EEEV). In some embodiments, the alphavirus is Western Equine Encephalitis virus (WEEV).
• VEEV Venezuelan equine encephalitis virus
• EEEV Eastern Equine Encephalitis virus
• WEEV Western Equine Encephalitis virus
• the alphavirus is Chikungunya virus (CHIKV).
• CHIKV strains suitable for the compositions and methods of the disclosure include CHIKV S27, CHIKV LR2006-OPY-1, CHIKV YO123223, CHIKV DRDE, CHIKV 37997, CHIKV 99653, CHIKV Ag41855, and Nagpur (India) 653496 strain.
• Virulent and avirulent CHIKV strains are both suitable.
• Additional examples of CHIKV strains suitable for the compositions and methods of the disclosure include but are not limited to those described in Afireen et al. Microbiol. Immunol.
• the modified CHIKV genome or replicon RNA (e.g., self-replicating RNA) is derived from CHIKV strain S27.
• the modified CHIKV genome or replicon RNA is derived from CHIKV strain DRDE.
• the modified CHIKV genome or replicon RNA is derived from CHIKV strain DRDE-06.
• the modified CHIKV genome or replicon RNA is derived from CHIKV strain DRDE-07.
• the alphavirus is Eastern Equine Encephalitis virus (EEEV).
• EEEV strains suitable for the compositions and methods of the disclosure include EEEV 792138, 783372, BeAn5122, BeAr300851, BeAr436087, C-49, FL91- 4679, FL93-939, GML903836, MP-9, PE6, and V105-00210.
• Virulent and avirulent EEEV strains are both suitable.
• Additional suitable EEEV strains include, but are not limited to those described in the Virus Pathogen Resource website (ViPR; which is publicly available at www.viprbrc.org/brc/vipr_genome_search.
• the modified EEEV genome or replicon RNA (e.g., self-replicating RNA) is derived from EEEV strain FL93-939.
• the alphavirus is Sindbis virus (SINV).
• the modified genome or RNA replicon e.g., self-replicating RNA
• SINV strains suitable for the compositions and methods of the disclosure include SINV strain AR339, AR86, and Girdwood.
• SINV strains suitable for the compositions and methods of the disclosure include, but are not limited to those described in Sammels et al. J. Gen. Virol. 1999, 80(3):739-748, Lundstrbm and Pfeffer Vector Borne Zoonotic Dis. 2010, 10(9):889-907, Sigei et al. Arch, of Virol.
• the modified genome or RNA replicon is of a SINV strain Girdwood.
• the modified genome or RNA replicon is of a SINV strain AR86.
• the modified SINV genome or replicon RNA is derived from SINV strain Girdwood. In some embodiments, the modified SINV genome or replicon RNA is derived from SINV strain AR86. In some embodiments, the at least one heterologous nsP or portion thereof of the modified genome or RNA replicon is derived from a SINV strain AR86. In some embodiments, the at least one heterologous nsP or portion thereof is nsPl, nsP3, nsP4, or a portion of any thereof, or a combination of any of the foregoing. In some embodiments, the modified genome or RNA replicon is of a SINV strain AR86.
• the alphavirus is Western Equine Encephalitis virus (WEEV).
• WEEV strains suitable for the compositions and methods of the disclosure include WEEV California, McMillan, IMP181, Imperial, Imperial 181, IMPR441, 71V-1658, AG80-646, BFS932, COA592, EP-6, E1416, BFS1703, BFS2005, BSF3060, BSF09997, CHLV53, KERN5547, 85452NM, Montana-64, S8-122, and TBT-235.
• WEEV strains suitable for the compositions and methods of the disclosure include 5614, 93A27, 93A30, 93A38, 93A79, B628(C1 15), CBA87, CNTR34, CO921356, Fleming, Lake43, PV012357A, PV02808A, PV72102, R02PV001807A, R02PV002957B, R02PV003422B, R05PV003422B, R0PV003814A and R0PV00384A.
• Virulent and avirulent WEEV strains are both suitable.
• Additional suitable WEEV strains include, but are not limited to those described in Bergren NA et al., J. Virol.
• the modified WEEV genome or srRNA is derived from WEEV strain Imperial. In some embodiments, the modified WEEV genome or srRNA is derived from WEEV strain McMillan.
• At least one heterologous nsP or a portion thereof is derived from another strain of the same alphavirus species. In some embodiments, at least one heterologous nsP or portion thereof is derived from another alphavirus species.
• at least one heterologous nsP or portion thereof of the modified SINV-AR86 genome or RNA replicon e.g., self-replicating RNA is derived from a SINV strain Girdwood. In some embodiments, at least one heterologous nsP or portion thereof of the modified SINV- AR86 genome or RNA replicon is derived from nsP2 of a SINV strain Girdwood.
• the structural proteins nsPl, nsP3, and nsP4 are from SINV strain AR86, while nsP2 is from SINV strain Girdwood.
• nsP4 is from AR86 strain
• nsPl, nsP2, and nsP3 are from Girdwood strain.
• nsP3 is from AR86 strain
• nsPl, nsP2, and nsP4 are from Girdwood strain.
• nsPl are from AR86 strain
• nsP2, nsP3, whereas nsP4 is from Girdwood (see, e.g., FIGS. 1 and 6).
• the modified viral genome or replicon RNA (e.g., self-replicating RNA) is devoid of the entire sequence encoding viral structural proteins, e.g., the modified viral genome or replicon RNA includes no nucleic acid sequence encoding the structural proteins of the viral unmodified genome or replicon RNA.
• the modified alphavirus genome or RNA replicon is devoid of at least a portion of the nucleic acid sequence encoding one or more viral structural proteins.
• the modified viral genome or RNA replicon is devoid of a substantial portion of the nucleic acid sequence encoding one or more viral structural proteins. In some embodiments, the modified viral genome or RNA replicon comprises no nucleic acid sequence encoding viral structural proteins.
• a substantial portion of a nucleic acid sequence encoding a viral structural polypeptide can include enough of the nucleic acid sequence encoding the viral structural polypeptide to afford putative identification of that polypeptide, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (see, for example, in “Basic Local Alignment Search Tool”; Altschul SF et al., J.
• a substantial portion of a nucleotide sequence comprises enough of the sequence to afford specific identification and/or isolation of a nucleic acid fragment comprising the sequence.
• a substantial portion of a nucleic acid sequence can include at least about 20%, for example, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% of the full-length nucleic acid sequence.
• the present disclosure provides nucleic acid molecules and constructs which are devoid of partial or complete nucleic acid sequences encoding one or more viral structural proteins.
• the nucleic acid constructs of the disclosure include a nucleic acid sequence encoding a modified alphavirus genome or replicon RNA (e.g., selfreplicating RNA), wherein a substantial portion of the nucleic acid sequence encoding one or more structural proteins of the modified alphavirus genome or replicon RNA has been removed, e.g., the modified alphavirus genome or replicon RNA does not include at least a portion of the coding sequence for one or more of the alphavirus structural proteins CP, El, E2, E3, and 6K.
• a modified alphavirus genome or replicon RNA e.g., selfreplicating RNA
• the modified alphavirus genome or replicon RNA does not include at least a portion of the coding sequence for one or more of the alphavirus structural proteins CP, El, E2, E3, and 6K.
• Non-limiting exemplary embodiments of the nucleic acid constructs of the disclosure can include one or more of the following features.
• at least a portion of the nucleic acid sequence encoding one or more of the viral structural proteins CP, El, E2, E3, and 6K of the unmodified viral genome or replicon RNA (e.g., self-replicating RNA) has been removed.
• a portion of or the entire sequence encoding CP has been removed.
• a portion of or the entire sequence encoding El has been removed.
• a portion of or the entire sequence encoding E2 has been removed.
• a portion of or the entire sequence encoding E3 has been removed.
• a portion of or the entire sequence encoding 6K has been removed. In some embodiments, a portion of or the entire sequence encoding a combination of CP, El, E2, E3, and 6K has been removed. In some embodiments, the entire sequence encoding viral structural proteins has been removed, e.g., the modified viral genome or replicon RNA includes no nucleic acid sequence encoding the structural proteins of the viral unmodified genome or replicon RNA.
• Non-limiting exemplary nucleic acid constructs of the present disclosure can comprise a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-4.
• nucleic acid constructs of the present disclosure can comprise a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of nucleic acid sequence selected from the group consisting of SEQ ID NO: 1.
• nucleic acid constructs of the present disclosure can comprise a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of nucleic acid sequence selected from the group consisting of SEQ ID NO: 2.
• nucleic acid constructs of the present disclosure can comprise a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of nucleic acid sequence selected from the group consisting of SEQ ID NO: 3.
• nucleic acid constructs of the present disclosure can comprise a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of nucleic acid sequence selected from the group consisting of SEQ ID NO: 4.
• the nucleic acid constructs (e.g., vectors or srRNA constructs) of the disclosure generally have a length of at least about 2 kb.
• the nucleic acid constructs (e.g., vectors or srRNAs) can have a length of at least about 2 kb, at least about 3 kb, at least about 4 kb, at least about 5 kb, at least about 6 kb, at least about 7 kb, at least about 8 kb, at least about 9 kb, at least about 10 kb, at least about 11 kb, at least about 12 kb or more than 12 kb.
• the nucleic acid constructs can have a length of about 4 kb to about 20 kb, about 4 kb to about 18 kb, about 5 kb to about 16 kb, about 6 kb to about 14 kb, about 7 kb to about 12 kb, about 8 kb to about 16 kb, about 9 kb to about 14 kb, about 10 kb to about 18 kb, about 11 kb to about 16 kb, about 5 kb to about 18 kb, about 6 kb to about 20 kb, about 5 kb to about 10 kb, about 5 kb to about 8 kb, about 5 kb to about 7 kb, about 5 kb to about 6 kb, about 6 kb to about 12 kb, about 6 kb to about 11 kb, about 6 kb to about 10 kb, about 6 kb to about to about 6 kb to about 6 kb to about 6 kb
• the nucleic acid constructs can have a length of about 6 kb to about 14 kb. In some embodiments, the nucleic acid constructs (e.g., vectors or srRNAs) can have a length of about 6 kb to about 16 kb.
• the nucleic acid constructs of the disclosure further include one or more expression cassettes.
• the nucleic acid constructs disclosed herein can generally include any number of expression cassettes.
• the nucleic acid constructs disclosed herein can include at least two, at least three, at least four, at least five, or at least six expression cassettes.
• expression cassette refers to a construct of genetic material that contains coding sequences and enough regulatory information to direct proper transcription and/or translation of the coding sequences in a cell, in vivo and/or ex vivo.
• the expression cassette can be inserted into a vector for targeting to a desired host cell and/or into a subject.
• the term expression cassette can be used interchangeably with the term “expression construct.”
• expression cassette refers to a nucleic acid construct that includes a gene encoding a protein or functional RNA operably linked to regulatory elements such as, for example, a promoter and/or a termination signal, and optionally, any or a combination of other nucleic acid sequences that affect the transcription or translation of the gene.
• At least one of the expression cassettes can include a promoter operably linked to a heterologous nucleic acid sequence.
• the nucleic acid constructs as provided herein can find use, for example, as an expression vector that, when including a regulatory element (e.g., a promoter) operably linked to a heterologous nucleic acid sequence, can affect expression of the heterologous nucleic acid sequence.
• at least one of the expression cassettes includes a subgenomic (sg) promoter operably linked to a heterologous nucleic acid sequence.
• the sg promoter is a 26S subgenomic promoter.
• the nucleic acid molecules of the disclosure further include one or more untranslated regions (UTRs).
• UTRs untranslated regions
• at least one of the UTRs is a heterologous UTR.
• the 5’ UTR sequence of the modified alphavirus genome or RNA replicon e.g., self-replicating RNA
• the 3’ UTR sequence of the modified alphavirus genome or RNA replicon is a heterologous 3’ UTR sequence.
• both 5’ UTR and 3’ UTR sequences of the modified alphavirus genome or RNA replicon are heterologous UTR sequences.
• the heterologous 5’ UTR and/or 3’ UTR sequences can be from Chikungunya virus.
• the heterologous 5’ UTR and/or 3’ UTR sequences can be from a Chikungunya strain S27.
• the heterologous 5’ UTR and/or 3’ UTR sequences can be from a Chikungunya strain DRDE.
• At least one of expression cassettes includes a coding sequence for a gene of interest (GO I).
• the coding sequence of the GOI is redesigned and/or optimized for a desired property, such as increased stability, potency, and expression (e.g., translation efficiency), which in turns can maximize the impact of producing, delivering, and administering biotherapeutics.
• the coding sequence of the GOI is optimized for expression at a level higher than the expression level of a reference coding sequence, for example, 20% higher, 30% higher, 40% higher, 50% higher, 60% higher, 70% higher, 80% higher, 90% higher, or 95% higher than a reference coding sequence.
• the reference coding sequence is a wild-type non-optimized sequence.
• degeneracy of the genetic code provides the possibility to substitute at least one base of the protein encoding sequence of a gene with a different base without causing the amino acid sequence of the polypeptide produced from the gene to be changed.
• the nucleic acid constructs of the present disclosure can also have any base sequence that has been changed from any polynucleotide sequence disclosed herein by substitution in accordance with degeneracy of the genetic code. References describing codon usage are readily publicly available.
• polynucleotide sequence variants can be produced for a variety of reasons, e.g., to optimize expression for a particular host (e.g., changing codon usage in the alphavirus mRNA to those preferred by other organisms such as human, non-human primates, hamster, mice, or monkey).
• the coding sequence of the GOI is optimized for expression at a level higher than the expression level of a reference coding sequence, such as, for example, a coding sequence that has not been codon-optimized in a target host cell through the use of codons optimized for expression.
• the codon-optimized sequence of the GOI results in an increased expression level by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% compared to a reference coding sequence that has not been codon-optimized. In some embodiments, the codon-optimized sequence of the GOI results in an increased expression level by at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold compared to a reference coding sequence that has not been codon-optimized. [0097] In some embodiments, the coding sequence of the GOI is optimized for enhanced RNA stability and/or expression.
• RNA stability generally relates to the “half-life” of RNA.
• “Half-life” relates to the period of time which is needed to eliminate half of the activity, amount, or number of molecules.
• the half-life of an RNA is indicative for the stability of said RNA.
• the half-life of RNA may influence the “duration of expression” of the RNA.
• replicons e.g., self-replicating RNAs
• GOI codon usage e.g., GOI codon usage
• replicon potency e.g., examine dsRNA in cells following transfection
• the polypeptide encoded by a GOI can generally be any polypeptide, and can be, for example a therapeutic polypeptide, a prophylactic polypeptide, a diagnostic polypeptide, a nutraceutical polypeptide, an industrial enzyme, and a reporter polypeptide.
• the GOI encodes a polypeptide selected from the group consisting of an antibody, an antigen, an immune modulator, an enzyme, a signaling protein, and a cytokine.
• the GOI encodes a polypeptide that can be an antibody, an antigen, an immune modulator, an enzyme, a signaling protein, or a cytokine.
• the GOI can encode microbial proteins, viral proteins, bacterial proteins, fungal proteins, mammalian proteins, and combinations of any thereof.
• Non-limiting examples of GOI include interleukins and interacting proteins, such as G-CSF, GM-CSF, IL-1, IL-10, IL-10-like, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-18BP, IL-l-like, IL-IRA, IL-la, IL-lp, IL-2, IL-20, IL-3, IL-4, IL-5, IL-6, IL-6-like, IL-7, IL-9, IL-21, IL-22, IL-33, IL-37, IL-38, LIF, and OSM.
• interleukins and interacting proteins such as G-CSF, GM-CSF, IL-1, IL-10, IL-10-like, IL-11, IL-12,
• Additional suitable GOIs include, but are not limited to, interferons (e.g., IFN-a, IFN-P, IFN-y), TNFs (e.g., CD154, LT-p, TNF-a, TNF-p, 4-1BBL, APRIL, CD70, CD153, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, and TRANCE), TGF-p (e.g., TGF-pl, TGF-p2, and TGF-P3), hematopoietins (e.g., Epo, Tpo, Flt-3L, SCF, M-CSF, MSP), chemokines and their receptors (e.g., XCL1, XCL2, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19
• Additional GO Is suitable for the compositions and methods of the disclosure include, but are not limited to, immunostimulatory gene products (e.g., CD27/CD70, CD40, CD40L, B7.1, BTLA, MAVS, 0X40, OX40L, RIG-I, and STING), drug resistant mutants/variants of genes, such as ABCB1, ABCC1, ABCG2, AKT1, ALK, BAFF, BCR-ABL, BRAF, CCND1, cMET, EGFR, ERBB2, ERBB3, ERK2, ESRI, GRB2, KRAS, MDR1, MRP1, NTRK1, PDC4, P-gp, PI3K, PTEN, RET, ROS1, RSK1, RSK2, SHIP, and STK11.
• immunostimulatory gene products e.g., CD27/CD70, CD40, CD40L, B7.1, BTLA, MAVS, 0X40, OX40L, RIG-I, and STING
• the GOI can encode an antibody or antibody variant (e.g. single chain Fv, bi-specifics, camelids, Fab, and HCAb).
• the antibody targets surface molecules associated or upregulated with cancers, or surface molecules associated with infectious disease.
• the antibody targets surface molecules having immunostimulatory function, or having immunosuppressive function.
• the GOI can encode an enzyme whose deficiency or mutation is associated with diseases or health conditions, such as, for example, agalsidase beta, agalsidase alfa, imiglucerase, taliglucerase alfa, velaglucerase alfa, alglucerase, sebelipase alpha, laronidase, idursulfase, elosulfase alpha, galsulfase, alglucosidase alpha, and CTFR.
• diseases or health conditions such as, for example, agalsidase beta, agalsidase alfa, imiglucerase, taliglucerase alfa, velaglucerase alfa, alglucerase, sebelipase alpha, laronidase, idursulfase, elosulfase alpha, galsulfase, alglucosidase alpha, and CT
• the GOI can encode a polypeptide selected from antigen molecules, biotherapeutic molecules, or combinations of any thereof.
• the GOI can encode a polypeptide selected from tumor-associated antigens, tumor-specific antigens, neoantigens, and combinations of any thereof.
• the GOI can encode a polypeptide selected from estrogen receptors, intracellular signal transducer enzymes, and human epidermal growth receptors.
• the GOI can encode a biotherapeutic polypeptide selected from immunomodulators, modulators of angiogenesis, modulators of extracellular matrix, modulators of metabolism, neurological modulators, and combinations of any thereof.
• the GOI can encode a cytokine selected from chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors.
• the GOI can encode an interleukins selected from IL-la, IL-ip, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, IL-15, IL-17, IL-23, IL-27, IL-35, IFNv and subunits of any thereof.
• the GOI can encode a biotherapeutic polypeptide is selected from IL-12A, IL-12B, IL-IRA, and combinations of any thereof.
• the nucleic acid construct of the disclosure may be incorporated within a vector.
• the vector of the disclosure may be singlestranded vector, e.g., ssDNA vector or ssRNA vector.
• the vector of the disclosure can be double-stranded vector, e.g., dsDNA vector or dsRNA vector.
• the vector of the disclosure can be a plasmid.
• the vector of the disclosure can be produced using recombinant DNA technology, e.g., polymerase chain reaction (PCR) amplification, rolling circle amplification (RCA), molecular cloning, etc., or chemical synthesis.
• the vector of the disclosure can be a fully synthetic vector, e.g., fully synthetic ssDNA vector. In some embodiments, the vector of the disclosure can be a fully synthetic dsDNA vector. In some embodiments, the vector of the disclosure can be a product of a PCR reaction. In some embodiments, the vector of the disclosure can be a product of an RCA reaction. In some embodiments, a vector can be a gene delivery vector. In some embodiments, a vector can be used as a gene delivery vehicle to transfer a gene into a cell.
• the polypeptide encoded by the GOI is a recombinant polypeptide.
• the GOI encodes an antigenic HA polypeptide of avian influenza A H5N1.
• the GOI encodes a protein relevant to oncology such as ESRI, HER2, and HER3 or a portion thereof.
• the GOI encodes a cytokine such as IL- IRA or IL- 12.
• the nucleic acid constructs of the disclosure include a nucleic acid sequence encoding a modified genome or RNA replicon (e.g, self-replicating RNA) of an alphavirus species having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-4.
• a modified genome or RNA replicon e.g, self-replicating RNA
• the nucleic acid constructs of the disclosure include a nucleic acid sequence encoding a modified genome or RNA replicon of an alphavirus species having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 1.
• the nucleic acid constructs of the disclosure include a nucleic acid sequence encoding a modified genome or RNA replicon of an alphavirus species having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 2.
• the nucleic acid constructs of the disclosure include a nucleic acid sequence encoding a modified genome or RNA replicon of an alphavirus species having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 3.
• the nucleic acid constructs of the disclosure include a nucleic acid sequence encoding a modified genome or RNA replicon of an alphavirus species having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 4.
• Nucleic acid sequences having a high degree of sequence identity e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to a sequence of a modified genome or RNA replicon e.g. self-replicating RNA) of an alphavirus species of interest can be identified and/or isolated by using the sequences identified herein (e.g., SEQ ID NOS: 1-4) or any others as they are known in the art, by genome sequence analysis, hybridization, and/or PCR with degenerate primers or gene-specific primers from sequences identified in the alphavirus species genome.
• the nucleic acid molecules are recombinant nucleic acid molecules.
• the term recombinant nucleic acid molecule means any nucleic acid molecule (e.g. DNA, RNA), that is, or results, however indirect, from human manipulation.
• a cDNA is a recombinant DNA molecule, as is any nucleic acid molecule that has been generated by in vitro polymerase reaction(s), or to which linkers have been attached, or that has been integrated into a vector, such as a cloning vector or expression vector.
• a recombinant nucleic acid molecule 1) has been synthesized or modified in vitro, for example, using chemical or enzymatic techniques (for example, by use of chemical nucleic acid synthesis, or by use of enzymes for the replication, polymerization, exonucleolytic digestion, endonucleolytic digestion, ligation, reverse transcription, transcription, base modification (including, e.g., methylation), or recombination (including homologous and site-specific recombination) of nucleic acid molecules; 2) includes conjoined nucleotide sequences that are not conjoined in nature; 3) has been engineered using molecular cloning techniques such that it lacks one or more nucleotides with respect to the naturally occurring nucleotide sequence; and/or 4) has been manipulated using molecular cloning techniques such that it has one or more sequence changes or rearrangements with respect to the naturally occurring nucleotide sequence.
• chemical or enzymatic techniques for example, by
• nucleic acid molecules disclosed herein are produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning, etc.) or chemical synthesis.
• Nucleic acid molecules as disclosed herein include natural nucleic acid molecules and homologs thereof, including, but not limited to, natural allelic variants and modified nucleic acid molecules in which one or more nucleotide residues have been inserted, deleted, and/or substituted, in such a manner that such modifications provide the desired property in effecting a biological activity as described herein.
• a nucleic acid molecule including a variant of a naturally-occurring nucleic acid sequence, can be produced using a number of methods known to those skilled in the art (see, for example, Sambrook et a!.. In: Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)).
• sequence of a nucleic acid molecule can be modified with respect to a naturally-occurring sequence from which it is derived using a variety of techniques including, but not limited to, classic mutagenesis techniques and recombinant DNA techniques, such as but not limited to site-directed mutagenesis, chemical treatment of a nucleic acid molecule to induce mutations, restriction enzyme cleavage of a nucleic acid fragment, ligation of nucleic acid fragments, PCR amplification and/or mutagenesis of selected regions of a nucleic acid sequence, recombinational cloning, and chemical synthesis, including chemical synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules, and combinations thereof.
• classic mutagenesis techniques and recombinant DNA techniques such as but not limited to site-directed mutagenesis
• chemical treatment of a nucleic acid molecule to induce mutations
• Nucleic acid molecule homologs can be selected from a mixture of modified nucleic acid molecules by screening for the function of the protein or the replicon (e.g., srRNA) encoded by the nucleic acid molecule and/or by hybridization with a wild-type gene or fragment thereof, or by PCR using primers having homology to a target or wild-type nucleic acid molecule or sequence.
• replicon e.g., srRNA
• nucleic acid construct e.g., vector or srRNA
• a nucleic acid construct of the present disclosure can be introduced into a host cell to produce a recombinant cell containing the nucleic acid construct and/or srRNA construct.
• the nucleic acid constructs of the present disclosure can be introduced into a host cell to produce a recombinant cell containing the nucleic acid construct.
• prokaryotic or eukaryotic cells that contain a nucleic acid construct encoding a modified genome or RNA replicon (e.g., self-replicating RNA) of an alphavirus species as described herein are also features of the disclosure.
• some embodiments disclosed herein relate to methods of transforming a cell which includes introducing into a host cell, such as an animal cell, a nucleic acid construct as provided herein, and then selecting or screening for a transformed cell.
• nucleic acid constructs of the disclosure into cells can be achieved by methods known to those skilled in the art such as, for example, viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)- mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, direct micro-injection, nanoparticle-mediated nucleic acid delivery, and the like.
• methods known to those skilled in the art such as, for example, viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)- mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, direct micro-injection, nanoparticle-mediated nucleic acid delivery, and the like.
• PEI polyethyleneimine
• some embodiments of the disclosure relate to recombinant cells, for example, recombinant eukaryotic cells, e.g., insect cells or animal cells that include a nucleic acid construct described herein.
• the nucleic acid construct can be stably integrated in the host genome, or can be episomally replicating, or present in the recombinant host cell as a mini-circle expression vector for a stable or transient expression. Accordingly, in some embodiments of the disclosure, the nucleic acid construct is maintained and replicated in the recombinant host cell as an episomal unit. In some embodiments, the nucleic acid construct is stably integrated into the genome of the recombinant cell.
• Stable integration can be completed using classical random genomic recombination techniques or with more precise genome-editing techniques such as using guide RNA directed CRISPR/Cas9 or TALEN genome editing.
• the nucleic acid construct present in the recombinant host cell as a mini-circle expression vector for a stable or transient expression.
• Host cells can be either untransformed cells or cells that have already been transfected with at least one nucleic acid molecule. Accordingly, in some embodiments, host cells can be genetically engineered (e.g, transduced or transformed or transfected) with at least one nucleic acid molecule.
• Suitable host cells for cloning or expression of the protein of interest as described herein include prokaryotic or eukaryotic cells described herein.
• the recombinant cell is a prokaryotic cell, such as the bacterium E. coh, or a eukaryotic cell, such as an insect cell (e.g, a mosquito cell or a Sf21 cell), or mammalian cells (e.g., COS cells, NIH 3T3 cells, or HeLa cells).
• the cell is in vivo, for example, a recombinant cell in a living body, e.g., cell of a transgenic subject.
• the subject is a vertebrate animal or an invertebrate animal. In some embodiments, the subject is an insect. In some embodiments, the subject is a mammalian subject. In some embodiments, the mammalian subject is a human subject. In some embodiments, the cell is ex vivo, e.g., has been extracted, as an individual cell or as part of an organ or tissue, from a living body or organism for a treatment or procedure, and then returned to the living body or organism. In some embodiments, the cell is in vitro, e.g., is obtained from a repository. In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the recombinant cell is an animal cell. In some embodiments, the animal cell is a vertebrate animal cell or an invertebrate animal cell. In some embodiments, the recombinant cell is a mammalian cell.
• suitable host cells can be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include insect cells. Vertebrate cells can also be used as hosts. In this regard, mammalian cell lines that are adapted to grow in suspension can be useful.
• the recombinant cell is an animal cell. In some embodiments, the animal cell is a vertebrate animal cell or an invertebrate animal cell. In some embodiments, the recombinant cell is a mammalian cell. In some embodiments, the animal cell is a human cell. In some embodiments, the animal cell is a non-human animal cell.
• the cell is a non-human primate cell.
• useful mammalian host cell lines include monkey kidney CV1 cells transformed by SV40 (COS-7), human embryonic kidney cells (e.g., HEK 293 or HEK 293 cell), baby hamster kidney cells (BEK), mouse sertoli cells (e.g., TM4 cells), human cervical carcinoma cells (HeLa), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3 A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor (MMT 060562), TRI cells, , FS4 cells, Chinese hamster ovary cells (CHO cell), African green monkey kidney cells (Vero cells), human A549 cells, human cervix cells, human CHME5 cells, human PER.C6 cells, NSO murine myeloma cells, human epidermoid larynx cells, human fibroblast cells, human HUH-7 cells, human MRC-5 cells, human muscle cells,
• COS-7
• the recombinant cell is selected from the group consisting of African green monkey kidney cell (Vero cell), baby hamster kidney (BHK) cell, Chinese hamster ovary cell (CHO cell), human A549 cell, human cervix cell, human CHME5 cell, human epidermoid larynx cell, human fibroblast cell, human HEK-293 cell, human HeLa cell, human HepG2 cell, human HUH-7 cell, human MRC-5 cell, human muscle cell, mouse 3T3 cell, mouse connective tissue cell, mouse muscle cell, and rabbit kidney cell.
• the recombinant cell is a BHK cell.
• the BHK cell is a BHK-21 cell.
• the recombinant cell is a Vero cell.
• the recombinant cell is an insect cell, e.g., cell of an insect cell line.
• the recombinant cell is a Sf21 cell.
• Additional suitable insect cell lines include, but are not limited to, cell lines established from insect orders Diptera, Lepidoptera and Hemiptera, and can be derived from different tissue sources.
• the recombinant cell is a cell of a lepidopteran insect cell line. In the past few decades, the availability of lepidopteran insect cell lines has increased at about 50 lines per decade. More information regarding available lepidopteran insect cell lines can be found in, e.g., Lynn D.E., Available lepidopteran insect cell lines.
• the recombinant cell is a mosquito cell, e.g., a cell of mosquito species within Anopheles An.), Culex (Cxi) and Aedes (Stegomyia) (Ae.) genera.
• mosquito cell lines suitable for the compositions and methods described herein include cell lines from the following mosquito species: Aedes aegypti, Aedes albopictus, Aedes pseudoscutellaris, Aedes triseriatus, Aedes vexans, Anopheles gambiae, Anopheles stephensi, Anopheles albimanus, Culex quinquefasciatus, Culex theileri, Culex tritaeniorhynchus, Culex bilaeniorhynchus. and Toxorhynchites amboinensis.
• Suitable mosquito cell lines include, but are not limited to, CCL-125, Aag-2, RML-12, C6/26, C6/36, C7-10, AP- 61, A t. GRIP-1, A t. GRIP-2, UM-AVE1, Mos.55, SualB, 4a-3B, Mos.43, MSQ43, and LSB- AA695BB.
• the mosquito cell is a cell of a C6/26 cell line.
• cell cultures including at least one recombinant cell as disclosed herein, and a culture medium.
• the culture medium can be any suitable culture medium for culturing the cells described herein. Techniques for transforming a wide variety of the above-mentioned host cells and species are known in the art and described in the technical and scientific literature. Accordingly, cell cultures including at least one recombinant cell as disclosed herein are also within the scope of this application. Methods and systems suitable for generating and maintaining cell cultures are known in the art.
• transgenic animals including a nucleic acid construct as described herein (e.g., vector or srRNA molecule).
• the transgenic animal is a vertebrate animal or an invertebrate animal.
• the transgenic animal is a mammal.
• the transgenic mammal is a non-human mammal.
• transgenic animals of the present disclosure can be any non-human animal known in the art.
• the non-human animals of the disclosure are non-human primates.
• animal species suitable for the compositions and methods of the disclosure include animals that are (i) suitable for transgenesis and (ii) capable of rearranging immunoglobulin gene segments to produce an antibody response.
• animals that are (i) suitable for transgenesis and (ii) capable of rearranging immunoglobulin gene segments to produce an antibody response.
• examples of such species include but are not limited to mice, rats, hamsters, rabbits, chickens, goats, pigs, sheep and cows.
• non-human animals suitable for the compositions and methods of the disclosure can include, without limitation, laboratory animals (e.g., mice, rats, hamsters, gerbils, guinea pigs, etc.), livestock (e.g., horses, cattle, pigs, sheep, goats, ducks, geese, chickens, etc.), non-human primates (e.g., apes, chimpanzees, orangutans, monkeys, etc.), fish, amphibians (e.g., frogs, salamanders, etc.), reptiles (e.g., snakes, lizards, etc.), and other animals (e.g., foxes, weasels, rabbits, mink, beavers, ermines, otters, sable, seals, coyotes, chinchillas, deer, muskrats, possums, etc.).
• laboratory animals e.g., mice, rats,
• the transgenic animal is an insect. In some embodiments, the insect is a mosquito. In some embodiments, the transgenic animals of the present disclosure are chimeric transgenic animals. In some embodiments, the transgenic animals of the present disclosure are transgenic animals with germ cells and somatic cells containing one or more (e.g., one or more, two or more, three or more, four or more, etc.) nucleic acid constructs of the present disclosure. In some embodiments, the one or more nucleic acid constructs are stably integrated into the genome of the transgenic animals. In some embodiments, the genomes of the transgenic animals of the present disclosure can comprise any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more copies of the one or more nucleic acid constructs of the present disclosure.
• transgenic non-human animals are known in the art. Exemplary methods include pronuclear microinjection, DNA microinjection, lentiviral vector mediated DNA transfer into early embryos and sperm-mediated transgenesis, adenovirus mediated introduction of DNA into animal sperm (e.g., in pig), retroviral vectors (e.g., avian species), somatic cell nuclear transfer (e.g., in goats).
• sperm e.g., in pig
• retroviral vectors e.g., avian species
• somatic cell nuclear transfer e.g., in goats.
• the transgenic non-human host animals of the disclosure are prepared using standard methods known in the art for introducing exogenous nucleic acid into the genome of a non-human animal.
• the transgenic animals of the disclosure can be generated using classical random genomic recombination techniques or with more precise techniques such as guide RNA-directed CRISPR/Cas genome editing, or DNA- guided endonuclease genome editing with NgAgo (Natronobacterium gregoryi Argonaute), or TALENs genome editing (transcription activator-like effector nucleases).
• the transgenic animals of the disclosure can be made using transgenic microinjection technology and do not require the use of homologous recombination technology and thus are considered to be easier to prepare and select than approaches using homologous recombination.
• the transgenic animal produces a protein of interest as described herein.
• compositions including pharmaceutical compositions.
• Such compositions generally include one or more of the nucleic acid constructs, recombinant cells, recombinant polypeptides described and provided herein, and a pharmaceutically acceptable excipient, e.g., carrier.
• the compositions of the disclosure are formulated for the prevention, treatment, or management of a health condition such as an immune disease or a microbial infection.
• compositions of the disclosure can be formulated as a prophylactic composition, a therapeutic composition, or a pharmaceutical composition comprising a pharmaceutically acceptable excipient, or a mixture thereof.
• the compositions of the present disclosure are formulated for use as a vaccine.
• the compositions of the present application are formulated for use as an adjuvant.
• compositions including a pharmaceutically acceptable excipient and: a) a nucleic acid construct (e.g., a vector or a srRNA molecule) of the disclosure; b) a recombinant cell of the disclosure; and/or c) a recombinant polypeptide of the disclosure.
• a nucleic acid construct e.g., a vector or a srRNA molecule
• a recombinant cell of the disclosure e.g., a recombinant cell of the disclosure
• a recombinant polypeptide of the disclosure e.g., a recombinant polypeptide of the disclosure.
• compositions including a nucleic acid construct e.g., a vector or a srRNA molecule as disclosed herein and a pharmaceutically acceptable excipient.
• compositions including a recombinant cell as disclosed herein and a pharmaceutically acceptable excipient.
• the compositions include a recombinant polypeptide of as disclosed herein and a pharmaceutically acceptable excipient.
• the nucleic acid constructs of the disclosure can be used in a naked form or formulated with a delivery vehicle.
• exemplary delivery vehicles suitable for the compositions and methods of the disclosure include, but are not limited to liposomes (e.g., neutral or anionic liposomes), microspheres, immune stimulating complexes (ISCOMS), lipid-based nanoparticles (LNP), solid lipid nanoparticles (SLN), polyplexes, polymer nanoparticles, viral replicon particles (VRPs), or conjugated with bioactive ligands, which can facilitate delivery and/or enhance the immune response.
• Adjuvants other than liposomes and the like are also used and are known in the art.
• Adjuvants may protect the antigen e.g., nucleic acid constructs, vectors, srRNA molecules) from rapid dispersal by sequestering it in a local deposit, or they may contain substances that stimulate the host to secrete factors that are chemotactic for macrophages and other components of the immune system. An appropriate selection can be made by those skilled in the art, for example, from those described below.
• a composition of the disclosure can include one or more of the following: physiologic buffer, a liposome, a lipid-based nanoparticle (LNP), a solid lipid nanoparticle (SLN), a polyplex, a polymer nanoparticle, a viral replicon particle (VRP), a microsphere, an immune stimulating complex (ISCOM), a conjugate of bioactive ligand, or a combination of any thereof.
• the compositions of the disclosure that formulated in a liposome.
• LNPs are generally less immunogenic than viral particles. While many humans have preexisting immunity to viral particles there is no preexisting immunity to LNP. In addition, adaptive immune response against LNP is unlikely to occur which enables repeat dosing of LNP.
• the lipids suitable for the compositions and methods described herein can be cationic lipids, ionizable cationic lipids, anionic lipids, or neutral lipids.
• the LNP of the disclosure can include one or more ionizable lipids.
• ionizable lipid refers to a lipid that is cationic or becomes ionizable (protonated) as the pH is lowered below the pKa of the ionizable group of the lipid, but is more neutral at higher pH values. At pH values below the pKa, the lipid is then able to associate with negatively charged nucleic acids (e.g., oligonucleotides).
• ionizable lipid includes lipids that assume a positive charge on pH decrease from physiological pH, and any of a number of lipid species that carry a net positive charge at a selective pH, such as physiological pH. Permanently cationic lipids such as DOTMA have proven too toxic for clinical use.
• the ionizable lipid can be present in lipid formulations according to other embodiments, preferably in a ratio of about 30 to about 70 Mol%, in some embodiments, about 30 Mol%, in other embodiments, about 40 Mol%, in other embodiments, about 45 Mol% in other embodiments, about 47.5 Mol% in other embodiments, about 50 Mol%, in still other embodiments, and about 60 Mol% in yet others (“Mol%” means the percentage of the total moles that is of a particular component). The term “about” in this paragraph signifies a plus or minus range of 5 Mol%.
• DODMA 1,2-di oleyl oxy-3 -dimethylaminopropane
• MC3 0-(Z,Z,Z,Z-heptatriaconta-6,9,26,29-tetraen-19-yl)-4-(N,N- dimethylamino) (“MC3”).
• Exemplary ionizable lipids suitable for the compositions and methods of the disclosure includes those described in PCT publications WO2020252589A1 and W02021000041A1, U.S. Patent Nos. 8,450,298 and 10,844,028, and Love K.T. et al., Proc Natl Acad Set USA, Feb. 2, 2010 107 (5) 1864-1869, all of which are hereby incorporated by reference in their entirety. Accordingly, in some embodiments, the LNP of the disclosure includes one or more lipid compounds described in Love K.T. et al. (2010 supra), such as Cl 6- 96, C14-110, and C12-200.
• the LNP includes an ionizable cationic lipid selected from the group consisting of ALC-0315, C12-200, LN16, MC3, MD1, SM-102, and a combination of any thereof.
• the LNP of the disclosure includes C 12-200 lipid.
• the structure of Cl 2-200 lipid is known in the art and described in, e.g., U.S. Patent Nos. 8,450,298 and 10,844,028, which are hereby incorporated by reference in their entirety.
• the Cl 2-200 is combined with cholesterol, C14-PEG2000, and DOPE.
• the C12-200 is combined with DSPC and DMG-PEG2000.
• the LNP of the disclosure includes one or more cationic lipids.
• ionizable cationic lipids include, but are not limited to, 98N12-5, C12-200, C14-PEG2000, DLin-KC2- DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1.
• a GalNAc moiety is attached to the outside of the LNP and acts as a ligand for uptake into the liver via the asialoglycoprotein receptor. Any of these cationic lipids can be used to formulate LNP for delivery of the srRNA constructs and nucleic acid constructs of the disclosure.
• the LNP of the disclosure includes one or more neutral lipids.
• neutral lipids suitable for the compositions and methods of the disclosure include DPSC, DPPC, POPC, DOPE, and SM.
• the LNP of the disclosure includes one or more ionizable lipid compounds described in PCT publications WO2020252589A1 and WO2021000041 AL [0131] A number of other lipids or combination of lipids that are known in the art can be used to produce a LNP.
• Non-limiting examples of lipids suitable for use to produce LNPs include DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP- DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG).
• Additional nonlimiting examples of cationic lipids include 98N12-5, C 12-200, C14-PEG2000, DLin-KC2- DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, 7C1, and a combination of any thereof
• Additional non-limiting examples of neutral lipids include DPSC, DPPC, POPC, DOPE, and SM.
• Non-limiting examples of PEG-modified lipids include PEG-DMG, PEG-CerC14, and PEG-CerC20.
• the mass ratio of lipid to nucleic acid in the LNP delivery system is about 100: 1 to about 3: 1, about 70: 1 to 10: 1, or 16: 1 to 4: 1. In some embodiments, the mass ratio of lipid to nucleic acid in the LNP delivery system is about 16: 1 to 4: 1. In some embodiments, the mass ratio of lipid to nucleic acid in the LNP delivery system is about 20: 1. In some embodiments, the mass ratio of lipid to nucleic acid in the LNP delivery system is about 8: 1.
• the lipid-based nanoparticles have an average diameter of less than about 1000 nm, about 500 nm, about 250 nm, about 200 nm, about 150 nm, about 100 nm, about 75 nm, about 50 nm, or about 25 nm. In some embodiments, the LNPs have an average diameter ranging from about 70 nm to 100 nm. In some embodiments, the LNPs have an average diameter ranging from about 88 nm to about 92 nm, from 82 nm to about 86 nm, or from about 80 nm to about 95 nm.
• compositions of the disclosure that formulated in a polymer nanoparticle.
• the compositions are immunogenic compositions, e.g., composition that can stimulate an immune response in a subject.
• the immunogenic compositions are formulated as a vaccine.
• the pharmaceutical compositions are formulated as an adjuvant.
• the immunogenic compositions are substantially non- immunogenic to a subject, e.g., compositions that minimally stimulate an immune response in a subject.
• the non-immunogenic or minimally immunogenic compositions are formulated as a biotherapeutic.
• the pharmaceutical compositions are formulated for one or more of intranasal administration, transdermal administration, intraperitoneal administration, intramuscular administration, intranodal administration, intratumoral administration, intraarticular administration, intravenous administration, subcutaneous administration, intravaginal administration, intraocular, rectal, and oral administration.
• compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
• suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM. (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS).
• the composition should be sterile and should be fluid to the extent that easy syringeability exists. It can be stable under the conditions of manufacture and storage, and can be preserved against the contaminating action of microorganisms such as bacteria and fungi.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
• the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants, e.g., sodium dodecyl sulfate.
• surfactants e.g., sodium dodecyl sulfate.
• Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
• isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, and/or sodium chloride in the composition.
• Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
• Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
• dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above.
• the pharmaceutical compositions of the disclosure are formulated for inhalation, such as an aerosol, spray, mist, liquid, or powder.
• Administration by inhalation may be in the form of either dry powders or aerosol formulations, which are inhaled by a subject (e.g., a patient) either through use of an inhalation device, e.g., a microspray, a pressurized metered dose inhaler, or nebulizer.
• the composition is formulated for one or more of intranasal administration, transdermal administration, intramuscular administration, intranodal administration, intratumoral administration, intraarticular administration, intravenous administration, intraperitoneal administration, oral administration, intravaginal, intraocular, rectal, or intra-cranial administration.
• the administered composition results in an increased production of interferon in the subject.
• one aspect of the present disclosure relates to, inter alia, methods of functionalizing alphaviruses by replacement of at least a portion of nsP- encoding sequence, methods of producing polypeptide of interest encoded by a gene of interest (GO I), methods of eliciting an immune response in a subject in need thereof, and methods of preventing and/or treating a health condition in a subject in need thereof.
• GO I gene of interest
• an aspect of the disclosure relates to a method for functionalizing/engineering an alphavirus genome or RNA replicon (e.g., self-replicating RNA).
• the methods includes (a) providing a non-functional alphavirus genome or RNA replicon; (b) replacing a nonstructural protein (nsP), or a portion thereof, of the non-functional alphavirus genome or RNA replicon with a heterologous coding sequence for the corresponding nsP or portion thereof derived from a different alphavirus strain to generate a modified alphavirus genome or RNA replicon; (c) assessing functionality of the modified alphavirus genome or RNA replicon; (d) identifying the modified alphavirus genome or RNA replicon as being functional if the modified alphavirus genome or RNA replicon is capable of RNA replication and/or expression.
• nsP nonstructural protein
• the heterologous nsP or portion thereof is derived from another strain of the same alphavirus species. In some embodiments, the heterologous nsP or portion thereof is derived from another alphavirus species. In some embodiments, the heterologous nsP or portion thereof is nsPl, nsP2, nsP3, nsP4, or a portion of any thereof. In some embodiments, the non-functionality of the alphavirus genome or RNA replicon (e.g., self- replicating RNA) is determined by a deficiency in self-replication within a host cell.
• RNA replicon e.g., self- replicating RNA
• RNA replication detection of viral protein expression
• CPE cytopathic effect
• heterologous transgene expression detection of heterologous transgene expression.
• a non-functional alphavirus can be identified as being incapable of self-replication within a cell culture or primary cell line, for example, but not limited to, BHK, VERO, or HEK293.
• non-functionality of an alphavirus can be determined when the deposited alphavirus sequence (e.g., sequences retrieved from public databases) is found insufficient when reproduced synthetically to selfreplicate.
• methods for producing a polypeptide of interest include culturing a recombinant cell including a nucleic acid construct as disclosed herein under conditions wherein the recombinant cell produces the polypeptide encoded by the GOI.
• methods for producing a polypeptide of interest in a subject wherein the methods include administering to the subject a nucleic acid construct as disclosed herein.
• the subject is vertebrate animal or an invertebrate animal.
• the subject is a mammalian subject.
• the mammalian subject is a human subject. Accordingly, the recombinant polypeptides produced by the method disclosed herein are also within the scope of the disclosure.
• Non-limiting exemplary embodiments of the disclosed methods for producing a recombinant polypeptide can include one or more of the following features.
• the methods for producing a recombinant polypeptide of the disclosure further include isolating and/or purifying the produced polypeptide.
• the methods for producing a polypeptide of the disclosure further include structurally modifying the produced polypeptide to increase half-life.
• the N-terminus of the produced polypeptide can be further chemically or enzymatically modified to increase half-life.
• the C-terminus of the produced polypeptide is chemically and/or enzymatically modified to increase half-life.
• Non-limiting examples of chemical and enzymatic modifications suitable for the methods described herein include PEGylation, XTENylation, PASylation®, ELPylation, and HAPylation. Techniques, systems, and reagents suitable for these modifications are known in the art.
• the polypeptide produced by the methods described herein can be PEGylated, XTENylated, PASylated, ELPylated, and/or HAPylated to increase half-life.
• the produced polypeptide is conjugated to another protein or peptide (e.g., serum albumin, an antibody Fc domain, transferrin, GLK, or CTP peptide) to increase half-life.
• methods for producing a polypeptide of interest comprises (i) rearing a transgenic animal of the present disclosure, or (ii) culturing a recombinant cell comprising a nucleic acid construct of the present disclosure under conditions wherein the recombinant cell produces the polypeptide encoded by the GOI.
• methods for producing a polypeptide of interest in a subject comprises administering to the subject a nucleic acid construct of the present disclosure.
• the subject is vertebrate animal or an invertebrate animal.
• the animal is an insect.
• the subject is a mammalian subject.
• the mammalian subject is a human subject.
• nucleic acid constructs e.g., vectors or srRNA molecules
• recombinant cells e.g., recombinant polypeptides, and/or pharmaceutical compositions
• relevant health conditions such as proliferative disorders (e.g., cancers), infectious diseases (e.g., acute infections, chronic infections, or viral infections), rare diseases, and/or autoimmune diseases, and/or inflammatory diseases.
• the nucleic acid constructs e.g., vectors or srRNA molecules
• recombinant cells e.g., recombinant cells
• recombinant polypeptides e.g., recombinant polypeptides
• pharmaceutical compositions as described herein can be incorporated into therapeutic agents for use in methods of treating an individual who has, who is suspected of having, or who may be at high risk for developing one or more relevant health conditions or diseases.
• Exemplary health conditions or diseases can include, without limitation, cancers, immune diseases, gene therapy, gene replacement, cardiovascular diseases, age-related pathologies, acute infection, and chronic infection.
• the individual is a patient under the care of a physician.
• the nucleic acid constructs e.g., vectors or srRNA molecules
• recombinant cells e.g., recombinant polypeptides, and/or pharmaceutical compositions as described herein
• the nucleic acid constructs e.g., vectors or srRNA molecules
• recombinant cells e.g., recombinant polypeptides, and/or pharmaceutical compositions as described herein
• a composition including: a) a nucleic acid construct of the disclosure (e.g., vector or srRNA molecule); b) a recombinant cell of the disclosure; c) a recombinant polypeptide of the disclosure; and/or d) a pharmaceutical composition of the disclosure.
• a nucleic acid construct of the disclosure e.g., vector or srRNA molecule
• compositions described herein for their capacity to confer a pharmacodynamic effect can be carried out in vivo and/or ex vivo.
• pharmacodynamic effects that can be analyzed include: immunogenicity effect (e.g., eliciting an immune response in vivo), a biomarker response, a therapeutic effect, a prophylactic effect, a desired effect, an undesired effect, an adverse effect, and effect in a disease model.
• the assessment of pharmacodynamic effects includes assessing induction of an immune response in vivo.
• the assessment of pharmacodynamic effects includes assessing induction of cytokine pathways that can potentiate an immune response and prevent angiogenesis and metastasis.
• a composition including: a) a nucleic acid construct of the disclosure (e.g., vector or srRNA molecule); b) a recombinant cell of the disclosure; c) a recombinant polypeptide of the disclosure; and/or d) a pharmaceutical composition of any one of the disclosure.
• a nucleic acid construct of the disclosure e.g., vector or srRNA molecule
• the health condition is a proliferative disorder or a microbial infection.
• the subject has or is suspected of having a condition associated with proliferative disorder or a microbial infection.
• the disclosed composition is formulated to be compatible with its intended route of administration.
• the nucleic acid constructs, recombinant cells, recombinant polypeptides, and/or pharmaceutical compositions of the disclosure can be given orally or by inhalation, but it is more likely that they will be administered through a parenteral route.
• parenteral routes of administration examples include, for example, intravenous, intranodal, intradermal, subcutaneous, transdermal (topical), transmucosal, intravaginal, and rectal administration.
• Solutions or suspensions used for parenteral application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
• a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol
• pH can be adjusted with acids or bases, such as mono- and/or dibasic sodium phosphate, hydrochloric acid or sodium hydroxide (e.g., to a pH of about 7.2-7.8, e.g., 7.5).
• acids or bases such as mono- and/or dibasic sodium phosphate, hydrochloric acid or sodium hydroxide (e.g., to a pH of about 7.2-7.8, e.g., 7.5).
• the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
• Dosage, toxicity and therapeutic efficacy of such subject nucleic acid constructs, recombinant cells, recombinant polypeptides, and/or pharmaceutical compositions of the disclosure can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g, for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
• the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
• Compounds that exhibit high therapeutic indices are generally suitable. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
• the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
• the dosage of such compounds lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity.
• the dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.
• the therapeutically effective dose can be estimated initially from cell culture assays.
• a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (e.g., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
• IC50 e.g., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
• levels in plasma can be measured, for example, by high performance liquid chromatography.
• compositions described herein can be administered one from one or more times per day to one or more times per week; including once every other day.
• the skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease, previous treatments, the general health and/or age of the subject, and other diseases present.
• treatment of a subject with a therapeutically effective amount of the subject multivalent polypeptides and multivalent antibodies of the disclosure can include a single treatment or, can include a series of treatments.
• the compositions are administered every 8 hours for five days, followed by a rest period of 2 to 14 days, e.g., 9 days, followed by an additional five days of administration every 8 hours.
• the therapeutically effective amount of a nucleic acid construct or recombinant polypeptide of the disclosure depends on the nucleic acid construct or recombinant polypeptide selected. For instance, single dose amounts in the range of approximately 0.001 to 0.1 mg/kg of patient body weight can be administered. In some embodiments, about 0.005, 0.01, 0.05 mg/kg can be administered. In some embodiments, single dose amounts in the range of approximately 0.03 pg to 300 pg/kg of patient body weight can be administered. In some embodiments, single dose amounts in the range of approximately 0.3 mg to 3 mg/kg of patient body weight can be administered.
• a therapeutically effective amount includes an amount of a therapeutic composition that is sufficient to promote a particular effect when administered to a subject, such as one who has, is suspected of having, or is at risk for a health condition, e.g., a disease or infection.
• an effective amount includes an amount sufficient to prevent or delay the development of a symptom of the disease or infection, alter the course of a symptom of the disease or infection (for example but not limited to, slow the progression of a symptom of the disease or infection), or reverse a symptom of the disease or infection. It is understood that for any given case, an appropriate effective amount can be determined by one of ordinary skill in the art using routine experimentation.
• the efficacy of a treatment including a disclosed therapeutic composition for the treatment of disease or infection can be determined by the skilled clinician. However, a treatment is considered effective treatment if at least any one or all of the signs or symptoms of disease or infection are improved or ameliorated. Efficacy can also be measured by failure of an individual to worsen as assessed by hospitalization or need for medical interventions (e.g., progression of the disease or infection is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and/or described herein.
• Treatment includes any treatment of a disease or infection in a subject or an animal (some non-limiting examples include a human, or a mammal) and includes: (1) inhibiting the disease or infection, e.g., arresting, or slowing the progression of symptoms; or (2) relieving the disease or infection, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of the development of symptoms.
• the nucleic acid constructs e.g., vectors or srRNA molecules
• recombinant cells e.g., recombinant cells
• recombinant polypeptides e.g., recombinant polypeptides
• pharmaceutical compositions of the disclosure can be administered to a subject in a composition having a pharmaceutically acceptable carrier and in an amount effective to stimulate an immune response.
• a subject can be immunized through an initial series of injections (or administration through one of the other routes described below) and subsequently given boosters to increase the protection afforded by the original series of administrations.
• the initial series of injections and the subsequent boosters are administered in such doses and over such a period of time as is necessary to stimulate an immune response in a subject, for example, by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% as compared to interferon production in a subject that has not been administered with the composition.
• the administered composition results in an increased production of interferon in the subject.
• the subject is a mammal. In some embodiments, the mammal is human.
• pharmaceutically acceptable carriers suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
• the composition must be sterile and must be fluid to the extent that easy syringeability exists.
• the composition must further be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils.
• the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• a coating such as lecithin
• surfactants for example, parabens, chlorobutanol, phenol, asorbic acid, thimerosal, and the like.
• Sterile injectable solutions can be prepared by incorporating the nucleic acid constructs (e.g., vectors or srRNA molecules), recombinant cells, and/or recombinant polypeptides in the required mount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
• nucleic acid constructs e.g., vectors or srRNA molecules
• recombinant cells e.g., recombinant cells
• polypeptides e.g., recombinant polypeptides
• nucleic acid constructs e.g., vectors or srRNA molecules
• recombinant cells e.g., recombinant polypeptides, and/or pharmaceutical compositions
• they can be orally administered, for example, with an inert diluent or an assimilable edible carrier.
• the nucleic acid constructs e.g., vectors or srRNA molecules
• recombinant cells, recombinant polypeptides, and/or pharmaceutical compositions and other ingredients can also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the individual's diet.
• the active compound can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
• the nucleic acid constructs e.g., vectors or srRNA molecules
• recombinant polypeptides of the disclosure can be delivered to a cell or a subject by a lipid-based nanoparticle (LNP).
• LNP are generally less immunogenic than viral particles. While many humans have preexisting immunity to viral particles there is no pre-existing immunity to LNP. In addition, adaptive immune response against LNP is unlikely to occur which enables repeat dosing of LNP.
• ionizable cationic lipids have been developed for use in LNP. These include C12-200, MC3, LN16, and MD1 among others.
• a GalNAc moiety is attached to the outside of the LNP and acts as a ligand for uptake in to the liver via the asialoglycoprotein receptor. Any of these cationic lipids can be used to formulate LNP for delivery of the nucleic acid constructs and recombinant polypeptides of the disclosure to the liver.
• a LNP refers to any particle having a diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm.
• a nanoparticle can range in size from 1-1000 nm, 1-500 nm, 1-250 nm, 25-200 nm, 25-100 nm, 35- 75 nm, or 25-60 nm.
• LNPs can be made from cationic, anionic, or neutral lipids.
• Neutral lipids such as the fusogenic phospholipid DOPE or the membrane component cholesterol, can be included in LNPs as ‘helper lipids’ to enhance transfection activity and nanoparticle stability.
• Limitations of cationic lipids include low efficacy owing to poor stability and rapid clearance, as well as the generation of inflammatory or anti-inflammatory responses.
• LNPs can also have hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids.
• lipids or combination of lipids that have been developed for use in LNP can be used to produce a LNP of the disclosure.
• Non-limiting examples of lipids suitable for use in production of LNPs include DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE- DMPE-polyethylene glycol (PEG).
• Non-limiting examples of cationic lipids suitable for use in production of LNPs include 98N12-5, C12-200, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1.
• Non-limiting examples of neutral lipids suitable for use in production of LNPs include DPSC, DPPC, POPC, DOPE, and SM.
• Non-limiting examples of PEG-modified lipids suitable for use in production of LNPs include PEG-DMG, PEG-CerC14, and PEG-CeraC20.
• the lipids can be combined in any number of molar ratios to produce a LNP.
• the mass ratio of lipid to nucleic acid in the LNP delivery system is about 100: 1 to about 3: 1, about 70: 1 to 10: 1, or 16: 1 to 4: 1.
• the mass ratio of lipid to nucleic acid in the LNP delivery system is about 16: 1 to 4: 1.
• the mass ratio of lipid to nucleic acid in the LNP delivery system is about 20: 1.
• the mass ratio of lipid to nucleic acid in the LNP delivery system is about 8: 1.
• the polynucleotide(s) can be combined with lipid(s) in a wide range of molar ratios to produce a LNP.
• the therapeutic compositions described herein e.g., nucleic acid constructs (e.g., vectors or srRNA molecules), recombinant cells, recombinant polypeptides, and/or pharmaceutical compositions are incorporated into therapeutic compositions for use in methods of preventing or treating a subject who has, who is suspected of having, or who can be at high risk for developing one or more relevant health conditions or diseases.
• exemplary health conditions or diseases can include, without limitation, cancers, immune diseases, autoimmune diseases, inflammatory diseases, gene therapy, gene replacement, cardiovascular diseases, age- related pathologies, rare disease, acute infection, and chronic infection.
• the nucleic acid constructs can be useful in the treatment and/or prevention of immune diseases, autoimmune diseases, or inflammatory diseases such as, for example, glomerulonephritis, inflammatory bowel disease, nephritis, peritonitis, psoriatic arthritis, osteoarthritis, Still’s disease, Familiar Mediterranean Fever, systemic scleroderma and sclerosis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, acute lung injury, meningitis, encephalitis, uveitis, multiple myeloma, glomerulonephritis, nephritis, asthma, atherosclerosis, leukocyte adhesion deficiency, multiple sclerosis, Raynaud's syndrome, Sjogren's syndromejuvenile
• Non-limiting examples of inflammatory suitable for the methods of the disclosure include inflammatory diseases such as asthma, inflammatory bowel disease (IBD), chronic colitis, splenomegaly, and rheumatoid arthritis.
• IBD inflammatory bowel disease
• chronic colitis splenomegaly
• rheumatoid arthritis rheumatoid arthritis
• autoimmune diseases suitable for the methods of the disclosure include, but are not limited to, rheumatoid arthritis, osteoarthritis, Still’s disease, Familiar Mediterranean Fever, systemic sclerosis, multiple sclerosis, ankylosing spondylitis, Hashimoto's thyroiditis, systemic lupus erythematosus, Sjogren's syndrome, diabetic retinopathy, diabetic vasculopathy, diabetic neuralgia, insulitis, psoriasis, alopecia areata, warm and cold autoimmune hemolytic anemia (AIHA), pernicious anemia, acute inflammatory diseases, autoimmune adrenalitis, chronic inflammatory demyelinating polyneuropathy (CIDP), Lambert-Eaton syndrome, lichen sclerosis, Lyme disease, Graves disease, Behcet's disease, Meniere's disease, reactive arthritis (Reiter's syndrome), Churg-Strauss syndrome, Cogan syndrome, CREST syndrome, pemph
• the therapeutic compositions described herein e.g., nucleic acid constructs (e.g., vectors or srRNA molecules), recombinant cells, recombinant polypeptides, and/or pharmaceutical compositions are incorporated into therapeutic compositions for use in methods of preventing or treating a subject who has, who is suspected of having, or who can be at high risk for developing a microbial infection (e.g., bacterial infection, micro-fungal infection, or viral infection).
• a microbial infection e.g., bacterial infection, micro-fungal infection, or viral infection.
• Non-limiting examples of infection suitable for the methods of the disclosure include infections with viruses such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis B virus (HCV), Cytomegalovirus (CMV), respiratory syncytial virus (RSV), human papillomavirus (HPV), Epstein-Barr virus (EBV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East Respiratory Syndrome (MERS), influenza virus, and Ebola virus.
• viruses such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis B virus (HCV), Cytomegalovirus (CMV), respiratory syncytial virus (RSV), human papillomavirus (HPV), Epstein-Barr virus (EBV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), severe acute respiratory syndrome coronavirus (S
• Additional infections suitable for the methods of the disclosure include infections with intracellular parasites such as Leishmania, Rickettsia, Chlamydia, Coxiella, Plasmodium, Brucella, mycobacteria, Listeria, Toxoplasma and Trypanosoma.
• the microbial infection is a bacterial infection.
• the microbial infection is a fungal infection.
• the microbial infection is a viral infection.
• the health condition is a rare disease, e.g., a disease or condition that affects less than 200,000 people in the United States, as defined by The Orphan Drug Act (www.fda.gov/patients/rare-diseases-fda) and/or an inflammatory and/or autoimmune disorder.
• the subject has or is suspected of having a condition associated with an inflammatory and/or autoimmune disorder and/or a rare disease (e.g. including but not limited to Familial Mediterranean Fever or adult onset Still’s disease).
• a composition according to the present disclosure is administered to the subject individually as a single therapy (monotherapy) or as a first therapy in combination with at least one additional therapies (e.g., second therapy).
• the second therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, targeted therapy, and surgery.
• the second therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy or surgery.
• the first therapy and the second therapy are administered concomitantly.
• the first therapy is administered at the same time as the second therapy.
• the first therapy and the second therapy are administered sequentially.
• the first therapy is administered before the second therapy. In some embodiments, the first therapy is administered after the second therapy. In some embodiments, the first therapy is administered before and/or after the second therapy. In some embodiments, the first therapy and the second therapy are administered in rotation. In some embodiments, the first therapy and the second therapy are administered together in a single formulation.
• kits for the practice of a method described herein provide kits for eliciting an immune response in a subject.
• kits for the prevention of a health condition in a subject in need thereof relate to kits for methods of treating a health condition in a subject in need thereof.
• kits that include one or more of the nucleic acid constructs e.g., vectors or srRNA molecules
• recombinant cells e.g., recombinant cells
• recombinant polypeptides e.g., as well as written instructions for making and using the same.
• kits of the disclosure further include one or more means useful for the administration of any one of the provided nucleic acid constructs (e.g., vectors or srRNA molecules), recombinant cells, recombinant polypeptides, and/or pharmaceutical compositions to a subject.
• the kits of the disclosure further include one or more syringes (including pre- filled syringes) and/or catheters (including pre-filled syringes) used to administer any one of the provided nucleic acid constructs (e.g., vectors or srRNA molecules), recombinant cells, recombinant polypeptides, and/or pharmaceutical compositions to a subject.
• a kit can have one or more additional therapeutic agents that can be administered simultaneously or sequentially with the other kit components for a desired purpose, e.g., for diagnosing, preventing, or treating a condition in a subject in need thereof.
• kits can further include one or more additional reagents, where such additional reagents can be selected from: dilution buffers, reconstitution solutions, wash buffers, control reagents, control expression vectors, negative controls, positive controls, reagents suitable for in vitro production of the provided nucleic acid constructs (e.g., vectors or srRNA molecules), recombinant cells, recombinant polypeptides, and/or pharmaceutical compositions of the disclosure.
• additional reagents can be selected from: dilution buffers, reconstitution solutions, wash buffers, control reagents, control expression vectors, negative controls, positive controls, reagents suitable for in vitro production of the provided nucleic acid constructs (e.g., vectors or srRNA molecules), recombinant cells, recombinant polypeptides, and/or pharmaceutical compositions of the disclosure.
• the components of a kit can be in separate containers. In some other embodiments, the components of a kit can be combined in a single container. Accordingly, in some embodiments of the disclosure, the kit includes one or more of the nucleic acid constructs (e.g., vectors or srRNA molecules), recombinant cells, recombinant polypeptides, and/or pharmaceutical compositions as provided and described herein in one container (e.g., in a sterile glass or plastic vial) and a further therapeutic agent in another container (e.g., in a sterile glass or plastic vial).
• the nucleic acid constructs e.g., vectors or srRNA molecules
• recombinant cells e.g., recombinant cells
• recombinant polypeptides e.g., recombinant polypeptides, and/or pharmaceutical compositions as provided and described herein in one container (e.g., in a sterile glass or plastic vial) and
• the kit includes a combination of the compositions described herein, including one or more nucleic acid constructs (e.g., vectors or srRNA molecules), recombinant cells, and/or recombinant polypeptides of the disclosure in combination with one or more additional therapeutic agents formulated together in a pharmaceutical composition and, optionally, in a single, common container.
• nucleic acid constructs e.g., vectors or srRNA molecules
• recombinant cells e.g., recombinant cells
• polypeptides of the disclosure in combination with one or more additional therapeutic agents formulated together in a pharmaceutical composition and, optionally, in a single, common container.
• the kit can include a device (e.g., an injection device or catheter) for performing such administration.
• the kit can include one or more hypodermic needles or other injection devices as discussed above containing one or more nucleic acid constructs (e.g., vectors or srRNA molecules), recombinant cells, and/or recombinant polypeptides of the disclosure.
• kits can further include instructions for using the components of the kit to practice the methods disclosed herein.
• the kit can include a package insert including information concerning the pharmaceutical compositions and dosage forms in the kit. Generally, such information aids patients and physicians in using the enclosed pharmaceutical compositions and dosage forms effectively and safely.
• the following information regarding a combination of the disclosure may be supplied in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdosage, proper dosage and administration, how supplied, proper storage conditions, references, manufacturer/distributor information and intellectual property information.
• the instructions for practicing the methods are generally recorded on a suitable recording medium.
• the instructions can be printed on a substrate, such as paper or plastic, etc.
• the instructions can be present in the kit as a package insert, in the labeling of the container of the kit or components thereof (e.g., associated with the packaging or subpackaging), etc.
• the instructions can be present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, flash drive, etc.
• the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g., via the internet), can be provided.
• An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions can be recorded on a suitable substrate.
• This Example describes the results of experiments performed to construct a number of base alphavirus vectors (e.g., without a heterologous gene) that were subsequently used for expression of a gene of interest (e.g., a hemagglutinin (HA) gene from influenza).
• a gene of interest e.g., a hemagglutinin (HA) gene from influenza.
• the Sindbis AR86-Girdwood hybrid 1 vector described in FIG. 2A was constructed as follows.
• the base SINV AR86-Girdwood hybrid 1 vector was synthesized de novo in four ⁇ 4 kb parts (Twist Bioscience) from an AR-86 reference sequence (Genbank U38305) with a unique restriction enzyme cut site (Spel, 5’-A’CTAG,T-3’) in place of the coding sequence of the SINV structural genes (where the 5’ A is the next nucleotide after a P2A sequence following nucleotide 93 of the structural polyprotein gene, and the 3’ T matches the location of the structural polyprotein’s stop codon TGA).
• a bacteriophage T7 RNA polymerase promoter (5’-
• TAATACGACTCACTATAG-3 was included upstream of the SINV genome sequence, and downstream contained a polyA sequence followed by a unique restriction enzyme site (SapI, 5’-GCTCTTC(N)I’(N)3,-3’) followed by a T7 terminator sequence (5’- AACCCCTCTCTAAACGGAGGGGTTTTTTT-3’; SEQ ID NO: 13) followed by a unique restriction enzyme cut site Notl, 5’-GC’GGCC,GC-3’).
• the parts were combined in a five-piece Gibson Assembly® reaction e.g., a linearized pYL backbone and the four synthesized fragments).
• the AR86 nsP2 gene was replaced with the Girdwood nsP2 gene (Genbank MF459683), resulting in the final SINV AR86-Girdwood hybrid 1 base vector (SEQ ID NO: 1).
• the Sindbis AR86-Girdwood hybrid 2 vector described in FIG. 3A was constructed as follows.
• the base SINV Girdwood vector was synthesized de novo in four ⁇ 4 kb parts (Twist Bioscience, Thermo Fisher GeneArt) from a Girdwood strain reference sequence (Genbank MF459683) with a unique restriction enzyme cut site (Spel, 5’-A’CTAG,T-3’) in place of the coding sequence of the SINV structural genes (where the 5’ A is the next nucleotide after a P2A sequence following nucleotide 93 of the structural polyprotein gene, and the 3’ T matches the location of the structural polyprotein’s stop codon TGA).
• a bacteriophage T7 RNA polymerase promoter (5’-TAATACGACTCACTATAG-3’; SEQ ID NO: 12) was included upstream of the SINV genome sequence, and downstream contained a polyA sequence followed by a unique restriction enzyme site (SapI, 5’-GCTCTTC(N)f(N)3,-3’) followed by a T7 terminator sequence (5’-AACCCCTCTCTAAACGGAGGGGTTTTTTT-3’; ; SEQ ID NO: 13) followed by a unique restriction enzyme cut site Notl, 5’-GC’GGCC,GC-3’).
• the parts were combined in a five-piece Gibson Assembly® reaction e.g., a linearized pYL backbone and the four synthesized fragments).
• the Girdwood nsP4 gene was replaced with the AR86 nsP4 gene, resulting in the final SINV AR86-Girdwood hybrid 2 base vector (SEQ ID NO: 2).
• Sindbis AR86-Girdwood hybrid 3 vector described in FIG. 4A was constructed as follows. Similar to the Sindbis AR86-Girdwood hybrid 2 base vector, the Sindbis AR86- Girdwood hybrid 3 base vector was constructed by instead replacing the Girdwood nsP3 gene with the AR86 nsP3 gene (SEQ ID NO: 3).
• the Sindbis AR86-Girdwood hybrid 4 vector described in FIG. 5A was constructed as follows. Similar to the Sindbis AR86-Girdwood hybrid 2 base vector, the Sindbis AR86- Girdwood hybrid 4 base vector was constructed by instead replacing the Girdwood nsPl gene with the AR86 nsPl gene (SEQ ID NO: 4).
• the alphavirus vectors in FIGS. 2B, 3B, 4B, and 5B were constructed by linearization of the empty base vector in FIGS. 2A, 3A, 4A, and 5A, respectively, by Spel endonuclease digestion.
• the hemagglutinin (HA) gene from influenza (Genbank #AY651334) was codon refactored for human expression in silico and synthesized (IDT).
• the synthetic product was amplified using the following primers which add 30 bp of flanking homology ends to the base vector on the PCR product.
• This Example describes the results of experiments performed to demonstrate that a defective (non-functional) RNA replicon (e.g. self-replicating RNA) can be functionalized e.g., caused to be functional) by replacement of a defective nsP sequence with a corresponding nsP sequence from a functional alphavirus.
• a defective (non-functional) RNA replicon e.g. self-replicating RNA
• srRNA Self-replicating RNA
• srRNA was prepared by in vitro transcription using a plasmid DNA template linearized by enzymatic digestion.
• the DNA was either linearized with Notl, which cuts downstream of the T7 terminator, or linearized with SapI, which cuts at the end of the poly(A).
• Bacteriophage T7 polymerase was used for in vitro transcription with either a 5’ ARCA cap (HiScribeTM T7 ARCA mRNA Kit, NEB) or by uncapped transcription (HiScribeTM T7 High Yield RNA Synthesis Kit, NEB) followed by addition of a 5’ cap 1 (Vaccinia Capping System, mRNA Cap 2 -O- Methyltransferase, NEB).
• srRNA was purified using phenol/chloroform extraction, LiCl precipitation, or column purification (Monarch® RNA Cleanup Kit, NEB). srRNA concentration was determined by absorbance at 260 nm (Nanodrop, Thermo Fisher Scientific).
• srRNA was transformed by electroporation into BHK-21 or Vero cells (e.g. 4D-NucleofectorTM, Lonza). At 17-20 h following transformation, the cells were fixed and permeabilized (eBioscienceTM Foxp3 / Transcription Factor Staining Buffer Set, Invitrogen) and stained using a PE-conjugated anti-double stranded RNA (dsRNA) mouse monocolonal antibody (J2, Scicons) to quantify the frequency of dsRNA+ cells and the mean fluorescence intensity (MFI) of dsRNA in individual cells by fluorescence flow cytometry.
• dsRNA PE-conjugated anti-double stranded RNA
• J2, Scicons PE-conjugated anti-double stranded RNA
• MFI mean fluorescence intensity
• RNA replicon fails to exhibit a signal after staining cells transfected with srRNA (see RNA replicons (e.g. self-replicating RNAs) marked with an X), where transfected functional RNA replicons (e.g. self-replicating RNAs) produce detectible dsRNA (see RNA replicons marked with a ‘ " symbol) which is indicative of RNA replication and is a necessary for 26S RNA transcription and subsequent transgene expression (FIG. 6).
• RNA replicons e.g. self-replicating RNAs marked with an X
• transfected functional RNA replicons e.g. self-replicating RNAs
• produce detectible dsRNA see RNA replicons marked with a ‘ " symbol) which is indicative of RNA replication and is a necessary for 26S RNA transcription and subsequent transgene expression (FIG. 6).
• This Example describes the results of in vitro experiments performed to evaluate expression levels of the modified alphavirus vector constructs described in Examples 1 and 2 above, and to investigate any differential behavior thereof (e.g., replication and protein expression).
• modified alphavirus srRNA vectors (SINV Girdwood with heterologous AR86 nsPl, or nsP2, or nsP3, or nsP4) encoding hemagglutinin precursor (HA) of the influenza A virus H5N1 (H5N1 HA) were also evaluated using the following assays.
• srRNA Self-replicating RNA
• srRNA was prepared by in vitro transcription using a plasmid DNA template linearized by enzymatic digestion.
• the DNA was either linearized with Notl, which cuts downstream of the T7 terminator, or linearized with Sap ., which cuts at the end of the poly(A).
• Bacteriophage T7 polymerase was used for in vitro transcription with either a 5’ ARCA cap (HiScribeTM T7 ARCA mRNA Kit, NEB) or by uncapped transcription (HiScribeTM T7 High Yield RNA Synthesis Kit, NEB) followed by addition of a 5’ cap 1 (Vaccinia Capping System, mRNA Cap 2 -0- Methyltransferase, NEB).
• srRNA was purified using phenol/chloroform extraction, LiCl precipitation, or column purification (Monarch® RNA Cleanup Kit, NEB). RNA concentration was determined by absorbance at 260 nm (Nanodrop, Thermo Fisher Scientific).
• srRNA was transformed by electroporation into BHK-21 or Vero cells (e.g. 4D-NucleofectorTM, Lonza). At 17-20 h following transformation, the cells were fixed and permeabilized (eBioscienceTM Foxp3 / Transcription Factor Staining Buffer Set, Invitrogen) and stained using a PE-conjugated anti-dsRNA mouse monoclonal antibody (J2, Scicons) to quantify the frequency of dsRNA+ cells and the mean fluorescence intensity (MFI) of dsRNA in individual cells by fluorescence flow cytometry.
• J2, Scicons PE-conjugated anti-dsRNA mouse monoclonal antibody
• APC-conjugated anti-HA mouse monoclonal antibody 2B7, Abeam; APC: allophycocyanin
• BHK-21 or Vero cells are pre-treated with a titrated curve of recombinant IFN prior to electroporation of RNA and impacts on replication and protein expression for each vector are measured using the above assays.
• a non-functional srRNA vector fails to exhibit a signal after staining srRNA- transfected cells to detect GOI expression, where functional srRNA vectors produce detectible GOI expression.
• GOI expression is quantified by mean fluorescence intensity (MFI) of cells that stained positive using the APC-conjugated anti-HA mouse monoclonal antibody.
• FIG. 7 illustrates that the base srRNA vectors which demonstrated RNA replication (FIG. 6) also exhibited expression after insertion of a GOI.
• This Example describes the results of in vivo experiments performed to evaluate any differential immune responses following vaccination with the modified alphavirus vector constructs described in Examples 1 and 2 above (e.g., both unformulated and LNP formulated vectors).
• mice Female C57BL/6 or BALB/c mice are purchased from Charles River Labs or Jackson Laboratories. On day of dosing, between 0.1-10 pg of material is injected intramuscularly split into both quadricep muscles. Vectors are administered either unformulated in saline, or LNP -formulated. Animals are monitored for body weight and other general observations throughout the course of the study. For immunogenicity studies, animals are dosed on Day 0 and Day 21. Spleens were collected at Day 35, and serum was isolated at Days 0, 14, and 35. For protein expression studies, animals are dosed on Day 0, and bioluminescence is assessed on Days 1, 3, and 7. In vivo imaging of luciferase activity is done using an IVIS system at the indicated time points.
• Replicon RNA e.g., self-replicating RNA
• lipid nanoparticles using a microfluidics mixer and analyzed for particle size, polydispersity using dynamic light scattering and encapsulation efficiency.
• Molar ratios of lipids used in formulating LNP particles is 30% C12-200, 46.5% Cholesterol, 2.5% PEG-2K and 16% DOPE.
• ELISpot To measure the magnitude of Influenza-specific T cell responses, IFNy ELISpot analysis is performed using Mouse IFNy ELISpot PLUS Kit (HRP) (MabTech) as per manufacturer’s instructions. In brief, splenocytes are isolated and resuspended to a concentration of 5 x 10 6 cells/mL in media containing peptides representing either CD4+ or CD8+ T cell epitopes to HA, PMA/ionomycin as a positive control, or DMSO as a mock stimulation. [0215] Intracellular cytokine staining.
• Spleens are isolated according to the methods outlined for ELISpots, and 1 x 10 6 cells are added to cells containing media in a total volume of 200 pL per well. Each well contains peptides representing either CD4+ or CD8+ T cell epitopes to HA, PMA/ionomycin as a positive control, or DMSO as a mock stimulation. After 1 hour, GolgiPlugTM protein transport inhibitor (BD Biosciences) is added to each well. Cells are incubated for another 5 hours. Following incubation, cells are surface stained for CD8+ (53-6.7), CD4+ (GK1.5), B220 (B238128), Gr-1 (RB6-8C5), CD16/32 (M93) using standard methods.
• CD8+ 53-6.7
• CD4+ GK1.5
• B220 B238128
• Gr-1 RB6-8C5
• CD16/32 M93
• Antibodies Antibody responses to measure total HA-specific IgG are measured using ELISA kits from Alpha Diagnostic International as per manufacturer’s instructions.
• This Example describes the results of in vitro experiments performed to evaluate expression levels of synthetic self-replicating RNAs (srRNAs) with a heterologous nonstructural protein and to investigate any differential behavior thereof (e.g., replication and protein expression).
• srRNAs synthetic self-replicating RNAs
• srRNAs derived from the SINV strain Girdwood and AR86 were designed and subsequently evaluated, including control VEEV srRNAs with irrelevant transgenes (RBI296, RBI298), VEEV srRNAs encoding both IL-IRA and IL-12 in two configurations (RBI299, RBI300), SINV AR86-Girdwood Hybrid 1 srRNAs encoding both IL-IRA and IL-12 in two configurations (RBI307, RBI308), and SINV Girdwood srRNAs encoding both IL-IRA and IL-12 in two configurations (RBI309, RBI310).
• srRNA was prepared by in vitro transcription from a Sapl- linearized plasmid template with bacteriophage T7 polymerase by uncapped transcription (HiScribeTM T7 High Yield RNA Synthesis Kit, NEB) followed by addition of a 5’ cap 1 (Vaccinia Capping System, mRNA Cap 2 '-O-Methyl transferase, NEB). srRNA was then purified by LiCl precipitation. srRNA concentration was determined by absorbance at 260 nm (Nanodrop, Thermo Fisher Scientific).
• srRNA was transformed by electroporation into BHK-21 cells (4D-NucleofectorTM, Lonza). At 24 and 48 hours following transformation, conditioned media was collected from the cells. Secreted IL-IRA was evaluated in a bioactivity assay by preincubating HEK-BlueTM IL-1R cells (InvivoGen) with a range of concentrations of recombinant IL-IRA (Peprotech) or conditioned media. Recombinant IL-1B (Invivogen) was added to the cells and incubated overnight then the SEAP reporter was quantified using QUANTI-BlueTM (Invivogen) (FIG. 8A).
• IL-12 was evaluated in a bioactivity assay by incubating a range of concentrations of recombinant IL-12 (Peprotech) or conditioned media on IL-12 bioassay cells (Promega) overnight in DMEM then the luciferase reporter was quantified using Bio-GioTM Luciferase (Promega) (FIG. 8B).
• This Example describes the results of in vivo experiments performed to evaluate any differential immune responses following vaccination with self-replicating RNAs (srRNAs) with a heterologous nonstructural protein, as both unformulated and LNP formulated vectors.
• srRNAs self-replicating RNAs
• mice Female C57BL/6 or BALB/c mice were purchased from Charles River Labs or Jackson Laboratories. On day of dosing, between 0.1-10 pg of material was injected intramuscularly split into both quadri cep muscles. Vectors were administered either unformulated in saline, or LNP-formulated. Animals were monitored for body weight and other general observations throughout the course of the study. For immunogenicity studies, animals were dosed on Day 0 and Day 21. Spleens were collected at Day 14 and/or 35, and serum was isolated at Days 14, and/or 35.
• LNP formulation For some studies, srRNA was formulated in lipid nanoparticles (LNPs) using a microfluidics mixer and analyzed for particle size, polydispersity using dynamic light scattering and encapsulation efficiency. LNP are composed of an ionizable lipid, cholesterol, PEG-2K, and DOPE.
• ELISpot To measure the magnitude of antigen-specific T cell responses, fFNy ELISpot analysis was performed using Mouse fFNy ELISpot PLUS Kit (HRP) (MabTech) as per manufacturer’s instructions. In brief, splenocytes are isolated and resuspended to a concentration of 2-5 x 10 6 cells/mL in media containing peptides representing either peptide pools corresponding to rabies virus glycoprotein G, ESRI, HER2, or HER3, PMA/ionomycin as a positive control, or DMSO as a mock stimulation.
• HRP Mouse fFNy ELISpot PLUS Kit
• Antibodies Neutralizing antibody responses to rabies virus are measured using rapid fluorescent focus inhibition test. In brief, serum dilutions are mixed with a standard amount of live rabies virus and incubated. If neutralizing anti -rabies antibodies are present, they will neutralize the virus. Next, cultured cells are added and the serum/virus/cells are incubated together. Uncoated rabies virus (i.e. that has not been neutralized by antibodies), will infect the cells and this can be visualized by microscopy. Calculation of the endpoint titer is made from the percent of virus infected cells observed on the slide.
• srRNA-based vaccines In addition to an infectious disease antigen, immunogenicity of srRNA-based vaccines to cancer antigens was assessed (FIG. 10).
• Each srRNA vaccine co-encoded sequences from ESRI, HER2, and HER3.
• Splenic T cell responses to these three antigens were determined using ELISpot analysis in mice having received two immunizations. Robust T cell responses were observed to all three targets, while the pattern of responses differed between srRNA vectors (FIG. 10).

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