WO2013148302A1

WO2013148302A1 - Delivery of packaged rna to mammalian cells

Info

Publication number: WO2013148302A1
Application number: PCT/US2013/031876
Authority: WO
Inventors: Stanislaw J. Kaczmarczyk; Deb K. Chatterjee
Original assignee: THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES, OFFICE OF TECHNOLOGY TRANSFER, NATIONAL INSTITUTES OF HEALTH
Priority date: 2012-03-26
Filing date: 2013-03-15
Publication date: 2013-10-03
Also published as: EP2831225A1; AU2013240248B2; US20150050243A1; US20160340653A1; EP3663395A1; JP2015527296A; AU2021240286A1; AU2019201911A1; JP2018186815A; EP3663395B1; US9506041B2; US20200102545A1; JP6585237B2; US10538743B2; AU2013240248A1

Abstract

Described herein are compositions relating to alphavirus-based virus-like particles (VLPs) and methods for making and using the described VLPs. The described compositions include VLPs and vectors and cells used to produce the VLPs. Also included are related methods to produce the VLPs, to transduce cells using the VLPs, and to produce a protein or polynucleotide of interest in a target cell using the VLPs. Also described are alphavirus-based replicons that allow for expression of proteins or polynucleotides of interest in a target cell without a cytopathic effect.

Description

DELIVERY OF PACKAGED RNA TO MAMMALIAN CELLS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of United States Provisional Application No. 61/615,687 filed March 26, 2012, which is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

[0002] The invention described herein relates to delivering and transcribing recombinant polynucleotides to mammalian cells using replication-defective virus-like particles.

BACKGROUND

[0003] Alphaviruses belong to the group IV Togaviridae family of viruses. The alphaviruses are small, spherical, enveloped viruses with a genome of a single positive sense strand RNA. The total genome length ranges between 1 1,000 and 12,000 nucleotides, and has a 5' cap, and 3' poly -A tail. The four non-structural protein genes (NSP genes) are encoded in the 5' two-thirds of the genome, while the three structural proteins are translated from a subgenomic mRNA colinear with the 3' one-third of the genome.

[0004] There are two open reading frames (ORFs) in the alphavirus genome, nonstructural and structural. The first includes NSP genes and encodes proteins (nsP l-nsP4) necessary for transcription and replication of viral RNA. The second encodes three structural proteins: 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.

[0005] The Sindbis (and VEEV) virus is an alphavirus whose genome comprises a positive mRNA strand of 11703 nucleotides. This virus infects a variety of vertebrate hosts. The genome of Sindbis virus encodes nonstructural (NS, replicon) and structural proteins (capsid and pH dependent fusogenic envelope) that are directly translated in the cytoplasm of the host cell. The alphaviruses also include Aura virus, Babanki virus, Barmah Forest virus, Bebaru virus, Cabassou virus, Chikungunya virus, Eastern equine encephalitis virus, Everglades virus, Fort Morgan virus, Getah virus, Highlands J virus, Kyzylagach virus, Mayaro virus, Me Tri virus, Middelburg virus, Mosso das Pedras virus, Mucambo virus, Ndumu virus, O'nyong-nyong virus, Pixuna virus, Rio Negro virus, Ross River virus, Salmon pancreas disease virus, Semliki Forest virus, Southern elephant seal virus, Tonate virus, Trocara virus, Una virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus, and Whataroa virus.

[0006] Infection of host cell with an alphavirus results in cytotoxicity culminating with apoptosis, This is mostly due to both: expression of alphavirus genomic RNA in large quantities triggering antiviral state in host cells and direct interaction of alphaviral non-structural proteins ( NSP2 of SIN or NC of VEEV) with cellular mRNA synthesis or translational shut-off causing cytophathic effect (CPE)on host cell host cell. A natural Sindbis virus variant containing a point mutation in one of the nonstructural proteins, NSP2 (at position 726) demonstrated sustained and noncytopathic growth in infected cells although the viral titer recovered from infected cells was substantially reduced (Frolov, I. et al, J. Virol. 3845-65 (May, 1999)).

[0007] Alphaviruses are of interest to gene therapy researchers. Ross River virus, Sindbis virus, Semliki Forest virus (SFV), and Venezuelan equine encephalitis virus (VEEV) have all been used to develop vectors for gene delivery. Pseudotyped viruses may be formed by combining alphaviral envelopes glycoproteins and retroviral capsids. Alphaviral envelope glycoproteins pseudotyped retroviruses or lentiviruses are able to integrate the genes that they carry into the potential host cells. The pseudotyped alphaviruses are recognized and infected by the alphaviral envelope proteins E2 and El. Stable integration of viral genes is mediated by retroviral interiors of these vectors.

[0008] There are limitations to the use of alphaviruses in the field of gene therapy due to their lack of specificity of targeting. However, through the introduction of variable antibody domains in a non-conserved loop in the structure of E2, specific populations of cells have been targeted. Furthermore, the use of whole alphaviruses for gene therapy is of limited efficacy both because several internal alphaviral proteins are involved in the induction of apoptosis upon infection and also because the alphaviral capsid mediates only the transient introduction of mRNA into host cells. Neither of these limitations extends to alphaviral envelope pseudotypes of retroviruses or lentiviruses.

SUMMARY

[0009] One aspect of the description is a virus-like particle (VLP) comprising an alphavirus replicon, wherein the alphavirus replicon comprises a recombinant polynucleotide, a retroviral gag protein, a fusogenic envelope protein, in which the VLP does not contain an alphavirus structural protein gene. The alphavirus replicon may be derived from Sindbis virus or VEEV nonstructural proteins NSP 1, NSP2, NSP3, and NSP4, and a retroviral packaging signal. The retroviral gag protein may be derived from Rous sarcoma virus or murine leukemia virus. The fusogenic envelope protein is selected from the group consisting of haemagglutinin, Rous sarcoma virus (RSV) fusion protein, an E protein of tick borne encephalitis virus and dengue fever virus, the El protein of SFV, baculovirus gp64, and Vesicular stomatitis (Indiana) virus-G (VSV-G) protein, preferably a glycoprotein, or fragment or derivative thereof, more preferably from a RNA virus or a retrovirus, or fragment or derivative thereof, most preferably VSV-G or EnvA, or an alteration of VSV-G. The VLP described herein may be capable of binding to a eukaryotic cell, preferably a human cell. The binding of the VLP may be specific to a target cell. The VLP described herein preferably replicates in the target cell. In some embodiments the VLP described herein is not cytopathic to the cell. The recombinant polynucleotide of the VLP may comprise a miRNA, shRNA or an antisense RNA, preferably a shRNA or antisense RNA that knocks down expression of a gene in the cell. The recombinant polynucleotide of the VLP may comprise an RNA encoding a protein that can be expressed by the cell.

[0010] Another embodiment of the description is a method of producing the VLP described herein, comprising the steps of co-transforming a eukaryotic cell with a first vector comprising a polynucleotide sequence encoding the alphavirus replicon, wherein the alphavirus replicon includes the polynucleotide of interest, a second vector comprising a polynucleotide sequence encoding the retroviral gag protein, and a third vector comprising a polynucleotide sequence encoding the fusogenic envelope protein; culturing the co-transformed cell under conditions suitable to cause each vector to produce its encoded product, thereby producing the VLP, and isolating the VLP from the cell, wherein neither the vectors nor the cell contain any alphavirus structural protein genes.

[0011] Another embodiment of the description is kit comprising a first vector comprising a polynucleotide sequence encoding an alphavirus replicon, wherein the alphavirus replicon includes the polynucleotide of interest; a second vector comprising a polynucleotide sequence encoding the retroviral gag protein; and a third vector comprising a polynucleotide sequence encoding the fusogenic envelope protein, such as VSV-G. In some embodiments, the vectors provided with the kits do not include alphavirus structural protein genes. Alternatively, in some embodiments one or more of the alphavirus replicon, retroviral gag protein and fusogenic envelope protein may be encoded by the same vector.

[0012] Another embodiment of the description is a method of expressing the recombinant polynucleotide in a eukaryotic cell comprising treating a cell with the VLP described herein. [0013] Another embodiment of the description is a method of delivering the recombinant polynucleotide described herein to a subject, comprising administering to said subject the VLP described herein.

[0014] Another embodiment of the description is a method of treating or preventing a disease or a disorder in a subject, comprising administering to a subject the VLP described herein. Preferably, expression of the gene of interest supplements deficient expression of an endogenous gene by said subject.

[0015] Another embodiment of the description is a pharmaceutical composition comprising the VLP described herein.

[0016] Another embodiment of the description is a eukaryotic cell produced by treating the cell with a VLP described herein.

[0017] To produce VLPS of this sort several components may be produced by transfecting or nucleofecting one or more vectors encoding these components into a cell line for in vitro production. In some embodiments, these components are encoded by separate vectors to reduce the likelihood that the resulting VLP will be replication competent. For example, a multi- vector system may be used where one vector encodes the genetic material, such as an alphavirus- based RNA polynucleotide, to be packaged by the VLP; another encodes the structural proteins of the VLP, such a gag protein; and another vector encodes a fusion protein, such as VSV-G, to facilitate fusion of the VLP to the membrane of a target cell. The alphavirus-based RNA polynucleotide can be derived from any alphavirus. In some embodiments, the RNA

polynucleotide is derived from Sindbis virus and encodes Sindbis virus nonstructural protein. In some embodiments, the RNA polynucleotide is derived from Venezuelan equine encephalitis virus (VEEV) and encodes VEEV nonstructural proteins. However, other alphavirus

nonstructural proteins may suffice for the RNA construct described herein. Suitable alphaviruses include Aura virus, Babanki virus, Barmah Forest virus, Bebaru virus, Cabassou virus,

Chikungunya virus, Eastern equine encephalitis virus, Everglades virus, Fort Morgan virus, Getah virus, Highlands J virus, Kyzylagach virus, Mayaro virus, Me Tri virus, Middelburg virus, Mosso das Pedras virus, Mucambo virus, Ndumu virus, O'nyong-nyong virus, Pixuna virus, Rio Negro virus, Ross River virus, Salmon pancreas disease virus, Semliki Forest virus, Southern elephant seal virus, Tonate virus, Trocara virus, Una virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus, and Whataroa virus.

[0018] Also described herein are cells comprising the vectors described to produce

VLPs. These cells may be used to produce the VLPs described herein by transcribing or expressing the polynucleotides of the vectors. For instance, a mammalian cell transfected with a vector having a polynucleotide sequence encoding an alphavirus RNA construct having a gene or polynucleotide of interest and a packaging signal, a vector encoding a retroviral gag protein, and a vector encoding a viral fusion protein could produce a VLP having the expressed viral fusion protein on its surface with one or two copies of the encoded alphavirus RNA construct housed inside the VLP. Furthermore, because none of these vectors encode alphavirus structural proteins the possibility of creating an infectious virus is substantially reduced compared to systems that do include alphavirus structural proteins.

[0019] VLPs produced using the vectors and cells are also described herein. The VLPs described herein will have four general characteristics: they will comprise one or two RNA molecules encoding an alphavirus replicon, and optionally a protein of interest; they will have a viral core comprising a retroviral gag protein, or, in some embodiments, a gag fusion protein; they will have a surface protein to facilitate fusion with a cell, and they will not contain a polynucleotide that encodes an alphavirus structural protein.

[0020] The VLPs described herein may be produced in a variety of ways, as will be apparent to those skilled in the art based on the provided disclosure. The commonality to these various methods is the expression of the described vectors in a cell capable of expressing the necessary proteins (gag and a fusion protein) and producing the alphavirus-based RNA replicon.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Fig. 1 illustrates the elements of the three vectors exemplified herein. Fig. 1A shows the plasmid pCMV-Sin Rep-protein of interest-2 (POI-2), which is a Sindbis virus-based replicon. Fig. IB shows the plasmid pGAG-protein of interest- 1 (POI-1), which encodes a retroviral gag protein. Fig. 1C shows the plasmid pEnvelope, which encodes the viral fusion protein, such as VSV-G.

[0022] Fig. 2 schematically shows an example of a vector according to the description corresponding to the plasmid depicted in Fig. 1A. The DNA sequence of the vector is provided in the Sequence Listing, appended hereto.

[0023] Fig. 3 schematically represents how the transducing VLPs described herein are produced.

[0024] Fig. 4 depicts the results of three separate VLP transduction experiments. Fig. 4A shows cells transduced with a GFP-expressing Sindbis virus replicon packaged into VLPs obtained from supernatants collected from Baby Hamster Kidney (BHK)-21 cells transfected with all three plasmids; Fig. 4B shows the absence of GFP expression in cells incubated with cell supernatants collected from BHK-21 cells nucleofected with only the pCMV-Sin Rep-POI-2 and pGAG-POI-1 plasmids in the absence of envelope protein. Fig. 4C shows transduction of human embryonic kidney (HEK293T) cells to demonstrate that the constructed VLPs can transduce human cells.

[0025] Fig. 5 shows that the VLPs described herein can successfully transduce cells following storage at 4 °C for 1 hour (A), 2 days (B), 4 days (C), and 8 days (D).

[0026] Fig. 6 shows the expression levels of Gaussia luciferase by cells transduced with VLPs having a VEEV replicon capable of expressing the Gaussia luciferase gene (Fig. 6A, condition 4). Conditions I, 2, and 3 of Fig. 6A show expression of Gaussia luciferase by cells transduced with VEEV replicons having no exogenous gene (condition 1) or with a gene encoding GFP (conditions 2 and 3). Fig. 6B provides an illustration of the expression kinetics of the luciferase protein during the first 4 hours after transduction.

[0027] Fig. 7 illustrates the genetic process by the cre/lox system can be used to alter gene expression in a cell by delivering ere recombinase to a cell via transduction with a VLP having a VEEV replicon with the ere recombinase gene (Fig. 7A). The gene expression profile for a cell line engineered to express GFP in the absence of ere recombinase and RFP in the presence of ere recombinase is shown before and after (days 5, 6, and 7) transduction with a VLP carrying a replicon capable of expressing ere recombinase. Fig. 7B shows results of delivering functional ere recombinase (red cells) to cells engineered to express GFP in the absence of ere recombinase.

[0028] Fig. 8 depicts expression of GFP following transduction of BHK-21 cells, 293T cells, or NIH3T3 cells with VLPs having either a Sindbis virus or VEEV-based replicon capable of expressing GFP. 24 and 48 hour time points are shown for all samples.

[0029] Fig. 9 Illustrates functional packaging of non-coding RNA into VLPs. Cells transduced with VLPs containing: VEE-rep-GFP (encoding GFP) (Fig. 9A), VEE-Rep miGFP (encoding microR A for GFP) (Fig. 9B), VEERep-GFP and VEERep miGFP (simultaneous transduction) (Fig. 9C), VEERep-GFP and VEERep miGFP (miRNA transduction occurred 4 hours prior to GFP transduction) (Fig. 9D), VEE-rep-Cre ( used in place of scrambled miRNA to demonstrate specificity of miRNA) (Fig. 9E), VEE-Rep-GFP and VEE-Rep -Cre (simultaneous transduction) (Fig. 9F), or VEERep-Cre, incubation of cells for 4 hr prior to transduction with VLPs containing VEE-Rep-GFP (Fig. 9G).

[0030] Fig. 10 shows a schematic representation of an alphavirus replicon having two different genes (encoding either HLA-DRl or CD80) the expression of which can occur in the same cell (Fig. 10A). Images of cells expressing both proteins following transduction with a

VLP having a replicon shown in Fig. 10A are provided in Fig. 10B, where HLA-DRl expression is visualized using immunospecific labeling with FITC (green) and CD80 is visualized with immunospecific labeling with phycoerythrin (red); a merged image is also shown to illustrate coexpression in the same cells.

[0031] Fig. 1 1 provides a schematic representation of pDest472-VEE-MCS.

[0032] Fig. 12 provides a schematic representation of pDest472-VEE.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0033] Various alphavirus-based expression vectors for transgene expression in target cells have been described (Xiong C, et al., 1989, Science 1188-91 ; and Bredenbeek P. et al, 1993, J. Virol. 6439-46). For safety considerations these expression systems usually comprise two plasmids. One plasmid contains the coding sequence of the viral replicon (i.e., non-structural proteins) and an internal promoter and transgene coding region, while the second plasmid encodes the viral structural genes. These plasmids are used to generate mRNA in vitro, which is then electroporated into host cells to generate one-round infectious virus particles. These viral particles are then used to infect target cells for transgene expression. These particles raise safety concerns, however, because recombination between the sequence elements encoding the nonstructural and the structural viral elements can yield replication-competent alphavirus particles having the ability to mediate a significant cytopathic effect in vivo.

[0034] A possible solution to this problem is to use unrelated VLPs to deliver alphavirus replicons to the cytoplasm of mammalian cells where they can replicate autonomously and express genes of interest without any nuclear involvement. These VLPs can be produced using three vectors. The first vector comprises the coding sequence for the alphavirus replicon under the control of a mammalian promoter (e.g., CMV), a retro viral-specific RNA packaging signal, and a gene or polynucleotide of interest. The gene may express a protein with therapeutic or research applications, or a shRNA or other regulatory nucleic acid. The second vector comprises retroviral Gag. The third vector would provide the suitable envelope glycoprotein for infection of target cells.

[0035] Upon co-transfection into an appropriate packing cell line, RNA molecules transcribed from the cellular promoter present in the first vector will be packaged into VLPs produced from the second vector. These VLPs can deliver the alphavirus-based replicon to a target cell based on the envelope glycoprotein present in the VLPs. Once inside the cell, the host translational machinery will translate the introduced alphavirus RNA and produce alphavirus replication proteins, which will in turn amplify the RNA and express the gene or polynucleotide of interest. Mutant replicons such as the one described above can greatly prolong the duration of expression with minimal impact on the host cell. Moreover, DNA encoding genes for alphavirus structural elements will be absent in the target cell, so the safety of the proposed system is greatly enhanced.

[0036] Described herein are compositions relating to VLPs and methods for making and using the described VLPs. The described compositions include VLPs, and vectors and cells used to produce the described VLPs. The related methods described herein relate to methods of producing the VLPs, methods of transducing cells using the VLPs, and methods of producing a protein or polynucleotide of interest in a target cell using the VLPs described herein. Also described are alphavirus-based replicons that allow for expression of proteins or polynucleotides of interest in a target cell without the risk of viral infection.

Definitions

[0037] When the terms "one," "a," or "an" are used in this disclosure, they mean "at least one" or "one or more," unless otherwise indicated.

[0038] The term "fusogenic protein" as used herein is meant to refer to a protein that can induce the fusion of the plasma membrane derived envelope of the VLP to the membrane of the recipient cell.

[0039] The terms "express" and "produce" are used synonymously herein, and refer to the biosynthesis of a gene product. These terms encompass the transcription of a gene into RNA. These terms also encompass translation of RNA into one or more polypeptides, and further encompass all naturally occurring post-transcriptional and post-translational modifications. The expression or production of an antibody or antigen-binding fragment thereof may be within the cytoplasm of the cell, or into the extracellular milieu such as the growth medium of a cell culture.

[0040] "Polynucleotide," synonymously referred to as "nucleic acid molecule," "nucleotides" or "nucleic acids," refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotides" include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single- stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, "polynucleotide" refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications may be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" also embraces relatively short nucleic acid chains, often referred to as oligonucleotides.

[0041] "Replicon" as used herein refers to a polynucleotide having the genetic elements necessary to facilitate replication of its sequence and while also being capable of undergoing translation.

[0042] "Virus-like particle" (VLP), as used herein, refers to a structure resembling a virus particle. In preferred embodiments, a VLP contains at least one fusogenic protein displayed on the surface of the particle. A virus-like particle in accordance with the invention lacks all or part of the replicative components of the viral genome. Typically, a virus-like particle in accordance with the invention does not carry genetic information encoding for the proteins of the virus-like particle.

Vectors

[0043] Described herein are vectors for use in producing VLPs carrying an alphavirus- based replicon that does not encode alphavirus structural proteins. To produce VLPS of this sort, several components may be produced by transfecting or nucleofecting one or more vectors encoding these components into a cell line for in vitro production. In some embodiments, these components are encoded by separate vectors to reduce the likelihood that the resulting VLP will be replication competent. For example, a multi-plasmid system may be used where one plasmid encodes the genetic material, such as an RNA polynucleotide encoding Sindbis virus or VEEV nonstructural proteins, to be packaged by the VLP; another encodes the structural proteins of the VLP, such as gag; and another plasmid encodes a fusion protein, such as VSV-G, to facilitate fusion of the VLP to the membrane of a target cell.

[0044] The vectors encoding the genetic material to be packaged by a host cell can take a variety of forms, such as selectable or inducible plasmids, but generally have some common characteristics. For example, vectors encoding an RNA alphavirus-based replicon described herein may include a promoter sequence, a retroviral packaging signal sequence, translation initiation sequences, nonstructural alphavirus proteins, a cloning site for inserting a gene or polynucleotide of interest, an inserted gene or polynucleotide of interest, a 3' untranslated region, and a poly-adenosine tail segment.

[0045] In some embodiments the described vectors include a promoter element that allows for segments of the vector to be transcribed by a host cell. In some embodiments the vector sequence may be transcribed into RNA to be packaged into a VLP. In most embodiments of the described vectors, the promoter sequence will be located upstream of all of the translatable elements included within the vector (see for example, Fig. 1(a) illustrating the location of the cytomegalovirus promoter "pCMV"). In some embodiments described herein the promoter sequence will be derived from a virus, such as cytomegalovirus (CMV), or simian virus 40 (SV40). Numerous other promoter sequences are well known to those skilled in the art and their use with the vectors described herein would be apparent based on the description provided.

[0046] In some embodiments the described vectors encoding the genetic material to be packaged by a host cell can include a polynucleotide sequence that encodes a retroviral packaging signal sequence (also known as a psi (Ψ) element) to allow one or two copies of the RNA sequence transcribed from the vector to be packaged into a VLP particle formed in a host cell. Most, if not all, retroviruses have a packaging sequence of this nature, thus these sequences, and their incorporation into the described vectors, will be readily apparent to those skilled in the art. In some embodiments the vectors described herein include a polynucleotide sequence that encodes a retroviral packaging sequence derived from Rous sarcoma virus, Moloney murine leukemia virus, simian immunodeficiency virus (SIV), HIV, human T-lymphotropic virus, and the like. In a particular embodiment, the retroviral packaging sequence is derived from Rous sarcoma virus. Alternatively, the retroviral packaging sequence is derived from murine leukemia virus.

[0047] Another aspect of the vectors encoding the genetic material to be packaged by a host cell described herein are translation initiation sequences, which allow the RNA sequence encoded by the vector to be translated in a host cell. In some embodiments the described translation initiation sequences may be capable of allowing for expression of alphavirus-based nonstructural proteins, which can replicate the RNA sequence carried by the described VLPs once it is delivered to the host cell. In some embodiments, the described translation initiation sequences may be capable of allowing for expression of a gene of interest. In some embodiments the translation initiation sequence may allow for the gene of interest to be translated by host cell translation complexes. In some embodiments the translation initiation sequences described herein may be derived from an alphavirus, such as Sindbis virus or VEEV. In other embodiments the translation initiation sequences may be derived from other genes, such as virus genes known to have translation initiation sequences capable of initiating translation of an RNA sequence by mammalian translation complexes. Alternatively, the translation initiation sequences may be derived from other genes, such as the native translation initiation sequence of the gene of interest inserted into the described alphavirus replicon. In some embodiments the translation initiation sequences described herein may be located at more than one location in the packaged RNA molecule, and thus may be encoded one or more times by the described vectors. For example, it may be desirable to translate the described Sindbis or VEEV nonstructural proteins separately from a gene of interest encoded by the package RNA molecule. In such an instance, both the polynucleotide(s) encoding the nonstructural proteins and the polynucleotide encoding the protein of interest will have separate translation initiation sequences located 5' of their position in the vector and packaged RNA. Based on this description, those skilled in the art will understand that a variety of translation initiation sequences capable of promoting the translation of RNA in a mammalian cell may be incorporated to the described VLP -packaged RNAs described herein.

[0048] The vectors encoding genetic material to be packaged by a host cell may also include polynucleotides that encode nonstructural alphavirus proteins, such as nonstructural proteins from Sindbis virus or VEEV. For example, in some embodiments the described vectors may include polynucleotides that encode one or more Sindbis virus nonstructural proteins. In some embodiments the described vectors may include polynucleotides that encode one or more VEEV nonstructural proteins. In some embodiments described vectors may include

polynucleotides that encode the Sindbis virus or VEEV nonstructural protein NSP1. In some embodiments described vectors may include polynucleotides that encode the Sindbis virus or VEEV nonstructural protein NSP2. In some embodiments described vectors may include polynucleotides that encode the Sindbis virus or VEEV nonstructural protein NSP3. In some embodiments described vectors may include polynucleotides that encode the Sindbis virus or VEEV nonstructural protein NSP4. In some embodiments described vectors may include polynucleotides that encode the Sindbis virus or VEEV nonstructural proteins NSP1, NSP2, NSP3, and NSP4. In some embodiments the polynucleotide of the described vector that encodes the alphavirus nonstructural proteins will be derived from the corresponding genomic sequence of an alphavirus genome, such as that of Sindbis virus or VEEV. In some embodiments, the polynucleotides encoding the alphavirus nonstructural proteins are void of any polynucleotides that encode the alphavirus structural proteins, regardless of whether the structural proteins are from the same or a different alphavirus than the nonstructural protein sequences present.

[0049] The vector described herein for incorporating genetic material to be packaged by a host cell may also contain a polynucleotide of interest that may be expressed in a host cell transduced by a VLP carrying the genetic material encoded by the vector. In some embodiments the described vectors may encode an RNA polynucleotide sequence to be packaged into a VLP, which can then be delivered to a host cell by VLP-mediated transduction of the cell. Once the

RNA polynucleotide sequence has been delivered to the target cell a polynucleotide of interest encoded by the RNA may provide for expression of a protein of interest. Accordingly, the vectors described herein are designed to encode an RNA for packaging into a VLP that can express a gene of interest once inside a target cell. Therefore, in some embodiments the described vectors will include a polynucleotide sequence of interest. In some embodiments of the described vector, the polynucleotide sequence of interest may encode a protein of interest. For example, the polynucleotide sequence of interest may encode GFP in some embodiments and serve a detectable marker of viral transduction of a target cell. In another embodiment, the polynucleotide sequence of interest may encode a functional version of a protein endogenous to the target cell. In another embodiment, the polynucleotide sequence of interest may encode a functional version of a protein endogenous to the target subject. In another embodiment, the polynucleotide sequence of interest may encode a protein that is foreign to the target cell. In another embodiment, the polynucleotide sequence of interest may encode a protein that is foreign to the target subject. In some embodiments the polynucleotide sequence of interest may encode a protein capable of having a therapeutic effect on a target cell. In some embodiments the polynucleotide sequence of interest may encode a protein capable of having a therapeutic effect on a target subject. In an alternative embodiment the polynucleotide sequence of interest may server as an interfering RNA molecule and function to regulate endogenous gene expression in a host cell. For example, in some embodiments the polynucleotide sequence of interest may comprise a sequence that provides for the formation of an RNA hairpin loop to initiate RNA interference. In addition, the polynucleotide of interest could be a positive or negative sense strand of RNA that can be transcribed by the RNA-dependent RNA polymerase complex formed by the alphavirus nonstructural proteins encoded by the packaged RNA molecule. Since this

RNA-dependent RNA polymerase can transcribe RNA in the positive-sense and negative-sense directions, an interfering RNA sequence, such as a miRNA or shRNA, may be inserted into the packaged RNA replicon and can be designed to encode an interfering polynucleotide in either direction. Those of skill in the art will appreciate the therapeutic characteristic of this aspect of the described transduction system, as it can allow for expression of selected proteins in a subject. In accordance with this aspect of the described vector, a cloning site having one or more restriction endonuclease sites may also be included in the vector, to facilitate insertion of a polynucleotide sequence of interest.

[0050] Another vector useful in the production of the VLPs described herein is a vector that encodes a virus structural protein. One such class of proteins is the retroviral group-specific antigen (gag) protein. The gag protein is the core structural protein of retroviruses and, in some instances, is capable of forming enveloped virus cores when expressed in eukaryotic cells. This property makes gag proteins particularly useful in the production of VLPs, because they can form the basic structural aspect of the particle and allow for packaging of RNA associated with a retroviral packaging signal sequence. Accordingly, described herein are vectors that include a polynucleotide that encodes a retroviral gag protein. In some embodiments, the described vectors include a polynucleotide that encodes a retroviral gag protein and a promoter polynucleotide sequence that allows for the gag gene sequence to be transcribed into mRNA by host cell RNA polymerase. In one embodiment, the promoter polynucleotide sequence is derived from a virus, such as SV40 or CMV. In some embodiments, the vector will further include a polynucleotide that encodes a protein of interest. Those skilled in the relevant art will understand that a polynucleotide sequence of a gag protein from any retrovirus may be used to produce the vectors and VLPs described herein. In some embodiments the polynucleotide sequence encoding the gag protein may be derived from Rous sarcoma virus. In some embodiments the polynucleotide sequence encoding the gag protein may be derived from murine leukemia virus. In some embodiments the polynucleotide sequence encoding the gag protein may be derived from SIV. In some embodiments the polynucleotide sequence encoding the gag protein may be derived from human T-lymphotropic virus.

[0051] Another vector useful in the production of the VLPs described herein is a vector that encodes a protein to mediate fusion between the VLP envelope and a host cell. A class of proteins suitable for this purpose is viral fusion proteins, which facilitate virus infection of cells by allowing an enveloped virus to fuse its membrane with that of a host cell. Many of viral fusion proteins also have known, or suspected, cellular receptor proteins that may allow for targeting of selected cell types, or in cases of more ubiquitous receptors, such as sialic acid for influenza virus, more generalized targeting may be desired. In some instances, viral fusion proteins work in conjunction with viral attachment proteins, ligands for cellular receptor, a receptor for a cell ligand, or accessory proteins, thus proteins of this sort may also be encoded by the described vectors, in addition to, or also by, the vector encoding a viral fusion protein. Alternatively, in some embodiments a viral fusion protein from one virus may be encoded by the described vector along with a viral attachment protein of another virus, a ligand of a cellular receptor, a receptor of a cell ligand, or an accessory protein to facilitate, or direct, targeting of a VLP to a desired cell type. In some embodiments the viral fusion protein, viral attachment protein, ligand of a cellular receptor, receptor of a cell ligand, or accessory protein will be a type- I membrane protein, which will allow the extracellular domain of the protein to be oriented extracellularly when present on the cell surface. This will also allow the fusion protein to be correctly oriented following budding of a VLP from a packaging cell. Expression of such proteins in a cell will typically result in the cell surface being coated with the proteins, so that budding of a VLP from any part of the cell membrane will provide the VLP with some amount of the protein(s) on its surface. In some embodiments, the described vectors include a polynucleotide that encodes a viral fusion protein and a promoter polynucleotide sequence that allows for the fusion protein gene sequence to be translated into mRNA by host cell RNA polymerase. In one embodiment, the promoter polynucleotide sequence is derived from a virus, such as SV40 or CMV. In some embodiments, the described vectors include a polynucleotide that encodes a viral attachment protein and a promoter polynucleotide sequence that allows for the attachment protein gene sequence to be translated into mRNA by host cell RNA polymerase. In one embodiment, the promoter polynucleotide sequence is derived from a virus, such as SV40 or CMV. In some embodiments the vectors described herein include a polynucleotide that encodes a vesicular stomatitis virus G protein. In some embodiments the vectors described herein include a polynucleotide that encodes the influenza hemaglutinin protein. In some embodiments the vectors described herein include a polynucleotide that encodes the influenza neuraminidase protein. In some embodiments the vectors described herein include a polynucleotide that encodes the respiratory syncytial virus fusion protein. In some embodiments the vectors described herein include a polynucleotide that encodes the rotavirus VP7 protein. Other such fusion proteins will be apparent to those skilled in the art based on desired tropism or cell target of the associated virus.

Cells expressing the described vectors

[0052] Provided herein are cells comprising the vectors described to produce VLPs. These cells may be used to produce the VLPs described herein by transcribing or expressing the polynucleotides of the vectors. For instance, a mammalian cell transfected with a vector having a polynucleotide sequence encoding an alphavirus RNA construct having a gene or polynucleotide of interest and a packaging signal, a vector encoding a retroviral gag protein, and a vector encoding a viral fusion protein could produce a VLP having the expressed viral fusion protein on its surface with one or two copies of the encoded alphavirus RNA construct housed inside the VLP. Furthermore, because none of these vectors encode alphavirus structural proteins the possibility of creating an infectious virus is substantially reduced compared to systems that do include alphavirus structural proteins.

[0053] The described cells may be any eukaryotic cell capable of transcribing, and where necessary (such as in the case of the gag and fusion proteins), translating the

polynucleotides of the described vectors. The cells will likely be mammalian cells in many embodiments. For example, rodent cells, such as murine, hamster (CHO or BHK-21), or rat cells could be used to express the described vectors; canine cells, such as Madin Darby canine kidney cells, could be used to express the described vectors; primate cells, such as vero cells, could be used to express the described vectors; and human cells, such as HEK293T cells (human kidney), Hep-2 cells (human airway), Caco-2 (intestine), HeLa (epithelium), and other such cell lines known in the art, could be used to express the described vectors. In some embodiments the described cells can be transfected and selected, using standard transfection and selection methods known in the art, to stably comprise one or more of the described vectors.

[0054] In some embodiments the cell lines described herein will contain a vector comprising a polynucleotide sequence encoding an alphavirus replicon wherein the alphavirus replicon encodes a protein of interest, a vector comprising a polynucleotide sequence encoding a gag protein, and a vector comprising a polynucleotide sequence encoding a heterologous fusogenic envelope protein, wherein neither the vectors nor the cell contain a gene encoding an alphavirus structural protein. In some embodiments the alphavirus replicon may be derived from Sindbis virus or VEEV. In some embodiments the alphavirus replicon may have polynucleotide sequences that encode Sindbis virus or VEEV nonstructural proteins NSP1, NSP2, NSP3, NSP4, and a retroviral packaging signal. In some embodiments the retroviral packaging signal may be derived from either Rous sarcoma virus or murine leukemia virus. In some embodiments the polynucleotide sequence encoding the gag protein is derived from Rous sarcoma virus. In some embodiments the polynucleotide sequence encoding the heterologous fusogenic envelope protein encodes VSV-G.

Virus-like particles

[0055] VLPs produced using the vectors and cells are also described herein. The VLPs described herein will have four general characteristics: they will comprise one or two RNA molecules encoding an alphavirus replicon, and optionally a protein of interest; they will have a viral core comprising a retroviral gag protein, or, in some embodiments, a gag fusion protein; they will have a surface protein to facilitate fusion with a cell, and they will not contain a polynucleotide that encodes an alphavirus structural protein.

[0056] The VLPs described herein will be useful in transducing cells in order to express a protein of interest therein. Accordingly, the described VLPs may incorporate one or two alphavirus-based RNA polynucleotides capable of encoding a protein of interest. To facilitate translation of the RNA sequence some embodiments of the described packaged RNA may include translation initiation sequences as described herein. In some embodiments the RNA sequence incorporated into the VLP will include a retroviral packaging sequence that will facilitate inclusion of the RNA into a forming VLP. In some embodiments the retroviral packaging sequence is derived from Rous sarcoma virus, Moloney murine leukemia virus, simian immunodeficiency virus (SIV), HIV, human T-lymphotropic virus, and the like. In a particular embodiment, the retroviral packaging sequence is derived from Rous sarcoma virus.

Alternatively, the retroviral packaging sequence may be derived from murine leukemia virus. In addition, the RNA sequences included in the VLP may be capable of encoding nonstructural alphavirus proteins. For example, in some embodiments the packaged RNA may encode one or more Sindbis virus or VEEV nonstructural proteins. In some embodiments the packaged RNA may encode the Sindbis virus or VEEV nonstructural protein NSP1. In some embodiments the packaged RNA may encode the Sindbis virus or VEEV nonstructural protein NSP2. In some embodiments the packaged RNA may encode the Sindbis virus or VEEV nonstructural protein

NSP3. In some embodiments the packaged RNA may encode the Sindbis virus or VEEV nonstructural protein NSP4. In some embodiments the packaged RNA may encode the Sindbis virus or VEEV nonstructural proteins NSP1, NSP2, NSP3, and NSP4. The packaged RNA may also include the polynucleotide sequence of a protein of interest. For example, the polynucleotide sequence of interest may encode GFP in some embodiments and serve a detectable marker of viral transduction of a target cell. In another embodiment, the polynucleotide sequence of interest may encode a functional version of a protein endogenous to the target cell. In another embodiment, the polynucleotide sequence of interest may encode a functional version of a protein endogenous to the target subject. In another embodiment, the polynucleotide sequence of interest may encode a protein that is foreign to the target cell. In another embodiment, the polynucleotide sequence of interest may encode a protein that is foreign to the target subject. In some embodiments the polynucleotide sequence of interest may encode a protein capable of having a therapeutic effect on a target cell. In some embodiments the polynucleotide sequence of interest may encode a protein capable of having a therapeutic effect on a target subject. Those of skill in the art will appreciate the therapeutic characteristic of this aspect of the described VLPs, as they can allow for expression of selected proteins in a cell or subject.

[0057] The described VLPs may also comprise a viral gag protein to provide a viral core structure to the particle. The gag protein is the core structural protein of retroviruses and, in some instances, is capable of forming enveloped virus cores when expressed in eukaryotic cells. This property makes gag proteins particularly useful in the production of VLPs, because they can form the basic structural aspect of the particle and allow for packaging of R A associated with a retroviral packaging signal sequence. Those skilled in the relevant art will understand that a gag protein from any retrovirus may be used to produce the vectors and VLPs described herein. In some embodiments the gag protein may be derived from Rous sarcoma virus. In some embodiments the gag protein may be derived from murine leukemia virus. In alternative embodiments the gag protein may be derived from SIV, HIV, human T-lymphotropic virus, or similar retrovirus.

[0058] Another component of the VLPs described herein is a protein to mediate fusion between the VLP envelope and a host cell. A class of proteins suitable for this purpose is viral fusion proteins, which facilitate virus infection of cells by allowing an enveloped virus to fuse its membrane with that of a host cell. Many of viral fusion proteins also have known, or suspected, cellular receptor proteins that may allow for targeting of selected cell types, or in cases of more ubiquitous receptors, such as sialic acid for influenza virus, more generalized targeting may be achieved. In some instances, viral fusion proteins may work in conjunction with viral attachment proteins, ligands of cellular receptors, receptors of cellular ligands, or accessory proteins, thus proteins of this sort may also be present on the VLP surface in addition to a viral fusion protein.

Alternatively, in some embodiments the described VLPs may have a viral fusion protein from one virus and a viral attachment protein of another virus, a ligand of a cellular receptor, a receptor of a cellular ligand, or an accessory protein to facilitate, or direct, targeting of a VLP to a desired cell type. Similarly, the described VLPs may be produced to have more than one fusion protein in the VLP envelope, as this may facilitate fusion to a select variety of cell types. In some embodiments the VLP surface protein(s) will be a type-I membrane protein, which will allow the extracellular domain of the protein to be oriented extracellularly when present on the cell surface. This will also allow the fusion protein to be correctly oriented following budding of a

VLP from a packaging cell. Expression of such proteins in a cell will typically result in the cell surface being coated with the proteins, so that budding of a VLP from any part of the cell membrane will provide the VLP with some amount of the fusion protein on its surface. In some embodiments the VLPs described herein include a vesicular stomatitis virus G protein (VSV-G) to mediate cell fusion. In some embodiments the VLPs described herein include an influenza hemaglutinin protein to mediate cell fusion. In some embodiments the VLPs described herein include an influenza neuraminidase protein to facilitate cell fusion. In some embodiments the VLPs described herein include respiratory syncytial virus fusion protein. In some embodiments the VLPs described herein include the rotavirus VP7 protein. Other such fusion proteins will be apparent to those skilled in the art based on desired tropism or cell target of the associated virus.

[0059] The VLPs described herein may comprise an alphavirus replicon, wherein the alphavirus replicon includes a polynucleotide of interest or encodes a protein of interest, retroviral gag protein, and heterologous fusogenic envelope protein; wherein the VLP does not contain an alphavirus structural protein gene. In some embodiments the alphavirus replicon of the VLP is derived from Sindbis virus or VEEV. For example, the VLPs described herein may have an alphavirus replicon encoding Sindbis virus or VEEV nonstructural proteins NSP1, NSP2, NSP3, and NSP4. In some embodiments the retroviral packaging signal associated with the packaged RNA in the described VLPs is derived from either Rous sarcoma virus or murine leukemia virus. Based on this description, those skilled in the art will readily understand that the described VLPs may be modified to incorporate aspects of viruses that may facilitate VLP stability, RNA packaging, or cell entry. Such modifications should be understood to be within the scope of the disclosures provided herein.

Methods of producing the described VLPs

[0060] The VLPs described herein may be produced in a variety of ways, as will be apparent to those skilled in the art based on the provided disclosure. The commonality to these various methods is the expression of the described vectors in a cell capable of expressing the necessary proteins (gag and a fusion protein) and producing the alphavirus-based RNA replicon. Accordingly, a method of producing a VLP described herein may include co-transforming, transfecting, or nucleofecting a eukaryotic cell with a vector comprising a polynucleotide sequence encoding an alphavirus replicon, wherein the alphavirus replicon includes a polynucleotide of interest or encodes a protein of interest; a vector comprising a polynucleotide sequence encoding a retroviral gag protein; and a vector comprising a polynucleotide sequence encoding a heterologous fusogenic envelope protein; and culturing the co-transformed cell under conditions suitable to cause each vector to produce its encoded product, thereby producing a virus-like particle. In some embodiments the polynucleotide sequence encoding the alphavirus replicon may be derived from Sindbis virus or VEEV. In some embodiments the alphavirus replicon may have polynucleotide sequences that encode Sindbis virus or VEEV nonstructural proteins NSPl, NSP2, NSP3, NSP4, and a retroviral packaging signal. In some embodiments the retroviral packaging signal may be derived from either Rous sarcoma virus or murine leukemia virus. In some embodiments the polynucleotide sequence encoding the gag protein is derived from Rous sarcoma virus. In some embodiments the polynucleotide sequence encoding the heterologous fusogenic envelope protein encodes VSV-G.

Compositions and methods of administration

[0061] Described herein are compositions comprising at least one described VLP and a pharmaceutically acceptable carrier. Such compositions are useful, for example, for

administration to subjects in need of expression of an exogenous protein or increased expression of a protein normally found in those of the same species as the subject. The compositions may be formulated as any of various preparations that are known and suitable in the art, including those described and exemplified herein. In some embodiments, the compositions are aqueous formulations. Aqueous solutions may be prepared by admixing the VLPs in water or suitable physiologic buffer, and optionally adding suitable colorants, flavors, preservatives, stabilizing and thickening agents and the like as desired. Aqueous suspensions may also be made by dispersing the VLPs in water or physiologic buffer with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.

[0062] The compositions may be formulated for injection into a subject. For injection, the compositions described may be formulated in aqueous solutions such as water or in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. The solution may contain one or more formulatory agents such as suspending, stabilizing or dispersing agents. Injection formulations may also be prepared as solid form preparations which are intended to be converted, shortly before use, to liquid form preparations suitable for injection, for example, by constitution with a suitable vehicle, such as sterile water, saline solution, or alcohol, before use.

[0063] The compositions may be formulated for aerosolized delivery to a subject. For aerosol delivery, the compositions described may be formulated in aqueous solutions such as water or in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. The solution may contain one or more formulatory agents such as suspending, stabilizing or dispersing agents. [0064] The compositions may be formulated in sustained release vehicles or depot preparations. Such long-acting formulations may be administered by implantation (for example subcutaneous ly or intramuscularly) or by intramuscular injection. Thus, for example, the compositions may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. Liposomes and emulsions are well-known examples of delivery vehicles suitable for use as carriers for hydrophobic drugs.

[0065] The following examples are provided for illustrative purposes and are meant to enhance, not limit, the preceding disclosure.

EXAMPLE 1 - Production of an Alphavirus-based Gene Expression System

[0066] An alphavirus gene expression system was designed to allow for VLP-mediated delivery an exogenous gene of interest (GOI) or protein of interest (POI) to a target cell with low risk of causing cytopathic viral infection. The expression system was designed using three vectors, which can be expressed in a packaging cell line to produce a transducing VLP. One vector codes for the alphavirus-based expression construct, another vector codes for a retroviral gag protein to facilitate VLP formation, and a third vector codes for a fusion protein to mediate VLP fusion to the host cell. In addition, the system was constructed to work without the need for alphavirus structural proteins being present.

[0067] To accomplish this, an alphavirus-based DNA plasmid was produced having a cytomegalovirus promoter (CMV); followed by a retroviral packaging signal of respective retroviral packaging protein GAG; followed by a Sindbis or VEE virus genes encoding nonstructural proteins NSP1, NSP2, NSP3, and NSP4; and finally, one or more subgenomic promoter (SGP; a promoter for virus-encoded RNA-dependent RNA polymerase, resulting in the formation of mRNA) to drive expression of a of a gene of interest (GOI), consisting of a recombinant polynucleotide, and inserted into a multiple cloning site; a 3 ' untranslated region (URT); and a polyA tail. Fig. 2 shows an example of such an alphavirus-based DNA plasmid. In another version of this expression vector, the retroviral packaging signal (GAG) is omitted.

[0068] Another plasmid was constructed to encode a retroviral gag protein and a second, optional protein of interest (POI). A third plasmid was constructed to provide expression of a VSV-G viral fusion protein. A schematic of an embodiment of these plasmids is provided in Fig. 1A-1C, respectively. Fig. 1A shows pCMV-Sin Rep-POI-2, Fig. IB shows pGAG-POI-1, and Fig. 1C shows pEnv for VSV-G. [0069] Once constructed the plasmids were tested for the ability to produce VLPs carrying a Sindbis virus replicon having a gene of interest. For these experiments, green fluorescent protein (GFP) was used as the gene of interest in order to facilitate detection of delivery and intracellular expression of the gene. To produce VLPs, each of the three plasmids described above were transfected into baby hamster kidney (BHK-21 ) cells using a standard nucleofection procedure with an Amaxa system according to manufacturer instructions (Lonza) (Fig. 3).

[0070] Briefly, the BHK-21 cells at 3xl0⁶ were re-suspended in 100 μΐ nucleofection solution L (Amaxa) and transferred to tube containing 4.5 μg of plasmid coding for GAG, 3 μg plasmid coding for VSV-G glycoprotein and 100 nanograms of plasmid coding for Sindbis alphavirus replicon or 2.5 micrograms for VEE replicon (in total volume of 10 μΐ). The mixture of cells and plasmids was transferred to nucleofection cuvette and nucleofected using Amaxa nucleofector II apparatus using settings for BHK-21. The nucleofected cells were re- suspended in 500 μΐ of completed culture medium and transferred to tissue culture plate containing culture medium solution and incubated at 37°C for period of 72-96 hr or for 72 hr at 32°C. After this time supernatants consisting of VLPs and encapsidated alphavirus replicon was clarified by centrifugation at 3000 RPM/10 min at 4°C, filtered by 0.45 um filter and exposed to 10 units of DNAse I ( turbo™-DNAse (Ambion)) for 30 min at RT. Processed VLPs were stored at 4°C or frozen on dry ice and transferred to -80°C. As a negative control (fusion-defective VLPs), BHK-21 cells were also nucleofected with only the pCMV-Sin Rep-POI-2 or VEEV- Rep-POI and pGAG-POI-1 plasmids, but not the pEnvelope plasmid encoding VSV-G.

Following transfection, the cells were incubated for 48-72 hours in tissue culture medium under normal growth conditions to allow for plasmid-driven production of VLPs. Once the transfected cells were finished incubating, the tissue culture supernatant, which should contain any produced VLPs, was collected. The collected cell supernatants were then added to cultured BHK-21 cells to determine if the cells could be successfully transduced with GFP. As shown in Fig. 4, cell supernatants collected from BHK-21 cells transfected with all three plasmids resulted in robust GFP expression when exposed to untransfected BHK-21 cells (Fig. 4A). Conversely, no GFP expression was observed for untransfected BHK-21 cells incubated with cell supernatants collected from BHK-21 cells transfected with only the pCMV-Sin Rep-POI-2 and pGAG-POI-1 plasmids (Fig. 4B). Similar experiments were also conducted using human embryonic kidney (HEK293T) cells to demonstrate that the constructed VLPs could transduce human cells (Fig. 4C). Furthermore, the constructed VLPs can also be stored at 4 °C for at least 30 days without losing the ability to transduce cells (Fig. 5A-5D). [0071] Experiments were also conducted to assess the ability of VEEV-based alpha virus replicon to express protein in cells. For these studies BHK-21 cells were transduced with VLPs having a Gaussia luciferase gene inserted into a VEEV replicon. Following transduction, cell supernatants monitored for expression of luciferase protein. As shown in Fig. 6, high amounts of luciferase were detected in the supernatants of cells transduced with the VEEV replicon having the Gaussia luciferase gene (Fig. 6A, condition 4), relative to control VEEV replicons without an exogenous gene (condition 1), or with a gene encoding GFP (conditions 2 and 3). Additionally, expression of the luciferase protein increased rapidly after transduction (Fig. 6B). Similar results were also observed in the context of delivering functional ere recombinase (red cells) to cells engineered to express GFP in the absence of ere recombinase (Fig. 7B).

[0072] Cells were transduced in parallel with either Sindbis-based VLPs encoding GFP or VEEV-based VLPs encoding GFP. As shown in Fig. 8, both alphavirus-based VLPs caused robust GFP expression, while the cells transduced with VEEV-based VLPs were observed to have the higher expression levels (Fig. 8).

EXAMPLE 2 - miRNA Expressed by a VEE Alphavirus Replicon Inhibits Protein Production

[0073] Experiments were conducted to assess whether an alphavirus replicon encoding an miRNA sequence could inhibit protein production. To assess this, BHK-21 cells were transduced with a VLP replicon encoding GFP, miRNA specific for GFP, or ere recombinase (Fig. 9). (See the vectors shown in Figs. 1 1 and 12, and SEQ ID NO: l in Table 1, below.) GFP expression was noticeable reduced when cells were first transduced with a replicon encoding miRNA for GFP and then subsequently transduced with a replicon encoding GFP (4 hours after the intial transduction), as compared to cells transduced with only a replicon encoding GFP (compare Fig. 9A with Fig. 9D). Conversely, when cells were first transduced with a replicon encoding ere recombinase and then subsequently transduced with a replicon encoding GFP (4 hours after the initial transduction), no significant reduction in GFP expression was apparent (Fig. 9G).

EXAMPLE 3 - Expression of Multiple Proteins in Target Cells Transduced with VEE VLPs [0074] To assess whether an alphavirus replicon can express two separate proteins in the same cell, experiments were performed using a VEE replicon having HLA-DRl under the control of one subgenomic promoter and CD80 under a another subgenomic promotor (Fig. 10A). Following production of VLPs having the described VEE replicon, cells were transduced and examined for expression of both proteins. As shown in Fig. 10B, transduced cells were able to express both proteins (immunolabled HLA-DRl is shown in green (FITC) and CD80 shown in red (phycoerythrin).

Table 1 : SEQ ID NO: 1

1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 781 gtaggcgtgt acggtgggag gtctatataa gcagagctcg tatggacata ttgtcgttag 841 aacgcggcta caattaatac ataaccttat gtatcataca catacgattt aggggacact 901 atagattgac ggcgtagtac acactattga atcaaacagc cgaccaattg cactaccatc 961 acaatggaga agccagtagt aaacgtagac gtagaccccc agagtccgtt tgtcgtgcaa 1021 ctgcaaaaaa gcttcccgca atttgaggta gtagcacagc aggtcactcc aaatgaccat 1081 gctaatgcca gagcattttc gcatctggcc agtaaactaa tcgagctgga ggttcctacc 1141 acagcgacga tcttggacat aggcagcgca ccggctcgta gaatgttttc cgagcaccag 1201 tatcattgtg tctgccccat gcgtagtcca gaagacccgg accgcatgat gaaatacgcc 1261 agtaaactgg cggaaaaagc gtgcaagatt acaaacaaga acttgcatga gaagattaag 1321 gatctccgga ccgtacttga tacgccggat gctgaaacac catcgctctg ctttcacaac 1381 gatgttacct gcaacatgcg tgccgaatat tccgtcatgc aggacgtgta tatcaacgct 1441 cccggaacta tctatcatca ggctatgaaa ggcgtgcgga ccctgtactg gattggcttc 1501 gacaccaccc agttcatgtt ctcggctatg gcaggttcgt accctgcgta caacaccaac 1561 tgggccgacg agaaagtcct tgaagcgcgt aacatcggac tttgcagcac aaagctgagt 1621 gaaggtagga caggaaaatt gtcgataatg aggaagaagg agttgaagcc cgggtcgcgg 1681 gtttatttct ccgtaggatc gacactttat ccagaacaca gagccagctt gcagagctgg 1741 catcttccat cggtgttcca cttgaatgga aagcagtcgt acacttgccg ctgtgataca 1801 gtggtgagtt gcgaaggcta cgtagtgaag aaaatcacca tcagtcccgg gatcacggga 1861 gaaaccgtgg gatacgcggt tacacacaat agcgagggct tcttgctatg caaagttact 1921 gacacagtaa aaggagaacg ggtatcgttc cctgtgtgca cgtacatccc ggccaccata 1981 tgcgatcaga tgactggtat aatggccacg gatatatcac ctgacgatgc acaaaaactt 2041 ctggttgggc tcaaccagcg aattgtcatt aacggtagga ctaacaggaa caccaacacc 2101 atgcaaaatt accttctgcc gatcatagca caagggttca gcaaatgggc taaggagcgc 2161 aaggatgatc ttgataacga gaaaatgctg ggtactagag aacgcaagct tacgtatggc 2221 tgcttgtggg cgtttcgcac taagaaagta cattcgtttt atcgcccacc tggaacgcag 2281 acctgcgtaa aagtcccagc ctcttttagc gcttttccca tgtcgtccgt atggacgacc 2341 tctttgccca tgtcgctgag gcagaaattg aaactggcat tgcaaccaaa gaaggaggaa 2401 aaactgctgc aggtctcgga ggaattagtc atggaggcca aggctgcttt tgaggatgct 2461 caggaggaag ccagagcgga gaagctccga gaagcacttc caccattagt ggcagacaaa 2521 ggcatcgagg cagccgcaga agttgtctgc gaagtggagg ggctccaggc ggacatcgga 2581 gcagcattag ttgaaacccc gcgcggtcac gtaaggataa tacctcaagc aaatgaccgt 2641 atgatcggac agtatatcgt tgtctcgcca aactctgtgc tgaagaatgc caaactcgca 2701 ccagcgcacc cgctagcaga tcaggttaag atcataacac actccggaag atcaggaagg 2761 tacgcggtcg aaccatacga cgctaaagta ctgatgccag caggaggtgc cgtaccatgg 2821 ccagaattcc tagcactgag tgagagcgcc acgttagtgt acaacgaaag agagtttgtg 2881 aaccgcaaac tataccacat tgccatgcat ggccccgcca agaatacaga agaggagcag 2941 tacaaggtta caaaggcaga gcttgcagaa acagagtacg tgtttgacgt ggacaagaag 3001 cgttgcgtta agaaggaaga agcctcaggt ctggtcctct cgggagaact gaccaaccct 3061 ccctatcatg agctagctct ggagggactg aagacccgac ctgcggtccc gtacaaggtc 3121 gaaacaatag gagtgatagg cacaccgggg tcgggcaagt cagctattat caagtcaact 3181 gtcacggcac gagatcttgt taccagcgga aagaaagaaa attgtcgcga aattgaggcc 3241 gacgtgctaa gactgagggg tatgcagatt acgtcgaaga cagtagattc ggttatgctc 3301 aacggatgcc acaaagccgt agaagtgctg tacgttgacg aagcgttcgc gtgccacgca 3361 ggagcactac ttgccttgat tgctatcgtc aggccccgca agaaggtagt actatgcgga 3421 gaccccatgc aatgcggatt cttcaacatg atgcaactaa aggtacattt caatcaccct 3481 gaaaaagaca tatgcaccaa gacattctac aagtatatct cccggcgttg cacacagcca 3541 gttacagcta ttgtatcgac actgcattac gatggaaaga tgaaaaccac gaacccgtgc 3601 aagaagaaca ttgaaatcga tattacaggg gccacaaagc cgaagccagg ggatatcatc 3661 ctgacatgtt tccgcgggtg ggttaagcaa ttgcaaatcg actatcccgg acatgaagta 3721 atgacagccg cggcctcaca agggctaacc agaaaaggag tgtatgccgt ccggcaaaaa 3781 gtcaatgaaa acccactgta cgcgatcaca tcagagcatg tgaacgtgtt gctcacccgc 3841 actgaggaca ggctagtgtg gaaaaccttg cagggcgacc catggattaa gcagcccact 3901 aacataccta aaggaaactt tcaggctact atagaggact gggaagctga acacaaggga 3961 ataattgctg caataaacag ccccactccc cgtgccaatc cgttcagctg caagaccaac 4021 gtttgctggg cgaaagcatt ggaaccgata ctagccacgg ccggtatcgt acttaccggt 4081 tgccagtgga gcgaactgtt cccacagttt gcggatgaca aaccacattc ggccatttac 4141 gccttagacg taatttgcat taagtttttc ggcatggact tgacaagcgg actgttttct 4201 aaacagagca tcccactaac gtaccatccc gccgattcag cgaggccggt agctcattgg 4261 gacaacagcc caggaacccg caagtatggg tacgatcacg ccattgccgc cgaactctcc 4321 cgtagatttc cggtgttcca gctagctggg aagggcacac aacttgattt gcagacgggg 4381 agaaccagag ttatctctgc acagcataac ctggtcccgg tgaaccgcaa tcttcctcac 4441 gccttagtcc ccgagtacaa ggagaagcaa cccggcccgg tcaaaaaatt cttgaaccag 4501 ttcaaacacc actcagtact tgtggtatca gaggaaaaaa ttgaagctcc ccgtaagaga 4561 atcgaatgga tcgccccgat tggcatagcc ggtgcagata agaactacaa cctggctttc 4621 gggtttccgc cgcaggcacg gtacgacctg gtgttcatca acattggaac taaatacaga 4681 aaccaccact ttcagcagtg cgaagaccat gcggcgacct taaaaaccct ttcgcgttcg 4741 gccctgaatt gccttaaccc aggaggcacc ctcgtggtga agtcctatgg ctacgccgac 4801 cgcaacagtg aggacgtagt caccgctctt gccagaaagt ttgtcagggt gtctgcagcg 4861 agaccagatt gtgtctcaag caatacagaa atgtacctga ttttccgaca actagacaac 4921 agccgtacac ggcaattcac cccgcaccat ctgaattgcg tgatttcgtc cgtgtatgag 4981 ggtacaagag atggagttgg agccgcgccg tcataccgca ccaaaaggga gaatattgct 5041 gactgtcaag aggaagcagt tgtcaacgca gccaatccgc tgggtagacc aggcgaagga 5101 gtctgccgtg ccatctataa acgttggccg accagtttta ccgattcagc cacggagaca 5161 ggcaccgcaa gaatgactgt gtgcctagga aagaaagtga tccacgcggt cggccctgat 5221 ttccggaagc acccagaagc agaagccttg aaattgctac aaaacgccta ccatgcagtg 5281 gcagacttag taaatgaaca taacatcaag tctgtcgcca ttccactgct atctacaggc 5341 atttacgcag ccggaaaaga ccgccttgaa gtatcactta actgcttgac aaccgcgcta 5401 gacagaactg acgcggacgt aaccatctat tgcctggata agaagtggaa ggaaagaatc 5461 gacgcggcac tccaacttaa ggagtctgta acagagctga aggatgaaga tatggagatc 5521 gacgatgagt tagtatggat tcatccagac agttgcttga agggaagaaa gggattcagt 5581 actacaaaag gaaaattgta ttcgtacttc gaaggcacca aattccatca agcagcaaaa 5641 gacatggcgg agataaaggt cctgttccct aatgaccagg aaagtaatga acaactgtgt 5701 gcctacatat tgggtgagac catggaagca atccgcgaaa agtgcccggt cgaccataac 5761 ccgtcgtcta gcccgcccaa aacgttgccg tgcctttgca tgtatgccat gacgccagaa 5821 agggtccaca gacttagaag caataacgtc aaagaagtta cagtatgctc ctccaccccc 5881 cttcctaagc acaaaattaa gaatgttcag aaggttcagt gcacgaaagt agtcctgttt 5941 aatccgcaca ctcccgcatt cgttcccgcc cgtaagtaca tagaagtgcc agaacagcct 6001 accgctcctc ctgcacaggc cgaggaggcc cccgaagttg tagcgacacc gtcaccatct 6061 acagctgata acacctcgct tgatgtcaca gacatctcac tggatatgga tgacagtagc 6121 gaaggctcac ttttttcgag ctttagcgga tcggacaact ctattactag tatggacagt 6181 tggtcgtcag gacctagttc actagagata gtagaccgaa ggcaggtggt ggtggctgac 6241 gttcatgccg tccaagagcc tgcccctatt ccaccgccaa ggctaaagaa gatggcccgc 6301 ctggcagcgg caagaaaaga gcccactcca ccggcaagca atagctctga gtccctccac 6361 ctctcttttg gtggggtatc catgtccctc ggatcaattt tcgacggaga gacggcccgc 6421 caggcagcgg tacaacccct ggcaacaggc cccacggatg tgcctatgtc tttcggatcg 6481 ttttccgacg gagagattga tgagctgagc cgcagagtaa ctgagtccga acccgtcctg 6541 tttggatcat ttgaaccggg cgaagtgaac tcaattatat cgtcccgatc agccgtatct 6601 tttccactac gcaagcagag acgtagacgc aggagcagga ggactgaata ctgactaacc 6661 ggggtaggtg ggtacatatt ttcgacggac acaggccctg ggcacttgca aaagaagtcc 6721 gttctgcaga accagcttac agaaccgacc ttggagcgca atgtcctgga aagaattcat 6781 gccccggtgc tcgacacgtc gaaagaggaa caactcaaac tcaggtacca gatgatgccc 6841 accgaagcca acaaaagtag gtaccagtct cgtaaagtag aaaatcagaa agccataacc 6901 actgagcgac tactgtcagg actacgactg tataactctg ccacagatca gccagaatgc 6961 tataagatca cctatccgaa accattgtac tccagtagcg taccggcgaa ctactccgat 7021 ccacagttcg ctgtagctgt ctgtaacaac tatctgcatg agaactatcc gacagtagca 7081 tcttatcaga ttactgacga gtacgatgct tacttggata tggtagacgg gacagtcgcc 7141 tgcctggata ctgcaacctt ctgccccgct aagcttagaa gttacccgaa aaaacatgag 7201 tatagagccc cgaatatccg cagtgcggtt ccatcagcga tgcagaacac gctacaaaat 7261 gtgctcattg ccgcaactaa aagaaattgc aacgtcacgc agatgcgtga actgccaaca 7321 ctggactcag cgacattcaa tgtcgaatgc tttcgaaaat atgcatgtaa tgacgagtat 7381 tgggaggagt tcgctcggaa gccaattagg attaccactg agtttgtcac cgcatatgta 7441 gctagactga aaggccctaa ggccgccgca ctatttgcaa agacgtataa tttggtccca 7501 ttgcaagaag tgcctatgga tagattcgtc atggacatga aaagagacgt gaaagttaca 7561 ccaggcacga aacacacaga agaaagaccg aaagtacaag tgatacaagc cgcagaaccc 7621 ctggcgactg cttacttatg cgggattcac cgggaattag tgcgtaggct tacggccgtc 7681 ttgcttccaa acattcacac gctttttgac atgtcggcgg aggattttga tgcaatcata 7741 gcagaacact tcaagcaagg cgacccggta ctggagacgg atatcgcatc attcgacaaa 7801 agccaagacg acgctatggc gttaaccggt ctgatgatct tggaggacct gggtgtggat 7861 caaccactac tcgacttgat cgagtgcgcc tttggagaaa tatcatccac ccatctacct 7921 acgggtactc gttttaaatt cggggcgatg atgaaatccg gaatgttcct cacacttttt 7981 gtcaacacag ttttgaatgt cgttatcgcc agcagagtac tagaagagcg gcttaaaacg 8041 tccagatgtg cagcgttcat tggcgacgac aacatcatac atggagtagt atctgacaaa 8101 gaaatggctg agaggtgcgc cacctggctc aacatggagg ttaagatcat cgacgcagtc 8161 atcggtgaga gaccacctta cttctgcggc ggatttatct tgcaagattc ggttacttcc 8221 acagcgtgcc gcgtggcgga tcccctgaaa aggctgttta agttgggtaa accgctccca 8281 gccgacgacg agcaagacga agacagaaga cgcgctctgc tagatgaaac aaaggcgtgg 8341 tttagagtag gtataacagg cactttagca gtggccgtga cgacccggta tgaggtagac 8401 aatattacac ctgtcctact ggcattgaga acttttgccc agagcaaaag agcattccaa 8461 gccatcagag gggaaataaa gcatctctac ggtggtccta aatagtcagc atagtacatt 8521 tcatctgact aatactacaa caccaccacc tctagagctt gccgccacca tggtgagcaa 8581 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtgaa 8641 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac 8701 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac 8761 cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt 8821 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga 8881 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat 8941 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta 9001 caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt 9061 gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca 9121 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac 9181 ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt 9241 cgtgaccgcc gccgggatca ctcacggcat ggacgagctg tacaagtaaa gcggccgtga 9301 gcatgcaggc cttgggccca atgatccgac cagcaaaact cgatgtactt ccgaggaact 9361 gatgtgcata atgcatcagg ctggtacatt agatccccgc ttaccgcggg caatatagca 9421 acactaaaaa ctcgatgtac ttccgaggaa gcgcagtgca taatgctgcg cagtgttgcc 9481 acataaccac tatattaacc atttatctag cggacgccaa aaactcaatg tatttctgag 9541 gaagcgtggt gcataatgcc acgcagcgtc tgcataactt ttattatttc ttttattaat 9601 caacaaaatt ttgtttttaa catttcaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 9661 aaagggaatt cctcgattaa ttaagcggcc gctcgagatg gcacacgtgt tacggtttta 9721 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat 9781 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 9841 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 9901 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 9961 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 10021 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 10081 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 10141 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 10201 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 10261 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 10321 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 10381 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 10441 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 10501 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 10561 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct 10621 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 10681 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 10741 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 10801 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 10861 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 10921 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt 10981 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt 11041 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag 11101 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct 11161 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt 11221 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc 11281 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt 11341 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg 11401 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg 11461 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct 11521 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc 11581 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt 11641 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt 11701 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg 11761 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat 11821 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg 11881 cgcacatttc cccgaaaagt gccacctgac gtc

//

Claims

WHAT IS CLAIMED:

1. A virus like particle (VLP) comprising:

a. an alphavirus replicon comprising a recombinant polynucleotide,

b. a retroviral gag protein, and

c. a fusogenic envelope protein,

wherein the VLP does not contain an alphavirus structural protein gene.

2. The VLP of claim 1, wherein the alphavirus replicon is derived from Sindbis virus or

Venezuelan equine encephalitis virus.

3. The VLP of claim 1 or 2, wherein the alphavirus replicon comprises genes encoding:

a. Sindbis virus or Venezuelan equine encephalitis virus nonstructural proteins NSP 1, NSP2, NSP3, and NSP4; and

b. a retroviral packaging signal.

4. The VLP of any one of claims 1 to 3, wherein the retroviral gag protein is derived from Rous sarcoma virus or murine leukemia virus.

5. The VLP of any one previous claim, wherein the retroviral gag protein is derived from Rous sarcoma virus.

6. The VLP of any one previous claim, wherein the fusogenic envelope protein is selected from the group consisting of haemagglutinin, Rous sarcoma virus fusion protein, an E protein of tick borne encephalitis virus and dengue fever virus, the El protein of Semliki Forest virus, baculovirus gp64, and VSV-G protein.

7. The VLP of any one previous claim, wherein the fusogenic envelope protein is a

glycoprotein, or fragment or derivative thereof.

8. The VLP of any one previous claim, wherein the fusogenic envelope protein is from a RNA virus or a retrovirus, or fragment or derivative thereof.

9. The VLP of any one previous claim, wherein the fusogenic envelope protein is VSV-G or VSV-EnvA.

10. The VLP of claim 9, wherein the VSV-G comprises an alteration.

1 1. The VLP of any one previous claim, wherein the VLP is capable of binding to a eukaryotic cell.

12. The VLP of claim 11, wherein the eukaryotic cell is a human cell.

13. The VLP of claim 11 wherein the binding is specific to a target cell.

14. The VLP of claim 1 1, wherein the replicon is reproduced in the cell.

15. The VLP of claim 14, wherein the VLP is not cytopathic to the cell.

16. The VLP of any one previous claim, wherein the alphavirus replicon comprises a promoter for a virus R A-dependent RNA polymerase operably linked to the recombinant polynucleotide.

17. The VLP of claim 16, wherein the alphavirus replicon comprises a cloning site for insertion of at least one recombinant polynucleotide.

18. The VLP of claim 17, wherein the recombinant polynucleotide codes for an antisense RNA.

19. The VLP of claim 18, wherein the recombinant polynucleotide codes for a shRNA or a miRNA.

20. The VLP of claim 19, wherein the shRNA or miRNA knocks down expression of a gene in the cell.

21. The VLP of claim 16, wherein the recombinant polynucleotide comprises an RNA that can be translated into a protein.

22. A method of producing the VLP of any one of claims 1-21 comprising:

a. co-transforming a eukaryotic cell with:

i. a first vector comprising a polynucleotide sequence encoding the alphavirus replicon, wherein the alphavirus replicon includes the polynucleotide of interest,

ii. a second vector comprising a polynucleotide sequence encoding the retroviral gag protein, and

iii. a third vector comprising a polynucleotide sequence encoding the fusogenic envelope protein; b. culturing the co-transformed cell under conditions suitable to cause each vector to produce its encoded product, thereby producing the VLP, and

c. isolating the VLP from the cell,

wherein neither the vectors nor the cell contain any alphavirus structural protein genes.

23. A kit comprising the first vector, the second vector, and the third vector of claim 22.

24. The kit of claim 23, wherein at least two of the vectors are contiguous.

25. A method of expressing at least one recombinant polynucleotide in a eukaryotic cell

comprising contacting a cell with the VLP of any one of claims 1 to 21.

26. A method of delivering the recombinant polynucleotide to a subject, comprising

administering to said subject the VLP of any one of claims 1 to 21.

27. A method of treating or preventing a disease or a disorder in a subject, comprising

administering to a subject the VLP of any one of claims 1 to 21.

28. The method of claim 25, wherein expression of the recombinant polynucleotide supplements deficient expression of an endogenous gene by said subject.

29. A pharmaceutical composition comprising the VLP of any one of claims 1-21.

30. A eukaryotic cell comprising: a. a plasmid comprising a polynucleotide sequence that codes for an alphavirus replicon wherein the alphavirus replicon includes a polynucleotide of interest, b. a plasmid comprising a polynucleotide sequence that codes for a gag protein, and c. a plasmid comprising a polynucleotide sequence that codes for a heterologous

fusogenic envelope protein, wherein neither the plasmids nor the cell contain a gene that codes for an alphavirus structural protein.

31. The eukaryotic cell of claim 30, wherein the polynucleotide sequence encoding the

alphavirus replicon is derived from Sindbis virus or Venezuelan equine encephalitis virus.

32. The eukaryotic cell of any one of claims 30 or 31, wherein the alphavirus replicon comprises genes encoding: a. Sindbis virus nonstructural proteins NSP 1, NSP2, NSP3, and NSP4; and b. a retroviral packaging signal.

33. The eukaryotic cell of claim 32, wherein the retroviral packaging signal is derived from

either Rous sarcoma virus or murine leukemia virus.

34. The eukaryotic cell of claim 30, wherein the polynucleotide sequence encoding the gag

protein is derived from Rous sarcoma virus.

35. The eukaryotic cell of claim 30, wherein the polynucleotide sequence encoding the

heterologous fusogenic envelope protein encodes VSV-G.

36. The method of claim 25, wherein two recombinant polynucleotides are expressed in said cell.

37. The method of claim 36, wherein the expression of the two recombinant polynucleotides is driven by separate subgenomic promoters.

38. The method of claim 22, wherein the vector comprising the polynucleotide sequence

encoding the alphavirus replicon is pDest472-VEE or pDest472-VEE-MCS.