In cellulo syntheses of targeting-ligand-conjugatable, RNA-specific, enveloped virus-like particles

Abstract

Embodiments of the invention disclosed herein involve the selective engineering of Sindbis virus so as to generate cells that make enveloped virus-like particles that contain therapeutic mRNAs, and whose membrane proteins have been mutated to serve as modular binding sites for cell-targeting ligands.

Description

[0001] IN CELLULO SYNTHESES OF TARGETING-LIGAND-CONJUGATABLE, RNA-SPECIFIC, ENVELOPED VIRUS-LIKE PARTICLES

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. Section 119(e) of copending and commonly-assigned U.S. Provisional Patent Application No. 63 / 739,260, filed December 27, 2024, entitled “IN CELLULO SYNTHESES OF TARGETINGLIGAND-CONJUGATABLE. RNA-SPECIFIC, ENVELOPED VIRUS-LIKE PARTICLES’’, which application is incorporated by reference herein.

[0004] TECHNICAL FIELD

[0005] The technical fields of the invention include molecular biology and medicine.

[0006] BACKGROUND OF THE INVENTION

[0007] Sindbis virus, a member of the alphavirus genus in the Togaviridae family, is a single-stranded, enveloped, positive-sense RNA virus. In nature, it is transmitted via mosquito bites to mammals. Genetically modified Sindbis virus has been shown to be useful to produce vectors that can transduce polynucleotides into cells.

[0008] There is a need in the art for improved Sindbis based vectors useful to transduce polynucleotides into mammalian cells

[0009] SUMMARY OF THE INVENTION

[0010] Embodiments of the invention disclosed herein involve the selective engineering of Sindbis virus so as to generate cells that make enveloped virus-like particles that contain therapeutic mRNA, and whose membrane proteins have been mutated to serve as modular binding sites for cell-targeting ligands. The basic components for in cellulo synthesis of these RNA-specific particles are derived from the Sindbis virus, the most highly ordered and the best characterized of the Alphavirusgenus. Embodiments of the invention include Sindbis viral particle compositions as well as methods for making and using them.

[0011] The invention disclosed herein has a number of embodiments. For example, embodiments of the invention include compositions of matter including a Sindbis viral particle formed from 240 Sindbis virus nucleocapsid proteins; wherein the Sindbis viral particle encapsulates a cargo consisting essentially of a heterologous polynucleotide. In these compositions, the Sindbis virus nucleocapsid proteins can be modified to comprise a polypeptide motif that selectively binds to a molecule such as one found on a polypeptide or cell, for example an avidin moiety that binds a biotinylated molecule. In certain embodiments of the invention, the heterologous polynucleotide comprises an mRNA that encodes a polypeptide of interest such as a therapeutic protein. In other embodiments of the invention, the heterologous polynucleotide comprises an untranslated RNA such a microRNA or a CRISPR guide RNA.

[0012] Embodiments of the invention include compositions of matter useful in making the Sindbis viral particles disclosed herein. Such embodiments of the invention include compositions of matter comprising a first polynucleotide comprising a genetically modified genome of a Sindbis virus, the genetically modified genome comprising a heterologous polynucleotide inserted into the Sindbis genome, wherein the heterologous polynucleotide is not inserted in a region of the Sindbis virus genome encoding Sindbis RNA-dependent RNA polymerase (RdRp) polyprotein. Such compositions further include a helper molecule, for example in the form of a second polynucleotide comprising a molecule that can be replicated by the RdRp encoded by the first polynucleotide and encodes polypeptides useful in these methods. In addition, such compositions can further include a Sindbis viral particle; wherein the Sindbis viral particle encapsulates a cargo comprising the heterologous polynucleotide.

[0013] Typically in the compositions of matter useful in making the Sindbis viral particles disclosed herein, the heterologous polynucleotide is inserted in a region ofthe Sindbis virus genome encoding Sindbis capsid and / or membrane proteins. In some embodiments of the invention, the heterologous polynucleotide encodes a polypeptide such as a Cas protein or a therapeutic polypeptide. In other embodiments of the invention, the heterologous polynucleotide comprises an untranslated RNA such as microRNA or a guide RNA. In certain embodiments of these compositions, the second polynucleotide encodes Sindbis capsid protein and Sindbis glyco-proteins El, E2, and E3; and / or the second polynucleotide comprises a promoter. Typically in these compositions, the second polynucleotide generates an mRNA upon synthesis of the full-length negative-sense strand followed by RdRp binding and transcription of the second polynucleotide. In these compositions, the Sindbis virus nucleocapsid proteins can be modified to comprise a polypeptide motif that selectively binds to a molecule such as one found on a polypeptide or cell, for example an avidin moiety that binds a biotinylated molecule. Optionally, these compositions are disposed within a mammalian cell.

[0014] Embodiments of the invention also include methods of making a Sindbis viral particle comprising a heterologous mRNA; wherein the Sindbis viral particle encapsulates a cargo comprising a heterologous mRNA. Typically these methods comprise transfecting a mammalian cell with a first polynucleotide comprising a genetically modified genome of a Sindbis virus, the genetically modified genome comprising a heterologous polynucleotide inserted into the Sindbis genome, wherein the heterologous polynucleotide is not inserted in a region of the Sindbis virus genome encoding Sindbis RNA-dependent RNA polymerase (RdRp) polyprotein; and also a second polynucleotide comprising a molecule that can be replicated by the RdRp encoded by the first polynucleotide. Such methods include allowing the cell to form the Sindbis viral particle such that the Sindbis viral particle encapsulates a cargo comprising the heterologous polynucleotide RNA. In certain of these methods, the heterologous polynucleotide encodes a polypeptide. In some embodiments of the invention, the heterologous polynucleotide comprises an untranslated RNA. Typically in these methods, the Sindbis virus nucleocapsid proteins of the viralparticle are modified to comprise a polypeptide motif that binds to a molecule on a mammalian cell. Embodiments of the invention also include Sindbis viral particles made by the methods disclosed herein.

[0015] Embodiments of the invention further include methods of delivering a polynucleotide into a mammalian cell. Typically these methods include combining the mammalian cell with a composition comprising a Sindbis viral particle; wherein the Sindbis viral particle encapsulates a cargo consisting essentially of a heterologous RNA; and allowing the Sindbis viral particle to contact the mammalian cell such that the polynucleotide is transduced into the mammalian cell. In these methods, the Sindbis virus nucleocapsid proteins can be modified to comprise a polypeptide motif that selectively binds to a molecule such as one found on a polypeptide or cell, for example an avidin moiety that binds a biotinylated molecule. In certain embodiments of the invention, the heterologous RNA comprises an mRNA that encodes a polypeptide of interest such as a therapeutic protein. In other embodiments of the invention, the heterologous RNA comprises an untranslated RNA such a microRNA or a CRISPR guide RNA.

[0016] Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating some embodiments of the present invention are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications.

[0017] BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 provides photographs of Sindbis virus. Cryo- EM reconstruction (left panel) of the Sindbis virion, showing the icosahedrally-ordered E1 / E2 glycoprotein heterodimers in its bilayer envelope, and a transmission image (right panel) showing the one-to-one correspondence between nucleocapsid and transmembrane proteins.Figure 2 provides schematics of the bicistronic Sindbis genome. In this figure, a modified Sindbis genome is shown, with its structural-protein ORF replaced by a gene of interest whose protein product is expressed only upon replication of the molecule to give its complementary strand, from which transcription at the subgenomic (sg) promoter yields the GOT mRNA. Tn this figure, “< “ and “>” denote start / stop codons respectively.

[0019] Figure 3 provides schematics where the top RNA is the Sindbis genome (see top of Figure 2) with its structural genes (capsid protein [CP] and glycoproteins [GPs], E3-E1) replaced by EYFP, and with the ORF encoding the nonstructural [NS] polyprotein fully maintained; the arrow / maroon portion is the sub-genomic promoter, and the orange portion near the 5’ end of the NS ORF is the packaging sequence for the Sindbis CP. The bottom RNA is the Sindbis genome whose replicase-related / non-structural genes (NS 1234) have been deleted.

[0020] DETAILED DESCRIPTION OF THE INVENTION

[0021] Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art. In the description of the preferred embodiment, reference may be made to the accompanying drawings which form a part hereof, and in which is shown by way of illustration a specific embodiment in which the invention may be practiced. It is to be understood that other embodiments may be utilized, and reagent substitutions may be made without departing from the scope of the present invention.

[0022] Embodiments of the invention disclosed herein involve the selective engineering of a particular mammalian virus so as to generate cells that make enveloped virus-like particles that contain therapeutic mRNA, and whose membrane proteins have been mutated to serve as modular binding sites for cell-targeting ligands. The basic components for in cellulo synthesis of these RNA-specific particles are derived from the Sindbis virus, the most highly ordered and the best characterized of the Alphavirus genus.Embodiments of the invention include compositions of matter comprising the engineered Sindbis virus particles disclosed herein as well as methods for making and using them. As discussed below, embodiments of the invention include a composition of matter comprising a Sindbis viral genome, wherein the OFR2 in the genome is deleted or truncated and replaced with an exogenous polynucleotide, for example one encoding a gene of interest such as a therapeutic molecule. Compositions of the invention include, for example, Sindbis vector particles that comprise: a mutant avidin-insert E2 glycoprotein that allows for modular binding of any biotinylated targeting ligand (e.g., antibody); and / or a self-rephcating mRNA cargo that simultaneously encodes for therapeutic (e.g., Cas) protein and regulatory (e.g., microRNA or guide RNA) sequence; and / or a therapeutic mRNA cargo that is replicated in producer cells to enhance yield and RNA specificity of particles but that - to avoid replication-induced cytotoxicity - is not replicated in target cells. In certain embodiments of the invention, the compositions comprise a defective helper (DHBB) agent or the like. Embodiments of the invention include methods of making such Sindbis vector particles compositions by genetically manipulating the Sindbis genomes as discussed above using this disclosure in combination with conventional molecular biology methods. Embodiments of the invention include methods of using such Sindbis vector particles compositions to deliver genes to cells such as human cells, the methods comprising combining the Sindbis vector particle compositions with human cells so that the human cells are transduced with a gene or interest carried by the Sindbis vector.

[0023] Embodiments of the invention include compositions of matter including a Sindbis viral particle formed from 240 Sindbis virus nucleocapsid proteins; wherein the Sindbis viral particle encapsulates a cargo consisting essentially of a heterologous RNA. In such embodiments, this Sindbis viral particle cannot reproduce Sindbis virus. In certain of these compositions, the Sindbis virus nucleocapsid proteins can be modified to comprise a polypeptide motif that selectively binds to a molecule such as one found on a polypeptide or cell, for example an avidin moiety that binds a biotinylated molecule. In certain embodiments of the invention, the heterologousRNA comprises an mRNA that encodes a polypeptide of interest such as a therapeutic protein (e.g. a human cytokine or growth factor). In other embodiments of the invention, the heterologous RNA comprises an untranslated RNA such a microRNA or a CRISPR guide RNA.

[0024] Embodiments of the invention include compositions of matter useful in making the Sindbis viral particles disclosed herein. Such embodiments of the invention include compositions of matter comprising a first polynucleotide comprising a genetically modified genome of a Sindbis virus, the genetically modified genome comprising a heterologous polynucleotide inserted into the Sindbis genome, wherein the heterologous polynucleotide is not inserted in a region of the Sindbis virus genome encoding Sindbis RNA-dependent RNA polymerase (RdRp) polyprotein. Such compositions can further include a helper molecule, for example in the form of a second polynucleotide comprising a molecule that can be replicated by the RdRp encoded by the first polynucleotide and encodes polypeptides useful in the methods. In addition, such compositions can further include a Sindbis viral particle; wherein the Sindbis viral particle encapsulates a cargo comprising the heterologous polynucleotide RNA.

[0025] Typically in the compositions of matter useful in making the Sindbis viral particles disclosed herein, the heterologous polynucleotide is inserted in a region of the Sindbis virus genome encoding Sindbis capsid and / or membrane proteins. In some embodiments of the invention, the heterologous polynucleotide encodes a polypeptide such as a Cas protein or a therapeutic polypeptide. In other embodiments of the invention, the heterologous polynucleotide comprises an untranslated RNA (e.g., one without a functional start codon) such as microRNA or a guide RNA. In certain embodiments of these compositions, the second polynucleotide encodes Sindbis capsid protein and Sindbis glyco-proteins El, E2, and E3; and / or the second polynucleotide comprises a promoter. Typically in these compositions, the second polynucleotide generates an mRNA upon synthesis of the full-length negative-sense strand followed by RdRp binding and transcription of the second poly nucleotide. Inthese compositions, the Sindbis virus nucleocapsid proteins can be modified to comprise a polypeptide motif that selectively binds to a molecule such as one found on a polypeptide or cell, for example an avidin moiety that binds a biotinylated molecule. Optionally, these compositions are disposed within a mammalian cell.

[0026] Embodiments of the invention also include methods of making Sindbis viral particles comprising a heterologous mRNA; wherein the Sindbis viral particle encapsulates a cargo comprising a heterologous mRNA (and lacks Sindbis genome). Typically these methods comprise transfecting a mammalian cell with a first polynucleotide comprising a genetically modified genome of a Sindbis virus, the genetically modified genome comprising a heterologous polynucleotide inserted into the Sindbis genome, wherein the heterologous polynucleotide is not inserted in a region of the Sindbis virus genome encoding Sindbis RNA-dependent RNA polymerase (RdRp) polyprotein; and also a second polynucleotide comprising a molecule that can be replicated by the RdRp encoded by the first polynucleotide. Such methods include allowing the cell to form the Sindbis viral particle such that the Sindbis viral particle encapsulates a cargo comprising the heterologous polynucleotide RNA. In certain of these methods, the heterologous polynucleotide encodes a polypeptide. In some embodiments of the invention, the heterologous polynucleotide comprises an untranslated RNA. Typically in these methods, the Sindbis virus nucleocapsid proteins of the viral particle are modified to comprise a polypeptide motif that binds to a molecule on a mammalian cell. Embodiments of the invention also include Sindbis viral particles made by the methods disclosed herein.

[0027] Embodiments of the invention further include methods of delivering polynucleotides into a mammalian cell using the Sindbis viral particles disclosed herein. Typically these methods include combining the mammalian cell with a composition comprising a Sindbis viral particle; wherein the Sindbis viral particle encapsulates a cargo consisting essentially of a heterologous (non-Sindbis) RNA; and allowing the Sindbis viral particle to contact the mammalian cell such that the polynucleotide is transduced into the mammalian cell. In these methods, the Sindbisvirus nucleocapsid proteins can be modified to comprise a polypeptide motif that selectively binds to a molecule such as one found on a polypeptide or cell, for example an avidin moiety that binds a biotinylated molecule. In certain embodiments of the invention, the heterologous RNA comprises an mRNA that encodes a polypeptide of interest such as a therapeutic protein. In other embodiments of the invention, the heterologous RNA comprises an untranslated RNA such a microRNA or a CRISPR guide RNA.

[0028] The cryo-EM reconstruction in Figure 1 (left) shows the T=4 Caspar-Klug icosahedral symmetry of the 240 heterodimers of El and E2 transmembrane glycoproteins that reflect perfectly the symmetry of the 240 nucleocapsid proteins (NCPs) - see rightmost image. This exceptional - indeed unique - situation for an enveloped virus is a direct consequence of the one-to-one strong noncovalent interactions between the subunits of the NCP and the inside domains of the transmembrane E2 proteins, and it is this interaction between capsid and envelope proteins that is the driving force for nucleocapsids acquiring their envelope and budding out of the cell as the final step in the viral life cycle. The genome of the Sindbis virus consists of a single positive-sense bicistronic mRNA molecule containing two open reading frames (ORFs), with the 2nd under the control of a subgenomic (sg) promoter - see Figure 2. The 1st ORF encodes an RNA-dependent RNA polymerase (RdRp) polyprotein that self-cleaves to generate the several repli case-related proteins, and the 2nd ORF encodes a polyprotein that self-cleaves to yield the structural (i.e., capsid and membrane) proteins.

[0029] In this context, subgenomic (Sg) control (and the associated subgenomic promoter noted above) in Sindbis virus refers to the mechanisms that regulate the production and translation of its subgenomic messenger RNA (sgRNA), which is essential for viral replication and structural protein synthesis. This control is crucial for the unique two-part replication cycle where the viral RNA-dependent RNA polymerase (RdRp) synthesizes both genomic RNA and a shorter sgRNA from the same positive-strand template. This process involves a subgenomic promoter thatinitiates the transcription of sgRNA, the viral polymerase nsP4 which has distinct binding sites for genomic and subgenomic promoters, and the translation of sgRNA even when host cell protein synthesis is shut down. In the mechanism of subgenomic control, the sgRNA is transcribed from an internal sg promoter on the viral genome. The efficiency of this sg promoter is highly regulated, and changes to its sequence can affect viral infectivity.

[0030] In embodiments of the invention, we replace the 2nd ORF polynucleotide with one or more exogenous polynucleotides, such as ones encoding therapeutic genes of interest (GOF). Alternatively this ORF can be swapped / replaced by non-coding / non-translated regulatory RNA sequence without a start codon. Significantly, the 2nd ORF of this non-infectious “replicon” is expressed under subgenomic (sg) control, i.e., its mRNA is generated only upon synthesis of the full-length negative-sense strand followed by the RdRp binding and transcribing it in the inter-ORF / sg region: see Figure 2. To compensate for deletion of the structural Sindbis genes, in embodiments of the invention, we provide them in the form of a “helper” molecule / agent that can be replicated “in trans” by the RdRp of the replicon (Rep). This is illustrated in Figure 3. for the case of enhanced yellow fluorescent protein (EYFP) as the gene of interest. Here NS 1234 denotes the RdRp poly protein that self-cleaves into replicase-related nonstructural (NS) proteins 1, 2, 3, and 4; the tan / orange-colored sequence in molecule B corresponds to the “packaging signal”; and the maroon sequence is the sg promoter. In the defective helper (DHBB) molecule C the structural genes encoding the capsid protein (CP) and glyco-proteins (GPs) El, E2, and E3 are inserted under the control of the sg promoter. The thick-black lines in constructs B and C denote the 5’ and 3’ untranslated regions (UTRs) that facilitate their replication by the nonstructural (NS) replicase-related proteins (NS 1234). As explained in the caption, the two constructs shown in Figure 3 are obtained by gutting, respectively, the structural and the nonstructural ORFs of the Sindbis genome shown at the top of Figure 2.Note that transfection of these two molecules in this embodiment of the invention results in specific packaging of the 1st (the “replicon”) by the CP gene product of the 2nd (the “helper”), followed by wrapping - and subsequent budding -of these nucleocapsids by the “helpe -molecule-encoded GPs. Significantly, the corresponding enveloped virus-like particles are identical to the Sindbis virions shown in Figure 1, except that the nucleocapsids now contain a non-infectious replicon molecule (EYFP-Rep) rather than the viral genome.

[0031] The gene delivery particles whose synthesis is described above - with a therapeutic gene of interest or non-coding RNA sequence replacing the EYFP - are qualitatively different from the lipid nanoparticle or exosomal vectors that have been developed recently for delivery of gene-editing reagents, because of their being monodisperse, thermodynamically-stable, ordered (indeed, stoichiometrically-precise). and their exploiting the highly-evolved RNA-packaging and delivery features of Sindbis, the most ordered, stable, and RNA-specific of enveloped mRNA-genome viruses. Further, the Sindbis vector particles that we describe making by transfecting cells with Sindbis replicon + helper molecules are different from those that have been reported previously by virtue of our optimizing replicon length and exploiting synonymous mutations for enhancing both their yield and their RNA specificity.

[0032] In addition, embodiments of the invention include the first syntheses of Sindbis vector particles that contain:

[0033] (i) a mutant avidin-insert E2 glycoprotein that allows for modular binding of any biotinylated targeting ligand (e.g., antibody);

[0034] (ii) a self-replicating mRNA cargo that simultaneously encodes for therapeutic (e.g., Cas) protein and regulatory' (e.g., microRNA or guide RNA) sequence; and (iii)a therapeutic mRNA cargo that is replicated in producer cells to enhance yield and RNA

[0035] specificity' of particles but that - to avoid replication -induced cytotoxicity - is not replicated in target cells.(i) can, for example, be achieved by insertion of an avidin sequence inserted with flanking flex regions as a replacement of E2 residues 71-72.

[0036] (ii) can, for example, be achieved by replacing the gene of interest (GOI) in the Sindbis replicon (see Figures 2 and 3) by the therapeutic gene followed by a stop codon and a “core catalytic sequence” (CCS) consisting of a regulatory sequence flanked by self-cleaving ribozymes. In certain embodiments, we introduce the CCS in its negative sense so that cleavage will not occur until the overall molecule has been amplified into its fully complementary sequence, in which context the ribozymes are active, with the consequent release of functional regulatory7RNA. At the same time, any “surviving” (wwcleaved) complementary strands will be amplified by7transcription back into the original construct, thereby enabling further rounds of amplification of the RNA.

[0037] Finally, (iii) can. for example, be achieved by deleting the nsP4 gene in the Sindbis therapeutic-gene replicon and by providing it in trans - in producer cells - so that the synthesized / secreted vector particles contain the nsP4-deleted molecule that will only be translated, and not replicated, in target cells.

[0038] Methods and materials describing the manipulation of the Sindbis virus can also be found in Bredenbeek et al., "Sindbis virus expression vectors: packaging of RNA replicons by using defective helper RNAs". J. Virology 67, 6439-46 (1993); Frolov et al., "Alphavirus-based expression vectors: strategies and applications", Proc. Natl. Acad. Sci. (USA) 93, 11371-7 (1996); and Odisse Azizgolshani "Sindbis Virus Budding: The Making of Enveloped Virus and Virus-Like Particles from Pre-assembled Nucleocapsids", a dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy7in Biochemistry and Molecular Biology , University of California Los Angeles, 2011; as well as WIPO publications W02005014017, W02006105279, W02010080570, WO2023045124, WO2024152852. WO2019173223, and W02021007276; and USPTO publications US20040102410, US20050031594, US5217879, and US20220401548, the contents of all of which are incorporated herein by reference.All publications mentioned herein are incorporated by reference to disclose and describe aspects, methods and / or materials in connection with the cited publications. Many of the techniques and procedures described or referenced herein are well understood and commonly employed by those skilled in the art.

[0039] Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0040] CONCLUSION

[0041] This concludes the description of the preferred embodiment of the present invention. The foregoing description of one or more embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teaching.

Claims

CLAIMS:

1. A composition of matter comprising:(a) a first polynucleotide comprising a genetically modified genome of a Sindbis virus, the genetically modified genome comprising a heterologous polynucleotide inserted into the Sindbis genome, wherein the heterologous polynucleotide is not inserted in a region of the Sindbis virus genome encoding Sindbis RNA-dependent RNA polymerase (RdRp) polyprotein;(b) a second polynucleotide comprising a molecule that can be replicated by the RdRp encoded by the first polynucleotide; and(c) a Sindbis viral particle; wherein the Sindbis viral particle encapsulates a cargo comprising the heterologous polynucleotide mRNA.

2. The composition of claim 1, wherein the heterologous polynucleotide is inserted in a region of the Sindbis virus genome encoding Sindbis capsid and / or membrane proteins.

3. The composition of claim 1, wherein the heterologous polynucleotide encodes a polypeptide.

4. The composition of claim 4, wherein the polypeptide comprises a Cas protein or a therapeutic polypeptide.

5. The composition of claim 1, wherein the heterologous polynucleotide comprises an untranslated RNA.

6. The composition of claim 5, wherein the untranslated RNA comprises a microRNA or a guide RNA.

7. The composition of claim 1, wherein:the second polynucleotide encodes Sindbis capsid protein and Sindbis glycoproteins El, E2, and E3; and / orthe second polynucleotide comprises a promoter.

8. The composition of claim 7, wherein the second polynucleotide generates an mRNA upon synthesis of the full-length negative-sense strand followed by RdRp binding and transcription of the second polynucleotide.

9. The composition of claim 1, wherein Sindbis virus nucleocapsid proteins are modified to comprise a polypeptide motif that binds to a molecule on a cell.

10. The composition of claim 9, wherein the polypeptide motif comprises an avidin moiety that binds a biotinylated molecule.

11. The composition of claim 1, wherein at least (a) and (b) are disposed within a mammalian cell.

12. A composition of matter comprising a Sindbis viral particle formed from 240 Sindbis virus nucleocapsid proteins; wherein the Sindbis viral particle encapsulates a cargo consisting essentially of a heterologous mRNA (non Sindbis genome), wherein the Sindbis viral particle is non infectious.

13. A method of making a Sindbis viral particle comprising a heterologous mRNA; wherein the Sindbis viral particle encapsulates a cargo comprising a heterologous mRNA (non Sindbis genome), the method comprising transfecting a mammalian cell with:(a) a first polynucleotide comprising a genetically modified genome of a Sindbis virus, the genetically modified genome comprising a heterologouspolynucleotide inserted into the Sindbis genome, wherein the heterologous polynucleotide is not inserted in a region of the Sindbis virus genome encoding Sindbis RNA-dependent RNA polymerase (RdRp) polyprotein;(b) a second polynucleotide comprising a molecule that can be replicated by the RdRp encoded by the first polynucleotide; andallowing the cell to form the Sindbis viral particle; wherein the Sindbis viral particle encapsulates a cargo comprising the heterologous polynucleotide mRNA.

14. The method of claim 13, wherein the heterologous polynucleotide encodes a polypeptide.

15. The method of claim 13, wherein the heterologous polynucleotide comprises an untranslated RNA.

16. The method of claim 13, wherein Sindbis virus nucleocapsid proteins of the viral particle are modified to comprise a polypeptide motif that binds to a molecule on a mammalian cell.

17. A Sindbis viral particle made by the method of claim 13.

18. A method of delivering a polynucleotide into a mammalian cell, the method comprising:combining the mammalian cell with a composition comprising a Sindbis viral particle; wherein the Sindbis viral particle encapsulates a cargo consisting essentially of a heterologous polynucleotide; and allowing the Sindbis viral particle to contact the mammalian cell such that the polynucleotide is transduced into the mammalian cell.

19. The method of claim 18, wherein the heterologous polynucleotide encodes a polypeptide.

20. The method of claim 18, wherein the heterologous polynucleotide comprises an untranslated RNA.

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