Alpharetrovirus-Based Particles for Delivery of RNA to Cells - Patent application

Abstract

The novel alpharetrovirus-based particle is suitable for highly efficient transient transduction of animal cells, such as human or mouse cells, and efficiently introduces the coding and non-coding RNA contained in the alpharetrovirus-based particle into target cells.Also, since the alpharetrovirus-based particle protects the incoming RNA from being degraded during the process, the particle provides highly efficient activity and / or integrity of the RNA introduced into animal cells.The transferred RNA may be non-coding RNA (e.g., single guide (sg) RNA, small hairpin (sh) RNA, micro RNA or long non-coding (lnc) RNA), or the RNA may code for proteins or peptides, such as receptors, transcription factors, cellular enzymes, antigens for use in vaccination, gene / protein therapy and / or gene editing nucleases, recombinases and transposases.

Description

[Technical field]

[0001] The present invention provides alpharetrovirus-based particles suitable for highly efficient transient transduction of animal cells, such as human or mouse cells, and efficiently introduces the coding and non-coding RNA contained therein into target cells.Also, since the alpharetrovirus-based particles protect the entering RNA from degradation during entry, the particles provide highly efficient activity and / or integrity of the RNA introduced into animal cells.The transferred RNA may be a non-coding RNA (e.g., single guide (sg)RNA, small hairpin (sh)RNA, microRNA or long non-coding (lnc)RNA), or the RNA may code for a protein or peptide, such as a receptor, a transcription factor, a cellular enzyme, an antigen for use in vaccination, a gene / protein therapy and / or a gene editing nuclease, a recombinase and a transposase.In addition, the transferred RNA may be a self-amplifying RNA replicon that codes for any of the mentioned RNA classes. Alpharetrovirus-based particle may contain at least one, for example at least two or three RNA constructs, and the RNA construct may be composed of coding or non-coding RNA or a combination of both.The gene editing nuclease may be selected, for example, from transcription activator-like effector (TALEN), zinc finger (ZFN), and preferably RNA-guided CRISPR (clustered regularly interspaced short palindromic repeats) / CRISPR-associated (Cas) nuclease, especially CRISPR / Cas9, and also in combination with sgRNA.The recombinase may be selected, for example, from Cre, Dre, Tre, Brec, Flp recombinase, and the transposase may be selected, for example, from Sleeping Beauty, PiggyBac transposon. [Background technology]

[0002] Knopp et al., Mol. Ther. Nucleic Acids 13, pp. 256-274 (2018), "Transient retrovirus-based CRISPR / Cas9 All-in-One Particles for efficient, targeted gene knock-out" (Non-Patent Document 1), describes gammaretrovirus-based particles that deliver CRISPR / Cas9 RNA for genetic engineering of human and mouse cells.

[0003] Hoffmann D. et al., Detailed comparison of retroviral vectors and promoter configurations for stable and high transgene expression in human induced pluripotent stem cells, Gene Ther. 2017 May;24(5):298-307 (Non-Patent Document 2) describes a reporter gene construct for EGFP (vector name=RRL.PPT.EFS.EGFP.PRE).

[0004] Heckl D, Kowalczyk MS, Yudovich D, Belizaire R, Puram RV, McConkey ME, Thielke A, Aster JC, Regev A, Ebert BL. Generation of mouse models of myeloid malignancy with combinatorial genetic lesions using CRISPR-Cas9 genome editing. Nat Biotechnol. 2014 Sep;32(9):941-6 (Non-Patent Document 3) describes a vector containing the RFP657.Tet2 reporter gene construct for Tet2 (vector name=LKO5d.SFFV.tRFP657(Tet2.4).iPAC).

[0005] Galla, Schambach, Baum, Synthetic Messenger RNA and Cell Metabolism Modulation: Methods and Protocols, Methods in Molecular Biology, Vol. 969 (2013), edited by Rabinovich, Chapter 10, “Retrovirus-Based mRNA Transfer for Transient Cell Manipulation” (Non-Patent Document 4), describes techniques for the production of retroviral vector particles. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Knopp et al., Mol. Ther. Nucleic Acids 13, pp. 256-274 (2018), “Transient retrovirus-based CRISPR / Cas9 All-in-One Particles for efficient, targeted gene knock-out” [Non-Patent Document 2] Hoffmann D. et al., Detailed comparison of retroviral vectors and promoter configurations for stable and high transgene expression in human induced pluripotent stem cells, Gene Ther. May 2017;24(5):298-307 [Non-Patent Document 3] Heckl D, Kowalczyk MS, Yudovich D, Belizaire R, Puram RV, McConkey ME, Thielke A, Aster JC, Regev A, Ebert BL. Generation of mouse models of myeloid malignancy with combinatorial genetic lesions using CRISPR-Cas9 genome editing. Nat Biotechnol. 2014 September;32(9):941~6 [Non-Patent Document 4] Galla, Schambach, Baum, Synthetic Messenger RNA and Cell Metabolism Modulation: Methods and Protocols, Methods in Molecular Biology Volume 969 (2013), edited by Rabinovich, Chapter 10, “Retrovirus-Based mRNA Transfer for Transient Cell Manipulation” Summary of the Invention [Problem to be solved by the invention]

[0007] The object of the present invention is to provide an alpharetrovirus-based particle that efficiently transduces animal cells, particularly human cells, and efficiently delivers at least one RNA construct to recipient cells in a transient manner.The retrovirus-based particle may simultaneously contain one or more types of RNA construct of interest, allowing the latter to be co-delivered in time and space to target cells (e.g., for transient expression from the RNA construct, for example, gene editing nuclease, alone or in combination with non-coding RNA, such as sgRNA, and / or transgene RNA construct for modifying target cell genome). Advantageously, the non-integrating alpharetroviral-based particles contain RNA constructs that do not cause insertional mutagenesis, and more preferably, the retroviral-based particles deliver RNA constructs for transient activity in recipient cells only, thereby also avoiding or reducing the potential for cytotoxicity mediated by stable and long-term overexpression of certain transgenes (e.g., SpCas9, Cre recombinase, ZFN nucleases, Sleeping Beauty DNA transposase) and / or other RNA species. [Means for solving the problem]

[0008] The present invention achieves the object by the features of the claims, inter alia, by providing a retroviral particle, which is an alpharetrovirus-based particle comprising or consisting of a protein component and at least one RNA construct associated (in particular packaged in the particle) via said protein component and a corresponding linked RNA hairpin structure. The alpharetrovirus-based protein component particle comprises or consists of an alpharetrovirus matrix protein (MA), an alpharetrovirus p2 protein (p2), an alpharetrovirus p10 protein (p10), an alpharetrovirus capsid protein (CA), an alpharetrovirus nucleocapsid protein (NC) and optionally an additional alpharetrovirus protease protein (PR), a linker which may be a viral protease site or may lack a protease site, and a domain which is an MS2 bacteriophage-derived MS2 coat protein dimer (2xMS2CP), preferably in that order from N-terminus to C-terminus, and preferably a pseudotyping protein, which may optionally comprise a targeting protein, a ligand or a peptide. During the production of alpharetrovirus-based particles, the pseudotyping protein is expressed, for example, from a separate expression cassette in cultured producer cells. The protein components between the domains contain protease sites, so that during the production of alpharetrovirus-based particles, one protein containing or consisting of the alpharetrovirus domains MA-p2-p10-CA-NC or MA-p2-p10-CA-NC-PR, each followed by 2xMS2CP, can be proteolyzed by viral proteases to generate separate proteins containing, for example, only one of the alpharetrovirus domains MA, p2, p10, CA or NC, or at least two connected alpharetrovirus domains. The protease sites located between each of the alpharetrovirus domains can be the natural protease sites of the alpharetrovirus.In the absence of protease activity in the producer cell, the Gag-MS2 protein component may be present in the alpharetrovirus-based particle as one single chain protein. It has been found that fully functional, e.g. transducing competent, alpharetrovirus-based particles according to the invention can be produced that contain a protein component containing a protease site between the alpharetroviral domains, the protein component being present as a single chain (e.g. comprising MA-p2-p10-CA-NC-2xMSCP). Alternatively, the protein component may lack a protease site between the domains, e.g. the alpharetroviral domains of the protein component may be connected by a linker that is not a protease site.

[0009] It has been found that during production of functional alpharetrovirus-based particles in producer cells, the viral protease site located between the C-terminal NC domain of the alpharetrovirus protein component and the MS2 coat protein dimer is not necessarily hydrolyzed. Optionally, therefore, the alpharetrovirus domain of MA-p2-p10-CA-NC or MA-p2-p10-CA-NC-PR can be linked to the MS2 coat protein, e.g., 2xMS2CP, by a linker that does not contain a protease site, in particular does not contain a viral protease site, e.g., a linker having a length of 6 to 25 amino acids, preferably up to 20 amino acids. Herein, the alpharetrovirus domain of MA-p2-p10-CA-NC or MA-p2-p10-CA-NC-PR (optionally including a protease site between each of these domains) is also referred to as a.Gag.

[0010] The pseudotyping protein can be expressed from an expression cassette separate from the expression cassette encoding the alpharetrovirus protein domain. The pseudotyping protein can be selected from, for example, VSVg, other strains of the VSV family, RD114, BaeV, MARAV, COCV, ecotropic or amphotropic MLV Env, GALV, measles envelope, Nipah V envelope. Measles and Nipah V envelope can be fused to targeting proteins, ligands, scFv or receptor binding domains in a blinded manner. It has been found that the target cell specificity of the alpharetrovirus-based particles of the present invention can be determined by its pseudotyping protein.

[0011] Alpharetrovirus-based particles have the advantage of containing at least one RNA construct in association with the protein component, which may be a non-coding RNA, such as shRNA, sgRNA, lncRNA, miRNA, shRNA / miRNA hybrid, or may be an RNA encoding at least one transgene, also called gene of interest (GOI), which may be, for example, an antigen for eliciting an immune response in the recipient, a coding sequence for a protein, encoding a transcription factor, or a coding sequence encoding a gene editing nuclease, such as Cas9, preferably, the coding sequence encoding the gene editing nuclease is combined with at least one additional RNA construct, such as an sgRNA, and optionally with at least two or three additional different sgRNAs. Generally, each sgRNA (single guide RNA) is provided to cooperate with a Cas9 nuclease.

[0012] For protein-encoding RNA constructs, the RNA construct preferably contains from 5' to 3' the following elements: Cap-GOI-2xTS-optionally PRE-polyA tail, where Cap is a cap structure consisting of an N7-methylated GTP molecule linked to the first transcribed nucleotide that serves as a ribosome start site, GOI is the coding sequence, and 2xTS are two adjacent MS2 target sites.

[0013] Alpharetrovirus-based particles encoding proteins that are antigens are vaccines, e.g., for use in eliciting an immune response and / or for generating immune protection against infectious agents, e.g., viruses or bacteria, and / or for the treatment of autoimmune and / or cancer diseases. Exemplary antigens are proteins of viral or bacterial origin for use in alpharetrovirus-based particles encoding antigens in generating immune protection, e.g., the spike protein of coronaviruses, e.g., SARS-CoV2, GP120 of HIV-1, Ag85 of Mycobacterium tuberculosis (e.g., according to https: / / pubmed.ncbi.nlm.nih.gov / 23035231 / ), OVA (ovalbumin), or for cancer therapy, encoding CEA as an embryonic cancer antigen, tumor neoantigens (e.g., NY-ESO-1, MAGE-A3) or the asparaginyl endopeptidase legumain, or cancer antigens.

[0014] Because the RNA constructs contained in the alpharetrovirus-based particles are produced as transcription products from coding or non-coding DNA sequences, generally herein the RNA constructs may also be referred to as transcripts.

[0015] The RNA constructs contained in the alpharetrovirus-based particles are bound and integrated via the protein component 2xMS2CP, which interacts with the corresponding MS2 target site (TS) present on the RNA construct. The TS comprises or consists of two genetically fused hairpin structures present on each RNA construct. In protein-encoding RNA constructs, the TS is preferably placed adjacent to the section encoding the gene of interest and 5' to the PRE element (downstream of which is adjacently placed a polyA tail).

[0016] For example, in the RNA construct that is sgRNA, shRNA, miRNA, shRNA / miRNA hybrid and / or lncRNA, the TS domain is incorporated within the sgRNA, shRNA, miRNA and / or lncRNA section, or is adjacent to and downstream of the sgRNA, shRNA, miRNA and / or lncRNA section, for example, 3'.Similarly, for other non-coding RNA constructs, the TS domain can be incorporated or adjacently placed.In addition, for example, a non-coding RNA, for example, sgRNA, can be placed between the hairpin sections of two TS domains, and for example, a sgRNA, shRNA, miRNA, lncRNA or a combination of at least two of them can link two TS hairpin sections, in particular link two stem sections of two TS hairpin sections.

[0017] Generally, the alpharetrovirus-based particles of the invention may have a lipid coat resulting from their derivation from a producer cell, e.g., due to budding, the alpharetrovirus-based particle may comprise a portion of the cell membrane of the producer cell. The alpharetrovirus-based particle comprises or consists of a.Gag MS2 protein component comprising or consisting of the domains of the alpharetrovirus MA-p2-p10-CA-NC-2xMS2CP or MA-p2-p10-CA-NC-PR-2xMS2CP, in each case optionally with a protease site between each of these domains, the a.Gag protein component preferably having at least 90%, preferably at least 95%, identity to the amino acid sequence encoded by nucleotides 1462 to 3207 of SEQ ID NO:1, e.g. the amino acid sequence encoded by nucleotides 1462 to 3186 of SEQ ID NO:1. In a preferred a.Gag protein component, the domains are separated by viral protease sites, for example a first linker which may be or contain a viral protease site having at least 90%, preferably at least 95%, identity to the amino acid sequence encoded by nucleotides 3187-3207 of SEQ ID NO:1, optionally an additional second linker between the first linker and the MS2 coat protein (2xMS2CP), for example a second linker having at least 90%, preferably at least 95% identity to the amino acid sequence encoded by nucleotides 3208-3222 of SEQ ID NO:1, and an MS2 coat protein dimer (2xMS2CP), preferably having at least 90%, preferably at least 95% identity to the amino acid sequence encoded by nucleotides 3223-3999 of SEQ ID NO:1, as well as a pseudotyping protein. At least one RNA construct, which is a transgene coding and / or non-coding RNA transcript in association with a protein component, is ligated to at least one target site (TS), preferably at least two target sites (2xTS).During production in the producer cell, at least one TS, preferably at least two TSs, of each RNA construct associates with an MS2 coat protein dimer (2xMS2CP), resulting in the RNA construct being associated with or packaged into a protein component. The pseudotyping protein VSVg may have an amino acid sequence encoded by nucleotides 1241 to 2776 of SEQ ID NO:4. In general, the present invention includes by reference all amino acid sequences and all nucleotide sequences of its priority application EP21216354.7.

[0018] Each of the MS2 target sites (TS) may have at least 90%, preferably at least 95%, nucleotide sequence with at least one sequence selected from nucleotides 1943-1965 and / or nucleotides 1982-2004 of SEQ ID NO:2, nucleotides 1681-1701 and / or nucleotides 1751-1771 of SEQ ID NO:3, nucleotides 1755-1775 and / or nucleotides 1794-1814 of SEQ ID NO:14, nucleotides 5697-5719 and / or nucleotides 5736-5758 of SEQ ID NO:5, nucleotides 2337-2359 and / or nucleotides 2376-2398 of SEQ ID NO:6. Therein, each of the at least two target sites may have the same or different nucleotide sequences. Preferably, the target sites are spaced 60-40 nucleotides (nt) apart, e.g., 50 nt apart, and the spacing nucleotides may comprise non-coding RNA, e.g., sgRNA, shRNA, miRNA. The two target sites (2xTS) may have, e.g., the nucleotide sequence of nucleotides 1912-2045 of SEQ ID NO:2, and / or nucleotides 1982-2004 of SEQ ID NO:2, or nucleotides 1681-1701, and may be spaced 60-40 nt apart, e.g., 50 nt apart, and may have the nucleotide sequence of nucleotides 1751-1771 of SEQ ID NO:3, e.g., nucleotides 1665-1800 of SEQ ID NO:3, or nucleotides 5666-5799 of SEQ ID NO:5, or nucleotides 2306-2439 of SEQ ID NO:6.

[0019] The alpharetrovirus-based particle according to the invention is suitable for use to introduce at least one RNA construct into a target cell, for example to express a gene of interest from its mRNA contained in the alpharetrovirus-based particle, and / or to introduce an RNA molecule, for example at least one, preferably at least two non-coding RNAs, such as non-coding RNAs selected from sgRNA, siRNA, shRNA, lncRNA, tRNA, rRNA or a combination of at least two of these, and / or RNAs encoding proteins. In addition, a self-amplifying RNA replicon encoding any of the above RNA species may be transferred.

[0020] In a process for producing an alpharetrovirus-based particle according to the invention, a Gag.MS2 protein component comprising the alpharetrovirus MA-p2-p10-CA-NC or MA-p2-p10-CA-NC-PR, a linker which may contain a viral protease site (pr), an MS2 coat protein dimer (2xMS2CP) having at least 90%, preferably at least 95%, identity to the amino acids encoded by nucleotides 1462 to 3208 of SEQ ID NO:1 (wherein MA-p2-p10-CA-NC-PR, the viral protease site (pr) and a linker which may contain a viral protease site (pr) are included. r), a linker that may contain an MS2 coat protein dimer (2xMS2CP) having at least 90%, preferably at least 95% identity to the amino acids encoded by nucleotides 1462 to 3594 of SEQ ID NO: 12), and a pseudotyping protein, and at least one RNA construct encoding a gene of interest and / or a coding or non-coding RNA species linked to at least one target site (TS), preferably linked to at least two target sites (2xTS), are co-expressed in the producer cell, e.g., from an expression cassette under the control of a polymerase II (Pol II) promoter element or under the control of a polymerase III (Pol III) promoter element, e.g., the CMV promoter, the PGK promoter, the EF1a promoter or the beta-actin promoter as examples of Pol II promoters, and hU6 and H1 as examples of Pol III promoters.

[0021] The expression cassettes encoding each a.Gag.MS2 protein component or encoding an RNA construct containing a GOI or protein coding sequence preferably encode at their 3' end a polyadenylation signal (pA) and, optionally, a PRE adjacent to the 5' end of the polyadenylation signal. At least one RNA construct containing a GOI or protein coding sequence, whose RNA is contained in an alpharetrovirus-based particle, has a Cap structure at its 5' end and a polyadenylation tail at its 3' end.

[0022] Surprisingly, it has been found that the a.Gag.MS2 protein component effectively packages an RNA construct, for example at least one, two or three different RNA constructs, into one alpharetrovirus-based particle (the protein component comprising or consisting of the alpharetrovirus domains MA, p2, p10, CA, NC, optionally PR, and MS2 coat protein dimer (2xMS2CP) and a pseudotyping protein, for example VSVg). Unexpectedly, the 2xMS2CP portion of the protein component, together with the alpharetrovirus MA, p2, p10, CA, NC and optionally PR protein domains and pseudotyping protein, forms an alpharetrovirus-based particle containing at least one RNA construct in a producer cell. Moreover, it was unexpected that these particles effectively associate with human or mouse target cells and efficiently transduce the target cells to deliver the RNA construct to the target cells. Generally, as used herein, a target cell is any cell to which the alpharetrovirus-based particle of the invention binds, the binding being determined, for example, by the choice of pseudotyping protein of the alpharetrovirus-based particle.

[0023] Generally, nucleic acid sequence elements are presented in their 5' to 3' orientation, and amino acid sequences are presented in their N-terminus to C-terminus orientation, unless otherwise indicated.

[0024] The invention will now be explained in more detail with reference to the drawings. [Brief description of the drawings]

[0025] FIG. 1A is a schematic diagram of a nucleic acid construct for the production of comparative gammaretrovirus-based g.Gag.MS2 particles. FIG. 1B is a schematic diagram of a nucleic acid construct for the generation of a.Gag.MS2 alpharetrovirus-based particles according to the present invention. - Figure 1C Schematic diagram of the FLP recombinase indicator gene cassette and the results of recombination of the reporter gene cassette in mouse SC1 cells after transduction by comparative analysis of three alpharetrovirus-based particles transferring the FLP recombinase according to the present invention (a.NC.MS2, a.NC.pr.MS2, a.PR.pr.MS2), the best-performing variant (a.NC.pr.MS2) is boxed and will also be used in the following figures. FIG. 1D shows the results of FLP-mediated recombination in reporter cells from FIG. 1C with a comparative gammaretrovirus-based particle (g.Gag.MS2) and with the best-performing alpharetrovirus-based particle according to the invention (a.NC.pr.MS2). FIG. 1E shows the results of FLP-mediated excision of a reprogramming gene (OKSM) cassette in human iPSCs after transduction with a comparative gammaretrovirus-based particle (g.Gag.MS2) and an alpharetrovirus-based particle according to the invention (a.NC.pr.MS2). FIG. 1F shows the results of firefly luciferase expression in human HT1080 cells after transduction with a comparative gammaretrovirus-based particle (g.Gag.MS2) and with an alpharetrovirus-based particle according to the invention (a.NC.pr.MS2). FIG. 1G shows the results of firefly luciferase expression in primary human neonatal foreskin fibroblasts (NuFF) after transduction with a comparative gammaretrovirus-based particle (g.Gag.MS2) and with an alpharetrovirus-based particle according to the invention (a.NC.pr.MS2). - Figure 2A shows the mRNA copy number in the supernatant of an alpharetrovirus-based particle according to the invention (a.NC.pr.MS2) and, for comparison, a gammaretrovirus-based particle (g.Gag.MS2). - FIG. 2B shows the titers (particles / μL) of supernatants from alpharetrovirus-based particles according to the invention (a.NC.pr.MS2) as well as gammaretrovirus-based supernatants (g.Gag.MS2) for comparison and non-retroviral extracellular vesicle controls (pcDNA3 only and VSVg only). - Figure 2C shows the results of particle size measurements of alpharetrovirus-based particles according to the invention (a.NC.pr.MS2) as well as gammaretrovirus-based particles (g.Gag.MS2) for comparison and non-retroviral extracellular vesicle controls (pcDNA3 only and VSVg only). - Figure 2D shows transmission electron micrographs of virus-like particles of the invention as well as comparative virus-like particles including control vector particles packaged with the respective wild-type (wt) gammaretrovirus or alpharetrovirus Gag-Pol (wt.g.Gag-Pol and wt.a.Gag-Pol). FIG. 2E shows the results of the determination of firefly luciferase transgene expression in target cells transduced with alpha retrovirus-like particles (a.NC.pr.MS2) and, for comparison, gamma retrovirus-like particles (g.Gag.MS2) as well as the respective integration controls. FIG. 3A Schematic showing the components used for the manufacture of a comparative gammaretrovirus-based particle (g.Gag.MS2) that delivers CRISPR / Cas9 components to target cells. FIG. 3B Schematic representation of the components used for the manufacture of an alpharetrovirus-based particle (a.NC.pr.MS2) delivering CRISPR / Cas9 RNA according to the present invention. - FIG. 3C shows RFP657.Tet2 reporter knockout rate by a.NC.pr.MS2-based CRISPR / Cas9 particles packaged with mouse Tet2-targeting sgRNAs with MS2 target sites within (TS.inc) or adjacent to (TS.adj) the sgRNA scaffold. - Figure 3D shows the copy number of CRISPR / Cas9 RNA (SpCas9.TS mRNA and Tet2.TS sgRNA transcripts) in the supernatant of alpharetrovirus-based particles according to the invention (a.NC.pr.MS2; here Tet2.TS.adj) and, for comparison, gammaretrovirus-based (g.Gag.MS2; here Tet2.TS.inc) supernatants. - Figure 3E shows the knockout rate of the RFP657.Tet2 reporter gene construct in human HT1080 target cells. - FIG. 3F shows the knockout rate of Tet2 target gene reporter constructs in human iPS cells. - Figure 4. Knockout of the endogenous CXCR4 gene in human Jurkat cells by gammaretrovirus-based (g.Gag.MS2) and alpharetrovirus-based (a.NC.pr.MS2) CRISPR / Cas9 all-in-one particles. In (A) a representative FACS result is shown, and in (B) an experiment in biological replicates compared to integrated lentiviral LIT.CXCR4 CRISPR / Cas9 all-in-one particles and non-targeted mouse Trp53 control Gag.MS2 particles. - Figures 5A-5D show alpha retrovirus-based (a.NC.pr.MS2) particle-mediated knockout rates of the endogenous human TP53 gene in (A) primary human NuFF cells, (B) primary human hepatocytes (PHH), (C) human CD34+ hematopoietic stem and progenitor cells (HSPCs) and (D) primary mouse embryonic fibroblasts (MEFs) by application of various multiplicities of infection (MOI), expressed as SpCas9.TS mRNA copies applied per cell. - Figures 6A and 6B show multiplexing experiments using alpharetrovirus-based particles delivering CRISPR / Cas9 containing two sgRNAs (Figure 6A, knockout of CXCR4 and RFP657.Tet2) or three sgRNAs (Figure 6B, in addition to A EGFP was also knocked out). - Figures 7A and 7B Figure 7A shows an exemplary expression plasmid for an alpharetrovirus-based particle containing a VEE self-amplifying RNA replicon (with nonstructural proteins nsP1, nsP2, NsP3, nsP4) and EGFP as a gene of interest. Figure 7B shows the results of transducing human HT1080 target cells with the alpharetrovirus-based particle of Figure 7A. - Figures 8A-8F Figures 8A-8C show mice as negative controls that were not treated with a.Gag.MS2 particles or PBS, but only with luciferin. Figures 8D and 8F show mice treated with viral particles and luciferin, and for comparison, Figure 8E shows a control mouse treated with PBS only and luciferin. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] A nucleic acid construct for producing an alpharetrovirus-based particle (a.Gag.MS2 particle) that delivers mRNA in a packaging cell according to the present invention is shown diagrammatically in Figure 1B, and Figure 1A shows a comparative nucleic acid construct for producing a gammaretrovirus-based particle (g.Gag.MS2 particle). In addition, Figures 1A and 1B show diagrammatically nucleic acid constructs for expressing wild-type alpharetrovirus particles (wt a.Gag-Pol) or gammaretrovirus particles (wt g.Gag-Pol).

[0027] FIG. 1A shows an expression construct for packaging of mRNA transfer gammaretrovirus Gag.MS2 (g.Gag.MS2) particles. 293T producer cells were transfected with an expression plasmid encoding g.Gag.MS2, an envelope glycoprotein (exemplified by VSVg) and an mRNA carrying a gene of interest (GOI). The latter also encodes two copies of the MS2 hairpin structure (TS), which allows specific packaging of the transcribed GOI.TS mRNA into the resulting particles by a genetically fused MS2 coat protein dimer (2xMS2CP) in g.Gag.MS2. Since the gammaretrovirus Pol enzyme containing viral protease (PR) is not added during g.Gag.MS2 particle production, the MS2CP dimer protein is separated from the gammaretrovirus nucleocapsid (NC) protein by a native viral protease site (pr, light grey box), which here functions as a linker. The structure of gammaretrovirus wild-type (wt) Gag-Pol is shown at the top. CMV: cytomegalovirus promoter; PRE: Woodchuck Hepatitis Virus posttranscriptional regulatory element; pA: polyA signal. MA: viral matrix protein, p12: viral p12 protein, CA: viral capsid protein, RT: viral reverse transcriptase, IN: viral integrase.

[0028] FIG. 1B shows an expression construct for the production of alpharetroviral Gag.MS2 (a.Gag.MS2) particles. Packaging of a.Gag.MS2 particles delivering GOI mRNA is shown for three different alpharetroviral a.Gag.MS2 variants, a.NC.MS2, a.NC.pr.MS2 and a.PR.pr.MS2. In the a.NC.MS2 variant, the MS2CP dimer is fused directly to the alpharetroviral NC domain, while in a.NC.pr.MS2, the NC and MS2CP dimers are separated by a linker, which in this case is the native viral protease site (pr) (including the extended linker) indicated by a light grey box. Particles packaged with a.PR.pr.MS2 additionally contain a viral PR that promotes particle maturation after budding. In this variant, the MS2CP dimer is separated by a PR at the native viral protease site (pr; light grey box) at this position. As shown diagrammatically in FIG. 1B, the alpharetrovirus-based a.Gag.MS2 protein component in the alpharetrovirus-based particle of the invention expressed as a translation product comprises or consists of a MA-p2-p10-CA-NC moiety, whose domains are linked by a protease recognition site, and a 2xMS2CP moiety, which are generated as one fusion protein (a.NC.pr.MS2) containing an extended viral protease site (pr) as a linker between the N-terminal MA-p2-p10-CA-NC and the C-terminal 2xMS2CP of one common coding sequence. The domains of the MA-p2-p10-CA-NC moiety in the presence of viral protease are separable at the protease site located between the domains. In this embodiment, the alpharetrovirus moiety is expressed as a translation product consisting of MA-p2-p10-CA-NC in a fusion protein that does not contain the PR domain of the wild-type Gag protein, but with the protease recognition site pr and the 2xMS2CP moiety.Furthermore, the alpharetrovirus portion, and respectively, the alpharetrovirus-based particle, does not contain viral enzyme proteins, e.g., does not contain reverse transcriptase or integrase and / or viral protease. One common coding sequence for the fusion protein is shown in FIG. 1B as a.Gag.MS2, which contains the coding sequence MA-p2-p10-CA-NC-viral protease site-2xMS2CP as DNA at nucleotides 1462-3207 of SEQ ID NO:1, including an extended linker containing a protease site and glycine, separating NC from MS2, and an MS2 coat protein dimer encoded by nucleotides 3223-3999 of SEQ ID NO:1, under the control of a promoter, exemplified by the CMV promoter at nucleotides 232-819 of SEQ ID NO:1, with a 3' terminal polyA signal (pA) at nucleotides 4078-4302 of SEQ ID NO:1.

[0029] As a further embodiment, FIG. 1B shows the arrangement of coding sequences for MA-p2-p10-CA-NC-PR-viral protease site-2xMS2CP (a.PR.pr.MS2), which contains a PR domain disposed between the NC domain and an extended viral protease site linker. The PR domain encodes an alpha retroviral protease. One common coding sequence for MA-p2-p10-CA-NC-PR-viral protease site-2xMS2CP is contained in SEQ ID NO: 12, where in the codon-optimized sequence, nucleotides 1462-3594 encode the polyprotein MA-p2-p10-CA-NC-PR-viral protease site, nucleotides 3595-3612 encode the adjacent linker, and nucleotides 3613-4389 encode adjacent MS2 coat protein dimers, and these coding sequences encode one fusion protein. The plasmid backbone of SEQ ID NO:12 is derived from pcDNA3.

[0030] FIG. 1B shows the arrangement of the coding sequence of MA-p2-p10-CA-NC-2xMS2CP (a.NC.MS2), which does not contain the extended viral protease recognition site linker (pr) between the NC domain and 2xMS2CP.

[0031] When producing alpharetroviral particles in producer cells, the DNA is transcribed into RNA, e.g., mRNA with a coding sequence. The polyA signal affects the addition of a polyadenylation tail to the mRNA, which is translated into protein components. In the presence of viral proteases, due to the viral protease sites located between the domains of MA-p2-p10-CA-NC, proteolysis occurs to generate multiple single domains, and enveloped / pseudotyped alpharetroviral-based particles are assembled, comprising the MS2 coat protein associated with an RNA molecule containing at least one, and preferably two or more target sites (TS). When producing alpharetroviral-based particles in the absence of proteolytic activity for the protease sites between the alpharetroviral domains (e.g., MA-p2-p10-CA-NC-viral protease site-2xMS2CP from SEQ ID NO: 1), it was found that the protein components are present as a single amino acid chain, with essentially no proteolytic processing observed in Western blots. Thus, in a protein component of the invention comprising or consisting of MA-p2-p10-CA-NC-viral protease site-2xMS2CP, the protease sites between the domains may be non-functional, e.g., the domains may be linked by a linker that does not contain a protease site, and thus the gag polyprotein component may exist as one single protein. This indicates that the protease sites between the alpharetroviral domains of the gag polyprotein component do not need to be proteolytically cleaved when producing a functional alpharetroviral-based Gag.MS2 particle of the invention. Of note, the presence of an extended linker 3' of NC, 5' of 2xMS2CP with an extended protease site linker (composed of a protease site and a glycine linker) was important for functionality (see FIG. 1C, shown by comparison of a.NC.MS2 and a.NC.pr.MS2).

[0032] In an embodiment of a protein component containing a protease PR, for example MA-p2-p10-CA-NC-PR-viral protease site-2xMS2CP (a.PR.pr.MS2) encoded by SEQ ID NO: 12, it was also found that the presence of a viral protease results in the production of functional alpharetrovirus-based particles of the invention (a.PR.pr.MS2 in Figures 1B and 1C). Preferably, the 2xMS2CP can be cleaved from the alpharetrovirus Gag (MA-p2-p10-CA-PR) portion. The efficient transduction of target cells and the activity of the transferred RNA indicate the efficient formation of functional a.Gag.MS2 particles from the a.NC.pr.MS2 and a.PR.pr.MS2 variants. The fact that both variants are capable of transferring functional RNA indicates that the protease and the ability to proteolytically cleave into the Gag.MS2 subdomain are not essential for functionality. Thus, in these a.Gag.MS2 particles, the viral protease sites between the viral Gag subdomains, and between Gag and 2xMS2CP, may be non-hydrolyzable (e.g. a.NC.pr.MS2) or may be optionally hydrolyzed (e.g. a.PR.pr.MS2) to generate separate or linked domains of MA, p2, p10, CA, NC, PR, preferably 2xMS2CP may be cleaved from the gag portion.

[0033] In the producer cell, the RNA construct is transcribed from DNA under the control of a promoter exemplified by the CMV promoter, and comprises or consists of the coding sequence of the gene of interest (GOI), at least one, preferably at least two target sites and a polyA signal, optionally with a PRE between the GOI and the polyA signal (GOI.TS). The resulting RNA consists, from 5' to 3', of the coding sequence of the GOI, at least one TS, preferably at least two TS, optionally a PRE, and preferably a polyadenylation tail. In the example, two target sites (TS) are shown that associate with the RNA construct (including packaging) and interact with the MS2CP protein to form a.Gag.MS2 particle. The GOI can be, for example, a gene editing nuclease, a transposase, a recombinase, an enzyme or other cellular protein, a non-coding RNA, for example, sgRNA, shRNA (small hairpin RNA), miRNA, lncRNA or a combination of coding and non-coding RNA. Preferably, the RNA containing the non-coding construct is transcribed from DNA under the control of a polymerase III promoter, such as the human U6 (hU6) or H1 promoter.

[0034] According to the present invention, an alpharetrovirus-based particle may contain at least one, two, three or more RNA coding and non-coding constructs, each RNA construct with a TS incorporated into the molecule (e.g., incorporated or adjacent), optionally a PRE (post-transcriptional regulatory element, e.g., the PRE of Woodchuck Hepatitis Virus), and, particularly for protein-coding RNAs and miRNAs, more preferably a 3'-terminal polyadenylation tail.

[0035] Generally, preferably, the coding GOI and optionally the non-coding GOI, such as lncRNA, miRNA and shRNA / miRNA hybrids, can be transcribed from DNA under the control of a Pol.II promoter, such as a CMV promoter, and preferably followed 3' of the GOI by at least one adjacent or embedded TS, polyA signal, and optionally PRE. The non-coding GOI, especially shRNA, sgRNA and other short non-coding RNAs, can be transcribed from DNA under the control of a Pol.III promoter, such as a hU6, H1 promoter, and preferably followed 3' of the non-coding RNA by a Pol.III termination signal (polyT stretch, TTTTT).

[0036] To generate a.Gag.MS2 particles, the addition of pseudotyped envelope protein is required. Thus, the producer cell contains a transfected or stably expressed construct for expressing a pseudotyped protein, such as VSVg (vesicular stomatitis virus glycoprotein), optionally an intron, and a 3'-end polyadenylation signal under the control of a promoter exemplified by the CMV promoter (CMV-VSVg-pA). When the pseudotyped protein is expressed in the producer cell, it is incorporated into the lipid envelope of the alpharetrovirus-based particle.

[0037] Preferably, the VSVg cDNA may be replaced by another envelope protein that is suitable for pseudotyping the vector with retroviruses, including gammaretroviruses, alpharetroviruses and lentiviruses, and may use the a.Gag.MS2 particles described herein (see list of envelope proteins provided above).

[0038] Example 1: Packaging and performance of alpharetrovirus-based particles delivering FLP recombinase or firefly luciferase mRNA The components used for the production of alpharetrovirus-based Gag.MS2 (a.Gag.MS2) particles are shown diagrammatically in FIG. 1B. 293T producer cells are transfected with an expression plasmid encoding a.Gag.MS2 (SEQ ID NO: 1), expressing the VSVg envelope glycoprotein from SEQ ID NO: 4, and transcribing mRNA with genes of interest (GOI) from SEQ ID NO: 2 (FLP) and SEQ ID NO: 6 (firefly luciferase). The mRNA encoding the FLP GOI also encodes two copies of the MS2 hairpin structure (TS) at nucleotides 1943-1965 and nucleotides 1982-2004 of SEQ ID NO: 2, which allow specific packaging of the transcribed GOI.TS mRNA into the MS2 coat protein dimer (2xMS2CP) of the resulting alpharetrovirus-based particle. Correspondingly, the mRNA encoding firefly luciferase also encodes two copies of the MS2 hairpin structure (TS) at nucleotides 2337-2359 and nucleotides 2376-2398 of SEQ ID NO:6.

[0039] For comparison, the gammaretrovirus protein component g.Gag.MS2 correlate with the mRNA encoding the same GOI is used to produce a comparative gammaretrovirus-like particle. As shown in Figure 1A, the comparative gammaretrovirus-based particle g.Gag.MS2 also contains an extended protease recognition site pr (light grey box) between the retroviral domain and the 2xMSCP, and was pseudotyped with VSVg.

[0040] CMV: cytomegalovirus promoter; PRE: woodchuck hepatitis virus posttranscriptional regulatory element; pA: polyA signal.

[0041] To produce alpharetrovirus-based Gag.MS2 particles, viral supernatants were generated after transient transfection of 293T producer cells using standard calcium phosphate DNA precipitation methods. Briefly, the day before transfection, 5–6x10 cells were plated per 10 cm diameter culture dish (Sarstedt, Nuembrekt, Germany). 6 Viral particles were generated by co-transfecting human embryonic kidney 293T (293T) cells with a plasmid encoding a.Gag.MS2 (SEQ ID NO: 1), GOI.TS containing the coding sequence for FLP recombinase with TS or firefly luciferase with TS (SEQ ID NO: 2 for FLP and SEQ ID NO: 6 for firefly luciferase) and a PRE and polyadenylation signal, and an expression plasmid containing the coding sequence for VSVg (SEQ ID NO: 4).

[0042] The alpharetrovirus-based Gag.MS2 particles were concentrated 100-fold by ultracentrifugation at 82,740 xg for 2 hours or at 13,238 xg overnight at 4°C and used as the alpharetrovirus-based particle supernatant.

[0043] For comparison, gammaretroviral particles were produced using the same protocol but using the coding sequence of the gammaretroviral Gag.MS2 expression construct of SEQ ID NO:13.

[0044] A cell-based FLP recombinase (FLP) reporter system was utilized to assess the functionality of each alpharetroviral Gag.MS2 particle and the activity of the RNA contained in the particle (Figure 1C). To this end, various a.Gag.MS2 particles delivering FLP recombinase (FLP) mRNA as the GOI.TS were produced as shown in Figure 1B and tested on SC1-based FLP reporter cells, where FLP-mediated recombination is indicated by conversion of EGFP to dTomato expression.

[0045] FLP reporter cells contained a reporter construct (FLP indicator cassette) in which EGFP (enhanced green fluorescent protein) was flanked by two FRT (FLP recognition target) sites and contained, 3', the red fluorescent dTomato as a second reporter gene lacking an ATG start codon. Thus, FLP activity results in the excision of EGFP and induces dTomato expression. Because the cells contained three copies of the FLP indicator cassette, incomplete recombination of all three alleles by FLP would also result in cells expressing both dTomato and EGFP. Both of them, and cells expressing only dTomato, were considered recombinant cells as indicated on the Y-axis of the graph.

[0046] Variants a.NC.pr.MS2 and a.PR.pr.MS2 performed better than variant a.NC.MS2. The best variant a.NC.pr.MS2 is boxed and used in the following analysis. This indicates that preferably within the a.Gag.MS2 protein component, the 2xMS2CP should be separated from the Gag subdomain by a linker region.

[0047] FIG. ID shows highly efficient transfer of FLP mRNA by the best alpharetrovirus-based Gag.MS2 particles (a.NC.pr.MS2; (filled circles)) compared to their gammaretrovirus-based counterparts (g.Gag.MS2, FIG. 1A, (open circles)). SC-1-based FLP reporter cells containing an FLP indicator cassette were transduced with the indicated volumes of g.Gag.MS2.FLP or a.NC.pr.MS2.FLP supernatants. The Y-axis shows the percentage of recombinant cells as revealed by FLP-mediated excision of EGFP flanked by FRT. Alpharetrovirus-based a.NC.pr.MS2.FLP particles show enhanced FLP-mediated recombination.

[0048] Figure 1E shows the results of highly efficient removal of a reprogramming vector cassette (SFFV.OKSM) from the genome of human induced pluripotent stem cells (iPSCs) by an exemplary a.Gag.MS2 particle (a.NC.pr.MS2) containing FLP recombinase as a gene of interest. Human iPSC cells with one SFFV.OKSM vector integration containing a reporter cassette were treated with gamma retrovirus-based (open circle) g.Gag.MS2 particle supernatant or alpha retrovirus-based (closed circle) a.Gag.MS2 particle supernatant for comparison.

[0049] Excision of SFFV.OKSM from the reporter cassette (FLP-mediated OKSM cassette excision), shown diagrammatically in FIG. 1E, was analyzed by quantitative real-time PCR using primers that detect the codon-optimized (co)OCT4 gene (O), which is part of the OKSM reprogramming cassette (VCN: vector copy number; LTR: long terminal repeat; SIN / FRT: self-inactivating LTR and FRT sites). The results show that delivery of FLP mRNA is much more efficient (97%) with alpharetrovirus-based particles compared to gammaretrovirus-based particles (42%).

[0050] As shown in Figures 1A and 1B, firefly luciferase mRNA was packaged as an additional GOI into alpharetrovirus-based a.Gag.MS2 particles, and into gammaretrovirus-based g.Gag.MS2 particles for comparison (GOI.TS, nucleotides 624-2439 of SEQ ID NO: 6). Results show excellent transfer of firefly luciferase mRNA by alpharetrovirus-based a.Gag.MS2 particles. Graphs show relative light units (RLU) normalized for total protein in human HT1080 cells (Figure 1F) or primary human neonatal foreskin fibroblasts (NuFF) (Figure 1G).

[0051] In the case of Gag.MS2 "mRNA only" delivery, the copy number of the coding mRNA in the 100-fold concentrated supernatant was determined by quantitative real-time RT (reverse transcription) PCR (qRT-PCR). To remove potential plasmid contamination, the supernatant was treated with 2 units of TURBO DNase (Ambion / Thermo Fisher Scientific) for 1 h at 37°C. RNA was subsequently extracted from Gag.MS2 particles using the QIAGEN RNeasy Micro Kit (QIAGEN, Hilden, Germany) according to the manufacturer's protocol but with an additional DNase digestion step on the column. The extracted RNA was reverse transcribed to cDNA and then qRT-PCR was performed using the QuantiTect Reverse Transcription Kit (QIAGEN). For PCR, primers specific for the wPRE present on the construct were used. Absolute copy numbers were calculated using serial dilutions of plasmid standards.

[0052] FIG. 2A shows the results of measuring the copy number of mRNA encoding FLP recombinase in alpharetrovirus-based a.Gag.MS2 (a.NC.pr.MS2 variant) particle-based supernatants according to the invention (filled circles), and the FLP recombinase mRNA content in gammaretrovirus-based g.Gag.MS2 supernatants for comparison (open circles). Each data point reflects one independently prepared particle supernatant produced using 293T producer cells. The results show a higher concentration of mRNA of the gene of interest encoding FLP recombinase for the supernatants containing alpharetrovirus-based Gag.MS2 particles.

[0053] Particle content was determined in the same supernatants by full-field interferometry (FFI; (open circles)) or by nanoparticle tracking analysis (NTA; (closed circles). As further controls, supernatants from 293T producer cells transfected alone with pcDNA3 or an expression plasmid encoding VSVg were analyzed.

[0054] FIG. 2B shows particle count results for concentrated supernatants from producer cells transfected with pcDNA3 (empty pcDNA3 plasmid) or VSVg-encoding plasmid (SEQ ID NO: 4) alone. Both preparations were used as controls for the estimation of the percentage of extracellular vesicles in alpha- and gamma-retrovirus-based Gag.MS2 supernatants. In addition, gamma- and alpha-retrovirus-based Gag.MS2 supernatants generated as described in FIG. 1A (g.Gag.MS2) or FIG. 1B (a.Gag.MS2, a.NC.pr.MS2 of SEQ ID NO: 1) were analyzed. The results show a higher concentration of virus-like particles composed of alpha-retrovirus-based Gag.MS2 protein components.

[0055] Figure 2C shows the results of viral particle size measurement of the same supernatants by NTA, revealing that alpharetrovirus-based Gag.MS2 particles (filled circles) consisting of a.NC.pr.MS2 and delivering FLP.TS mRNA exhibit a similar size to the gammaretrovirus-based particles (open circles) for comparison.

[0056] FIG. 2D shows transmission electron micrographs (TEM photographs, same magnification, size bar is 100 nm) of an alpharetrovirus-based Gag.MS2 particle (a.NC.pr.MS2) containing the protein components MA-p2-p10-CA-NC-viral protease site-2xMS2CP and mRNA encoding FLP recombinase with 3'-terminal TS (FLP.TS), also used in the constructs of FIGS. 1C-1E and 2A-2C, and a comparative gammaretrovirus-based Gag.MS2 particle (g.Gag.MS2) containing the same mRNA construct. For comparison, wild-type viral vector particles with complete Gag-Pol are shown for alpharetrovirus (wt a.Gag-Pol) and gammaretrovirus (wt g.Gag-Pol) derived vector particles. White arrows point to electron-dense capsids or parts thereof. Black arrows highlight membrane bulges visible in areas where the electron-dense capsid appears to be incomplete.

[0057] FIG. 2E shows the results of determining Gag.MS2-mediated transgene expression, which is shown to be transient. The expression of firefly luciferase after transduction of HT1080 cells with firefly luciferase mRNA transfer Gag.MS2 particles was monitored over time. Each alpha and gamma retrovirus integrative transfer vector particle (g.RIT and a.RIT) was used as a positive control. In this experiment, the transient expression of firefly luciferase from alpha retrovirus-based Gag.MS2 particles was found to be consistently higher than that from gamma retrovirus-based Gag.MS2 particles. The comparative integrative constructs g.RIT and a.RIT show continuous high expression. Prior to this assay, the content of reporter mRNA (firefly luciferase) in the supernatant was determined by qRT-PCR, and the volume of the supernatant was adjusted to apply an equal amount of firefly luciferase mRNA copies to the target cells. Interestingly, in addition to the transient nature of both Gag.MS2 particle systems, a.Gag.MS2 particles composed of a.NC.pr.MS2 showed higher luciferase expression 1 hour after particle application, suggesting more functional particles per supernatant volume, more efficient cell entry, and / or other advantages during target cell transduction for a.Gag.MS2 particles.

[0058] Example 2: Packaging and performance of alpharetrovirus-based a.Gag.MS2 particles delivering CRISPR / Cas9 RNA (SpCas9 mRNA and respective sgRNA) The components used for the production of alpharetrovirus-based a.Gag.MS2 particles are shown diagrammatically in Figure 3B. Particles containing the expression cassette shown in Figure 3B, encoding MA-p2-p10-CA-NC-viral protease site-2xMS2CP (a.Gag.MS2, nucleotides 1462-3999 of SEQ ID NO: 1) under the control of the CMV promoter, and another expression vector encoding VSVg (SEQ ID NO: 4) under the control of the CMV promoter to produce mRNA for Streptococcus pyogenes Cas9 (SpCas9) fused to the coding sequence for the P2A protease site of porcine teschovirus-1, followed by the coding sequence for EGFP, two copies of the MS2 target site hairpin (TS), a PRE element and a polyadenylation signal (pA), were produced in 293T producer cells. The latter construct is designated SpCas9.TS (SEQ ID NO:5). This construct encodes the mRNA of SpCas9-P2A-EGFP linked to TS, PRE and pA. From this construct, SpCas9 (gene of interest) and EGFP (reporter) are transcribed as a single mRNA transcript and separated during translation by peptide bond skipping mediated by the P2A site.

[0059] The single guide RNA (sgRNA) sgRNA TS.adj or sgRNA TS.inc for cooperation with SpCas9 enzyme in target cells is expressed using Pol III human polymerase promoter U6 (hU6) promoter, linked to two MS2 TS copies, and terminated by a poly-T stretch, preferably by the sequence TTTTT or TTTT. In addition, these constructs express DsRedexp under the control of the CMV promoter and terminated by a polyadenylation signal (pA). The sgRNA TS.inc construct (SEQ ID NO:3) has a TS copy integrated into the sgRNA backbone, while in the sgRNA TS.adj (SEQ ID NO:14) construct, the TS copy is placed 3' (adjacent) to the sgRNA backbone. Both sgRNA constructs were designed to allow for the easy replacement of their respective protospacer sequences, i.e., the portion of the sgRNA that confers binding to the intended DNA target site in the recipient cell genome (without affecting the respective sgRNA.TS backbone).

[0060] In these sgRNA constructs with an additional coding sequence of the GOI represented by the reporter gene DsRedexp, the sgRNA is placed between the GOI and pA. These nucleic acid constructs contain the human codon-optimized coding sequence of DsRedexp (Discosoma sp. Red Fluorescent Protein, DsRed-Express, available from BD Clontech, product number 6995-1) as a reporter gene for expression in transfected target cells, which is linked to the promoter hU6 that controls the transcription of the sgRNA in transfected target cells.

[0061] Also, the mRNA of the GOI exemplified by DsRedexp encodes two copies of the MS2 hairpin structure (TS) at nucleotides 1681-1701 and 1751-1771 of SEQ ID NO: 3, which allows specific packaging of the transcribed GOI.TS mRNA into the MS2 coat protein dimer (2xMS2CP) of the resulting alpharetroviral particle. The protein components are encoded as a fusion protein within MA-p2-p10-CA-NC-viral protease site-2xMS2CP (a.Gag.MS2). This embodiment shows that an sgRNA, as an example of a small non-coding RNA, can be incorporated into the region of the TS, e.g., the sgRNA is placed between the two hairpin structures and / or immediately adjacent to the 5' of the hairpin structure. As an example, SEQ ID NO: 3 contains a section called BsmBI-stuffer at nucleotides 1560-1668, part of which or which may be completely replaced by the protospacer sequence of the sgRNA. Exemplary sgRNA sequences are shown as SEQ ID NO: 7 for a sgRNA to target mouse methylcytosine dioxygenase Tet2, SEQ ID NO: 8 for a sgRNA targeting mouse cell tumor antigen p53, SEQ ID NO: 9 for a sgRNA targeting human cell tumor suppressor gene p53, SEQ ID NO: 10 for a sgRNA targeting human CXC chemokine receptor 4, and SEQ ID NO: 11 for a sgRNA targeting enhanced green fluorescent protein (EGFP).

[0062] FIG. 3A shows a schematic of the components used to produce comparative gammaretrovirus-based Gag.MS2 particles, which contain each g.Gag.MS2 variant, the same pseudotyping protein VSVg, and a functionally identical RNA nucleic acid construct, in contrast to a.Gag.MS2.

[0063] Figure 3C shows that alpharetrovirus-based a.NC.pr.MS2 CRISPR / Cas9 particles packaged with TS.inc sgRNA directed against the mouse Tet methylcytosine dioxygenase 2 gene (Tet2) outperform their sgRNA.Tet.TS.adj counterparts. Successful CRISPR / Cas9-mediated knockout of RFP657.Tet2 in human HT1080 cells (Heckl et al., cited above) is indicated by loss of RFP657 expression. Thus, a.NC.pr.MS2 CRISPR / Cas9 and sgRNA.TS.inc transcripts were used in the following figures. The reporter cassette is shown diagrammatically, with the Tet2 recognition site located within the coding sequence of RFP657 (immediately downstream of the ATG start codon), so that CRISPR / Cas9 induces a DNA double-strand break and imprecise DNA repair leads to a frameshift and therefore loss of RFP657 expression. SFFV: promoter from spleen focus forming virus.

[0064] FIG. 3D shows the number of mRNA transcripts contained in alpharetrovirus-based Gag.MS2 particles (a.Gag.MS2) and comparative gammaretrovirus-based Gag.MS2 particles (g.Gag.MS2) for SpCas9-encoding mRNA linked to TS and mouse Tet methylcytosine dioxygenase 2 gene (Tet2)-targeting sgRNA linked to TS. The results show that alpharetrovirus-based Gag.MS2 particles contain a higher number of SpCas9 mRNAs, approximately 8-fold higher, but contain approximately 2.3-fold lower numbers of sgRNAs, respectively, compared to gammaretrovirus-based Gag.MS2 particles. a.Gag.MS2 particles were packaged with Tet2.TS.inc, while g.Gag.MS2 particles were packaged with Tet2.TS.adj sgRNA.

[0065] The activity of the SpCas9 enzyme in combination with sgRNA in various target cells transduced with alpharetrovirus-based Gag.MS2 particles or, for comparison, Gag.MS2 gammaretrovirus-based particles, is shown in Figures 3E, 3F.

[0066] The sgRNA is specific for the mouse Tet methylcytosine dioxygenase 2 gene (Tet2) (protospacer, see SEQ ID NO: 7) and, in cooperation with the SpCas9 enzyme encoded by the mRNA of the virus-based particle, results in knockout of RFP657 in target cells containing the RFP657 reporter gene with the corresponding Tet2 recognition site (SEQ ID NO: 7) in the 5' coding region (downstream of the ATG start codon), also used in Figure 3C. This reporter gene is expressed by the spleen focus forming virus (SFFV) promoter (Figures 3C and 3E) or by the CBX.EFS promoter (an epigenetic silencing resistant promoter consisting of the minimal ubiquitous chromatin-opening element CBX3 and the elongation factor 1 alpha short (EFS) promoter) (Figure 3F).

[0067] We found superior knockout activity of the reporter gene RFP657 in human HT1080 fibroblasts (Figure 3E) and human iPSCs (Figure 3F) containing a reporter cassette with a Tet2 recognition site, or of the endogenous CXCR4 target gene in Jurkat cells (Example 3, Figure 4) by the alpharetrovirus-based particle a.Gag.MS2 containing a nucleic acid construct encoding CRISPR / Cas9, compared to otherwise similar gammaretrovirus-based particles. Successful CRISPR / Cas9-mediated knockout of RFP657.Tet2 in target cells is indicated by the loss of RFP657 expression.

[0068] Example 3: Packaging and performance of alpharetrovirus-based Gag.MS2 particles delivering CRISPR / Cas9 RNA to manipulate endogenous genes As an example of an endogenous gene, the human CXCR4 gene was targeted by alpharetrovirus-based Gag.MS2 particles according to the present invention. Alpha and gammaretrovirus-based Gag.MS2 particles were packaged with an endogenous human CXCR4-targeting sgRNA.TS transcript containing an sgRNA comprising the protospacer sequence of SEQ ID NO: 10. Each particle containing an sgRNA.TS transcript directed against the mouse Trp53 gene (with the corresponding protospacer shown in SEQ ID NO: 8) was used as a non-targeting negative control. The results in Figures 4A and 4B show that alpharetrovirus-based particles are also more effective in manipulating the natural endogenous CXCR4 target gene than gammaretrovirus-based particles. Interestingly, a.Gag.MS2 particles outperformed the lentivirus-integrated CRISPR / Cas9 all-in-one vector control (LIT.CXCR4).

[0069] Figure 4A shows representative FACS data of knockout of endogenous CXCR4 gene in Jurkat cells. Non-transduced cells were used as mock controls. Alpha and gamma retrovirus-based Gag.MS2 particles were packaged with sgRNA.TS transcripts targeting CXCR4 (g.Gag.MS2:CXCR4.TS.adj; a.Gag.MS2 CXCR4.TS.inc). Particles containing each sgRNA.TS transcript directed against mouse Trp53 gene were used as negative controls. The inset in Figure 4A shows that knockout of endogenous CXCR4 gene targeted by each sgRNA.TS was only 15% by gamma retrovirus-based particles (g.Gag.MS2) but 91% by alpha retrovirus-based particles of the present invention (a.NC.pr.MS2). FIG. 4B graphically illustrates this higher rate of genetic engineering of the endogenous CXCR4 target gene by alpharetroviral-based particles.

[0070] Example 4: Performance of alpharetrovirus-based Gag.MS2 CRISPR / Cas9 particles targeting the endogenous human tumor suppressor gene TP53 in various primary human and mouse cell types (Figure 5) The protein components of the alpharetrovirus-based Gag.MS2 particle were those described in the above examples. Each alpharetrovirus-based Gag.MS2 particle contains an mRNA encoding SpCas9, followed by two copies of a TS hairpin (SpCas9.TS), optionally a PRE, and a polyA tail, and another sgRNA transcript specific for the human TP53 gene and a TS hairpin (TS.inc) incorporated into the sgRNA backbone. The nucleic acid construct is SEQ ID NO:5 for the mRNA encoding SpCas9 nuclease, and SEQ ID NO:9 for the protospacer replacing the BsmBI stuffer section of SEQ ID NO:3.

[0071] Alpharetrovirus-based Gag.MS2 particles were produced in 293T producer cells and the particle-containing supernatant was used to transduce target cells. Optionally, the supernatant was concentrated by ultracentrifugation. Supernatants were analyzed for the concentration of SpCas9.TS mRNA by qRT PCR, and volumes of alpharetrovirus-based Gag.MS2 particle supernatants containing the copy numbers of mRNA shown in the figures were used to transduce human NuFF (Figure 5A), primary human hepatocytes (PHH) (Figure 5B), human umbilical cord blood-derived CD34+ hematopoietic stem and progenitor cells (CD34+ HSPC) (Figure 5C) or mouse embryonic fibroblasts (MEFs; derived from CF1 or C3H mice) (Figure 5D). The applied SpCas9.TS mRNA dose (copies / cell) is shown on the X-axis, where 2x4150 (see FIG. 5A) or 2x4800 (see FIG. 5D) represent two doses of alpharetroviral-based a.Gag.MS2 particles, each with 4150 or 4800 mRNA copies, respectively. The Y-axis represents the percentage of deletion in the TP53 gene of the target cells.

[0072] These results demonstrate that alpharetrovirus-based Gag.MS2 particles can effectively transduce human and mouse primary cells, and that the encoded gene editing nuclease, e.g., SpCas9, in combination with sgRNA, effectively interacts with the target cell genome. Furthermore, these results demonstrate that the activity of alpharetrovirus-based Gag.MS2 particles against target cells is dose-dependent.

[0073] Example 5: Performance of alpharetrovirus-based Gag.MS2 particles encoding CRISPR / Cas9 to simultaneously edit two or three genes / target sites in target cells This example demonstrates that the alpharetrovirus-based Gag.MS2 particles of the invention are effective in specifically engineering, ie, genetically engineering or co-editing, two or three target genes in a cell.

[0074] The protein components of the alpharetrovirus-based Gag.MS2 particle are those described in the examples above, with a packaged mRNA encoding SpCas9 mRNA (SpCas9.TS, SEQ ID NO:5) linked upstream to a TS, and a combination of a first sgRNA.TS.inc construct containing a protospacer sequence specific for the human CXCR4 gene (SEQ ID NO:10) and a second sgRNA.TS.inc construct containing a protospacer sequence specific for the mouse Tet2 gene (SEQ ID NO:7) as one embodiment of an alpharetrovirus-based Gag.MS2 particle containing nucleic acid constructs for simultaneously editing two genes in a target cell (Figure 6A).

[0075] As a further embodiment, alpharetrovirus-based Gag.MS2 particles for simultaneous editing of three genes / target sites in target cells. In addition to SpCas9.TS.mRNA, alpharetrovirus-based particles were produced that contain a first sgRNA.TS.inc construct containing a protospacer sequence specific for the human CXCR4 gene (SEQ ID NO: 10), a second sgRNA.TS.inc construct containing a protospacer sequence specific for the mouse Tet2 gene (SEQ ID NO: 7), and a third sgRNA.TS.inc construct encoding a protospacer sequence specific for the EGFP gene (SEQ ID NO: 11). As an alternative or in addition to the sgRNA specific for knocking out the target gene, or in addition to SpCas9.TS.mRNA, mRNAs encoding additional GOIs can be introduced by the alpharetrovirus-based particles.

[0076] Each embodiment consisted of the same protein components, namely, alpharetroviral domains MA, p2, p10, CA and NC linked via a linker to NC (which linker may contain a viral protease site), the MS2 coat protein dimer (2xMS2CP) expressed from SEQ ID NO:1, and VSVg expressed from SEQ ID NO:4.

[0077] The target cells were Jurkat cells that endogenously express CXCR4 and were transduced by lentiviral vectors with nucleic acid constructs containing an expression cassette for EGFP and / or an expression cassette for the RFP657.Tet2 reporter gene (according to Hoffmann et al., cited above; Heckl et al., cited above; and Knopp et al., cited above).

[0078] FIG. 6A shows results for alpharetrovirus-based Gag.MS2 particles containing the coding mRNA SpCas9.TS in combination with sgRNA.TS.inc specific for CXCR4 and sgRNA.TS.inc specific for Tet2. Non-transduced / untreated cells were used as a negative control (sham). Representative FACS plots show that both the CXCR4 gene and the RFP657.Tet2 reporter gene (CXCR4 / Tet2) were effectively knocked out by alpharetrovirus-based Gag.MS2 CRISPR / Cas9 particles. Columns representing FACS results of three biological replicates show the number of % knockouts in cells, demonstrating the dose-dependence and positive correlation of alpharetrovirus-based Gag.MS2 particles, expressed as increasing SpCas9.TS mRNA copies per cell, and the amount of gene-edited cells in the treated cultures.

[0079] FIG. 6B shows FACS results of Jurkat target cells after transduction with alpharetrovirus-based Gag.MS2 particles containing mRNA encoding the gene editing nuclease SpCas9 in combination with three additional sgRNA.TS.inc transcripts targeting human CXCR4, mouse Tet2 and EGFP. Untreated mock cells were used as a negative control (mock), and as shown in the representative FACS plots shown, untreated mock cells were highly positive for all three reporter genes, i.e., CXCR4, Tet2, EGFP. In contrast, target cells transduced with alpharetrovirus-based Gag.MS2 particles showed effective knockout for all three targeted reporter genes. Cells negative for CXCR4 by 64% showed an additional knockout of 58% for RFP657 and EGFP reporter genes. The bar graph shows that increasing amounts of alpharetrovirus-based Gag.MS2 particles, determined as SpCas9.TS mRNA copies per target cell, resulted in an increasing percentage of triple-negative target cells genetically engineered with SpCas9 and all three sgRNA.TS.inc transcripts, e.g., approximately 25% at 68 SpCas9.TS mRNA copies / cell and approximately 30% at 170 SpCas9.TS mRNA copies / cell.

[0080] Example 6: Transduction of cells with self-amplifying RNA by alpharetrovirus-based particles As a further embodiment of RNA, a self-amplifying RNA replicon derived from Venezuelan equine encephalitis virus (VEE) is introduced into cells by the alpharetrovirus-based particle of the present invention. In this case, EGFP is transferred as GOI. However, the transferred RNA can also be a non-coding RNA, such as miRNA or lncRNA.

[0081] Figure 7A shows a schematic of the nucleic acid construct of alpharetrovirus-based a.Gag.MS2 particles (a.NC.pr.MS2) transfecting EGFP and encoding a self-amplifying RNA replicon derived from Venezuelan equine encephalitis virus (VEE). At the top, the expression plasmid for the generation of the VEE self-amplifying RNA replicon is shown. nsP1-4: VEE nonstructural proteins; RBZ: HDV antigenomic ribozyme; VEE promoter: VEE subgenomic promoter. Figure 7B shows a representative FACS plot of HT1080 cells transduced with a.NC.pr.MS2-based VEE replicon particles. On the right, a summary of multiple biological replicates is shown. Increasing amounts of 100-fold concentrated supernatant resulted in an increase in the percentage of EGFP-positive cells.

[0082] This shows that the alpharetrovirus-based particles of the invention may contain a self-amplifying RNA replicon comprising a GOI, exemplified here by the coding mRNA for EGFP.

[0083] Example 6: In vivo transduction with alpharetrovirus-based particles As a representative example of a gene of interest contained in an alpharetroviral particle of the invention, an alpharetroviral-based firefly luciferase-encoding a.Gag.MS2 particle (schematically shown in FIG. 1B, a.NC.pr.MS2, and GOI.TS encoding luciferase as the GOI), used in the results of FIGS. 1F and 1G, was injected into Balb / C mice at 4×10 9 In mRNA copies, 6x10 in Figure 8F 9 The mRNA copies were injected intraspleenically.

[0084] Eight hours later, the firefly luciferase substrate D-luciferin was administered intraperitoneally and the mice were examined by bioluminescence imaging. Strikingly, the background signal in control mice treated with PBS alone (0.1x10 on the color scale in Figure 8E) was 0.01x0.01. 7A stronger liver-specific signal could be detected for mice treated with a.Gag.MS2 particles (larger area and higher radiance, approximately 0.18x10 on the color scale in FIG. 8D ) compared to mice treated with a.Gag.MS2 particles (radiance values ​​below 0.18x10 on the color scale in FIG. 8D ). 7 ~0.25x10 7 , and for even larger areas and higher radiances, approximately 0.3x10 on the color scale in FIG. 8F. 7 ~0.6x10 7 Values ​​of 0.01 and 0.15 were obtained. Moreover, the intensity of the signal correlated with the dose of particles applied. The negative controls in Figures 8A-C, treated with luciferin only, did not show any bioluminescence.

[0085] This example demonstrates that the alpharetroviral particles of the invention, upon administration to a mammal, result in expression of a gene of interest encoded within the RNA construct contained in the alpharetroviral particle.

Claims

1. An alpharetrovirus-based (Gag.MS2) particle comprising an alpharetroviral domain consisting of MA-p2-p10-CA-NC-PR, a protein component comprising a linker of 7 to 25 amino acids and at least two MS2 coat proteins (2xMS2CP), a pseudotyping protein, and at least one RNA construct associated with the protein component, the RNA construct encoding a gene of interest linked to at least one MS2 target site (TS).

2. 2. The alpharetrovirus-based particle of claim 1, wherein said protein component consists of MA-p2-p10-CA-NC.

3. 3. An alpharetrovirus-based particle as described in claim 1 or 2, characterized in that the alpharetrovirus domain is separated from the at least two MS2 coat proteins by hydrolysis of the linker.

4. 3. An alpharetrovirus-based particle as described in claim 1 or 2, characterized in that a protease site is located between each of the alpharetrovirus domains.

5. 3. An alpharetrovirus-based particle as described in claim 1 or 2, characterized in that the linker between the alpharetrovirus domain and the at least two MS2 domains is a viral protease site.

6. the alpharetroviral domain has an amino acid sequence that has at least 90% identity to the amino acid sequence (MA-p2-p10-CA-NC-PR) encoded by nucleotides 1462 to 3573 of SEQ ID NO: 12 or to the amino acid sequence encoded by nucleotides 1462 to 3186 of SEQ ID NO: 1; the linker of 7 to 25 amino acids has an amino acid sequence that is at least 90% identical to the amino acid sequence encoded by nucleotides 3187 to 3207 of SEQ ID NO: 1 or the amino acid sequence encoded by nucleotides 3574 to 3594 of SEQ ID NO: 12, or is a glycine linker; the at least two MS2 coat proteins comprise an amino acid sequence having at least 90% identity to the amino acid sequence encoded by nucleotides 3613 to 4389 of SEQ ID NO: 12 or the amino acid sequence encoded by nucleotides 3223 to 3999 of SEQ ID NO: 1; An alpharetrovirus-based particle according to claim 1 or 2, characterized in that it

7. 3. The alpharetrovirus-based particle of claim 1, wherein the RNA construct comprises coding and / or non-coding RNA or contains a self-amplifying RNA replicon, and wherein the RNA construct is selected from a single guide (sg) RNA linked to at least one target site (TS), a small hairpin (sh) RNA, a microRNA, an shRNA / miRNA hybrid, a self-amplifying RNA replicon, a coding sequence for a DNA recombinase, a coding sequence for an enzyme, a coding sequence for a receptor, a coding sequence for a transcription factor, a gene editing nuclease, a recombinase, a coding sequence for a transposon, a coding sequence for an antigen, or a combination of at least two of these.

8. 3. An alpharetrovirus-based particle according to claim 1 or 2, characterized in that the RNA construct is a non-coding RNA and has a poly-T stretch linked to its 3' end.

9. An alpharetrovirus-based particle as described in claim 1 or 2, characterized in that it comprises at least two different sgRNAs, each linked to at least one target site (TS).

10. 3. The alpharetrovirus-based particle of claim 1 or 2, wherein the RNA construct contains an sgRNA integrated with at least two target sites (TS), the target sites being spaced 60 to 40 nucleotides apart, the sgRNA being positioned between the hairpin sections of two TS domains, or the RNA construct contains an sgRNA positioned 5' to at least two directly adjacent target sites (TS).

11. 3. An alpharetrovirus-based particle according to claim 1 or 2, characterized in that the target site (TS) has at least 90%, preferably at least 95%, nucleotide sequence identity with at least one sequence selected from nucleotides 1943 to 1965 and / or nucleotides 1982 to 2004 of SEQ ID NO:2, nucleotides 1681 to 1701 and / or nucleotides 1751 to 1771 of SEQ ID NO:3, nucleotides 1755 to 1775 and / or nucleotides 1794 to 1814 of SEQ ID NO:14, nucleotides 5697 to 5719 and / or nucleotides 5736 to 5758 of SEQ ID NO:5, nucleotides 2337 to 2359 and / or nucleotides 2376 to 2398 of SEQ ID NO:6, and the 2xMS2CP has at least 90%, preferably at least 95%, identity with the amino acid sequence encoded by nucleotides 3223 to 3999 of SEQ ID NO:

1.

12. An alpharetrovirus-based particle as described in claim 1 or 2, characterized in that it is pseudotyped with an envelope glycoprotein, thereby allowing the particle to be targeted to a specific cell type and to direct its target range.

13. An alpharetrovirus-based particle according to claim 1 or 2 for use in medical treatment.

14. 14. An alpharetrovirus-based particle for use according to claim 13 for introducing at least one RNA construct into a cell.

15. 3. A method for producing an alpharetrovirus-based particle according to claim 1 or 2 by expressing in a producer cell protein components comprising or consisting of, from N- to C-terminus, the alpharetrovirus MA-p2-p10-CA-NC or MA-p2-p10-CA-NC-PR, a linker, and at least one MS2 coat protein dimer (2xMS2CP), as well as expressing a pseudotyping protein, and expressing at least one RNA construct encoding a gene of interest linked to at least one MS2 target site (TS).

16. 16. The method of claim 15, wherein the protein is proteolyzed at its protease site to separate the alpharetroviral domain from the at least one MS2 coat protein.

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