WO2023143606A1

WO2023143606A1 - Fully human endogenous gene delivery system

Info

Publication number: WO2023143606A1
Application number: PCT/CN2023/073840
Authority: WO
Inventors: Cheng Wang; Xiyan WANG
Original assignee: Innovec Biotherapeutics
Priority date: 2022-01-30
Filing date: 2023-01-30
Publication date: 2023-08-03

Abstract

Provided is a fully human endogenous gene delivery system, which comprising a human endogenous enveloping protein, a human endogenous capsid protein, and a gene of interest. The endogenous gene delivery can be applied to CRISPR system, gene therapy,vaccine manufacturing and the like.

Description

Fully Human Endogenous Gene Delivery System

This application claims priority to PCT application No. PCT/CN2022/075171, filed January 30, 2022, the contents of which are hereby incorporated by reference in its entirety.

Field of the Invention

The present invention relates to a fully human endogenous gene delivery system. Specifically, the present invention relates to a fully human endogenous gene delivery system comprising a human endogenous enveloping protein, a human endogenous capsid protein, and a gene of interest, which has the advantage of being safe and efficient and can be applied to CRISPR systems, gene therapy, vaccine manufacturing, and the like.

Background of the Invention

Biologic nucleotide delivery vectors are applied for gene therapy in a series of disease to eliminate genetic disorders or to restore deficient gene expression. Lentiviral vectors derived from self-inactivated HIV-1 have been identified as efficient gene transfer vectors for stable long-term gene expression in host cells by integrating viral RNA genome into host chromatin. Lentiviral transduction depends on virus-cell membrane fusion which is mediated by envelope glycoprotein-extracellular receptor interaction. Vesicular stomatitis G glycoprotein (VSVg) is the most widely-utilized enveloping element in diverse lentiviral packaging system for its ideal viral particle stability, and outstanding gene transduction efficiency to host cells derived from different original tissue types. However, lentiviral vectors showed significant immunogenic properties during in vivo studies which have reported unexpected immune responses. Moreover, the non-site-specific integrity of lentiviral genome into host DNA also brought up risk and limitation for further application of lentiviral vectors, which requires improvement of gene delivery system with lower immunogenicity or integrating activity.

Recently, virus-like particles (VLPs) are employed into gene therapy field for design and construction of safety, specific and efficient mRNA delivery systems. Human paternally expressed 10 (hPEG10) functions as capsid-like properties and recognizes its own mRNA at not protein-coding sequence but 5'/3' UTR regions, therefore formulates capsid-mRNA VLP-like structure. The specific recognition of hPEG10 mRNA UTR regions and package of entire mRNA fragment into hPEG10 capsid allows the replacement of protein-coding sequence with gene of interest (GOI) , thus enables hPEG10 to specifically package mRNA of GOI cargoed with hPEG10 mRNA UTR (cargoRNA) into its capsid particle upon applicable design. Furthermore, hPEG10 VLP is capable to entry host cells by co-packaging with fusogens, result in an engineered mRNA delivery VLP system. VSVg and mouse syncytin a (Syna) are characterized fusogens when co-expressed with hPEG10 and cargoRNA in HEK 293 cell line, and VSVg/Syna-pseudotyped hPEG10 VLPs exhibited considerable transduction efficiency and diverse gene functions (GFP, Cre recombinase, Cas9 nuclease) in human and mouse cells in vitro. Additionally, hPEG10 VLP showed less immunogenic than lentiviral vectors due to its human endogenous capsid and might be a more safety mRNA delivery vector for the non-integrating property of cargoRNA. These results indicates that gene delivery systems consisted of highly or fully human endogenous elements would be a promising vector for in vivo gene transduction in human body.

In summary, notwithstanding the existing need, no gene delivery system consisting of highly or fully endogenous human elements has been developed to date. Thus, the technical problem underlying the present invention can be seen as the provision of means and methods which allow for safe and efficient gene delivery in a fully human endogenous way. The technical problem is solved by the embodiments characterized in the claims and herein below.

Summary of the Invention

The present invention relates to gene delivery system, production method and use thereof.

In a first aspect, the present invention provides a fully human endogenous gene delivery system comprising a human endogenous enveloping protein, a human endogenous capsid protein, and a gene of interest.

In a specific embodiment, the human endogenous enveloping protein can be selected from the group consisting of hERV-K, hERV-H, hERV-W, hERV-FRD, hERV-E, and a variant thereof. More specifically, hERV-K can be selected from the group consisting of hERV-K108, hERV-K109, hERV-K113, hERV-K115; hERV-H can be selected from the group consisting of hERV-H/env59, hERV-H/env60, hERV-H/env62; hERV-W can be ERVW-1; and hERV-FRD can be ERVFRD-1.

In a specific embodiment, the human endogenous enveloping protein can be c-terminal truncated ERVFRD-1. Preferably, the c-terminal truncated ERVFRD-1 has an amino acid sequence as set forth in any one of SEQ ID NOs.: 2-7, or a variant having at least 90%homology thereto. More specifically, the c-terminal truncated ERVFRD-1 is tERVFRD-1 Del515 having an amino acid sequence as set forth in SEQ ID NO.: 2, or a variant thereof, the c-terminal truncated ERVFRD-1 is tERVFRD-1 Del514 having an amino acid sequence as set forth in SEQ ID NO.: 3, or a variant thereof, the c-terminal truncated ERVFRD-1 is tERVFRD-1 Del513 having an amino acid sequence as set forth in SEQ ID NO.: 4, or a variant thereof, the c-terminal truncated ERVFRD-1 is tERVFRD-1 Del512 having an amino acid sequence as set forth in SEQ ID NO.: 5, or a variant thereof, the c-terminal truncated ERVFRD-1 is tERVFRD-1 Del511 having an amino acid sequence as set forth in SEQ ID NO.: 6, or a variant thereof, the c-terminal truncated ERVFRD-1 is tERVFRD-1 Del510 having an amino acid sequence as set forth in SEQ ID NO.: 7, or a variant thereof.

In a specific embodiment, the human endogenous capsid protein is selected from the group consisting of hPEG10, hArc, and a variant thereof.

In a specific embodiment, the human endogenous capsid protein is hPEG10 having an amino acid sequence as set forth in SEQ ID NO.: 8, or a variant thereof.

In a specific embodiment, the gene of interest comprises DNA or RNA, preferably RNA. More specifically, the gene of interest comprises but is not limited to mRNA, circle RNA, gRNA, siRNA, miRNA, shRNA, ASO (antisense oligonucleotide) , RNA scaffold, and non-coding RNA.

In a second aspect, the present invention provides a packaging system for producing a fully human endogenous gene delivery system of the present invention, which comprises at least at least an enveloping plasmid comprising CDS for a human endogenous enveloping protein, a packaging plasmid comprising CDS for a human endogenous capsid protein, and a transfer plasmid comprising a gene of interest.

In a specific embodiment, the human endogenous enveloping protein can be c-terminal truncated ERVFRD-1. Preferably, the c-terminal truncated ERVFRD-1 has an amino acid sequence as set forth in any one of SEQ ID NOs.: 2-7, or a variant thereof. More specifically, the c-terminal truncated ERVFRD-1 is tERVFRD-1 Del515 having an amino acid sequence as set forth in SEQ ID NO.: 2, or a variant thereof, the c-terminal truncated ERVFRD-1 is tERVFRD-1 Del514 having an amino acid sequence as set forth in SEQ ID NO.: 3, or a variant thereof, the c-terminal truncated ERVFRD-1 is tERVFRD-1 Del513 having an amino acid sequence as set forth in SEQ ID NO.: 4, or a variant thereof, the c-terminal truncated ERVFRD-1 is tERVFRD-1 Del512 having an amino acid sequence as set forth in SEQ ID NO.: 5, or a variant thereof, the c-terminal truncated ERVFRD-1 is tERVFRD-1 Del511 having an amino acid sequence as set forth in SEQ ID NO.: 6, or a variant thereof, the c-terminal truncated ERVFRD-1 is tERVFRD-1 Del510 having an amino acid sequence as set forth in SEQ ID NO.: 7, or a variant thereof.

In a specific embodiment, the CDS for a human endogenous enveloping protein has a nucleotide sequence as set forth in any one of SEQ ID NOs.: 10-15, or a variant thereof. More specifically, the CDS for tERVFRD-1 Del515 has a nucleotide sequence as set forth in SEQ ID NO.: 10, or a variant thereof, the CDS for tERVFRD-1 Del514 has a nucleotide sequence as set forth in SEQ ID NO.: 11, or a variant thereof, the CDS for tERVFRD-1 Del513 has a nucleotide sequence as set forth in SEQ ID NO.: 12, or a variant thereof, the CDS for tERVFRD-1 Del512 has a nucleotide sequence as set forth in SEQ ID NO.: 13, or a variant thereof, the CDS for tERVFRD-1 Del511 has a nucleotide sequence as set forth in SEQ ID NO.: 14, or a variant thereof, the CDS for tERVFRD-1 Del510 has a nucleotide sequence as set forth in SEQ ID NO.: 15, or a variant thereof.

In a specific embodiment, the CDS for hPEG10 has a nucleotide sequence as set forth in SEQ ID NO.: 16, or a variant thereof.

In a third aspect, the present invention provides a kit for produce a fully human endogenous gene delivery system of the present invention, which comprises a packaging system of the present invention, and a packaging cell line.

In a fourth aspect, the present invention provides a method to produce a fully human endogenous gene delivery system of the present invention, which comprises the following steps:

a) transfecting a package cell line with a packaging system comprising at least (1) an enveloping plasmid comprising CDS for a human endogenous enveloping protein, (2) a packaging plasmid comprising CDS for a human endogenous capsid protein, and (3) a transfer plasmid comprising a gene of interest.

b) incubating the transfected cells obtained in step a) to produce the fully human endogenous gene delivery system of the present invention; and

c) harvesting and concentrating the fully human endogenous gene delivery system obtained in step b) ,

preferably wherein step c) further comprises centrifuging and/or purifying the fully human endogenous gene delivery system obtained in step b) .

In a fifth aspect, the present invention provides a fully human endogenous gene delivery system for use in delivering Cas9 mRNA and gRNA to a subject for enabling gene editing and gene therapy, or in carrying mRNA expressing tumor antigens and viral antigens for vaccines, or in expressing cellular reprogramming factors for making pluripotent stem cells, modifying cellular function, or in delivering chimeric antigen receptor mRNA for cellular use in immunotherapy, or in expressing intact mRNA, small RNA, and RNP for gene therapy.

In a sixth aspect, the present invention provides use of a fully human endogenous gene delivery system in delivering Cas9 mRNA and gRNA to a subject for enabling gene editing and gene therapy, or in carrying mRNA expressing tumor antigens and viral antigens for vaccines, or in expressing cellular reprogramming factors for making pluripotent stem cells, modifying cellular function, or in delivering chimeric antigen receptor mRNA for cellular use in immunotherapy, or in expressing intact mRNA, small RNA, and RNP for gene therapy.

In a seventh aspect, the present invention provides a pharmaceutical composition comprising a fully human endogenous gene delivery system of the present invention, a packaging system of the present invention, or a kit of the present invention; and a pharmaceutically acceptable carrier.

Brief Description of the Drawings

In order to describe more clearly the object, technical solutions and beneficial effects of the present invention, the following accompanying drawings are provided.

Fig. 1 is a graph showing design and strategy for identification of hPEG10 VLPs pseudotyped with different fusogens. Fig. 1A shows biological structure of VLP, and fusogen, capsid and cargoRNA are annotated with icons. Fig. 1B shows key ORFs of VLP packaging plasmid system.

Fig. 2 is a graph showing that c-terminal truncated ERVFRD-1 resulted in comparable production and less fusogenic properties during fully human VLP transduction. Fig. 2A shows that fusogen induced cell-to-cell fusion at 48 h after co-transfection of packaging plasmids. Fig. 2B shows statistics of VLP production, n=3. p<0.05, *, p<0.01, **. Fig. 2C shows cell fusogenicity and GFP expression 24 h after hPEG10 fully human VLP treatment.

Fig. 3 is a graph illustrating the bioinformatics workflow to search candidate genes in human genome which may encode capsid and envelop proteins, and selected capsid (CA) and envelop (ENV) encoding genes.. Fig. 3A shows the bioinformatics workflow to search candidate genes in human genome, HHpred and blast were used as the primary searching algorithms. Fig. 3B shows the expression of CA genes in different human tissues. Fig. 3C shows the expression of ENV genes in different human tissues. Fig. 3D shows the maximum likelihood phylogenetic tree of the investigated CA genes. Fig. 3E shows the maximum likelihood phylogenetic tree of the investigated ENV genes.

Fig. 4 is a graph showing the purification and transduction to diverse cell lines of fully human VLP. Fig. 4A shows the yield of hPEG10 VLPs enveloped with different fusogens. Fig. 4B shows the transduction of GFP mRNA to different cell lines by fully human VLP. Fig. 4C shows statistics of fully human VLP transducted GFP positive cells analyzed by FACS.

Fig. 5 is a graph showing the transduction of CRISPR RNAs by fully human VLP. Fig. 5A shows the design of CRISPR edit-on system on HEK 293T genome. Fig. 5B shows the in-frame fluorescent protein mCherry expression after the transduction of SpCas9 mRNA and gRNA by fully human VLP.

Detailed Description of the Invention

Preferred embodiments of the invention will be described in detail below in conjunction with the accompanying drawings. Experimental methods for which specific conditions are not indicated in the embodiments usually follow conventional conditions, or follow the conditions recommended by the manufacturer. It will be understood that the specific embodiments described herein are intended only to explain the invention and not to limit it.

Fusogen is a class of proteins which mediate virus-cell membrane fusion by receptor-ligand interaction during viral infection process. Lentivirus, psuedotyped lentivirus and VLPs enveloped with different fusogens (for example, VSVg, Syna, ERVW-1, ERVFRD-1) are practically employed to transfer therapeutic RNA fragment into host cells in vitro. Yet, the non-human components of gene transfer vectors remain immunogenic risks for in vivo utilization. Endogenous retrovirus group FRD member 1 (ERVFRD-1) is identified as human endogenous expressing gene and a homologue of mouse syncytin b (Synb) , ERVFRD-1-pseudotyped lentivirus induces strong cellular membrane fusion during infection of host cells. Full-length ERVFRD-1 fusogen induced cellular membrane fusion results in severe cell death and poor production of viral particles.

In view of this, and in order to reduce the immune risk induced by the use of gene delivery systems in humans, the inventors conceived a fully human endogenous gene delivery system that minimizes the non-human components of the gene delivery system.

As used herein, the term “fully human endogenous gene delivery system” refers to a gene delivery system in which both the enveloping and capsid proteins are human endogenous proteins, or fusions or truncated proteins derived from human endogenous proteins. It should be understood by those of skill in the art that some components/elements of non-human origin may still be comprised in the fully human endogenous gene delivery system of the present invention. Specifically, the RNA delivered by the system of the present invention may comprise non-human origin sequences, such as optimized codons for amino acid, transcriptional regulatory elements, small interfering RNA (siRNA) , guide RNA (gRNA) , and the like.

The inventors first performed a bioinformatic screen for potential candidate capsid protein encoding genes and enveloping protein encoding genes in the human genome.

This bioinformatics screening process is depicted in FIG. 4. First, a gene collection was constructed that included well characterized human endogenous genes encoding capsid (CA) and enveloping (ENV) proteins. CA gene collection includes the following genes: ARC, PNMA3, PNMA5, ZCH12, PNMA1, PNMA6A, MOAP1, APRV1, PEG10, RTL10, and ENV gene collection includes: ERV3-1, ERVK-6, ERVMER34-1, Syncytin-1, Syncytin-2. The bait sequences were aligned and the aligned CA and ENV domain sequences were extracted, respectively. Two routes were then used to fish additional candidate genes. First, by using HHpred algorithm, the CA and ENV homologues were searched in human protein database (PDB_mmCIF30_12_Oct and PDB_mmCIF30_12_Oct) , and the HHpred search results were filtered (hhpred probability > 90 and homologues length > 100) . In the second route, all human open reading frames (ORFs) were extract from human reference genome (GRCh38) and the DNA sequences were converted into protein sequences. A blast database was constructed for the extracted ORFs. The aligned CA and ENV bait sequences were blasted against the database. The blast results were filtered if the aligned protein sequence length > 100, aligned sequence identity > 50%and blast e value < 0.001. The identified candidate genes were combined to generate the final list.

The candidate CA encoding genes and ENV encoding genes after the screening are shown in Figs. 3D and 3E. The candidate CA encoding genes comprise MOAP1, PNMA1, PNMA2, PNMA3, PNMA6A, PNMA5, ZCH12, ZCC18, RTL1, ZSC9, ZN274, RTL9, ARC APRV1, PEG10, NOL3, BOP, RTL4, RTL3, RTL5, and RTL6. The candidate ENV encoding genes comprises ERVV1, ERVV2, ENVT1, EFC2, SYCY2, SYCY1, ENK6, ENK19, ENR1, OCC1, ERB1, MER34, and FGL1.

The proteins encoded by the aforesaid genes, or fusions or truncated proteins derived therefrom can be used in the fully human endogenous gene delivery system of the present invention.

The inventors designed lentivirus/VLP packaging system with truncated ERVFRD-1 expressing vectors, tERVFRD-1 Del 515 (SEQ ID NO.: 2) , tERVFRD-1 Del 514 (tERVFRD-1 V1, SEQ ID NO.: 3) , tERVFRD-1 Del 513 (SEQ ID NO.: 4) , tERVFRD-1 Del 512 (SEQ ID NO.: 5) and tERVFRD-1 Del511 (SEQ ID NO.: 6) , tERVFRD-1 Del 510 (tERVFRD-1 V2, SEQ ID NO.: 7) , which are partially expressed with deletion of C-terminal residual amino acids respectively. Truncated ERVFRD-1 proteins showed significant weaker membrane fusion during lentiviral/VLP packaging in HEK 293T cell line as well as the similar lentiviral/VLP production to VSVg-pseudotyped packaging systems. The inventors packaged VSVg/ERVFRD-1/tERVFRD-1 V1 (tERVFRD-1 D514) /tERVFRD-1 V2 (tERVFRD-1 Del510) -pseudotyped hPEG10 VLP carrying GFP mRNA and treated HEK 293T cell with VLP containing supernatant, found that all VLPs mediated efficient GFP mRNA transduction and expression in HEK 293T cell, whereas tERVFRD-1 V2-hPEG10 VLP exhibited comparable cell fusion and higher mRNA transfer efficiency than VSVg-hPEG10 VLP. C-terminal truncated ERVFRD-1 pseudotyped lentiviral particle/VLP is a novel designed and identified gene delivery system.

The present invention provides a fully human endogenous gene delivery system comprising a human endogenous enveloping protein, a human endogenous capsid protein, and a gene of interest.

In a specific embodiment, the human endogenous enveloping protein is selected from the group consisting of hERV-K, hERV-H, hERV-W, hERV-FRD, hERV-E, and a variant thereof. More specifically, hERV-K can be selected from the group consisting of hERV-K108, hERV-K109, hERV-K113, hERV-K115; hERV-H can be selected from the group consisting of hERV-H/env59, hERV-H/env60, hERV-H/env62; hERV-W can be ERVW-1; and hERV-FRD can be ERVFRD-1 (or named Syncytin-2) .

As used herein, when the term “avariant” of a protein is mentioned, it should be understood that that the variant can have an amino acid sequence that is substantially identical, for example, at least about 70 to 99%identical, and all integers thereof to the original amino sequence of the protein, and which form functionally equivalent three-dimensional structures and retain the biological activity of the protein. It is well known in the biological field that specific amino acid substitutions can be formed in the protein sequence without affecting the function of the protein. Substitution mutations, insertion and deletion variants of the disclosed amino acid sequences can be readily prepared by methods well known in the art. These variants can be used in the same manner as the deliberately exemplified sequences, provided that the variants have essential sequence identity with the deliberately exemplified sequences of the present invention and maintain the desired functionality.

Preferably, the human endogenous enveloping protein is a fragment of the protein of hERV family including but not limited to hERV-K, hERV-H, hERV-W, hERV-FRD, hERV-E. Specifically, the human endogenous enveloping protein is c-terminal truncated ERVFRD-1. Preferably, the c-terminal truncated ERVFRD-1 is obtained by removing 20-30 amino acid residues from c-terminal of the full-length ERVFRD-1, for example, 22-30, 23-30, 23-29, or 23-28 amino acid residues, or any number in the above ranges, for example, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid residues. More preferably, the c-terminal truncated ERVFRD-1 is tERVFRD-1 Del515 having an amino acid sequence as set forth in SEQ ID NO.: 2, or a variant thereof, the c-terminal truncated ERVFRD-1 is tERVFRD-1 Del514 having an amino acid sequence as set forth in SEQ ID NO.: 3, or a variant thereof, the c-terminal truncated ERVFRD-1 is tERVFRD-1 Del513 having an amino acid sequence as set forth in SEQ ID NO.: 4, or a variant thereof, the c-terminal truncated ERVFRD-1 is tERVFRD-1 Del512 having an amino acid sequence as set forth in SEQ ID NO.: 5, or a variant thereof, the c-terminal truncated ERVFRD-1 is tERVFRD-1 Del511 having an amino acid sequence as set forth in SEQ ID NO.: 6, or a variant thereof, the c-terminal truncated ERVFRD-1 is tERVFRD-1 Del510 having an amino acid sequence as set forth in SEQ ID NO.: 7, or a variant thereof.

Preferably, the variant of tERVFRD-1 D515 has about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99%identity to SEQ ID NO.: 2. Preferably, the variant of tERVFRD-1 D515 contains 1 to 10, or 1 to 8, or 1 to 6, or 1 to 4, or 1, 2, 3, 4 amino acid residue addition, deletion or substitution to the amino acid sequence of SEQ ID NO.: 2. Preferably, the variant of tERVFRD-1 D514 has about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99%identity to SEQ ID NO.: 3. Preferably, the variant of tERVFRD-1 D514 contains 1 to 10, or 1 to 8, or 1 to 6, or 1 to 4, or 1, 2, 3, 4 amino acid residue addition, deletion or substitution to the amino acid sequence of SEQ ID NO.: 3. Preferably, the variant of tERVFRD-1 D513 has about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99%identity to SEQ ID NO.: 4. Preferably, the variant of tERVFRD-1 D513 contains 1 to 10, or 1 to 8, or 1 to 6, or 1 to 4, or 1, 2, 3, 4 amino acid residue addition, deletion or substitution to the amino acid sequence of SEQ ID NO.: 4. Preferably, the variant of tERVFRD-1 D512 has about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99%identity to SEQ ID NO.: 5. Preferably, the variant of tERVFRD-1 D512 contains 1 to 10, or 1 to 8, or 1 to 6, or 1 to 4, or 1, 2, 3, 4 amino acid residue addition, deletion or substitution to the amino acid sequence of SEQ ID NO.: 5. Preferably, the variant of tERVFRD-1 D511 has about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99%identity to SEQ ID NO.: 6. Preferably, the variant of tERVFRD-1 D511 contains 1 to 10, or 1 to 8, or 1 to 6, or 1 to 4, or 1, 2, 3, 4 amino acid residue addition, deletion or substitution to the amino acid sequence of SEQ ID NO.: 6. Preferably, the variant of tERVFRD-1 Del510 has about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99%identity to SEQ ID NO.: 7. Preferably, the variant of tERVFRD-1 Del510 contains 1 to 10, or 1 to 8, or 1 to 6, or 1 to 4, or 1, 2, 3, 4 amino acid residue addition, deletion or substitution to the amino acid sequence of SEQ ID NO.: 7.

In a specific embodiment, the full human endogenous gene delivery system is a pseudotyped viral particle, preferably a pseudotyped lentiviral particle, or a VLP.

As used herein, the term "viral vector" refers to a non-replicating, non-pathogenic virus engineered to deliver genetic material into a cell. In viral vectors, viral genes essential for replication and virulence have been replaced by heterologous target genes.

As used herein, the term "viral particle" refers to an extracellular form of a non-pathogenic virus, in particular a viral vector, which consists of genetic material made from DNA or RNA and surrounded by a protein shell (called a capsid) , which in some cases is derived from a host cell. In some cases, it is surrounded by an envelope that is derived from a portion of the host cell membrane and includes viral glycoproteins.

As used herein, the term "virus-like particle" or "VLP" refers to a self-assembling, non-replicating, non-pathogenic, genome-free particle that is similar in size and configuration to an intact infectious virus particle.

Preferably, the human endogenous capsid protein is hPEG10 having an amino acid sequence as set forth in SEQ ID NO.: 8, or a variant thereof.

Preferably, the variant of hPEG10 has about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99%identity to SEQ ID NO.: 8. Preferably, the variant of hPEG10 contains 1 to 10, or 1 to 8, or 1 to 6, or 1 to 4, or 1, 2, 3, 4 amino acid residue addition, deletion or substitution to the amino acid sequence of SEQ ID NO.: 8.

As is well known to those skilled in the art, the fully human endogenous gene delivery system of the present invention may also include those constituent parts commonly included in common lentiviral vector particles, VLPs in the art, including but not limited to lipid bilayers, and the like.

In a specific embodiment, the fully human endogenous gene delivery system of the present invention delivers DNA or RNA, preferably RNA. Specifically, the RNAs to be delivered include but are not limited to stranded mRNA or circle RNA for encoding proteins, gRNA for gene editing systems, siRNA/miRNA/shRNA or ASO (antisense oligonucleotide) for gene silencing/interference, RNA scaffolds or non-coding RNAs for protein factor recruitment, transcriptional regulatory functions, and the like.

The present invention provides a packaging system for producing a fully human endogenous gene delivery system of the present invention, which comprises at least at least an enveloping plasmid comprising CDS (coding sequence) for a human endogenous enveloping protein, a packaging plasmid comprising CDS for a human endogenous capsid protein, and a transfer plasmid comprising a gene of interest.

The human endogenous enveloping protein, the human endogenous capsid protein and the gene of interest can be used in the packing system are also described as aforesaid.

In a specific embodiment, the CDS for a human endogenous enveloping protein is CDS for tERVFRD-1 D515 as set forth in SEQ ID NO.: 10, or a variant thereof, CDS for tERVFRD-1 D514 as set forth in SEQ ID NO.: 11, or a variant thereof, CDS for tERVFRD-1 D513 as set forth in SEQ ID NO.: 12, or a variant thereof, CDS for tERVFRD-1 D512 as set forth in SEQ ID NO.: 13, or a variant thereof, CDS for tERVFRD-1 D511 as set forth in SEQ ID NO.: 14, or a variant thereof, CDS for tERVFRD-1 D510 as set forth in SEQ ID NO.: 15, or a variant thereof.

Preferably, the variant of CDS for tERVFRD-1 D515 has about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99%identity to SEQ ID NO.: 10. Preferably, the variant of CDS for tERVFRD-1 D515 contains 1 to 10, or 1 to 8, or 1 to 6, or 1 to 4, or 1, 2, 3, 4 nucleotides addition, deletion or substitution to the amino acid sequence of SEQ ID NO.: 10. Preferably, the variant of CDS for tERVFRD-1 D514 has about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99%identity to SEQ ID NO.: 11.Preferably, the variant of CDS for tERVFRD-1 D514 contains 1 to 10, or 1 to 8, or 1 to 6, or 1 to 4, or 1, 2, 3, 4 nucleotides addition, deletion or substitution to the amino acid sequence of SEQ ID NO.: 11. Preferably, the variant of CDS for tERVFRD-1 D513 has about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99%identity to SEQ ID NO.: 12. Preferably, the variant of CDS for tERVFRD-1 D513 contains 1 to 10, or 1 to 8, or 1 to 6, or 1 to 4, or 1, 2, 3, 4 nucleotides addition, deletion or substitution to the amino acid sequence of SEQ ID NO.: 12. Preferably, the variant of CDS for tERVFRD-1 D512 has about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99%identity to SEQ ID NO.: 13. Preferably, the variant of CDS for tERVFRD-1 D512 contains 1 to 10, or 1 to 8, or 1 to 6, or 1 to 4, or 1, 2, 3, 4 nucleotides addition, deletion or substitution to the amino acid sequence of SEQ ID NO.: 13. Preferably, the variant of CDS for tERVFRD-1 D511 has about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99%identity to SEQ ID NO.: 14.Preferably, the variant of CDS for tERVFRD-1 D511 contains 1 to 10, or 1 to 8, or 1 to 6, or 1 to 4, or 1, 2, 3, 4 nucleotides addition, deletion or substitution to the amino acid sequence of SEQ ID NO.: 14. Preferably, the variant of CDS for tERVFRD-1 Del510 has about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99%identity to SEQ ID NO.: 15. Preferably, the variant of CDS for tERVFRD-1 Del510 contains 1 to 10, or 1 to 8, or 1 to 6, or 1 to 4, or 1, 2, 3, 4 nucleotides addition, deletion or substitution to the amino acid sequence of SEQ ID NO.: 15.

In a specific embodiment, the CDS for a human endogenous capsid protein is CDS for hPEG10 as set forth in SEQ ID NO.: 16, or a variant thereof.

Preferably, the variant of CDS for hPEG10 has about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99%identity to SEQ ID NO.: 16. Preferably, the variant of CDS for hPEG10 contains 1 to 10, or 1 to 8, or 1 to 6, or 1 to 4, or 1, 2, 3, 4 nucleotides addition, deletion or substitution to the amino acid sequence of SEQ ID NO.: 16.

In a specific embodiment, the packaging system may further comprise in any one of the plasmids at least one element selected from but not limited to: a 5' LTR, a ψelement, an RRE element, a cPPT/CTS element, a polyclonal site for insertion of a nucleotide sequence encoding a polypeptide of interest, a WPRE element, a 3' LTR, and a UTR, a cytomegalovirus (CMV) enhancer/promoter sequence, a terminator, an internal enhancer, an internal promoter, all of which are commonly known to those skilled in the art.

Construction of the plasmid can be accomplished using any suitable genetic engineering techniques known in the art, including, without limitation, the standard techniques of restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing, for example as described in Sambrook et al. (1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, N.Y. ) , Coffin et al. (Retroviruses. Cold Spring Harbor Laboratory Press, N. Y. (1997) ) and "RNA Viruses: A Practical Approach" (Alan J. Cann, Ed., Oxford University Press, (2000) , each of the foregoing which is incorporated herein by reference in its entirety.

The packaging system comprises a gene of interest that is desirable to express in target cells. Typically, the gene of interest is located between the 5' LTR and 3' LTR sequences. Further, the gene of interest is preferably in a functional relationship with other genetic elements, for example transcription regulatory sequences including promoters or enhancers, to regulate expression of the gene of interest in a particular manner. In certain instances, the useful transcriptional regulatory sequences are those that are highly regulated with respect to activity, both temporally and spatially. Expression control elements that may be used for regulating the expression of the components are known in the art and include, but are not limited to, inducible promoters, constitutive promoters, secretion signals, enhancers and other regulatory elements.

In a specific embodiment, the transfer plasmid comprises a UTR sequence upstream of the gene of interest, and/or a UTR sequence downstream of the gene of interest. The UTR sequences can be same or different.

The present invention provides a kit for produce a fully human endogenous gene delivery system of the present invention, which comprises a packaging system of the present, and a packaging cell line.

The present invention provides a method to produce a fully human endogenous gene delivery system of the present invention, which comprises the following steps:

preferably wherein step c) further comprises centrifuging and/or purifying the fully human endogenous gene delivery system obtained in step b)

The packaging cell line comprises cultured cells capable of packaging a lentiviral vector/VLP. In some cases, the packaging cell line is the HEK-293T cell line. Similar results can be achieved in other cell lines, including but not limited to mammalian gene expression cell lines such as Vero and CHO. Other cell lines that can be transfected in vitro and are capable of producing high titers of lentiviral vectors/VLPs may be used.

The present disclosure also provides for genetic engineering of packaging cell lines to otherwise improve the immune properties of the lentiviral vectors and particles of the present disclosure, the other means including but not limited to adding genes, deleting genes and introducing point mutations into genes.

The present invention provides a fully human endogenous gene delivery system for use in delivering Cas9 mRNA and gRNA to a subject for enabling gene editing and gene therapy, or in carrying mRNA expressing tumor antigens and viral antigens for vaccines, or in expressing cellular reprogramming factors for making pluripotent stem cells, modifying cellular function, or in delivering chimeric antigen receptor mRNA for cellular use in immunotherapy, or in expressing intact mRNA, small RNA, and RNP for gene therapy.

The present invention provides use of a fully human endogenous gene delivery system in delivering Cas9 mRNA and gRNA to a subject for enabling gene editing and gene therapy, or in carrying mRNA expressing tumor antigens and viral antigens for vaccines, or in expressing cellular reprogramming factors for making pluripotent stem cells, modifying cellular function, or in delivering chimeric antigen receptor mRNA for cellular use in immunotherapy, or in expressing intact mRNA, small RNA, and RNP for gene therapy.

The fully human endogenous gene delivery system of the present invention may be used in a therapeutic treatment in human or animal subjects. The treatment may be a vaccine for immunization, wherein the vaccine is prophylactic or therapeutic. The vaccine comprises a fully human endogenous gene delivery system with a pharmaceutically acceptable carrier. Alternatively, the fully human endogenous gene delivery system may be administered in vitro to cells, including the step of mixing the cells with any of the above fully human endogenous gene delivery system.

Also provided herein are pharmaceutical compositions comprising a packaging system, a host cell line, a kit or a fully human endogenous gene delivery system as described above. Such compositions comprise therapeutically effective amounts of the above packaging system, host cell line, kit or fully human endogenous gene delivery system, as well as a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically acceptable carrier" refers to a solid or liquid diluent, filler, antioxidant, stabilizer, or other substance that can be safely administered without undue adverse side effects and is suitable for maintaining the viability of the drug or active agent located therein. In accordance with the route of administration, a variety of different carriers well known in the art may be used, including, but not limited to, sugars, starches, cellulose and derivatives thereof, maltose, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffers, emulsifiers, isotonic saline, and/or pyrogen-free water, and the like.

If necessary, the pharmaceutical compositions may also contain small amounts of wetting agents or emulsifiers or pH buffers. These pharmaceutical compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like.

The pharmaceutical compositions provided herein can be made in clinically acceptable dosage forms such as powders, injectables, etc. The pharmaceutical compositions of the present invention may be administered to a subject using any suitable route, for example, by oral, intravenous infusion, intramuscular, subcutaneous, subperitoneal, rectal, sublingual, or via inhalation, transdermal, etc.

In preferred embodiments, the pharmaceutical composition is formulated according to conventional procedures suitable for intravenous or intramuscular administration. Typically, the pharmaceutical composition for intravenous or intramuscular administration is a solution in a sterile isotonic aqueous buffer. Where necessary, said pharmaceutical composition may also comprise a solubilizing agent and a local anesthetic such as lidocaine to relieve pain at the subject's injection site.

As used herein, the term "subject" means an animal, such as a mammal, including but not limited to a human, rodent, ape, feline, canine, equine, bovine, porcine, sheep, goat, mammalian laboratory animal, mammalian farm animal, mammalian sport animal, and mammalian pet. Subjects may be male or female and may be of any age, including infants, juveniles, young adults, adults, and elderly subjects. In some embodiments, a subject is an individual in need of treatment for a disease or condition. In some embodiments, the subject receiving the treatment may be a patient who has a condition associated with the treatment or is at risk of developing the condition. In particular embodiments, the subject is a human, such as a human patient. The term is often used interchangeably with "patient" , "test subject" , "treatment subject" , and the like.

In an embodiment, the packaging system, or the fully human endogenous gene delivery system of the present invention may be delivered in a vesicle, particularly a liposome. In yet another embodiment, the nucleic acid sequence, the packaging system, or the fully human endogenous gene delivery system of the present invention may be delivered in a controlled release system.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

The articles “a” , “an” , and “the” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

The use of alternatives (e.g., "or" ) should be understood to mean one, two, or any combination thereof of the alternatives.

The term "and/or" is to be understood as referring to one or both alternatives.

As used herein, the term "approximately" or "about" means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length range of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%with respect to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

Unless otherwise specified, any concentration range, percentage range, ratio range, or integer range shall be understood to include any integer value within said range and, where appropriate, fractions thereof (such as tenths and hundredths of an integer) . When immediately preceded by a numeric or numeric value, the term "approximately" refers to plus or minus 10 percent of the numeric or numeric range.

Throughout the specification, unless the context otherwise requires, the term "comprise/comprises/comprising" should be understood to mean the inclusion of a specified step, or element, or group of steps or elements, but does not exclude any other step, or element, or group of steps or elements. In particular embodiments, the terms "include" , "have" , "contain" and "comprise" are used synonymously.

All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent were specifically and separately indicated as not incorporated by reference. In the event of conflict, this application (including any definitions herein) shall control. However, any references, articles, publications, patents, patent publications and patent applications cited herein are not and shall not be deemed to be admissions or suggestions of any kind which constitute valid prior art or form part of the common knowledge of any country in the world.

The present invention is further described in the following examples, which are not limiting the scope of the present invention as described in the claims.

Example

Materials

HEK 293T cell line (ATCC, CRL-3216)

Fetal bovine serum (Gibco, A3161002C)

Dulbecco's Modified Eagle Medium (Gibco, 11965092)

Penicillin-Streptomycin (Gibco, 15140122)

0.05%Trypsin-EDTA (Gibco, 25300062)

Opti-MEM (Gibco, 31985070)

Lipofectamine 3000 transfection reagent (Invitrogen, L3000015)

D-PBS, with Ca²⁺, Mg²⁺ (Corning, 21-030-CV)

24-well TC-treated culture plate (Costar, 3524)

100 mm TC-treated culture dish (Jet Bio-Filtration, TCD010100)

15 mL Centrifuge Tube (Axygen, SCT-15ML-25-S)

Serological Pipets (Jet Bio-Filtration)

Benzonase (Sigma-Aldrich, E8263-25KU)

EasyPure Viral DNA/RNA Kit (Transgen, ER201-02)

TransScript II One-Step RT-PCR SuperMix (Transgen, AH411-02)

HIV1 p24 ELISA Kit (Abcam, ab218268)

Example 1: Vector design &construction

ERVFRD-1/tERVFRD-1 Del515/tERVFRD-1 Del514 (tERVFRD-1 V1) /tERVFRD-1 Del513/tERVFRD-1 Del512/tERVFRD-1 Del511/tERVFRD-1 Del510 (tERVFRD-1 V2) CDS were synthesized and cloned separately into pCMV-VSVg plasmid (purchased from CellBiolabs) with Gibson Assembly, while VSVg CDS was removed by EcoRI treatment to make the pFusogen plasmids. pCapsid plasmids was inserted into pCMV-VSVg by replacing the VSVg with synthesized hPEG10 CDS or other human endogenous Gag-pol used in this invention. CargoRNA plasmids were constructed with pSMPUW-IRES-Puro (purchased from CellBiolabs) plasmid by the insertion corresponding UTR Cargo (GFP or Cas9) UTR downstream of CMV promoter for strong mRNA transcription. Fig. 1A provides a schematic diagram of the structure of VLPs according to the examples and comparative examples of the present invention, wherein cargoRNA represents an example of the gene of interest to be delivered. FIG. 1B provides a schematic diagram of the structures of plasmids according to the examples and comparative examples of the present invention. The pFusogen represents an example of enveloping plasmid, the pCapsid represents an example of packaging plasmid, and the pCargoRNA represents an example of transfer plasmid.

Example 2: VLP packaging, purification and titration

HEK 293T cells were seeded in 100 mm dish and grown to 70%～80%confluent for plasmid transfection. For VLP packaging, 5 μg pSMPUW-hPEG10, 10 μg pCargoRNA-GFP and 5 μg pFusogen plasmids were co-transfected with Lipofectamine 3000 transfection reagent into HEK 293T cells, cell culture supernatant was harvested at 48 h after transfection. VLPs were purified and concentrated by ultracentrifugation involving a 20%sucrose cushion at 80000 g for 2 h, the Beckman ultracentrifuge was set to 4℃ during VLP ultracentrifugation. Purified VLPs were titrated by RT-qPCR and were stored at -80℃.

Isolated cell culture supernatant containing lentiviral particle/VLP or purified VLPs was pre-treated with 25 U/mL benzonase at 37℃ for 1 h to remove free DNA/RNA, then benzonase was inactivated by 56℃ incubation for 10 min. Lentiviral/VLP genomic RNA were extracted with EasyPure Viral DNA/RNA Kit (Transgen Biotech) , and VLP genomic RNA were subsequently analyzed by RT-qPCR with TransScript II One-Step RT-PCR SuperMix (Transgen Biotech) .

Example 3 VLP transduction Assay

Fig. 2A shows that fusogen induced cell-to-cell fusion at 48 h after co-transfection of packaging plasmids, tERVFRD-1 V2 exhibited significant less fusogenic effect than other human envelope candidates. Fig. 2B illustrates that both tERVFRD-1 V1 and tERVFRD-1 V2 result in normal VLP production. For testing the ability of VLP transduction, 5×10⁴ HEK 293T cells were planted in 24-well plate for 12～16 h, VLP solution was gently added into cell culture supernatant. VLPs were quickly settled and attached to adherent HEK 293T cells with 1000 g centrifuging at room temperature for 30 min, cell culture medium was renewed afterwhile. GFP expression efficiency was detected and analyzed with Cyto-FLEX S flow cytometer at 24 h after VLP infection. Fig. 2C shows all ERVFRD-1 envelopes mediated comparable GFP transduction to VSVg. However, VLP containing ERVFRD-1 or tERVFRD-1 V1 induced cell to cell fusion, which will limit the usage of those VLPs since cell fusion might lead to cell death or carcinogenesis.

Example 4: Discovery of endogenous Gags and Envelopes.

Fig. 3A shows the workflow to search candidate genes in human genome which may encode capsid and envelop proteins. Results with contact CA or Env open reading frame are showing in Figs. 3B-3E. Several Envelopes were moved to next step to test their VLP packaging and transduction capability.

Example 5: Production and transduction of VLP with more human endogenous envelopes

Different envelops could mediated significant VLP particle formation (Fig. 4A) . However only VSVg and tERVFRD-1 V2 mediated GFP transduction in several cell lines (Figs. 4B and 4C) . we did not detect GFP transduction of VLPs with other human endogenous envelopes. One of the reasons might be the lack of receptors on those cells.

Examples 6: VLP mediating Cas9 transduction

To detect Cas9 transduction, the 293T edit-on cell line was constructed by infection of a lentivirus carrying a GFP gRNA sequence upstream a frameshifted mCherry. Indels induced by Cas9-gGFP complex may turn on the mCherry by put it on frame.

24 h before transfection of VLP-Cas9 packaging plasmids, a plasmid with GFP sgRNA were transfected into HEK 293T cell to serve gGFP RNA during VLP production. 24 h after VLP transduction, the cells were analyzed by FACS. The mCherry was turned on, indicating that Cas9 was transduced by VLP (Fig. 5) .

Sequence Listings

Amino acid sequence of ERVFRD-1 (SEQ ID NO.: 1)

Amino acid sequence of ERVFRD-1 Del515 (SEQ ID NO.: 2)

Amino acid sequence of tERVFRD-1 Del514 (SEQ ID NO.: 3)

Amino acid sequence of ERVFRD-1 Del513 (SEQ ID NO.: 4)

Amino acid sequence of ERVFRD-1 Del512 (SEQ ID NO.: 5)

Amino acid sequence of ERVFRD-1 Del511 (SEQ ID NO.: 6)

Amino acid sequence of tERVFRD-1 Del510 (SEQ ID NO.: 7)

Amino acid sequence of hPEG10 (SEQ ID NO.: 8)

Nucleotide sequence of CDS for ERVFRD-1 (SEQ ID NO.: 9)

Nucleotide sequence of CDS for ERVFRD-1 Del515 (SEQ ID NO.: 10)

Nucleotide sequence of CDS for tERVFRD-1 Del514 (SEQ ID NO.: 11)

Nucleotide sequence of CDS for ERVFRD-1 Del513 (SEQ ID NO.: 12)

Nucleotide sequence of CDS for ERVFRD-1 Del512 (SEQ ID NO.: 13)

Nucleotide sequence of CDS for ERVFRD-1 Del511 (SEQ ID NO.: 14)

Nucleotide sequence of CDS for tERVFRD-1 Del510 (SEQ ID NO.: 15)

Nucleotide sequence of CDS for hPEG10 (SEQ ID NO.: 16)

Claims

A fully human endogenous gene delivery system, which comprises (1) a human endogenous enveloping protein, (2) a human endogenous capsid protein, and (3) a gene of interest.
The fully human endogenous gene delivery system according to claim 1, wherein the human endogenous enveloping protein is selected from the group consisting of hERV-K, hERV-H, hERV-W, hERV-FRD, hERV-E, and a variant thereof.
The fully human endogenous gene delivery system according to claim 1 or 2, wherein the human endogenous enveloping protein is c-terminal truncated ERVFRD-1.
The fully human endogenous gene delivery system according to any one of claims 1 to 3, wherein the human endogenous capsid protein is selected from the group consisting of hPEG10, hArc, and a variant thereof.
The fully human endogenous gene delivery system according to any one of claims 1 to 4, wherein the human endogenous capsid protein is hPEG10 having amino acid sequence as set forth in SEQ ID NO.: 8 or a variant having at least 90%homology to SEQ ID NO.: 8.
The fully human endogenous gene delivery system according to any one of claims 1 to 5, wherein the gene of interest is DNA or RNA, preferably RNA, more preferably mRNA.
A packaging system for producing a fully human endogenous gene delivery system according to any one of claims 1-6, which comprises at least (1) an enveloping plasmid comprising CDS for a human endogenous enveloping protein, (2) a packaging plasmid comprising CDS for a human endogenous capsid protein, and (3) a transfer plasmid comprising a gene of interest.
A kit for produce a fully human endogenous gene delivery system according to any one of claims 1-6, which comprises a packaging system according to claim 7, and a packaging cell line.
A method to produce a fully human endogenous gene delivery system according to any one of claims 1 to 6, which comprises the following steps:

a) transfecting a package cell line with a packaging system comprising at least (1) an enveloping plasmid comprising CDS for a human endogenous enveloping protein, (2) a packaging plasmid comprising CDS for a human endogenous capsid protein, and (3) a transfer plasmid comprising a gene of interest.

b) incubating the transfected cells obtained in step a) to produce the fully human endogenous gene delivery system according to any one of claims 1 to 6; and

c) harvesting and concentrating the fully human endogenous gene delivery system obtained in step b) ,

preferably wherein step c) further comprises centrifuging and/or purifying the fully human endogenous gene delivery system obtained in step b) .
A fully human endogenous gene delivery system according to any one of claims 1-6 for use in delivering Cas9 mRNA and gRNA to a subject for enabling gene editing and gene therapy, or in carrying mRNA expressing tumor antigens and viral antigens for vaccines, or in expressing cellular reprogramming factors for making pluripotent stem cells, modifying cellular function, or in delivering chimeric antigen receptor mRNA for cellular use in immunotherapy, or in expressing intact mRNA, small RNA, and RNP for gene therapy.
Use of a fully human endogenous gene delivery system according to any one of claims 1-6 in delivering Cas9 mRNA and gRNA to a subject for enabling gene editing and gene therapy, or in carrying mRNA expressing tumor antigens and viral antigens for vaccines, or in expressing cellular reprogramming factors for making pluripotent stem cells, modifying cellular function, or in delivering chimeric antigen receptor mRNA for cellular use in immunotherapy, or in expressing intact mRNA, small RNA, and RNP for gene therapy.