Retrotransposon-based delivery vehicles and methods of use thereof

Abstract

The present disclosure provides a gene delivery system, the gene delivery system comprising: a) R2 retrotransposon R2 polypeptide, or a first nucleic acid comprising a nucleotide sequence encoding the R2 polypeptide; and b) a nucleic acid comprising a heterologous nucleotide sequence encoding one or more heterologous gene products, wherein the heterologous nucleotide sequence is flanked by the R2 retrotransposon 3' untranslated region (UTR) and the R2 retrotransposon 5'UTR, and wherein the heterologous nucleotide sequence has a length of at least 200 nucleotides. The present disclosure provides a method for delivering one or more target gene products to eukaryotic cells, the method comprising contacting the cells with the gene delivery vehicle system.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 697,829, filed on July 13, 2018, which is incorporated herein by reference in its entirety.

[0003] introduction

[0004] Gene therapy is becoming an increasingly successful technology for treating human diseases. Gene therapy involves the delivery of a nucleic acid comprising a coding region encoding a target gene product, wherein the gene product can provide functional gain or functional loss to correct abnormal behavior in a specific cell. Delivery is generally divided into two main categories: viral-mediated and non-viral-mediated delivery. In addition, the delivery of nucleic acids produces transient expression of all or part of the nucleic acid or irreversible integration into the DNA of the host cell. Virus-mediated integration methods are most commonly used for dividing cells, where delivery is mediated, for example, by using lentiviruses and retroviruses engineered to carry therapeutic DNA into cells. Such viruses can be effectively and stably integrated into the genome of the host cell; however, they have many disadvantages. Integration into random locations in the cell genome can lead to disruption of cell function, and expression may even eventually be silenced by the cell's own mechanisms. In addition, when the size of the coding region to be delivered exceeds 6 kilobases (kb) or 8kb for retroviruses and lentiviruses, respectively, viral methods suffer serious damage in terms of their ability to package and deliver the coding region encoding the target gene product. Although many gene therapies involve coding regions within packaging constraints, the length of the coding region of the gene product is approaching the maximum limit of 8 kb, and the addition of larger cDNAs, regulatory elements, or multiple genes will increase the insert size to a size larger than current viral delivery vehicles can accommodate.

[0005] Class II transposons, such as piggyBac, Sleeping Beauty, and Tol2, can integrate larger payloads; however, such transposons have disadvantages. For example, DNA transposons can integrate at specific but common sites in the genome (notably, transposable elements typically make up 45% of the genome), and some tend to integrate in regions where active transcription occurs.

[0006] There is a need in the art for delivery vehicles for the delivery of larger coding regions. Summary of the invention

[0007] The present disclosure provides a gene delivery system, the gene delivery system comprising: a) a first nucleic acid, the first nucleic acid comprising a nucleotide sequence encoding an R2 retrotransposon R2 polypeptide; and b) a second nucleic acid, the second nucleic acid comprising a heterologous nucleotide sequence encoding one or more heterologous gene products, wherein the heterologous nucleotide sequence is flanked by the R2 retrotransposon 3' untranslated region (UTR) and the R2 retrotransposon 5'UTR, and wherein the heterologous nucleotide sequence has a length of up to about 15 kilobases. The present disclosure provides a method of delivering one or more target gene products to a eukaryotic cell, the method comprising contacting the cell with the gene delivery vehicle system. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1A-1B Description: Figure 1A ) Schematic diagram of a dual vector integration system encoding a transgene flanked by an R2UTR targeting sequence; and ( Figure 1B )The mechanism of the R2 integration mechanism proposed by

[0009] Figure 2 Depicted is analysis of genomic integration junctions in 28S rDNA for genes delivered using the gene delivery system of the present disclosure.

[0010] Figure 3 Depicted is analysis of genomic integration junctions in 28S rDNA for genes delivered using the gene delivery system of the present disclosure.

[0011] Figure 4 A protocol for cell transfection and passaging is depicted, and data showing green fluorescent protein (GFP) expression following transfection with the gene delivery system of the present disclosure is presented.

[0012] Figure 5A and 5B The effect of optimized R2 ("OR2") on transgene expression is depicted.

[0013] Fig. 6A and 6B Depicted is the expression of a chimeric antigen receptor (CAR) following transfection of cells with the gene delivery system of the present disclosure, wherein the heterologous nucleic acid comprises a nucleotide sequence encoding the CAR.

[0014] Figure 7 The amino acid sequence of the R2 polypeptide is provided (SEQ ID NO: 37).

[0015] Figure 8 The nucleotide sequence of the 5'UTR is provided (SEQ ID NO: 38).

[0016] Fig. 9The nucleotide sequence of the 3'UTR is provided (SEQ ID NO: 39).

[0017] Fig.10 Depicted is the percentage of HEK293 cells stably expressing green fluorescent protein (GFP) 14 days after transfection with the gene delivery system of the present disclosure.

[0018] Fig.11 Depicted are the number of hygromycin-resistant HEK293 colonies 14 days after transfection with the gene delivery system of the present disclosure.

[0019] Fig.12 Depicts c-myc 14 days after transfection with the gene delivery system of the present disclosure. + Percentage of HEK293 cells.

[0020] definition

[0021] As used herein, "heterologous" refers to a nucleotide or polypeptide sequence that does not occur in a native (eg, naturally occurring) nucleic acid or protein, respectively.

[0022] The terms "polynucleotide" and "nucleic acid" used interchangeably herein refer to a polymeric form of nucleotides (ribonucleotides or deoxyribonucleotides) of any length. Thus, this term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases.

[0023] "Operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.

[0024] The terms "polypeptide", "peptide" and "protein" used interchangeably herein refer to a polymeric form of amino acids of any length, which may include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. The terms include fusion proteins, including but not limited to fusion proteins with heterologous amino acid sequences, fusions with heterologous and homologous leader sequences, with or without an N-terminal methionine residue; immunolabeled proteins; etc.

[0025] The terms "chimeric antigen receptor" and "CAR" used interchangeably herein refer to artificial multi-module molecules capable of triggering or inhibiting the activation of immune cells, which molecules generally but not exclusively include an extracellular domain (e.g., a ligand / antigen binding domain), a transmembrane domain, and one or more intracellular signaling domains. The term "CAR" is not specifically limited to CAR molecules, but also includes CAR variants. CAR variants include separated CARs, wherein the extracellular portion (e.g., ligand binding portion) and the intracellular portion (e.g., intracellular signaling portion) of CAR are present on two separate molecules. CAR variants also include ON-switch CARs as conditionally activatable CARs, for example, comprising separated CARs, wherein the conditional heterodimerization of the two parts of the separated CAR is controlled pharmacologically. CAR variants also include bispecific CARs, which include a secondary CAR binding domain that can amplify or inhibit the activity of primary CAR. CAR variants also include inhibitory chimeric antigen receptors (iCARs), which can be used, for example, as components of a bispecific CAR system, wherein the binding of the secondary CAR binding domain results in the inhibition of primary CAR activation. CAR molecules and their derivatives (i.e., CAR variants) are described in, for example, PCT Application No. US2014 / 016527; Fedorov et al. Sci Transl Med (2013); 5(215):215ra172; Glienke et al. Front Pharmacol (2015) 6:21; Kakarla and Gottschalk 52 Cancer J (2014) 20(2):151-5; Riddell et al. Cancer J (2014) 20(2):141-4; Pegram et al. Cancer J (2014) 20(2):127-33; Cheadle et al. Immunol Rev (2014) 257(1):91-106; Barrett et al. Annu Rev Med (2014) 65:333-47; Sadelain et al. Cancer Discov (2013) 3(4):388-98; Cartellieri et al., J Biomed Biotechnol (2010) 956304, the disclosures of which are incorporated herein by reference in their entirety.

[0026] "Single-chain Fv" or "sFv" antibody fragments comprise the V H and V L domains, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a V H Domain and V LA polypeptide linker between the domains that enables the sFv to form the structure required for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, ed., Springer-Verlag, New York, pp. 269-315 (1994).

[0027] As used herein, the term "nanobody" (Nb) refers to the smallest antigen-binding fragment or single variable domain (V HH ), and are known to those skilled in the art. They are derived from heavy chain-only antibodies found in camelids (Hamers-Casterman et al., 1993; Desmyter et al., 1996). In the "Camelid" family, immunoglobulins without light polypeptide chains are found. "Camelids" include Old World camelids (Bactrian camels and dromedaries) and New World camelids (e.g., alpacas (Llama paccos), llamas (Llama glama), guanicoes (Llama guanicoe), and llamas (Llama vicugna)). Single variable domain heavy chain antibodies are referred to herein as nanobodies or V HH Antibody.

[0028] As used herein, the terms "treatment", "treating", "treat" and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of complete or partial prevention of a disease or its symptoms and / or therapeutic in terms of partial or complete cure of a disease and / or adverse effects attributable to the disease. "Treatment" as used herein encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed as having the disease; (b) inhibiting the disease, i.e., arresting its development; and (c) alleviating the disease, i.e., causing regression of the disease.

[0029] The terms "subject," "subject," "host," and "patient," used interchangeably herein, refer to mammals, including but not limited to murines (rats, mice), non-human primates, humans, canines, felines, ungulates (e.g., equines, bovines, ovines, porcines, caprines), lagomorphs, and the like. In some cases, the subject is a human. In some cases, the subject is a non-human primate. In some cases, the subject is a rodent, such as a rat or mouse. In some cases, the subject is a lagomorph, such as a rabbit.

[0030] Before further describing the present invention, it should be understood that the present invention is not limited to the specific embodiments described, and thus, of course, may vary. It should also be understood that the terminology used herein is only used for the purpose of describing specific embodiments, and is not intended to be limiting, because the scope of the present invention will be limited only by the appended claims.

[0031] When providing the range of values, it should be understood that unless the context clearly indicates otherwise, each intermediate value (to one tenth of the lower limit unit) between the upper and lower limits of the range and any other specified value or intermediate value in the specified range are all included in the scope of the present invention. The upper and lower limits of these smaller ranges can be independently included in the smaller range and are also included in the present invention, with any clearly excluded limit in the specified range as a condition. When the specified range includes one or two limits, the scope of any one or two of the included limits is also included in the present invention.

[0032] 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 invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials associated with the cited publications.

[0033] It must be noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to an "R2 polypeptide" includes a plurality of such polypeptides, and reference to a "heterologous gene product" includes one or more heterologous gene products and equivalents thereof known to those skilled in the art, and so forth. It should also be noted that the claims may be drafted to exclude any optional elements. Likewise, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "only", "only", etc., in conjunction with the recitation of claim elements, or for use of a "negative" limitation.

[0034] It should be understood that certain features of the invention described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention described in the context of a single embodiment for brevity may also be provided individually or in any suitable subcombination. The present invention specifically encompasses all combinations of embodiments related to the present invention, and is disclosed herein as if each combination were individually and explicitly disclosed. In addition, the present invention also specifically encompasses all subcombinations of various embodiments and elements thereof, and is disclosed herein as if each such subcombination were individually and explicitly disclosed herein.

[0035] The publications discussed herein are provided only as of the date of filing of the present application. Any content herein should not be construed as admitting that the present invention is not entitled to precede such publications by virtue of prior invention. In addition, the publication date provided may be different from the actual publication date, which may require independent confirmation. DETAILED DESCRIPTION

[0036] The present disclosure provides a gene delivery system, the gene delivery system comprising: a) R2 retrotransposon R2 polypeptide, or a first nucleic acid comprising a nucleotide sequence encoding the R2 polypeptide; and b) a nucleic acid comprising a heterologous nucleotide sequence encoding one or more heterologous gene products, wherein the heterologous nucleotide sequence is flanked by the R2 retrotransposon 3' untranslated region (UTR) and the R2 retrotransposon 5'UTR, and wherein the heterologous nucleotide sequence has a length of at least 200 nucleotides. The R2 polypeptide, 5'UTR and 3'UTR insert the heterologous nucleic acid into the 28S region of the eukaryotic cell genome. The present disclosure provides a method for delivering one or more target gene products to eukaryotic cells, the method comprising contacting the cell with the gene delivery vehicle system.

[0037] R2 protein recognizes 5' and 3' sites of DNA sequences that are conserved in many species in 28S rDNA. R2 protein interacts and binds to the transcribed RNA 5' and 3' of the R2 coding sequence. Based on whether the R2 protein binds to 5'UTR or 3'UTR, it will then interact with the 28S DNA upstream or downstream of the target site. The current integration model is that the R2 protein bound to the 3'UTR RNA will bind to the upstream of the cleavage site and form a nick at the 3' end via the endonuclease domain in the protein. From here, R2 will start the process of reverse transcription (TPRT) initiated by the target, and as proposed by Eickbush et al., synthesize the 3' strand of DNA in the 28S region (Fig. 1). Eickbush, et al. Microbiol. Spectr. 3, MDNA3-0011-2014 (2015).

[0038] Once completed, the downstream associated R2 protein or native replication machinery initiates second strand synthesis. The end result is integration of the transgene of interest ("heterologous nucleic acid" or "heterologous nucleotide sequence") into the native 28S rDNA locus.

[0039] Gene delivery system

[0040] The present disclosure provides a gene delivery system, the gene delivery system comprising: a) a first nucleic acid comprising a nucleotide sequence encoding an R2 retrotransposon R2 polypeptide; and b) a second nucleic acid comprising a heterologous nucleotide sequence encoding one or more heterologous gene products, wherein the heterologous nucleotide sequence is flanked by an R2 retrotransposon 3'UTR and an R2 retrotransposon 5'UTR, and wherein the heterologous nucleotide sequence has a length of at least 200 nucleotides. The first and second nucleic acids may be RNA. The first and second nucleic acids may be DNA.

[0041] In some cases, the second nucleic acid comprises, in 5' to 3' order: i) R2 5'UTR; ii) a promoter; iii) a heterologous nucleotide sequence encoding one or more heterologous gene products; and iv) R2 3'UTR. In some cases, the second nucleic acid comprises, in 5' to 3' order: i) R2 5'UTR; ii) a promoter; iii) a heterologous nucleotide sequence encoding one or more heterologous gene products; iv) a polyadenylation (polyA) sequence; and v) R2 3'UTR. In some cases, for example, when the promoter is an RNA polymerase II promoter, the heterologous nucleotide sequence encoding the one or more heterologous gene products is in an opposite (reverse) orientation relative to the R2 5'UTR and the R2 3'UTR; that is, the heterologous nucleotide sequence encoding the one or more heterologous gene products is in a 3' to 5' orientation. In some cases, for example, when the promoter is an RNA polymerase I promoter, the heterologous nucleotide sequence encoding one or more heterologous gene products is in the same orientation as the R2 5'UTR and the R2 3'UTR; that is, the heterologous nucleotide sequence encoding one or more heterologous gene products is in a 5' to 3' orientation. The promoter is operably linked to the heterologous nucleotide sequence encoding one or more heterologous gene products. In some cases, the promoter is heterologous to the nucleotide sequence encoding one or more heterologous gene products.

[0042] The present disclosure provides a gene delivery system, the gene delivery system comprising: a) an R2 retrotransposon R2 polypeptide; and b) a nucleic acid comprising a heterologous nucleotide sequence encoding one or more heterologous gene products, wherein the heterologous nucleotide sequence is flanked by an R2 retrotransposon 3'UTR and an R2 retrotransposon 5'UTR, and wherein the heterologous nucleotide sequence has a length of at least 200 nucleotides.

[0043] R2 polypeptide

[0044] The R2 polypeptide encoded by the first nucleic acid of the gene delivery system of the present disclosure (wherein the gene delivery system comprises the first nucleic acid and the nucleic acid), or the R2 polypeptide present in the gene delivery system of the present disclosure (wherein the gene delivery system comprises the R2 polypeptide and the nucleic acid) may comprise Figure 7 The R2 amino acid sequence depicted in the embodiment of the present invention has an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity. The R2 polypeptide may have a length of 1105 amino acids to 1125 amino acids, such as about 1105 amino acids to about 1110 amino acids, about 1110 amino acids to about 1115 amino acids, about 1115 amino acids to about 1120 amino acids, or about 1120 amino acids to about 1125 amino acids. In some cases, the R2 polypeptide has a length of 1114 amino acids.

[0045] 5'UTR and 3'UTR

[0046] A suitable 5'UTR is any 5'UTR of the R2 retrotransposon. The nucleotide sequences of the 5'UTRs of the R2 retrotransposon are known in the art; and any such 5'UTR may be included in the gene delivery system of the present disclosure.

[0047] In some cases, a suitable 5'UTR comprises Figure 8 In some cases, the nucleotide sequence depicted in the present invention has a nucleotide sequence with at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% nucleotide sequence identity. In some cases, a suitable 5'UTR has a length of about 1000 nucleotides (nt) to about 1100nt, such as about 1000nt to about 1025nt, about 1025nt to about 1050nt, about 1050nt to about 1075nt or about 1075nt to about 1100nt. In some cases, a suitable 5'UTR has a length of about 1050nt to about 1060nt. In some cases, a suitable 5'UTR has a length of 1056nt.

[0048] In some cases, a suitable 3'UTR comprises Fig. 9In some cases, the nucleotide sequence depicted in the nucleotide sequence has a nucleotide sequence with at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% nucleotide sequence identity. In some cases, the suitable 3'UTR has a length of about 475nt to about 550nt, such as about 475nt to about 500nt, about 500nt to about 525nt, or about 525nt to about 550nt. In some cases, the suitable 3'UTR has a length of about 500nt to about 510nt. In some cases, the suitable 3'UTR has a length of 502nt.

[0049] Heterologous nucleotide sequence

[0050] As described above, the second nucleic acid of the gene delivery system of the present disclosure comprises a heterologous nucleotide sequence encoding one or more heterologous gene products (also referred to herein as a "heterologous nucleic acid"), wherein the heterologous nucleotide sequence has a length of at least 200 nucleotides (nt). For example, in some cases, the heterologous nucleotide sequence has a length of about 200nt to about 300nt, about 300nt to about 400nt, about 400nt to about 500nt, about 500nt to about 750nt, about 750nt to about 1 kilobase (kb), about 1kb to about 1.5kb, about 1.5kb to about 2kb, about 2kb to about 2.5kb, about 2.5kb to about 3kb, or about 3kb to about 3.5kb. As another example, in some cases, the heterologous nucleotide sequence has a length of about 3.5 kb to about 4 kb, about 4 kb to about 4.5 kb, about 4.5 kb to about 5 kb, about 5 kb to about 5.5 kb, about 5.5 kb to about 6 kb, about 6 kb to about 6.5 kb, about 6.5 kb to about 7 kb, about 7 kb to about 8 kb, about 8 kb to about 9 kb, about 9 kb to about 10 kb, about 10 kb to about 11 kb, about 11 kb to about 12 kb, about 12 kb to about 13 kb, about 13 kb to about 14 kb, or about 14 kb to about 15 kb. In some cases, the heterologous nucleotide sequence has a length of about 200 nt to about 1 kb. In some cases, the heterologous nucleotide sequence has a length of about 1 kb to about 5 kb. In some cases, the heterologous nucleotide sequence has a length of about 3.5 kb to about 6 kb. In some cases, the heterologous nucleotide sequence has a length of about 6 kb to about 8 kb. In some cases, the heterologous nucleotide sequence has a length of about 8 kb to about 15 kb. In some cases, the heterologous nucleotide sequence has a length of about 9 kb to about 15 kb. In some cases, the heterologous nucleotide sequence has a length of about 10 kb to about 15 kb.

[0051] In some cases, when the heterologous gene product is a polypeptide, the heterologous nucleotide sequence can encode a single heterologous gene product of more than 50 amino acids in length. In some cases, when the heterologous gene product is a polypeptide, the heterologous nucleotide sequence can encode a single heterologous gene product of more than 200 amino acids in length. In some cases, when the heterologous gene product is a polypeptide, the heterologous nucleotide sequence can encode a single heterologous gene product having a length of about 50 amino acids (aa) to about 100 aa, about 100 aa to about 200 aa, about 200 aa to about 300 aa, about 300 aa to about 400 aa, about 400 aa to about 500 aa, about 500 aa to about 750 aa, about 750 aa to about 1000 aa, about 1000 aa to about 1500 aa, about 1500 aa to about 2000 aa, about 2000 aa to about 2500 aa, or about 2500 aa to about 3000 aa. In some cases, when the heterologous gene product is a polypeptide, the heterologous nucleotide sequence can encode a single heterologous gene product having a length of up to 3000 amino acids. In some cases, when the heterologous gene product is a polypeptide, the heterologous nucleotide sequence may encode a single heterologous gene product of about 3000 aa to about 5,000 aa in length. When the heterologous gene product is two or more polypeptides, the heterologous nucleotide sequence may encode two or more heterologous gene products of up to 3000 amino acids (aa), up to 4000 aa, or up to 5000 aa in combined length. When the heterologous gene product is two or more polypeptides, the heterologous nucleotide sequence may encode two or more heterologous gene products of more than 5,000 aa in combined length. In the case where the heterologous gene product is a nucleic acid, the heterologous nucleotide sequence may encode a single heterologous gene product of at least 200 nt in length (e.g., about 200 nt to about 500 nt, about 500 nt to about 1 kb, about 1 kb to about 3.5 kb, about 3.5 kb to about 6 kb, about 6 kb to about 10 kb, or about 10 kb to about 15 kb). Where the heterologous gene products are two or more nucleic acids, the heterologous nucleotide sequences can encode heterologous gene products having a combined length of at least 200 nt (e.g., about 200 nt to about 500 nt, about 500 nt to about 1 kb, about 1 kb to about 3.5 kb, about 3.5 kb to about 6 kb, about 6 kb to about 10 kb, or about 10 kb to about 15 kb).Where the heterologous nucleotide sequence encodes a first gene product that is a nucleic acid and a second gene product that is a polypeptide, the heterologous nucleotide sequence can encode, for example, any combination of the lengths of the gene products such that the total combined length of the coding sequences encoding both gene products is at least 200 nt (e.g., about 200 nt to about 500 nt, about 500 nt to about 1 kb, about 1 kb to about 3.5 kb, about 3.5 kb to about 6 kb, about 6 kb to about 10 kb, or about 10 kb to about 15 kb).

[0052] In some cases, the second nucleic acid of the gene delivery system of the present disclosure comprises a nucleotide sequence encoding a single heterologous polypeptide. In some cases, the second nucleic acid of the gene delivery system of the present disclosure comprises a nucleotide sequence encoding a single heterologous nucleic acid. In some cases, the second nucleic acid of the gene delivery system of the present disclosure comprises: a) a first nucleotide sequence encoding a first heterologous polypeptide; and b) a second nucleotide sequence encoding a second heterologous polypeptide. In some cases, the second nucleic acid of the gene delivery system of the present disclosure comprises: a) a first nucleotide sequence encoding a first heterologous polypeptide; b) a second nucleotide sequence encoding a second heterologous polypeptide; and c) a third nucleotide sequence encoding a third heterologous polypeptide. In some cases, the second nucleic acid of the gene delivery system of the present disclosure comprises: a) a first nucleotide sequence encoding a heterologous polypeptide; and b) a second nucleotide sequence encoding a heterologous nucleic acid. In some cases, the second nucleic acid of the gene delivery system of the present disclosure comprises: a) a first nucleotide sequence encoding a heterologous polypeptide; b) a second nucleotide sequence encoding a heterologous nucleic acid; and c) a third nucleotide sequence encoding a third heterologous nucleic acid.

[0053] When the heterologous nucleotide sequence encodes two or more gene products (for example, when the heterologous nucleotide sequence comprises a first nucleotide sequence encoding a first heterologous gene product and a second nucleotide sequence encoding a second heterologous gene product, etc.), in some cases, a nucleic acid linker is provided between the first nucleotide sequence and the second nucleotide sequence (or between any two nucleotide sequences encoding the gene products). The nucleic acid linker may be an internal ribosome entry site (IRES). The nucleic acid linker may comprise a nucleotide sequence encoding a self-cleaving 2A peptide (such as P2A, T2A, E2A or F2A) that connects the 3' end of the first nucleotide sequence to the 5' end of the second nucleotide sequence.

[0054] In some cases, the second nucleic acid of the gene delivery system of the present disclosure comprises: a) a first nucleotide sequence encoding a first heterologous polypeptide; b) a second nucleotide sequence encoding a second heterologous polypeptide; wherein the first and second nucleotide sequences are under the control of a single promoter. In some cases, the promoter is operably connected to the 5' end of the first nucleotide sequence, and there is a nucleic acid linker selected from the group consisting of: IRES and a nucleic acid encoding a self-cleaving 2A peptide (such as P2A, T2A, E2A or F2A) that connects the 3' end of the first nucleotide sequence to the 5' end of the second nucleotide sequence, wherein the first nucleotide sequence and the second nucleotide sequence are transcribed into a single RNA under the control of the promoter. In some cases, the promoter is operably connected to the 5' end of the second nucleotide sequence, and there is a nucleic acid linker selected from the group consisting of: IRES and a nucleic acid encoding a self-cleaving 2A peptide (such as P2A, T2A, E2A or F2A) that connects the 3' end of the second nucleotide sequence to the 5' end of the first nucleotide sequence, wherein the first nucleotide sequence and the second nucleotide sequence are transcribed into a single RNA under the control of the promoter. In some cases, the promoter is inducible.

[0055] Suitable self-cleaving viral 2A peptides for use include viral 2A peptides, which are porcine teschovirus-1 (P2A), foot-and-mouth disease virus (F2A), lycopodiella virus (T2A), equine rhinitis A virus (E2A), and porcine teschovirus 1 (P2A) peptides. P2A (GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 1)), T2A (GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 2)), E2A (GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 3)) and F2A (GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 4)) can be considered "proteolytic cleavage sites" or "ribosomal skipping signals" (CHYSEL) See, e.g., Kim et al. (2011) PLoS ONE 6: e18556. The mechanism for producing the encoded polypeptide as two polypeptide chains may be by self-cleavage of the linker, by ribosome skipping or translational shunting.

[0056] As an example, in some cases, the second nucleic acid of the gene delivery system of the present disclosure comprises: a) a first nucleotide sequence encoding a first heterologous polypeptide as the first chain of a heterodimer; b) a second nucleotide sequence encoding a second heterologous polypeptide as the second chain of a heterodimer; wherein the first and second nucleotide sequences are under the control of a single promoter. In some cases, the second nucleic acid of the gene delivery system of the present disclosure comprises: a) a first nucleotide sequence encoding a first heterologous polypeptide as the first chain of a heterodimer; b) a nucleic acid linker selected from an IRES and a nucleic acid encoding a self-cleaving 2A peptide; and c) a second nucleotide sequence encoding a second heterologous polypeptide as the second chain of a heterodimer; wherein the first and second nucleotide sequences are under the control of a single promoter.

[0057] In some cases, the heterologous nucleotide sequence encodes a single polypeptide chain that is cleaved post-translationally to produce two polypeptide chains. For example, in some cases, the heterologous nucleotide sequence encodes a single polypeptide chain that comprises, in N-terminal to C-terminal order: i) a first polypeptide; ii) a proteolytically cleavable linker; and iii) a second polypeptide.

[0058] The proteolytically cleavable linker may include a protease recognition sequence recognized by a protease selected from the group consisting of alanine carboxypeptidase, Armillaria astaxanthin, bacterial leucyl aminopeptidase, cancer procoagulant, cathepsin B, clostripain, cytosolic alanyl aminopeptidase, elastase, endoproteinase Arg-C, enterokinase, pepsin, gelatinase, Gly-X carboxypeptidase, glycyl endopeptidase, human rhinovirus 3C protease, dermatophytin C, IgA-specific serine endopeptidase, leucyl aminopeptidase, leucyl endopeptidase, lysC, lysosomal pro-X carboxypeptidase, lysyl aminopeptidase, methylthiothreitol Aminoacyl aminopeptidase, myxobacter, nardilysin, pancreatic endopeptidase E, picornain endopeptidase 2A, picornain endopeptidase 3C, proendopeptidase, prolyl aminopeptidase, proprotein convertase I, proprotein convertase II, russellysin, saccharopepsin, semenogelase, T-plasminogen activator, thrombin, tissue kallikrein, tobacco etch virus (TEV), togavirin, tryptophanyl aminopeptidase, U-plasminogen activator, V8, venombin A, venombin AB, and Xaa-pro aminopeptidase.

[0059] For example, the proteolytically cleavable linker may comprise a matrix metalloproteinase cleavage site, such as a cleavage site of a MMP selected from the group consisting of collagenase-1, -2 and -3 (MMP-1, -8 and -13), gelatinase A and B (MMP-2 and -9), stromelysin 1, 2 and 3 (MMP-3, -10 and -11), matrix lytic factor (MMP-7) and membrane metalloproteinase (MT1-MMP and MT2-MMP). For example, the cleavage sequence of MMP-9 is Pro-XX-Hy (wherein X represents any residue; Hy, a hydrophobic residue; SEQ ID NO: 5), such as Pro-XX-Hy-(Ser / Thr) SEQ ID NO: 6, such as Pro-Leu / Gln-Gly-Met-Thr-Ser (SEQ ID NO: 7) or Pro-Leu / Gln-Gly-Met-Thr (SEQ ID NO: 8). Another example of a protease cleavage site is a plasminogen activator cleavage site, such as uPA or tissue plasminogen activator (tPA) cleavage site. Another example of a suitable protease cleavage site is a prolactin cleavage site. Specific examples of cleavage sequences of uPA and tPA include sequences comprising Val-Gly-Arg. Another example of a protease cleavage site that may be included in a proteolytically cleavable linker is a tobacco etch virus (TEV) protease cleavage site, such as ENLYTQS (SEQ ID NO: 9) where the protease cleavage site is cleaved between glutamine and serine. Another example of a protease cleavage site that may be included in a proteolytically cleavable linker is an enterokinase cleavage site, such as DDDDK (SEQ ID NO: 10) where cleavage occurs after a lysine residue. Another example of a protease cleavage site that may be included in a proteolytically cleavable linker is a thrombin cleavage site, such as LVPR (SEQ ID NO: 11). Additional suitable linkers comprising a protease cleavage site include linkers comprising one or more of the following amino acid sequences: LEVLFQGP (SEQ ID NO: 12), a fusion protein comprising human rhinovirus 3C protease and glutathione-S-transferase; Walker et al. (1994) Biotechnol.12:601) cleavage; thrombin cleavage sites, such as CGLVPAGSGP (SEQ ID NO: 13); SLLKSRMVPNFN (SEQ ID NO: 14) or SLLIARRMPNFN (SEQ ID NO: 15), cleaved by cathepsin B; SKLVQASASGVN (SEQ ID NO: 16) or SSYLKASDAPDN (SEQ ID NO: 17), cleaved by Epstein-Barr virus protease; RPKPQQFFGLMN (SEQ ID NO: 18) cleaved by MMP-3 (stromelysin); SLRPLALWRSFN (SEQ ID NO: 19) cleaved by MMP-7 (matrilytic factor); SPQGIAGQRNFN (SEQ ID NO: 20) cleaved by MMP-9; DVDERDVRGFASFL SEQ ID NO: 21, cleaved by thermolysin-like MMP NO:21); SLPLGLWAPNFN (SEQ ID NO:22) cleaved by matrix metalloproteinase 2 (MMP-2); SLLIFRSWANFN (SEQ ID NO:23) cleaved by cathepsin L; SGVVIATVIVIT (SEQ ID NO:24) cleaved by cathepsin D; SLGPQGIWGQFN (SEQ ID NO:25) cleaved by matrix metalloproteinase 1 (MMP-1); KKSPGRVVGGSV (SEQ ID NO:26) cleaved by urokinase-type plasminogen activator; PQGLLGAPGILG (SEQ ID NO:27) cleaved by type 1 membrane matrix metalloproteinase (MT-MMP); HGPEGLRVGFYESDVMGRGHARLVHVEEPHT (SEQ ID NO:28) cleaved by stromelysin 3 (or MMP-11), thermolysin, fibroblast collagenase and stromelysin-1. NO:28); GPQGLAGQRGIV (SEQ ID NO:29) cleaved by matrix metalloproteinase 13 (collagenase-3); GGSGQRGRKALE (SEQ ID NO:30) cleaved by tissue plasminogen activator (tPA); SLSALLSSDIFN (SEQ ID NO:31) cleaved by human prostate-specific antigen; SLPRFKIIGGFN (SEQ ID NO:32) cleaved by kallikrein (hK3); SLLGIAVPGNFN (SEQ ID NO:33) cleaved by neutrophil elastase; and FFKNIVTPRTPP (SEQ ID NO:34) cleaved by calpain (calcium-activated neutral protease). .

[0060] In some cases, the second nucleic acid of the gene delivery system of the present disclosure comprises: a) a first nucleotide sequence encoding a first heterologous polypeptide; and b) a second nucleotide sequence encoding a second heterologous polypeptide; wherein the first and second nucleotide sequences are under the control of two different promoters. For example, in some cases, the second nucleic acid of the gene delivery system of the present disclosure comprises: a) a first nucleotide sequence encoding a first heterologous polypeptide, wherein the first nucleotide sequence is under the control of a first promoter; and b) a second nucleotide sequence encoding a second heterologous polypeptide, wherein the second nucleotide sequence is under the control of a second promoter. In some cases, both the first promoter and the second promoter are regulatable (e.g., inducible) promoters. In some cases, both the first promoter and the second promoter are constitutive promoters. In some cases, the first promoter is inducible, and the second promoter is constitutive. In some cases, the first promoter is constitutive, and the second promoter is.

[0061] Transcriptional control elements

[0062] The heterologous nucleotide sequence present in the second nucleic acid of the gene delivery system of the present disclosure can be operably linked to one or more transcription control elements. In some cases, the transcription control element is inducible. In some cases, the transcription control element is constitutive. In some cases, the transcription control element is a promoter. In some cases, the promoter is functional in eukaryotic cells. In some cases, the promoter is a cell type specific promoter. In some cases, the promoter is a tissue specific promoter. In some cases, the promoter is constitutively active. In some cases, the promoter is a regulatable promoter.

[0063] A promoter can be a constitutively active promoter (i.e., a promoter that is constitutively in the active / "ON" state), it can be an inducible promoter (i.e., a promoter whose state (active / "ON" or inactive / "OFF") is controlled by an external stimulus such as the presence of a specific temperature, compound, or protein), it can be a spatially restricted promoter (i.e., transcriptional control elements, enhancers, etc.) (e.g., tissue-specific promoters, cell type-specific promoters, etc.), and it can be a temporally restricted promoter (i.e., the promoter is in the "ON" state or the "OFF" state during a specific stage of embryonic development or during a specific stage of a biological process (e.g., the hair follicle cycle in mice)).

[0064] Suitable promoters and enhancer elements are known in the art. For expression in eukaryotic cells, suitable promoters include, but are not limited to, light chain and / or heavy chain immunoglobulin gene promoters and enhancer elements; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoters present in the long terminal repeat sequence of retrovirus; mouse metallothionein-I promoter; and various tissue-specific promoters known in the art.

[0065] Suitable reversible promoters, including reversible inducible promoters, are known in the art. Such reversible promoters can be isolated from and derived from many organisms, such as eukaryotes and prokaryotes. Modification of a reversible promoter derived from a first organism (e.g., a first prokaryote and a second eukaryote, a first eukaryote and a second prokaryote, etc.) for a second organism is well known in the art. Such reversible promoters and systems based on such reversible promoters but also comprising additional control proteins include, but are not limited to, alcohol-regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoters, promoters responsive to alcohol transactivator proteins (AlcR), etc.), tetracycline-regulated promoters (e.g., promoter systems including Tet activators, TetON, TetOFF, etc.), steroid-regulated promoters (e.g., rat glucocorticoid receptor promoter system, human estrogen receptor promoter system, retinoids Promoter system, thyroid promoter system, ecdysone promoter system, mifepristone promoter system, etc.), metal-regulated promoters (e.g., metallothionein promoter system, etc.), pathogen-related regulated promoters (e.g., salicylic acid-regulated promoters, ethylene-regulated promoters, benzothiadiazole-regulated promoters, etc.), temperature-regulated promoters (e.g., heat shock-inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.)), light-regulated promoters, synthetic inducible promoters, etc. Suitable promoters may be RNA pol I promoters. Suitable promoters may be RNA pol II promoters.

[0066] Inducible promoters suitable for use include any inducible promoters described herein or known to those of ordinary skill in the art. Examples of inducible promoters include, but are not limited to, chemical / biochemically regulated promoters and physically regulated promoters, such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline-responsive promoter systems, including tetracycline repressor protein (tetR), tetracycline operating sequence (tetO) and tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g., based on rat glucocorticoid receptor, human estrogen receptor, moth hormone receptor, thymidine ... In some embodiments, the promoters of the promoters are regulated by the promoters of the steroid / retinoid / thyroid receptor superfamily, the promoters of the ecdysone receptor, the promoters of the steroid / retinoid / thyroid receptor superfamily, the promoters of the metal-regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and chelate metal ions) genes from yeast, mouse, and human), the promoters of the pathogen-regulated promoters (e.g., promoters inducible by salicylic acid, ethylene, or benzothiadiazole (BTH)), the promoters of the temperature / heat-inducible promoters (e.g., heat shock promoters), and the promoters of the light-regulated promoters (e.g., light-responsive promoters from plant cells).

[0067] In some cases, the promoter is a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or a NK-specific promoter. For example, the CD4 gene promoter can be used. See, for example, Salmon et al. (1993) Proc. Natl. Acad. Sci. USA 90: 7739; and Marodon et al. (2003) Blood 101: 3416. As another example, the CD8 gene promoter can be used. NK cell-specific expression can be achieved by using the Ncr1 (p46) promoter; see, for example, Eckelhart et al. (2011) Blood 117: 1565.

[0068] In some cases, the promoter is a cardiomyocyte-specific promoter. In some cases, the promoter is a smooth muscle cell-specific promoter. In some cases, the promoter is a neuron-specific promoter. In some cases, the promoter is an adipocyte-specific promoter. Other cell type-specific promoters are known in the art and are suitable for use herein.

[0069] Heterologous gene product

[0070] Any of a plurality of heterologous gene products may be encoded by a heterologous nucleotide sequence present in a second nucleic acid of a gene delivery system of the present disclosure. The heterologous gene product may be a single heterologous polypeptide. The heterologous gene product may be a single nucleic acid. The heterologous gene product may be two or more heterologous polypeptides. The heterologous gene product may be two or more heterologous nucleic acids. The heterologous gene product may be: i) a heterologous polypeptide; and ii) a heterologous nucleic acid.

[0071] When the heterologous gene product is a polypeptide, suitable polypeptides include, but are not limited to, receptors, enzymes, antibodies, homodimeric polypeptides, heterodimeric polypeptides, polypeptide hormones, extracellular matrix proteins, proteoglycans, nucleases, RNA CRISPR / Cas effector polypeptides, chimeric polypeptides, fusion polypeptides, etc. In some cases, the heterologous gene product is CAR. In some cases, the heterologous gene product is a synNotch polypeptide. In some cases, the heterologous gene product is a synNotch polypeptide and a CAR.

[0072] When the heterologous gene product is a nucleic acid, suitable nucleic acids include, but are not limited to, RNA comprising a nucleotide sequence encoding a polypeptide; microRNA; ribozyme; inhibitory RNA; RNA comprising a first segment complementary to a nucleotide sequence in a target nucleic acid and a second segment that binds to an effector polypeptide of the RNA, etc. In some cases, the heterologous gene product is a short interfering RNA (siRNA), a short hairpin RNA (shRNA), a ribozyme, a microRNA (miRNA), a small sequential RNA (stRNA), an antisense RNA, a small RNA-induced gene activation (RNAa), or a small activating RNA (saRNA).

[0073] Chimeric Antigen Receptor

[0074] As a non-limiting example, the heterologous polypeptide is a chimeric antigen receptor (CAR). In some cases, the CAR is a single polypeptide chain CAR. In other cases, the CAR is a heterodimeric CAR comprising two polypeptide chains. The single polypeptide chain CAR may include: i) an antigen binding domain; ii) a transmembrane domain; and iii) an intracellular signaling domain. The single polypeptide chain CAR may include: i) an antigen binding domain; ii) a transmembrane domain; iii) an immunomodulatory domain; and iv) an intracellular signaling domain.

[0075] The antigen binding portion of CAR can be an antibody or an antibody fragment. "Antibody fragment" includes a part of a complete antibody, such as an antigen binding region or variable region of a complete antibody. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; single-chain Fv (scFv); double antibodies; linear antibodies (Zapata et al., Protein Eng.8 (10): 1057-1062 (1995)); chimeric antibodies; humanized antibodies; single-chain antibodies (scAb); single-domain antibodies (dAb); single-domain heavy chain antibodies; single-domain light chain antibodies; nanobodies; bispecific antibodies; multispecific antibodies; and fusion proteins comprising antigen binding (also referred to herein as antigen binding) portions of antibodies and non-antibody proteins. In some cases, the antigen binding domain is scFv. In some cases, the antigen binding domain is a nanobody. Other antibody-based antigen binding domains (cAb VHH (camelid antibody variable domain) and humanized versions, IgNAR VH (shark antibody variable domain) and humanized versions, sdAb VH (single domain antibody variable domain) as well as "camelized" antibody variable domains are suitable for use.

[0076] The antigen binding domain of CAR may have a variety of antigen binding specificities. In some cases, the antigen binding domain is specific to an epitope present in an antigen (i.e., a cancer cell-associated antigen) expressed (synthesized by it) by a cancer cell. Cancer cell-associated antigens may be antigens associated with the following cells: such as breast cancer cells, B cell lymphomas, Hodgkin lymphoma cells, ovarian cancer cells, prostate cancer cells, mesothelioma, lung cancer cells (e.g., small cell lung cancer cells), non-Hodgkin B cell lymphoma (B-NHL) cells, ovarian cancer cells, prostate cancer cells, mesothelioma cells, lung cancer cells (e.g., small cell lung cancer cells), melanoma cells, chronic lymphocytic leukemia cells, acute lymphocytic leukemia cells, neuroblastoma cells, gliomas, glioblastomas, medulloblastomas, colorectal cancer cells, etc. Cancer cell-associated antigens may also be expressed by noncancerous cells.

[0077] Non-limiting examples of antigens to which the antigen binding domain of CAR can bind include, for example, CD19, CD20, CD38, CD30, Her2 / neu, ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight melanoma-associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, etc.

[0078] Suitable immunomodulatory domains (also referred to as "costimulatory domains" or "costimulatory polypeptides") include, for example, 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM.

[0079] Suitable intracellular signaling domains include, for example, polypeptides containing one or more immunoreceptor tyrosine-based activation motifs (ITAMs). The ITAM motif is YX1X2L / I, where X1 and X2 are independently any amino acids. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to: DAP12; FCER1G (Fcε receptor Iγ chain); CD3D (CD3δ); CD3E (CD3ε); CD3G (CD3γ); CD3Z (CD3ζ); and CD79A (antigen receptor complex-associated protein α chain).

[0080] In some cases, CAR is a heterodimer CAR comprising two polypeptide chains. See, for example, U.S. Patent Nos. 9,821,012 and 9,587,020. For example, in some cases, heterodimer CAR comprises: a) a first polypeptide chain, the first polypeptide chain comprising: i) an extracellular antigen binding domain that specifically binds to an antigen on a target cell; ii) a transmembrane domain; and iii) a first member of a dimerization pair; and b) a second polypeptide, the second polypeptide comprising: i) a transmembrane domain; ii) a second member of a dimerization pair; and iii) an intracellular signaling domain comprising ITAM, wherein the intracellular signaling domain provides signal transduction activity. The first polypeptide, the second polypeptide, or the first and second polypeptides of the heterodimer CAR all comprise a co-stimulatory polypeptide. When present in a eukaryotic cell membrane, the first polypeptide of the CAR binds to an antigen, and the CAR dimerizes in the presence of a small molecule dimerizer.

[0081] In some cases, the heterodimeric CAR comprises: a) a first polypeptide, which comprises, in N-terminal to C-terminal order: i) an antigen binding domain (e.g., an antigen binding single-chain Fv (scFv) or a nanobody); ii) a transmembrane domain; and iii) a first member of a dimerization pair; and b) a second polypeptide, which comprises, in N-terminal to C-terminal order: i) a transmembrane domain; ii) a second member of the dimerization pair; and iii) an intracellular signaling domain comprising an ITAM, wherein the intracellular signaling domain provides signal transduction activity, wherein the first polypeptide, the second polypeptide, or both the first and second polypeptides comprise a co-stimulatory polypeptide inserted between the transmembrane domain and the member of the dimerization pair. In some cases, the co-stimulatory polypeptide is a 4-1BB polypeptide. In some cases, the co-stimulatory polypeptide is a CD28 polypeptide. In some cases, the co-stimulatory polypeptide is an OX-40 polypeptide. In some cases, the first polypeptide comprises a hinge region between an antigen binding domain (e.g., scFv, a nanobody, etc.) and a transmembrane domain. In some cases, the ITAM-containing intracellular signaling domain is selected from the group consisting of CD3-ζ and ZAP70.

[0082] Suitable dimerization pairs include, for example, a) FK506 binding protein (FKBP) and FKBP; b) FKBP and calcineurin catalytic subunit A (CnA); c) FKBP and cyclophilin; d) FKBP and FKBP rapamycin-related protein (FRB); e) gyrase B (GyrB) and GyrB; f) dihydrofolate reductase (DHFR) and DHFR; g) DmrB and DmrB; h) PYL and ABI; i) Cry2 and CIB1; and j) GAI and GID1.

[0083] Suitable dimerizing factors ("dimerizing agents") that can dimerize a first member of a dimerizing factor-binding pair and a second member of a dimerizing factor-binding pair include, for example (where the dimerizing factor is in parentheses after the dimerizing factor-binding pair): a) FKBP and FKBP (rapamycin); b) FKBP and CnA (rapamycin); c) FKBP and cyclophilin (rapamycin); d) FKBP and FRG (rapamycin); e) GyrB and GyrB (coumarin); f) DHFR and DHFR (methotrexate); g) DmrB and DmrB (AP20187); h) PYL and ABI (abscisic acid); i) Cry2 and CIB1 (blue light); and j) GAI and GID1 (gibberellin).

[0084] synNotch

[0085] As another non-limiting example, a heterologous nucleotide sequence may encode a synNotch polypeptide (also referred to herein as a "chimeric Notch receptor polypeptide". Suitable synNotch polypeptides are described, for example, in U.S. Pat. No. 9,670,281; Morsut et al. (2016) Cell 164:780; and Roybal et al. (2016) Cell 167:419. A synNotch polypeptide comprises: i) an antigen binding domain; ii) a portion of a Notch polypeptide; and iii) an intracellular domain (e.g., a transcription factor (e.g., a transcription activator or a transcription repressor), a site-specific nuclease, etc.). A synNotch polypeptide does not bind to δ (a naturally occurring ligand of Notch). Instead, a synNotch polypeptide binds to an antigen bound by an antigen binding domain present in a synNotch polypeptide. Binding of the antigen binding domain to an antigen (e.g., an antigen present on a cell, such as a cancer cell) induces cleavage of the S2 proteolytic cleavage site and / or the S3 proteolytic cleavage site in the Notch polypeptide, thereby releasing the intracellular domain.

[0086] In some cases, a synNotch polypeptide comprises: i) an antigen binding domain; ii) a Notch regulatory region comprising a Lin 12-Notch repeat sequence, a heterodimerization domain comprising an S2 proteolytic cleavage site, and a transmembrane domain comprising an S3 proteolytic cleavage site; and iii) an intracellular domain heterologous to the Notch regulatory region, the intracellular domain comprising a transcriptional activator comprising a DNA binding domain, wherein trans binding of the antigen binding domain to an antigen induces cleavage at the S2 and S3 proteolytic cleavage sites, thereby releasing the intracellular domain.

[0087] In some cases, a synNotch polypeptide comprises: i) an antigen binding domain; ii) a Notch regulatory region comprising a Lin 12-Notch repeat sequence, an S2 proteolytic cleavage site, and a transmembrane domain comprising an S3 proteolytic cleavage site; and iii) an intracellular domain comprising a transcriptional activator or transcriptional repressor that is heterologous to the Notch regulatory region, wherein binding of the first member of the specific binding pair to the second member of the specific binding pair present on a cell or other solid support induces cleavage at the S2 and S3 proteolytic cleavage sites, thereby releasing the intracellular domain.

[0088] The antigen binding portion of the synNotch polypeptide can be an antibody or an antibody fragment. An "antibody fragment" includes a portion of a complete antibody, such as an antigen binding region or variable region of a complete antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; single-chain Fv (scFv); diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); chimeric antibodies; humanized antibodies; single-chain antibodies (scAb); single-domain antibodies (dAb); single-domain heavy chain antibodies; single-domain light chain antibodies; nanobodies; bispecific antibodies; multispecific antibodies; and fusion proteins comprising antigen binding (also referred to herein as antigen binding) portions of antibodies and non-antibody proteins. In some cases, the antigen binding domain is a scFv. In some cases, the antigen binding domain is a nanobody. Other antibody-based antigen binding domains (cAb VHH (camelid antibody variable domain) and humanized versions, IgNAR VH (shark antibody variable domain) and humanized versions, sdAb VH (single domain antibody variable domain) as well as "camelized" antibody variable domains are suitable for use.

[0089] The antigen binding domain of the synNotch polypeptide may have a variety of antigen binding specificities. In some cases, the antigen binding domain is specific for an epitope present in an antigen (i.e., a cancer cell-associated antigen) expressed (synthesized) by a cancer cell. Cancer cell-associated antigens may be antigens associated with the following cells: for example, breast cancer cells, B cell lymphomas, Hodgkin lymphoma cells, ovarian cancer cells, prostate cancer cells, mesothelioma cells, lung cancer cells (e.g., small cell lung cancer cells), non-Hodgkin B cell lymphoma (B-NHL) cells, ovarian cancer cells, prostate cancer cells, mesothelioma cells, lung cancer cells (e.g., small cell lung cancer cells), melanoma cells, chronic lymphocytic leukemia cells, acute lymphocytic leukemia cells, neuroblastoma cells, gliomas, glioblastomas, medulloblastomas, colorectal cancer cells, etc. Cancer cell-associated antigens may also be expressed by non-cancerous cells.

[0090] Non-limiting examples of antigens to which the antigen binding domain of a synNotch polypeptide can bind include, for example, CD19, CD20, CD38, CD30, Her2 / neu, ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight melanoma-associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, and the like.

[0091] In some cases, the antigen binding domain of synNotch is a scFv. As another example, in some cases, the antigen binding domain of a synNotch polypeptide is a nanobody.

[0092] In some cases, the Notch polypeptide present in the synNotch polypeptide comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity to the following sequence: PPQIEEACELPECQVDAGNKVCNLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDGFDCQLTEGQCNPLYDQYCKDHFSDGHCDQGCNSAECEWDGLDCAEHVPERLAAGTLVLVVLLPPDQLRNNSFHFLRELSHVLHTNVVFKRDAQGQQMIFPYYGHEEELRKHPIKRSTVGWATSSLLPGTSGGRQRRELDPMDIRGSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGALASLGSLNIPYKIEAVKSEPVEPPLPSQLHLMYVAAAAFVLLFFVGCGVLLS (SEQ ID NO: 1) NO:35). In some cases, the Notch polypeptide present in the synNotch polypeptide comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity to the following sequence: PCVGSNPCYNQGTCEPTSENPFYRCLCPAKFNGLLCHILDYSFTGGAGRDIPPPQIEEACELPECQVDAGNKVCNLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDGFDCQL TEGQCNPLYDQYCKDHFSDGHCDQGCNSAECEWDGLDCAEHVPERLAAGTLVLVVLLPPDQLRNNSFHLFRELSHVLHTNVVFKRDAQGQQMIFPYYGHEEELRKHPIKRSTVG WATSSLLPGTSGGRQRRELDPMDIRGSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGALASLGSLNIPYKIEAVKSEPVEPPLPSQLHLMYVAAAAFVLLFFVGCGVLLS(SEQ ID NO:36).

[0093] In some cases, the intracellular domain is a transcription factor. Suitable transcription factors include, for example, ASCL1, BRN2, CDX2, CDX4, CTNNB1, EOMES, JUN, FOS, HNF4a, HOXAs (e.g., HOXA1, HOXA2, HOXA3, HOXA4, HOXA5, HOXA10, HOXA11, HOXA13), HOXBs (e.g., HOXB9), HOXCs (e.g., HOXC4, HOXC5, HOXC6, HOXC8, HOXC9, HOXC10, HOXC11, HOXC12, HOXC13), HOXDs (e.g., HOXD1, HOXD3, HOXD4, HOXD8, HOXD9, HOXD10, HOX XD11, HOXD12, HOXD13), SNAI1-3, MYOD1, MYOG, NEUROD1-6 (e.g., NEUROD1, NEUROD2, NEUROD4, NEUROD6), PDX1, PU.1, SOX2, Nanog, Klf4, BCL-6, SOX9, STAT1-6, TBET, TCF, TEAD1-4 (e.g., TEAD1, TEAD2, TEAD3, TEAD4), TAF6L, CLOCK, CREB, GATA3, IRF7, MycC, NFkB, RORyt, RUNX1, SRF, TBX21, NFAT, MEF2D, and FoxP3.

[0094] In some cases, the intracellular domain is a transcription factor that has a regulatory effect in one or more immune cells (i.e., an immune cell regulatory transcription factor). Suitable immune cell regulatory transcription factors include, for example, 2210012G02Rik, Akap81, Appl2, Arid4b, Arid5b, Ash11, Atf7, Atm, C430014K11Rik, Chd9, Dmtf1, Fos, Foxo1, Foxp1, Hmbox1, Kdm5b, Klf2, Mga, Mll1, Mll3, Myst4, Pcgf6, Rev31, Scml4, Scp2, Smarca2, Ssbp2, Suhw4, Tcf7, Tfdp2, Tox, Zbtb20, Zbtb44, Zeb1, Zfml , Zfp1, Zfp319, Zfp329, Zfp35, Zfp386, Zfp445, Zfp518, Zfp652, Zfp827, Zhx2, Eomes, Arntl, Bbx, Hbp1, Jun, Mef2d, Mterfd1, Nfat5, Nfe2l2, Nr1d2, Phf21a, Taf4b, Trf, Zbtb25, Zfp326, Zfp451, Zfp58, Zfp672, Egr2, Ikzf2, Taf1d, Chrac1, Dnajb6, Aplp2, Batf, Bhlhe40, Fosb, Hist1h 1c, Hopx, Ifih1, Ikzf3, Lass4, Lin54, Mxd1, Mxi1, Prdm1, Prf1, Rora, Rpa2, Sap30, Stat2, Stat3, Taf9b, Tbx21, Trps1, Xbp1, Zeb2, Atf3, Cenp c1, Lass6, Rb1, Zbtb41, Crem, Fosl2, Gtf2b, Irf7, Maff, Nr4a1, Nr4a2, Nr4a3, Obfc2a, Rbl2, Rel, Rybp, Sra1, Tgif1, Tnfaip3, Uhrf2, Zbtb1, C cdc124, Csda, E2f3, Epas1, H1f0, H2afz, Hif1a, Ikzf5, Irf4, Nsbp1, Pim1, Rfc2, Swap70, Tfb1m, 2610036L11Rik, 5133400G04Rik, Apitd1, Blm , Brca1, Brip1, C1d, C79407, Cenpa, Cfl1, Clspn, Ddx1, Dscc1, E2f7, E2f8, Ercc6l, Ezh2, Fen1, Foxm1, Gen1, Gsg2, H2afx, Hdac1, Hdgf, Hells,Hist1h1e, Hist3h2a, Hjurp, Hmgb2, Hmgb3, Irf1, Irf8, Kif22, Kif4, Lig1, Lmo2, Lnp, Mbd4, Mcm2, Mcm3, Mcm4, Mcm5, Mcm6, Mcm7, Mybl2, Neil3, Nusap1, Orc6 l, Pola1, Pola2, Pole, Pole2, Polh, Polr2f, Polr2j, Ppp1r8, Prim2, Psmc3ip, Rad51, Rad51c, Rad54l, Rfc3, Rfc4, Rnps1, Rpa1, Smarcc1, Spic, Ssrp1, Taf9 , Tfdp1, Tmpo, Topbp1, Trdmt1, Uhrf1, Wdhd1, Whsc1, Zbp1, Zbtb32, Zfp367, Car1, Polg2, Atr, Lef1, Myc, Nucb2, Satb1, Taf1a, Ift57, Apex1, Chd7, Chtf8, C tnnb1, Etv3, Irf9, Myb, Mybbp1a, Pms2, Preb, Sp110, Stat1, Trp53, Zfp414, App, Cdk9, Ddb1, Hsf2, Lbr, Pa2g4, Rbms1, Rfc1, Rfc5, Tada2l, Tex261, Xrcc6, etc. ,

[0095] RNA effector peptides and RNA

[0096] As another non-limiting example, the heterologous nucleotide sequence may encode an effector polypeptide of an RNA. As another non-limiting example, the heterologous nucleotide sequence may include: i) a first nucleotide sequence encoding an effector polypeptide of an RNA; and ii) a second nucleotide sequence encoding an RNA. As another non-limiting example, the heterologous nucleotide sequence may include: i) a first nucleotide sequence encoding an effector polypeptide of an RNA; ii) a second nucleotide sequence encoding a first RNA; and iii) a third nucleotide sequence encoding a second RNA.

[0097] The effector polypeptide of a suitable RNA includes, for example, a CRISPR / Cas endonuclease (e.g., a Class 2 CRISPR / Cas endonuclease, such as a Type II, Type V, or Type VI CRISPR / Cas endonuclease). The effector polypeptide of a suitable RNA is a CRISPR / Cas endonuclease (e.g., a Class 2 CRISPR / Cas endonuclease, such as a Type II, Type V, or Type VI CRISPR / Cas endonuclease). In some cases, the effector polypeptide of the RNA is a Class 2 CRISPR / Cas endonuclease. In some cases, the effector polypeptide of a suitable RNA is a Class 2 Type II CRISPR / Cas endonuclease (e.g., a Cas9 protein). In some cases, the effector polypeptide of a suitable RNA is a Class 2 Type V CRISPR / Cas endonuclease (e.g., a Cpf1 protein, a C2c1 protein, or a C2c3 protein). In some cases, the effector polypeptide of a suitable RNA is a Class 2 Type VI CRISPR / Cas endonuclease (e.g., a C2c2 protein; also referred to as a "Cas13a" protein). Also suitable for use is the CasX protein. Also suitable for use is the CasY protein. Also suitable for use is an effector polypeptide of RNA, which has reduced nuclease activity, but still retains target nucleic acid binding activity when complexed with RNA. Also suitable for use is an effector polypeptide of RNA, which has substantially no nuclease activity, but still retains target nucleic acid binding activity when complexed with RNA. Also suitable for use is an effector polypeptide of RNA that exhibits nickase activity. Also suitable for use is an effector polypeptide of RNA that cleaves RNA.

[0098] Examples of various Cas9 proteins (and Cas9 domain structures) and Cas9 RNAs (as well as information regarding requirements for protospacer adjacent motif (PAM) sequences present in the target nucleic acid) can be found in the art, e.g., see Jinek et al., Science. 2012 Aug 17;337(6096):816-21; Chylinski et al., RNA Biol. 2013 May;10(5):726-37; Ma et al., Biomed Res Int. 2013;2013:270805; Hou et al., Proc Natl Acad Sci USA. 2013 Sep 24;110(39):15644-9; Jinek et al., Elife. 2013;2:e00471; Pattanayak et al., Nat Biol. 2013;2:e00472; Pattanayak et al., Nat Biol. 2013;2:e00473; Pattanayak et al., Nat Biol. 2013;2:e00474; Pattanayak et al., Nat Biol. 2013;2:e00475; Pattanayak et al., Nat Biol. 2013;2:e00476; Pattanayak et al., Nat Biol. 2013;2:e00477; Pattanayak et al., Nat Biol. 2013;2:e00478; Pattanayak et al., Nat Biol. Biotechnol. 2013 Sep;31(9):839-43; Qi et al. Cell. 2013 Feb 28;152(5):1173-83; Wang et al. Cell. 2013 May 9;153(4):910-8; Auer et al. Genome Res. 2013 Oct 31; Chen et al. Nucleic Acids Res. 2013 Nov 01;41(20):e19; Cheng et al. Cell Res. 2013 Oct;23(10):1163-71; Cho et al. Genetics. 2013 Nov;195(3):1177-80; DiCarlo et al. Nucleic Acids Res. 2013 Apr;41(7):4336-43; Dickinson et al., Nat Methods. 2013 Oct;10(10):1028-34; Ebina et al., Sci Rep. 2013;3:2510; Fujii et al., Nucleic Acids Res. 2013 Nov 1;41(20):e187; Hu et al., Cell Res. 2013 Nov;23(11):1322-5; Jiang et al., Nucleic Acids Res. 2013 Nov 1;41(20):e188; Larson et al., Nat Protoc. 2013 Nov;8(11):2180-96; Mali et al., Nat Methods. 2013 Oct;10(10):957-63; Nakayama et al., Genesis. 2013 Dec;51(12):835-43; Ran et al., Nat Protoc.2013 Nov;8(11):2281-308; Ran et al. Cell. 2013 Sep 12;154(6):1380-9; Upadhyay et al. G3 (Bethesda). 2013 Dec 9;3(12):2233-8; Walsh et al. Proc Natl Acad Sci USA. 2013 Sep 24;110(39):15514-5; Xie et al. Mol Plant. 2013 Oct 9; Yang et al. Cell. 2013 Sep 12;154(6):1370-9; Briner et al. Mol Cell. 2014 Oct 23;56(2):333-9; Shmakov et al. Nat Rev Microbiol.2017 Mar;15(3):169-182; and U.S. patents and patent applications: 8,906,616; 8,895,308; 8,889,418; 8,889,356; 8,871,445; 8,865,406; 8,795,965; 8,771,945; 8,697,359; 20140068797; 20140170753; 20140179006; 20140179770; 2014018 6843; 20140186919; 20140186958; 20140189896; 20140227787; 20140234972; 20140242664; 20140242699; 20140242700; 20140242702; 20140248702; 20140256046; 20140273037; 20140273226; 20140273230; 2014027 3231; 20140273232; 20140273233; 20140273234; 20140273235; 20140287938; 20140295556; 20140295557; 20140298547; 20140304853; 20140309487; 20140310828; 20140310830; 20140315985; 20140335063; 201403 35620; 20140342456; 20140342457; 20140342458; 20140349400; 20140349405; 20140356867; 20140356956; 20140356958; 20140356959; 20140357523; 20140357530; 20140364333; and 20140377868; each of which is hereby incorporated by reference in its entirety. .

[0099] In some cases, the effector polypeptide of a suitable RNA is a variant Cas9 protein. When compared to the amino acid sequence of the corresponding wild-type Cas9 protein, the variant Cas9 protein has an amino acid sequence that is different from at least one amino acid (e.g., with a deletion, insertion, substitution, fusion). In some instances, the variant Cas9 protein has an amino acid change (e.g., a deletion, insertion, or substitution) that reduces the nuclease activity of the Cas9 protein. For example, in some cases, the variant Cas9 protein has 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 1% or less of the nuclease activity of the corresponding wild-type Cas9 protein. In some cases, the variant Cas9 protein has substantially no nuclease activity. When the Cas9 protein is a variant Cas9 protein that has substantially no nuclease activity, it may be referred to as a nuclease-deficient Cas9 protein or a "dCas9" for a "dead" Cas9. Proteins that cleave one strand of a double-stranded target nucleic acid but not the other strand (e.g., Class 2 CRISPR / Cas proteins, such as Cas9 proteins) are referred to herein as "nickases" (e.g., "nickase Cas9").

[0100] Also suitable for use are effector polypeptides fused to RNA, wherein the effector polypeptide fused to RNA comprises: a) an effector polypeptide of RNA; and b) a heterologous fusion partner. In some cases, the fusion partner has an enzymatic activity that modifies a target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA). Examples of enzymatic activities that can be provided by the fusion partner include, but are not limited to: nuclease activity, such as that provided by a restriction enzyme (e.g., FokI nuclease); methyltransferase activity, such as that provided by a methyltransferase (e.g., HhaI DNA m5c-methyltransferase (M.HhaI), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants), etc.); demethylase activity, such as that provided by a demethylase (e.g., 10-11 translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1, etc.); DNA repair activity; DNA damage activity; deamination activity, such as that provided by a deaminase ( For example, cytosine deaminase, such as rat APOBEC1) provided activity; dismutase activity; alkylation activity; depurination activity; oxidation activity; pyrimidine dimer formation activity; integrase activity, such as provided by integrase and / or resolvase (e.g., Gin convertase such as Gin convertase hyperactive mutant GinH106Y, human immunodeficiency virus type 1 integrase (IN), Tn3 resolvase, etc.) activity; transposase activity; recombinase activity, such as provided by recombinase (e.g., the catalytic domain of Gin recombinase); polymerase activity; ligase activity; helicase activity; photolyase activity and glycosylase activity). In some cases, the fusion partner is a nuclease, such as FokI nuclease. In some cases, the heterologous fusion partner is a deaminase. Suitable deaminases include cytidine deaminase and adenosine deaminase.

[0101] In some cases, the effector polypeptide of the RNA or the effector polypeptide of the fusion RNA comprises one or more nuclear localization signals (NLS). In some cases, the effector polypeptide of the RNA or the effector polypeptide of the fusion RNA comprises a cell penetrating peptide. In some cases, the effector polypeptide of the RNA or the effector polypeptide of the fusion RNA comprises an endosomal lytic peptide.

[0102] In some cases, the RNA comprises two separate nucleic acid molecules: an "activator" and a "target", and is referred to herein as a "dual RNA", "bi-molecule RNA", "two-molecule RNA", or "dgRNA". In some cases, the RNA is one molecule (e.g., for some Class 2 CRISPR / Cas proteins, the corresponding RNA is a single molecule; and in some cases, the activator and the target are covalently linked to each other, e.g., via intervening nucleotides), and the RNA is referred to as a "single RNA", "single-molecule guide RNA", "one-molecule guide RNA", or simply "sgRNA".

[0103] Composition

[0104] The present disclosure provides a composition comprising the gene delivery system of the present disclosure.

[0105] The compositions of the present disclosure include: a) a gene delivery system of the present disclosure; and b) at least one additional component, wherein suitable additional components include, for example, salts, buffers, protease inhibitors, nuclease inhibitors, lipids, etc. In some cases, the compositions of the present disclosure include: a) a gene delivery system of the present disclosure; and b) lipids. In some cases, the compositions of the present disclosure include: a) a gene delivery system of the present disclosure; and b) liposomes, hydrogels, microparticles, nanoparticles, or block copolymer micelles.

[0106] The composition of the present disclosure may comprise: a) the gene delivery system of the present disclosure; and b) one or more of the following: a buffer, a surfactant, an antioxidant, a hydrophilic polymer, a dextrin, a chelating agent, a suspending agent, a solubilizing agent, a thickening agent, a stabilizer, a bacteriostat, a wetting agent, and a preservative. Suitable buffers include, but are not limited to, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (BIS-Tris), N-(2-hydroxyethyl)piperazine-N'3-propanesulfonic acid (EPPS or HEPPS), glycylglycine, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 3-(N-morpholino)propanesulfonic acid (MOPS), piperazine-N,N'-bis(2-ethane-sulfonic acid) (PIPES), sodium bicarbonate, 3-(N-tris(hydroxymethyl)-methyl-amino)-2-hydroxy-propanesulfonic acid) TAPSO, (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), N-tris(hydroxymethyl)methyl-glycine (Tricine), tris(hydroxymethyl)-aminomethane (Tris), etc. Suitable salts include, for example, NaCl, MgCl2, KCl, MgSO4, and the like.

[0107] The composition of the present disclosure may include: a) a gene delivery system of the present disclosure; and b) a pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients have been fully described in a variety of publications, including, for example, A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy", 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) HC Ansel et al., ed., 7th edition, Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) AK Kibbe et al., ed., 3rd edition Amer. Pharmaceutical Assoc.

[0108] In some cases, the gene delivery system of the present disclosure is located in or associated with a particle.The terms "particle" and "nanoparticle" may be used interchangeably as appropriate.

[0109] The gene delivery system of the present disclosure can be present in or associated with a particle, such as a delivery particle comprising a lipid or lipidoid and a hydrophilic polymer (e.g., a cationic lipid and a hydrophilic polymer); for example, wherein the cationic lipid comprises 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 1,2-diisodecanoyl-sn-glycero-3-phosphocholine (DMPC), and / or wherein the hydrophilic polymer comprises ethylene glycol or polyethylene glycol (PEG); and / or wherein the particle further comprises cholesterol (e.g., particles from Formulation 1 = DOTAP 100, DMPC 0, PEG 0, cholesterol 0; Formulation No. 2 = DOTAP 90, DMPC 0, PEG 10, cholesterol 0; Formulation No. 3 = DOTAP 90, DMPC 0, PEG 5, cholesterol 5). For example, particles can be formed using a multi-step process in which the gene delivery system of the present disclosure is mixed together, e.g., in a 1:1 molar ratio, e.g., at room temperature, e.g., for 30 minutes, e.g., in sterile nuclease-free 1× phosphate buffered saline (PBS); and DOTAP, DMPC, PEG, and cholesterol suitable for the formulation are separately dissolved in alcohol (e.g., 100% ethanol); and the two solutions are mixed together to form particles containing the gene delivery system of the present disclosure).

[0110] The gene delivery system of the present disclosure can be part of a nanoparticle. For example, a nanoparticle having a biodegradable core-shell structure of a poly(β-amino ester) (PBAE) core encapsulated by a phospholipid bilayer shell can be used. In some cases, particles / nanoparticles based on self-assembling bioadhesive polymers are used; such particles / nanoparticles can be applied to oral delivery, intravenous delivery, and intranasal delivery.

[0111] In some cases, the composition of the present disclosure comprises a gene delivery system of the present disclosure and a poly(β-amino alcohol) (PBAA). US Patent Publication No. 20130302401 relates to a class of poly(β-amino alcohols) (PBAA) prepared using combinatorial polymerization.

[0112] In some cases, compositions of the present disclosure include gene delivery system of the present disclosure and one or more lipid nanoparticles (LNP).Negatively charged polymer (such as RNA) can be loaded into LNP at low pH (for example, pH 4), wherein ionizable lipids show positive charge.However, at physiological pH, LNP shows a low surface charge compatible with a longer circulation time.Four kinds of ionizable cationic lipids have been concerned, i.e. 1,2-dilinoleyl-3-dimethylammonium-propane (DLinDAP), 1,2-dilinoleyloxy-3-N, N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-keto-N, N-dimethyl-3-aminopropane (DLinKDMA) and 1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA). Preparation of LNPs is described, for example, in Rosin et al. (2011) Molecular Therapy 19: 1286-2200). Cationic lipids 1,2-dilinoleoyl-3-dimethylammonium-propane (DLinDAP), 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA), 1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA), (3-o-[2"-(methoxypolyethylene glycol 2000) succinyl]-1,2-dimyristoyl-sn-ethylene glycol (PEG-S-D MG), and R-3-[(ω-methoxy-poly(ethylene glycol) 2000)carbamoyl]-1,2-dimyristoyloxypropyl-3-amine (PEG-C-DOMG). Nucleic acids (e.g., guide RNA; nucleic acids of the present disclosure, etc.) can be encapsulated in LNPs containing DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA (cationic lipid: DSPC: CHOL: PEGS-DMG or PEG-C-DOMG molar ratio of 40:10:40:10). In some cases, 0.2% SP-DiOC18 is used.

[0113] In some cases, the compositions of the present disclosure comprise a gene delivery system of the present disclosure and a spherical nucleic acid (SNA). TM) constructs or other nanoparticles (particularly gold nanoparticles). See, for example, Cutler et al., J. Am. Chem. Soc. 2011 133:9254-9257; Hao et al., Small. 2011 7:3158-3162; Zhang et al., ACS Nano. 2011 5:6962-6970; Cutler et al., J. Am. Chem. Soc. 2012 134:1376-1391; Young et al., Nano Lett. 2012 12:3867-71; Zheng et al., Proc. Natl. Acad. Sci. USA. 2012 109:11975-80; Mirkin, Nanomedicine 2012 7:635-638; Zhang et al., J. Am. Chem. Soc. 2012 134:16488-1691; Weintraub, Nature 2013 495:S14-S16; Choi et al., Proc. Natl. Acad. Sci. USA. 2013 110(19):7625-7630; Jensen et al., Sci. Transl. Med. 5, 209ra152 (2013); and Mirkin, et al., Small, 10:186-192.

[0114] In some cases, the gene delivery system of the present disclosure is present in nanoparticles or associated with nanoparticles. In general, "nanoparticles" refer to any particles with a diameter less than 1000nm. In some cases, the nanoparticles suitable for the gene delivery system of the present disclosure to be delivered to the target cell have a diameter of 500nm or less, for example, 25nm to 35nm, 35nm to 50nm, 50nm to 75nm, 75nm to 100nm, 100nm to 150nm, 150nm to 200nm, 200nm to 300nm, 300nm to 400nm or 400nm to 500nm. In some cases, the nanoparticles suitable for the gene delivery system of the present disclosure to be delivered to the target cell have a diameter of 25nm to 200nm. In some cases, the nanoparticles suitable for the gene delivery system of the present disclosure to be delivered to the target cell have a diameter of 100nm or less. In some cases, the nanoparticles suitable for the gene delivery system of the present disclosure to be delivered to the target cell have a diameter of 35nm to 60nm.

[0115] Nanoparticles can be provided in various forms, for example, as solid nanoparticles (e.g., metals such as silver, gold, iron, titanium), non-metals, lipid-based solids, polymers), suspensions of nanoparticles, or combinations thereof. Metallic, dielectric, and semiconductor nanoparticles can be prepared, as well as hybrid structures (e.g., core-shell nanoparticles). If the nanoparticles made of semiconductor materials are small enough (usually less than 10 nm) so that quantization of the electron energy levels occurs, they can also be labeled with quantum dots. Such nanoscale particles are used as drug carriers or imaging agents in biomedical applications, and can be adapted for similar purposes in the present disclosure.

[0116] Semisolid and soft nanoparticles are also suitable for inclusion in the compositions of the present disclosure comprising the gene delivery systems of the present disclosure.A prototypical nanoparticle with a semisolid nature is a liposome.

[0117] In some cases, the composition of the present disclosure comprises a gene delivery system and a liposome of the present disclosure. Liposomes are spherical vesicle structures, which are composed of a monolayer or multilayer lipid bilayer surrounding an internal aqueous compartment and a relatively impermeable external lipophilic phospholipid bilayer. Liposomes can be made of several different types of lipids; however, phospholipids are most commonly used to generate liposomes. Although liposome formation is spontaneous when the lipid film is mixed with an aqueous solution, the formation of liposomes can also be accelerated by applying force in the form of shaking using a homogenizer, an ultrasonic crusher or an extrusion device. Several other additives can be added to liposomes to change their structure and properties. For example, cholesterol or sphingomyelin can be added to the liposome mixture to help stabilize the liposome structure and prevent leakage of the liposome contents (inner cargo). Liposome preparations can be mainly composed of natural phospholipids and lipids, such as 1,2-distearoyl-sn-glyceryl-3-phosphatidylcholine (DSPC), sphingomyelin, egg phosphatidylcholine and monosialic acid ganglioside.

[0118] In some cases, the composition of the present disclosure comprises a gene delivery system of the present disclosure and a stable nucleic acid-lipid particle (SNALP). The SNALP formulation may contain 2:40:10:48 molar percentages of lipid 3-N-[(methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristoyloxy-propylamine (PEG-C-DMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-distearoyl-sn-glyceryl-3-phosphocholine (DSPC) and cholesterol. The size of the resulting SNALP liposome may be about 80-100 nm. SNALP may comprise synthetic cholesterol (Sigma-Aldrich, StLouis, Mo., USA), dipalmitoylphosphatidylcholine (Avanti Polar Lipids, Alabaster, Ala., USA), 3-N-[(w-methoxypoly(ethylene glycol) 2000)carbamoyl]-1,2-dimyristoyloxypropylamine and cationic 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane. SNALP may comprise synthetic cholesterol (Sigma-Aldrich), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC; Avanti Polar Lipids Inc.), PEG-cDMA and 1,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMA).

[0119] Other cationic lipids, such as amino lipid 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) may be included in the compositions of the present disclosure. Preformed vesicles with the following lipid composition may be considered: amino lipids, distearoylphosphatidylcholine (DSPC), cholesterol, and (R)-2,3-bis(octadecyloxy)propyl-1-(methoxypoly(ethylene glycol) 2000)propylcarbamate (PEG-lipid) in a molar ratio of 40 / 10 / 40 / 10 and a FVII siRNA / total lipid ratio of about 0.05 (w / w). In order to ensure a narrow particle size distribution in the range of 70-90nm and a low polydispersity index of 0.11.+-.0.04 (n=56), the particles may be extruded up to three times through an 80nm membrane before adding the gene delivery system. Particles containing the highly potent amino lipid 16 can be used, wherein the molar ratios of the four lipid components 16, DSPC, cholesterol, and PEG-lipid (50 / 10 / 38.5 / 1.5) can be further optimized to enhance in vivo activity.

[0120] Lipid can be prepared to form lipid nanoparticles (LNP) together with system of the present disclosure or one or more components thereof or nucleic acid encoding it. Suitable lipids include but are not limited to DLin-KC2-DMA4, C12-200 and colipids (colipid) distearoylphosphatidylcholine, cholesterol and PEG-DMG can be prepared with spontaneous vesicle formation procedure together with system of the present disclosure or its components. Component molar ratio can be about 50 / 10 / 38.5 / 1.5 (DLin-KC2-DMA or C12-200 / distearoylphosphatidylcholine / cholesterol / PEG-DMG).

[0121] The gene delivery system of the present disclosure may be encapsulated in PLGA microspheres, such as those further described in US Published Applications 20130252281, 20130245107, and 20130244279.

[0122] In some cases, the compositions of the present disclosure include a gene delivery system of the present disclosure and a supercharged protein. Supercharged proteins are a class of engineered or naturally occurring proteins that have an abnormally high positive or negative net theoretical charge. Both supernegatively charged proteins and superpositively charged proteins exhibit the ability to tolerate thermally or chemically induced aggregation. Superpositively charged proteins are also able to penetrate mammalian cells. Associating cargo with these proteins (such as plasmid DNA, RNA or other proteins) can contribute to the functional delivery of these macromolecules to mammalian cells in vitro and in vivo.

[0123] The present disclosure also provides an implantable device comprising a gene delivery system of the present disclosure. An implantable device may include a container (e.g., a reservoir, a matrix, etc.), and the container includes a gene delivery system of the present disclosure, for example, comprising a composition containing a gene delivery system of the present disclosure. Suitable implantable devices may include, for example, a polymer substrate (such as a matrix) used as a device body, and include other support materials (such as metals or other polymers) in some cases, and enhance visibility and imaging materials. An implantable delivery device may be local and provide release aspects in a long period of time, and the nucleic acid to be delivered is directly released to a target site, such as an extracellular matrix (ECM), the vascular system around a tumor, diseased tissue, etc.

[0124] Suitable implantable delivery device comprises the device that is applicable to be delivered to the cavity such as peritoneal cavity and / or any other application type that delivery system is not anchored or attached thereto, and the device comprises biostable and / or degradable and / or bioabsorbable polymer substrate, and it is for example optionally matrix.In some cases, suitable implantable delivery device comprises degradable polymer, and wherein main release mechanism is overall erosion (bulk erosion).In some cases, suitable implantable delivery device comprises non-degradable or slowly degradable polymer, and wherein main release mechanism is diffusion rather than overall erosion, makes outer part as film and its inner part as reservoir, in fact, described drug reservoir will not be affected by surrounding environment for a long time (for example, about one week to about several months).Also can optionally use the combination of different polymers with different release mechanisms.

[0125] In some cases, the implantable delivery system is designed to protect the nucleotide-based therapeutic (gene delivery system of the disclosure) from degradation, either chemically or due to attack by enzymes and other factors within the subject's body.

[0126] Pill Box

[0127] The present disclosure provides a medicine box, the medicine box comprises: a) a first nucleic acid, the first nucleic acid comprises a nucleotide sequence encoding an R2 retrotransposon R2 polypeptide; and b) a second nucleic acid, the second nucleic acid comprises an insertion site for inserting a heterologous nucleotide sequence encoding one or more heterologous gene products, wherein the insertion site is flanked by the R2 retrotransposon 3' untranslated region (UTR) and the R2 retrotransposon 5'UTR, and wherein the heterologous nucleotide sequence has a length of at least 200 nucleotides. Therefore, the second nucleic acid allows the insertion of a heterologous nucleotide sequence encoding any desired heterologous gene product. In some cases, the first nucleic acid and the second nucleic acid are located in separate containers. In some cases, the second nucleic acid also comprises a transcription control element at 5' of the insertion site. The transcription control element is positioned relative to the insertion site so that once the heterologous nucleotide sequence is inserted into the second nucleic acid, the transcription control element is operably connected to the heterologous nucleotide sequence. Suitable transcription control elements are as described above.

[0128] The present disclosure provides a kind of, described medicine box comprises: a) R2 retrotransposon R2 polypeptide; And b) nucleic acid, described nucleic acid comprises the insertion site for inserting heterologous nucleotide sequence encoding one or more heterologous gene products, wherein described insertion site is flanked by R2 retrotransposon 3'UTR and R2 retrotransposon 5'UTR, and wherein described heterologous nucleotide sequence has the length of at least 200 nucleotides. Therefore, described nucleic acid allows the insertion of heterologous nucleotide sequence encoding any desired heterologous gene product. In some cases, described R2 polypeptide and described nucleic acid are located in separate containers. In some cases, described nucleic acid also comprises the transcription control element of insertion site 5'. Described transcription control element is positioned relative to described insertion site, so that once heterologous nucleotide sequence is inserted into described nucleic acid, described transcription control element is operably connected to described heterologous nucleotide sequence. Suitable transcription control element is as described above.

[0129] As described above, the nucleic acid that allows the insertion of a heterologous nucleotide sequence encoding any desired heterologous gene product provides for the insertion of a heterologous nucleotide sequence of at least 200 nucleotides (nt). For example, in some cases, the heterologous nucleotide sequence has a length of about 200 nt to about 300 nt, about 300 nt to about 400 nt, about 400 nt to about 500 nt, about 500 nt to about 750 nt, about 750 nt to about 1 kilobase (kb), about 1 kb to about 1.5 kb, about 1.5 kb to about 2 kb, about 2 kb to about 2.5 kb, about 2.5 kb to about 3 kb, or about 3 kb to about 3.5 kb. As another example, in some cases, the heterologous nucleotide sequence has a length of about 3.5 kb to about 4 kb, about 4 kb to about 4.5 kb, about 4.5 kb to about 5 kb, about 5 kb to about 5.5 kb, about 5.5 kb to about 6 kb, about 6 kb to about 6.5 kb, about 6.5 kb to about 7 kb, about 7 kb to about 8 kb, about 8 kb to about 9 kb, about 9 kb to about 10 kb, about 10 kb to about 11 kb, about 11 kb to about 12 kb, about 12 kb to about 13 kb, about 13 kb to about 14 kb, or about 14 kb to about 15 kb. In some cases, the heterologous nucleotide sequence has a length of about 200 nt to about 1 kb. In some cases, the heterologous nucleotide sequence has a length of about 1 kb to about 5 kb. In some cases, the heterologous nucleotide sequence has a length of about 3.5 kb to about 6 kb. In some cases, the heterologous nucleotide sequence has a length of about 6 kb to about 8 kb. In some cases, the heterologous nucleotide sequence has a length of about 8 kb to about 15 kb. In some cases, the heterologous nucleotide sequence has a length of about 9 kb to about 15 kb. In some cases, the heterologous nucleotide sequence has a length of about 10 kb to about 15 kb.

[0130] Methods for delivering one or more gene products to eukaryotic host cells

[0131] The present disclosure provides a method for delivering one or more target gene products to eukaryotic cells, the method comprising contacting the cells with the gene delivery vehicle system of the present disclosure.The R2 polypeptide, 5'UTR and 3'UTR insert the heterologous nucleic acid into the 28S region of the eukaryotic cell genome.

[0132] In some cases, the eukaryotic cell is in vitro. In some cases, the eukaryotic cell is in vivo. In some cases, the eukaryotic cell is ex vivo.

[0133] Suitable eukaryotic cells include, for example, human cells, non-human animal cells, plant cells, vertebrate cells, invertebrate cells, bird cells, arthropod cells, arachnid cells, insect cells, reptile cells, amphibian cells, etc. In some cases, the eukaryotic cell is a human cell. In some cases, the eukaryotic cell is a non-human animal cell. In some cases, the eukaryotic cell is a plant cell. In some cases, the eukaryotic cell is an invertebrate cell. In some cases, the cell is a diseased cell.

[0134] Suitable cells include stem cells (e.g., embryonic stem (ES) cells, induced pluripotent stem (iPS) cells; germ cells (e.g., oocytes, sperm, oogonia, spermatogonia, etc.); somatic cells, such as fibroblasts, oligodendrocytes, glial cells, hematopoietic cells, neurons, muscle cells, bone cells, hepatocytes, pancreatic cells, etc.

[0135] Suitable cells include human embryonic stem cells, embryonic cardiomyocytes, myofibroblasts, mesenchymal stem cells, cardiomyocytes, adipocytes, totipotent cells, pluripotent cells, blood stem cells, myoblasts, adult stem cells, bone marrow cells, mesenchymal cells, embryonic stem cells, parenchymal cells, epithelial cells, endothelial cells, mesothelial cells, fibroblasts, osteoblasts, chondrocytes, exogenous cells, endogenous cells, stem cells, hematopoietic stem cells, bone marrow-derived progenitor cells, cardiomyocytes, skeletal cells, fetal cells, undifferentiated cells, multipotent progenitor cells, unipotent progenitor cells, monocytes, cardiac myoblasts, skeletal myoblasts, macrophages, capillary endothelial cells, xenogeneic cells, allogeneic cells, and postpartum stem cells. Suitable cells include peripheral blood mononuclear cells (PBMC).

[0136] In some cases, the cell is an immune cell, a neuron, an epithelial cell and an endothelial cell or a stem cell. In some cases, the immune cell is a T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell or a macrophage. In some cases, the immune cell is a cytotoxic T cell. In some cases, the immune cell is a helper T cell. In some cases, the immune cell is a regulatory T cell (Treg).

[0137] In some cases, the cell is a stem cell. Stem cells include adult stem cells. Adult stem cells are also called somatic stem cells.

[0138] Adult stem cells reside in differentiated tissues but retain the properties of self-renewal and the ability to generate a variety of cell types, often typical of the tissue in which the stem cell resides. Many examples of somatic stem cells are known to those skilled in the art, including muscle stem cells; hematopoietic stem cells; epithelial stem cells; neural stem cells; mesenchymal stem cells; mammary stem cells; intestinal stem cells; mesoderm stem cells; endothelial stem cells; olfactory stem cells; neural crest stem cells, etc.

[0139] Target stem cells include mammalian stem cells, wherein the term "mammal" refers to any animal classified as a mammal, including humans; non-human primates; livestock and farm animals; and zoo, laboratory, sports or pet animals, such as dogs, horses, cats, cows, mice, rats, rabbits, etc. In some cases, the stem cells are human stem cells. In some cases, the stem cells are rodent (e.g., mouse; rat) stem cells. In some cases, the stem cells are non-human primate stem cells.

[0140] The stem cells may express one or more stem cell markers, such as SOX2, OCT4, NANOG, NESTIN, SOX1, PAX6, KLF4, SOX9, KRT19, KRT7, LGR5, CA9, FXYD2, CDH6, CLDN18, TSPAN8, BPIFB1, OLFM4, CDH17, and PPARGC1A.

[0141] In some cases, the stem cell is a hematopoietic stem cell (HSC). HSCs are mesoderm-derived cells that can be isolated from bone marrow, blood, umbilical cord blood, fetal liver, and yolk sac. HSCs are characterized by CD34 + and CD3 -HSCs can regenerate erythroid, neutrophil-macrophage, megakaryocyte, and lymphoid hematopoietic cell lineages in vivo. In vitro, HSCs can be induced to undergo at least some self-renewing cell divisions, and can be induced to differentiate into the same lineages as seen in vivo. Thus, HSCs can be induced to differentiate into one or more of erythroid, megakaryocyte, neutrophil, macrophage, and lymphocyte.

[0142] In other cases, stem cells are neural stem cells (NSC). Neural stem cells (NSC) can differentiate into neurons and glial cells (including oligodendrocytes and astrocytes). Neural stem cells are multipotent stem cells that can divide multiple times, and under certain conditions, daughter cells as neural stem cells can be produced, or neural progenitor cells that can be used as neuroblasts or glial cells, for example, cells that are dedicated to becoming neurons and glial cells of one or more types, respectively. Methods for obtaining NSC are known in the art.

[0143] In other cases, the stem cell is a mesenchymal stem cell (MSC). MSCs are originally derived from embryonic mesoderm and isolated from adult bone marrow and can differentiate into muscle, bone, cartilage, fat, bone marrow stroma and tendon. Methods for isolating MSCs are known in the art; and any known method can be used to obtain MSCs. See, for example, U.S. Patent No. 5,736,396, which describes the isolation of human MSCs.

[0144] In some cases, the cell is a plant cell. The plant cell can be a cell of a monocot. The cell can be a cell of a dicot.

[0145] In some cases, the cell is an arthropod cell. For example, the cell can be a cell of the following suborder, family, subfamily, group, subpopulation, or species: for example, Chelicerata, Myriapodia, Hexipodia, Arachnida, Insecta, Archaeognatha, Thysanura, Palaeoptera, Ephemeroptera, Odonata, Anisoptera, Zygoptera, Neoptera, Exopterygota, Plecoptera, Embioptera ), Orthoptera, Zoraptera, Dermaptera, Dictyoptera, Notoptera, Grylloblattid ae, Mantophasmatidae, Phasmatodea, Blattaria, Isoptera, Mantodea, Parapneuroptera, Psocoptera, Thysanoptera, Phthiraptera, Hemiptera, Endopterygota or Holometabola, Hymenoptera, Coleoptera, Strepsiptera, Raphidioptera, Megaloptera, Neuroptera, Mecoptera, Siphonaptera, Diptera, Trichoptera or Lepidoptera.

[0146] In some cases, the cell is an insect cell. For example, in some cases, the cell is a cell of a mosquito, grasshopper, hemiptera, fly, flea, bee, wasp, ant, louse, moth, or beetle.

[0147] The gene delivery system of the present disclosure can be introduced into eukaryotic cells by any of a variety of methods, many of which are known in the art. Suitable methods include, for example, viral infection, transfection, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, etc.

[0148] Well-developed transfection techniques can be used (see, e.g., Angel and Yanik (2010) PLoS ONE 5(7):e11756); and commercially available from Qiagen Reagents, Stemfect commercially available from Stemgent TM RNA transfection kits and commercially available from Mirus Bio LLC Transfection kits introduce nucleic acids into eukaryotic cells. See also Beumer et al. (2008) PNAS 105(50):19821-19826.

[0149] In some cases, the gene delivery system of the present disclosure is administered to an individual in need. The gene delivery system of the present disclosure can be administered to an individual by any of a variety of administration routes. Conventional and pharmaceutically acceptable administration routes include intratumoral, peritumoral, intramuscular, intratracheal, intracranial, subcutaneous, intradermal, topical, intravenous, intraarterial, rectal, nasal, oral and other enteral and parenteral administration routes. If desired, the administration route can be combined, or adjusted according to the gene delivery system and / or the desired effect. The gene delivery system of the present disclosure can be administered in single doses or multiple doses.

[0150] In some embodiments, the gene delivery system of the present disclosure is administered intravenously. In some cases, the gene delivery system of the present disclosure is administered intramuscularly. In some cases, the gene delivery system of the present disclosure is administered topically. In some cases, the gene delivery system of the present disclosure is administered intratumorally. In some cases, the gene delivery system of the present disclosure is administered peritumorally. In some cases, the gene delivery system of the present disclosure is administered intracranially. In some cases, the gene delivery system of the present disclosure is administered subcutaneously.

[0151] In some cases, the target cell or target cell colony is removed (obtained) from the individual; The target cell or target cell colony is contacted in vitro with the gene delivery system of the present disclosure to produce a genetically modified target cell or a genetically modified target cell colony; The genetically modified target cell or genetically modified target cell colony is applied to the individual from which the target cell or the target cell colony is obtained. Therefore, in some cases, the method of the present disclosure includes: a) contacting the target cell or target cell colony with the gene delivery system of the present disclosure in vitro, thereby producing a genetically modified target cell or a genetically modified target cell colony, wherein the target cell or target cell colony is obtained from an individual in need of treatment; and b) applying the genetically modified target cell or genetically modified target cell colony to the individual, thereby treating the individual. In some cases, the method of the present disclosure includes: a) obtaining a target cell or a target cell colony from an individual; b) contacting the target cell or target cell colony with the gene delivery system of the present disclosure in vitro, thereby producing a genetically modified target cell or a genetically modified target cell colony; and c) applying the genetically modified target cell or genetically modified target cell colony to an individual. As a non-limiting example, the cell can be a T cell; and the one or more heterologous polypeptides can be CAR (e.g., a single polypeptide chain CAR; or a heterodimeric CAR). As another example, the cell can be a diseased cell, and the heterologous gene product can be: i) an effector polypeptide of RNA, such as a Cas9 polypeptide; ii) RNA.

[0152] Examples of non-limiting aspects of the present disclosure

[0153] Aspects of the above-described subject matter (including embodiments) may be beneficial alone or in combination with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the present disclosure, numbered 1-33, are provided below. It will be apparent to those skilled in the art upon reading this disclosure that each individually numbered aspect may be used or combined with any preceding or succeeding individually numbered aspect. This is intended to provide support for all such combinations of aspects, and is not limited to the combinations of aspects explicitly provided below:

[0154] Aspect 1. A gene delivery vehicle system, comprising: a) a first nucleic acid and a second nucleic acid, wherein: i) the first nucleic acid comprises a nucleotide sequence encoding an R2 retrotransposon R2 polypeptide; and ii) the second nucleic acid comprises a heterologous nucleotide sequence encoding one or more heterologous gene products, wherein the heterologous nucleotide sequence is flanked by an R2 retrotransposon 3' untranslated region (UTR) and an R2 retrotransposon 5'UTR, and wherein the heterologous nucleotide sequence has a length of at least 200 nucleotides; or b) a polypeptide and a nucleic acid, wherein: i) the polypeptide is an R2 retrotransposon R2 polypeptide; and ii) the nucleic acid comprises a heterologous nucleotide sequence encoding one or more heterologous gene products, wherein the heterologous nucleotide sequence is flanked by an R2 retrotransposon 3'UTR and an R2 retrotransposon 5'UTR, and wherein the heterologous nucleotide sequence has a length of at least 200 nucleotides.

[0155] Aspect 2. The gene delivery vehicle system of aspect 1, wherein the R2 polypeptide comprises Figure 7 An amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence depicted in .

[0156] Aspect 3. The gene delivery vehicle system of aspect 1 or aspect 2, wherein the heterologous nucleotide sequence encodes a single heterologous gene product.

[0157] Aspect 4. The gene delivery vehicle system of aspect 3, wherein the single heterologous gene product is a polypeptide.

[0158] Aspect 5. The gene delivery vehicle system of aspect 3, wherein the single heterologous gene product is RNA.

[0159] Aspect 6. The gene delivery vehicle system of aspect 1 or aspect 2, wherein the heterologous nucleotide sequence encodes at least a first heterologous gene product and a second heterologous gene product.

[0160] Aspect 7. The gene delivery vehicle system of aspect 6, wherein the first heterologous gene product is a polypeptide, and wherein the second heterologous gene product is RNA.

[0161] Aspect 8. The gene delivery vehicle system according to aspect 4, wherein the polypeptide is a chimeric antigen receptor.

[0162] Aspect 9. The gene delivery vehicle system of aspect 6, wherein the first heterologous gene product is a first heterologous polypeptide, and wherein the second heterologous gene product is a second heterologous polypeptide.

[0163] Aspect 10. The gene delivery vehicle system of aspect 9, wherein the heterologous nucleotide sequence comprises, in 5' to 3' order: i) a nucleotide sequence encoding the first heterologous polypeptide; ii) an internal ribosome entry site or a nucleotide sequence encoding a self-cleaving polypeptide; and iii) a nucleotide sequence encoding the second heterologous polypeptide.

[0164] Aspect 11. The gene delivery vehicle system of aspect 9, wherein the first polypeptide and the second polypeptide together form a heterodimeric chimeric antigen receptor.

[0165] Aspect 12. The gene delivery vehicle system according to aspect 7, wherein the polypeptide is an effector polypeptide of RNA, and wherein the RNA is an RNA that binds to the effector polypeptide of RNA.

[0166] Aspect 13. The gene delivery vehicle system of any one of aspects 1-12, wherein the nucleotide sequence encoding the R2 polypeptide is codon optimized for expression in eukaryotic cells.

[0167] Aspect 14. The gene delivery vehicle system of any one of aspects 1-13, wherein the heterologous nucleotide sequence encoding one or more heterologous gene products is operably linked to a transcriptional control element.

[0168] Aspect 15. The gene delivery vehicle system of aspect 14, wherein the transcriptional control element is a regulatable promoter.

[0169] Aspect 16. The gene delivery vehicle system of aspect 14, wherein the transcriptional control element is a constitutive promoter.

[0170] Aspect 17. The gene delivery vehicle system of any one of aspects 1-16, wherein the heterologous nucleotide sequence has a length of at least 3 kb.

[0171] Aspect 18. The gene delivery vehicle system of any one of aspects 1-16, wherein the heterologous nucleotide sequence has a length of about 5 kb to about 10 kb.

[0172] Aspect 19. The gene delivery vehicle system of any one of aspects 1-16, wherein the heterologous nucleotide sequence has a length of about 10 kb to about 15 kb.

[0173] Aspect 20. A medicine kit, comprising:

[0174] a1) a first nucleic acid comprising a nucleotide sequence encoding an R2 retrotransposon R2 polypeptide; and

[0175] b1) a second nucleic acid comprising an insertion site for inserting a heterologous nucleotide sequence encoding one or more heterologous gene products, wherein the insertion site is flanked by an R2 retrotransposon 3' untranslated region (UTR) and an R2 retrotransposon 5' UTR, and wherein the heterologous nucleotide sequence has a length of at least 200 nucleotides; or

[0176] a2) R2 retrotransposon R2 polypeptide; and

[0177] b2) a nucleic acid comprising an insertion site for inserting a heterologous nucleotide sequence encoding one or more heterologous gene products, wherein the insertion site is flanked by an R2 retrotransposon 3'UTR and an R2 retrotransposon 5'UTR, and wherein the heterologous nucleotide sequence has a length of at least 200 nucleotides.

[0178] Aspect 21. The kit of aspect 20 (a1 and b1), wherein the first nucleic acid and the second nucleic acid are in separate containers.

[0179] Aspect 22. The kit of aspect 20(a1), wherein the nucleotide sequence encoding the R2 polypeptide is codon-optimized for expression in eukaryotic cells.

[0180] Aspect 23. The kit of aspect 20(b1), wherein the second nucleic acid further comprises a transcriptional control element 5' to the insertion site.

[0181] Aspect 24. The kit of aspect 20 (a2 and b2), wherein the R2 polypeptide and the nucleic acid are in separate containers.

[0182] Aspect 25. The kit of aspect 20(b2), wherein the nucleic acid further comprises a transcriptional control element 5' to the insertion site.

[0183] Aspect 26. A method for delivering one or more target gene products to a eukaryotic cell, the method comprising contacting the cell with a gene delivery vehicle system as described in any of Aspects 1-19, wherein the R2 polypeptide, the 5'UTR and the 3'UTR insert the heterologous nucleic acid into the 28S region of the genome of the eukaryotic cell.

[0184] Aspect 27. The method of aspect 26, wherein the contacting is in vitro.

[0185] Aspect 28. The method of aspect 26, wherein the contacting is in vivo.

[0186] Aspect 29. The method of aspect 26, wherein the contacting is ex vivo.

[0187] Aspect 30. The method of any one of Aspects 26-29, wherein the eukaryotic cell is a non-human animal cell.

[0188] Aspect 31. The method of any one of Aspects 26-29, wherein the eukaryotic cell is a human cell.

[0189] Aspect 32. The method of any one of Aspects 26-29, wherein the eukaryotic cell is a plant cell.

[0190] Aspect 33. The method of any one of aspects 26-29, wherein the eukaryotic cell is an invertebrate cell.

[0191] Example

[0192] The following examples are given so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numerical values ​​used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure. Standard abbreviations may be used, for example, bp, base pair; kb, kilobase; pl, picoliter; s or sec, second; min, minute; h or hr, hour; aa, amino acid; kb, kilobase; bp, base pair; nt, nucleotide; im, intramuscular (intramuscular); ip, intraperitoneal (intraperitoneal); sc, subcutaneous (subcutaneous), etc.

[0193] Example 1

[0194] Two DNA plasmids were generated based on DNA from the silkworm Bombyx mori. One plasmid contained an optimized version of the coding sequence for protein R2 (OR2Bm), while the second plasmid contained the transgene flanked by 5' and 3' untranslated regions (UTRs), oriented in a 3' to 5' direction), all under the control of a single rDNA promoter that controls transcription of the complete rDNA transcript.

[0195] HEK 293 cells and HEK 293T cells were PEI transfected with both plasmid constructs and genomic DNA was extracted 48 hours later and sequenced through the 5' junction of integration to ensure that the transgene was integrated at the correct site and full-length integration occurred ( Figure 2). The transgenic cassette was also amplified to include the hygromycin resistance gene and then used to quantify the integration frequency and titrated to determine the most efficient ratio. Transfection of a plasmid containing the UTR and the transgene resulted in 1% integration efficiency via homology driven recombination (HDR). When co-transfected with the OR2 plasmid, the efficiency was increased to 4% of the total cell population.

[0196] Figure 2 : Genomic DNA was amplified across the integration junction in 28S rDNA and specific bands were shown when a transgene flanked by 5' and 3' UTRs under the CMV promoter was amplified with OR2Bm (lane 1) and without (lane 3). The transgenic cassette under the control of the RNA pol I promoter (lane 2) showed specific integration bands only in the presence of OR2Bm and not in the absence of OR2Bm (lane 4).

[0197] Figure 3 It is shown that when cells were transfected with only the transgene flanked by UTRs without OR2, the full-length amplicon was amplified without bands from genomic DNA.

[0198] Cells were transfected with a transgene flanked by UTRs or a transgene flanked by UTRs and OR2. Figure 4 The protocol is schematically depicted. After three days, the cells were passaged at a ratio of 1:10 and a portion was taken for screening via flow cytometry. After 6 days, the cells were passaged again at a ratio of 1:10 and on day 14, the cells were screened via flow cytometry for stable expression of the transgene (GFP). Data are presented in Figure 4 GFP expression was compared between the two samples and showed that OR2 helped mediate the generation of a stable population of 3.4% GFP positive cells. Cells transfected with OR2 had 4-fold more GFP expression. * indicates p<0.005

[0199] Cells were transfected with different amounts of OR2 and transgene. Cells were then kept under antibiotic selection (200 μg / mL hygromycin B) for two weeks, and surviving colonies were counted after several washes. The data are presented in Figure 5. As shown in Figure 5, it was found that OR2 mediated a higher level of sustained expression. * indicates p<0.005

[0200] Cells were transfected with CAR-T receptors and OR2 and passaged within two weeks. Figure 6 schematically depicts the scheme. Afterwards, cells were labeled with antibodies against the receptor and screened via flow cytometry. Genomic DNA was then extracted and complete transcripts were amplified to further confirm integration. Data are presented in Figure 6. * indicates p<0.005.

[0201] Example 2

[0202] Two gene delivery constructs were prepared. The first construct is a recombinant expression vector comprising a nucleotide sequence encoding an R2 retrotransposon R2 polypeptide. The second construct is a recombinant expression vector comprising a heterologous nucleotide sequence oriented in a 3' to 5' direction, and the heterologous nucleotide sequence encodes 2 polypeptide chains of a conditionally active CAR. The heterologous nucleotide sequence is flanked by R2 5'UTR and R2 3'UTR. The R2 polypeptide is an optimized R2 (OR2) polypeptide. The first polypeptide chain of the conditionally active CAR comprises: i) an extracellular antigen binding domain that specifically binds to an antigen on a target cell (eg, a cancer cell); ii) a transmembrane domain; and iii) a first member of a dimerization pair. The second polypeptide chain of the conditionally active CAR comprises: i) a transmembrane domain; ii) a second member of a dimerization pair; and iii) an intracellular signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM), wherein the intracellular signaling domain provides signal transduction activity. The first polypeptide of the conditionally active CAR, the second polypeptide of the conditionally active CAR, or both the first polypeptide and the second polypeptide of the conditionally active CAR comprise an intracellular co-stimulatory polypeptide.

[0203] Two gene delivery constructs were introduced into T cells (e.g., CD8 + The genetically modified T cells are administered to an individual, such as an individual with cancer, wherein the cancer includes cells expressing an antigen recognized by the conditionally active CAR.

[0204] Example 3

[0205] Two gene delivery constructs were prepared. The first construct is a recombinant expression vector comprising a nucleotide sequence encoding an R2 retrotransposon R2 polypeptide. The second construct is a recombinant expression vector comprising a heterologous nucleotide sequence oriented in a 3' to 5' direction, and the heterologous nucleotide sequence encodes a CAR and a synNotch polypeptide. The heterologous nucleotide sequence is flanked by R2 5'UTR and R2 3'UTR. The R2 polypeptide is an optimized R2 (OR2) polypeptide. The CAR is operably connected to a promoter activated by the intracellular signaling domain of the synNotch polypeptide.

[0206] Example 4

[0207] Human embryonic kidney (HEK) 293 cells were transfected with: 1) 2.5 μg of a plasmid containing a GFP expression construct (comprising a nucleotide sequence encoding GFP, wherein the nucleotide sequence is operably linked to a cytomegalovirus promoter) flanked by R2 5'UTR and R2 3'UTR and oriented in a 3' to 5' direction relative to the UTRs; and ii) 5 μg of a plasmid containing an expression cassette encoding OR2 or 5 μg of a plasmid containing a stuffer DNA (control DNA that does not encode an OR2 polypeptide). The transfected HEK293 cells were then grown for 14 days to dilute and degrade the unintegrated plasmid. After 14 days, the transfected HEK293 cells were quantified via flow cytometry to determine the stable cell population that had integrated the GFP transgene.

[0208] The results are Fig.10 On average, 5.6% of cells transfected with a construct containing OR2 and a GFP transgene stably expressed GFP. In contrast, on average, 1.3% of cells transfected with a stuffer fragment and a GFP transgene expressed GFP.

[0209] Example 5

[0210] HEK293 cells were transfected with: i) 0.5 μg of a plasmid containing a hygromycin B (HygB) resistance cassette (comprising a nucleotide sequence encoding hygromycin B phosphotransferase, wherein the nucleotide sequence is operably linked to an SV40 promoter), the resistance cassette being flanked by R2 5'UTR and R2 3'UTR and oriented in a 3' to 5' direction relative to the UTR; and ii) 0.5 μg of a plasmid containing an expression cassette encoding OR2 or 0.5 μg of a plasmid containing a stuffer DNA (control DNA not encoding an OR2 polypeptide). The transfected HEK293 cells were in a culture medium containing 200 μg / m hygromycin. The transfected HEK293 cells were grown in a culture medium containing hygromycin for 14 days to dilute and degrade the unintegrated plasmid. The HEK293 cells were stained with methylene blue and then manually counted to quantify the number of colonies, each of which would represent an integration event.

[0211] The results are Fig.11 An average of 177 integration events occurred in cells transfected with OR2 and the HygB resistance cassette, and an average of 39.33 integration events occurred in cells transfected with the stuffer fragment and the HygB resistance cassette.

[0212] Example 6

[0213] HEK293 cells were transfected with: i) 2.5 μg of an expression construct containing a chimeric antigen receptor (CAR) (CAR-c-myc) encoding a c-myc tag (wherein the expression construct comprises a nucleotide sequence encoding CAR-c-myc, wherein the nucleotide sequence is operably linked to the EF1-α promoter), the expression construct is flanked by the R2 untranslated region (R2 5'UTR and R2 3'UTR) and oriented in the 3' to 5' direction relative to the UTR; and ii) 5 μg of a plasmid containing an expression cassette for OR2 or 5 μg of a plasmid containing a stuffer DNA (control DNA that does not encode an OR2 polypeptide). The transfected HEK293 cells were then grown for 14 days to dilute and degrade the unintegrated plasmid. After 14 days, the transfected HEK293 cells were quantified by flow cytometry by staining for cell surface expression of the c-myc tag to determine the stable cell population that had integrated the transgene.

[0214] Data in Fig.12 On average, 1.85% of cells transfected with constructs containing OR2 and CAR-c-myc transgenes stably expressed the c-myc tag. In contrast, on average, 0.87% of cells transfected with the stuffer fragment and GFP transgene expressed the c-myc tag.

[0215] Although the present invention has been described with reference to its specific embodiments, it will be appreciated by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the present invention. In addition, many modifications may be made to adapt specific circumstances, materials, compositions of matter, processes, one or more process steps to the purpose, spirit and scope of the present invention. All of these modifications are intended to fall within the scope of the appended claims. Sequence Listing <110> The Regents of the University of California Schaffer, David V. Barnes, Christopher <120> Retrotransposon-based delivery vehicles and methods of use thereof <130> BERK-392WO <150> US62 / 697,829 <151> 2018-07-13 <160> 39 <170> PatentIn version 3.5 <210> 1 <211> twenty two <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 1 Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val 1 5 10 15 Glu Glu Asn Pro Gly Pro 20 <210> 2 <211> twenty one <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 2 Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu 1 5 10 15 Glu Asn Pro Gly Pro 20 <210> 3 <211> twenty three <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 3 Gly Ser Gly Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp 1 5 10 15 Val Glu Ser Asn Pro Gly Pro 20 <210> 4 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 4 Gly Ser Gly Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala 1 5 10 15 Gly Asp Val Glu Ser Asn Pro Gly Pro 20 25 <210> 5 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <220> <221> Misc_feature <222> (2)..(3) <223> Xaa can be any amino acid residue <220> <221> Misc_feature <222> (4) <223> Xaa is a hydrophobic amino acid residue <400> 5 Pro Xaa Xaa Xaa 1 <210> 6 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <220> <221> Misc_feature <222> (2)..(3) <223> Xaa can be any amino acid residue <220> <221> Misc_feature <222> (4) <223> Xaa is a hydrophobic amino acid residue <220> <221> Misc_feature <222> (5) <223> Xaa is a Ser or Thr residue <400> 6 Pro Xaa Xaa Xaa Xaa 1 5 <210> 7 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <220> <221> Misc_feature <222> (2) <223> Xaa is Lue or Gln amino acid residue <400> 7 Pro Xaa Gly Met Thr Ser 1 5 <210> 8 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <220> <221> Misc_feature <222> (2) <223> Xaa is a Leu or Gln residue <400> 8 Pro Xaa Gly Met Thr 1 5 <210> 9 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 9 Glu Asn Leu Tyr Thr Gln Ser 1 5 <210> 10 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 10 Asp Asp Asp Asp Lys 1 5 <210> 11 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 11 Leu Val Pro Arg 1 <210> 12 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 12 Leu Glu Val Leu Phe Gln Gly Pro 1 5 <210> 13 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 13 Cys Gly Leu Val Pro Ala Gly Ser Gly Pro 1 5 10 <210> 14 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 14 Ser Leu Leu Lys Ser Arg Met Val Pro Asn Phe Asn 1 5 10 <210> 15 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 15 Ser Leu Leu Ile Ala Arg Arg Met Pro Asn Phe Asn 1 5 10 <210> 16 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 16 Ser Lys Leu Val Gln Ala Ser Ala Ser Gly Val Asn 1 5 10 <210> 17 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 17 Ser Ser Tyr Leu Lys Ala Ser Asp Ala Pro Asp Asn 1 5 10 <210> 18 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 18 Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Met Asn 1 5 10 <210> 19 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 19 Ser Leu Arg Pro Leu Ala Leu Trp Arg Ser Phe Asn 1 5 10 <210> 20 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 20 Ser Pro Gln Gly Ile Ala Gly Gln Arg Asn Phe Asn 1 5 10 <210> twenty one <211> 14 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> twenty one Asp Val Asp Glu Arg Asp Val Arg Gly Phe Ala Ser Phe Leu 1 5 10 <210> twenty two <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> twenty two Ser Leu Pro Leu Gly Leu Trp Ala Pro Asn Phe Asn 1 5 10 <210> twenty three <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> twenty three Ser Leu Leu Ile Phe Arg Ser Trp Ala Asn Phe Asn 1 5 10 <210> twenty four <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> twenty four Ser Gly Val Val Ile Ala Thr Val Ile Val Ile Thr 1 5 10 <210> 25 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 25 Ser Leu Gly Pro Gln Gly Ile Trp Gly Gln Phe Asn 1 5 10 <210> 26 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 26 Lys Lys Ser Pro Gly Arg Val Val Gly Gly Ser Val 1 5 10 <210> 27 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 27 Pro Gln Gly Leu Leu Gly Ala Pro Gly Ile Leu Gly 1 5 10 <210> 28 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 28 His Gly Pro Glu Gly Leu Arg Val Gly Phe Tyr Glu Ser Asp Val Met 1 5 10 15 Gly Arg Gly His Ala Arg Leu Val His Val Glu Glu Pro His Thr 20 25 30 <210> 29 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 29 Gly Pro Gln Gly Leu Ala Gly Gln Arg Gly Ile Val 1 5 10 <210> 30 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 30 Gly Gly Ser Gly Gln Arg Gly Arg Lys Ala Leu Glu 1 5 10 <210> 31 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 31 Ser Leu Ser Ala Leu Leu Ser Ser Asp Ile Phe Asn 1 5 10 <210> 32 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 32 Ser Leu Pro Arg Phe Lys Ile Ile Gly Gly Phe Asn 1 5 10 <210> 33 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 33 Ser Leu Leu Gly Ile Ala Val Pro Gly Asn Phe Asn 1 5 10 <210> 34 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 34 Phe Phe Lys Asn Ile Val Thr Pro Arg Thr Pro Pro 1 5 10 <210> 35 <211> 306 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 35 Pro Pro Gln Ile Glu Glu Ala Cys Glu Leu Pro Glu Cys Gln Val Asp 1 5 10 15 Ala Gly Asn Lys Val Cys Asn Leu Gln Cys Asn Asn His Ala Cys Gly 20 25 30 Trp Asp Gly Gly Asp Cys Ser Leu Asn Phe Asn Asp Pro Trp Lys Asn 35 40 45 Cys Thr Gln Ser Leu Gln Cys Trp Lys Tyr Phe Ser Asp Gly His Cys 50 55 60 Asp Ser Gln Cys Asn Ser Ala Gly Cys Leu Phe Asp Gly Phe Asp Cys 65 70 75 80 Gln Leu Thr Glu Gly Gln Cys Asn Pro Leu Tyr Asp Gln Tyr Cys Lys 85 90 95 Asp His Phe Ser Asp Gly His Cys Asp Gln Gly Cys Asn Ser Ala Glu 100 105 110 Cys Glu Trp Asp Gly Leu Asp Cys Ala Glu His Val Pro Glu Arg Leu 115 120 125 Ala Ala Gly Thr Leu Val Leu Val Val Leu Leu Pro Pro Asp Gln Leu 130 135 140 Arg Asn Asn Ser Phe His Phe Leu Arg Glu Leu Ser His Val Leu His 145 150 155 160 Thr Asn Val Val Phe Lys Arg Asp Ala Gln Gly Gln Gln Met Ile Phe 165 170 175 Pro Tyr Tyr Gly His Glu Glu Glu Leu Arg Lys His Pro Ile Lys Arg 180 185 190 Ser Thr Val Gly Trp Ala Thr Ser Ser Leu Leu Pro Gly Thr Ser Gly 195 200 205 Gly Arg Gln Arg Arg Glu Leu Asp Pro Met Asp Ile Arg Gly Ser Ile 210 215 220 Val Tyr Leu Glu Ile Asp Asn Arg Gln Cys Val Gln Ser Ser Ser Gln 225 230 235 240 Cys Phe Gln Ser Ala Thr Asp Val Ala Ala Phe Leu Gly Ala Leu Ala 245 250 255 Ser Leu Gly Ser Leu Asn Ile Pro Tyr Lys Ile Glu Ala Val Lys Ser 260 265 270 Glu Pro Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu Met Tyr Val 275 280 285 Ala Ala Ala Ala Phe Val Leu Leu Phe Phe Val Gly Cys Gly Val Leu 290 295 300 Leu Ser 305 <210> 36 <211> 358 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 36 Pro Cys Val Gly Ser Asn Pro Cys Tyr Asn Gln Gly Thr Cys Glu Pro 1 5 10 15 Thr Ser Glu Asn Pro Phe Tyr Arg Cys Leu Cys Pro Ala Lys Phe Asn 20 25 30 Gly Leu Leu Cys His Ile Leu Asp Tyr Ser Phe Thr Gly Gly Ala Gly 35 40 45 Arg Asp Ile Pro Pro Pro Gln Ile Glu Glu Ala Cys Glu Leu Pro Glu 50 55 60 Cys Gln Val Asp Ala Gly Asn Lys Val Cys Asn Leu Gln Cys Asn Asn 65 70 75 80 His Ala Cys Gly Trp Asp Gly Gly Asp Cys Ser Leu Asn Phe Asn Asp 85 90 95 Pro Trp Lys Asn Cys Thr Gln Ser Leu Gln Cys Trp Lys Tyr Phe Ser 100 105 110 Asp Gly His Cys Asp Ser Gln Cys Asn Ser Ala Gly Cys Leu Phe Asp 115 120 125 Gly Phe Asp Cys Gln Leu Thr Glu Gly Gln Cys Asn Pro Leu Tyr Asp 130 135 140 Gln Tyr Cys Lys Asp His Phe Ser Asp Gly His Cys Asp Gln Gly Cys 145 150 155 160 Asn Ser Ala Glu Cys Glu Trp Asp Gly Leu Asp Cys Ala Glu His Val 165 170 175 Pro Glu Arg Leu Ala Ala Gly Thr Leu Val Leu Val Val Leu Leu Pro 180 185 190 Pro Asp Gln Leu Arg Asn Asn Ser Phe His Phe Leu Arg Glu Leu Ser 195 200 205 His Val Leu His Thr Asn Val Val Phe Lys Arg Asp Ala Gln Gly Gln 210 215 220 Gln Met Ile Phe Pro Tyr Tyr Gly His Glu Glu Glu Leu Arg Lys His 225 230 235 240 Pro Ile Lys Arg Ser Thr Val Gly Trp Ala Thr Ser Ser Leu Leu Pro 245 250 255 Gly Thr Ser Gly Gly Arg Gln Arg Arg Glu Leu Asp Pro Met Asp Ile 260 265 270 Arg Gly Ser Ile Val Tyr Leu Glu Ile Asp Asn Arg Gln Cys Val Gln 275 280 285 Ser Ser Ser Gln Cys Phe Gln Ser Ala Thr Asp Val Ala Ala Phe Leu 290 295 300 Gly Ala Leu Ala Ser Leu Gly Ser Leu Asn Ile Pro Tyr Lys Ile Glu 305 310 315 320 Ala Val Lys Ser Glu Pro Val Glu Pro Pro Leu Pro Ser Gln Leu His 325 330 335 Leu Met Tyr Val Ala Ala Ala Ala Phe Val Leu Leu Phe Phe Val Gly 340 345 350 Cys Gly Val Leu Leu Ser 355 <210> 37 <211> 1114 <212> PRT <213> Bombyx mori <400> 37 Met Met Ala Ser Thr Ala Leu Ser Leu Met Gly Arg Cys Asn Pro Asp 1 5 10 15 Gly Cys Thr Arg Gly Lys His Val Thr Ala Ala Pro Met Asp Gly Pro 20 25 30 Arg Gly Pro Ser Ser Leu Ala Gly Thr Phe Gly Trp Gly Leu Ala Ile 35 40 45 Pro Ala Gly Glu Pro Cys Gly Arg Val Cys Ser Pro Ala Thr Val Gly 50 55 60 Phe Phe Pro Val Ala Lys Lys Ser Asn Lys Glu Asn Arg Pro Glu Ala 65 70 75 80 Ser Gly Leu Pro Leu Glu Ser Glu Arg Thr Gly Asp Asn Pro Thr Val 85 90 95 Arg Gly Ser Ala Gly Ala Asp Pro Val Gly Gln Asp Ala Pro Gly Trp 100 105 110 Thr Cys Gln Phe Cys Glu Arg Thr Phe Ser Thr Asn Arg Gly Leu Gly 115 120 125 Val His Lys Arg Arg Ala His Pro Val Glu Thr Asn Thr Asp Ala Ala 130 135 140 Pro Met Met Val Lys Arg Arg Trp His Gly Glu Glu Ile Asp Leu Leu 145 150 155 160 Ala Arg Thr Glu Ala Arg Leu Leu Ala Glu Arg Gly Gln Cys Ser Gly 165 170 175 Gly Asp Leu Phe Gly Ala Leu Pro Gly Phe Gly Arg Thr Leu Glu Ala 180 185 190 Ile Lys Gly Gln Arg Arg Arg Glu Pro Tyr Arg Ala Leu Val Gln Ala 195 200 205 His Leu Ala Arg Phe Gly Ser Gln Pro Gly Pro Ser Ser Gly Gly Cys 210 215 220 Ser Ala Glu Pro Asp Phe Arg Arg Ala Ser Gly Ala Glu Glu Ala Gly 225 230 235 240 Glu Glu Arg Cys Ala Glu Asp Ala Ala Ala Tyr Asp Pro Ser Ala Val 245 250 255 Gly Gln Met Ser Pro Asp Ala Ala Arg Val Leu Ser Glu Leu Leu Glu 260 265 270 Gly Thr Gly Arg Arg Arg Ala Cys Arg Ala Met Arg Pro Lys Thr Ala 275 280 285 Gly Arg Arg Asn Asp Leu His Asp Asp Arg Thr Ala Ser Ala His Lys 290 295 300 Thr Ser Arg Gln Lys Arg Arg Ala Val Tyr Ala Arg Val Gln Glu Leu 305 310 315 320 Tyr Lys Lys Cys Arg Ser Arg Ala Ala Ala Glu Val Ile Asp Gly Ala 325 330 335 Cys Gly Gly Val Gly His Ser Leu Glu Glu Met Glu Thr Tyr Trp Arg 340 345 350 Pro Ile Leu Glu Arg Val Ser Asp Ala Pro Gly Pro Thr Pro Glu Ala 355 360 365 Leu His Ala Leu Gly Arg Ala Glu Trp His Gly Gly Asn Arg Asp Tyr 370 375 380 Thr Gln Leu Trp Lys Pro Ile Ser Val Glu Glu Thr Lys Ala Ser Arg 385 390 395 400 Phe Asp Trp Arg Thr Ser Pro Gly Pro Tyr Gly Ile Arg Ser Gly Gln 405 410 415 Trp Arg Ala Val Pro Val His Leu Lys Ala Glu Met Phe Asn Ala Trp 420 425 430 Met Ala Arg Gly Glu Ile Pro Glu Val Leu Arg Gln Cys Arg Thr Val 435 440 445 Phe Val Pro Lys Val Glu Arg Pro Gly Gly Pro Gly Glu Tyr Arg Pro 450 455 460 Ile Ser Ile Ala Ser Ile Pro Leu Arg His Phe His Ser Ile Leu Ala 465 470 475 480 Arg Arg Leu Leu Ala Cys Cys Pro Pro Asp Ala Arg Gln Arg Gly Phe 485 490 495 Ile Cys Ala Asp Gly Thr Leu Glu Asn Ser Ala Val Leu Asp Ala Val 500 505 510 Leu Gly Asp Ser Arg Lys Lys Leu Trp Glu Cys His Val Ala Val Leu 515 520 525 Asp Phe Ala Lys Ala Phe Asp Thr Val Ser His Glu Ala Leu Val Glu 530 535 540 Leu Leu Arg Leu Arg Gly Met Pro Val Gln Phe Cys Gly Tyr Ile Ala 545 550 555 560 His Leu Tyr Asp Thr Ala Ser Thr Thr Leu Ala Val Asn Asn Glu Met 565 570 575 Ser Ser Pro Val Lys Val Gly Arg Gly Val Arg Gln Gly Asp Pro Leu 580 585 590 Ser Pro Ile Leu Phe Asn Val Val Met Asp Leu Ile Leu Ala Ser Leu 595 600 605 Pro Glu Arg Val Gly Tyr Arg Leu Glu Met Glu Pro Val Ser Ala Leu 610 615 620 Ala Tyr Ala Asp Asp Leu Val Leu Leu Ala Gly Ser Lys Val Gly Met 625 630 635 640 Gln Glu Ser Ile Ser Ala Val Asp Cys Val Gly Arg Gln Met Gly Leu 645 650 655 Arg Leu Asn Cys Arg Lys Ser Ala Val Leu Ser Met Ile Pro Gly Gly 660 665 670 His Arg Lys Lys His His Tyr Leu Thr Glu Arg Thr Phe Asn Ile Gly 675 680 685 Gly Lys Pro Leu Arg Gln Val Ser Cys Val Glu Arg Trp Arg Tyr Leu 690 695 700 Gly Val Asp Phe Glu Ala Ser Gly Cys Val Thr Leu Glu His Ser Ile 705 710 715 720 Ser Ser Ala Leu Asn Asn Ile Ser Arg Ala Pro Leu Lys Pro Gln Gln 725 730 735 Arg Leu Glu Ile Leu Arg Ala His Leu Ile Pro Arg Phe Gln His Gly 740 745 750 Phe Val Leu Gly Asn Ile Ser Asp Asp Arg Leu Arg Met Leu Asp Val 755 760 765 Gln Ile Arg Lys Ala Val Gly Gln Trp Leu Arg Leu Pro Ala Asp Val 770 775 780 Pro Lys Ala Tyr Tyr His Ala Ala Val Gln Asp Gly Gly Leu Ala Ile 785 790 795 800 Pro Ser Val Arg Ala Thr Ile Pro Asp Leu Ile Val Arg Arg Phe Gly 805 810 815 Gly Leu Asp Ser Ser Pro Trp Ser Val Ala Arg Ala Ala Ala Lys Ser 820 825 830 Asp Lys Ile Arg Lys Lys Leu Arg Trp Ala Trp Lys Gln Leu Arg Arg 835 840 845 Phe Ser Arg Val Asp Ser Thr Thr Gln Arg Pro Ser Val Arg Leu Phe 850 855 860 Trp Arg Glu His Leu His Ala Ser Val Asp Gly Arg Glu Leu Arg Glu 865 870 875 880 Ser Thr Arg Thr Pro Thr Ser Thr Lys Trp Ile Arg Glu Arg Cys Ala 885 890 895 Gln Ile Thr Gly Arg Asp Phe Val Gln Phe Val His Thr His Ile Asn 900 905 910 Ala Leu Pro Ser Arg Ile Arg Gly Ser Arg Gly Arg Arg Gly Gly Gly 915 920 925 Glu Ser Ser Leu Thr Cys Arg Ala Gly Cys Lys Val Arg Glu Thr Thr 930 935 940 Ala His Ile Leu Gln Gln Cys His Arg Thr Arg Gly Gly Arg Ile Leu 945 950 955 960 Arg His Asn Lys Ile Val Ser Phe Val Ala Lys Ala Met Glu Glu Asn 965 970 975 Lys Trp Thr Val Glu Leu Glu Pro Arg Leu Arg Thr Ser Val Gly Leu 980 985 990 Arg Lys Pro Asp Ile Ile Ala Ser Arg Asp Gly Val Gly Val Ile Val 995 1000 1005 Asp Val Gln Val Val Ser Gly Gln Arg Ser Leu Asp Glu Leu His 1010 1015 1020 Arg Glu Lys Arg Asn Lys Tyr Gly Asn His Gly Glu Leu Val Glu 1025 1030 1035 Leu Val Ala Gly Arg Leu Gly Leu Pro Lys Ala Glu Cys Val Arg 1040 1045 1050 Ala Thr Ser Cys Thr Ile Ser Trp Arg Gly Val Trp Ser Leu Thr 1055 1060 1065 Ser Tyr Lys Glu Leu Arg Ser Ile Ile Gly Leu Arg Glu Pro Thr 1070 1075 1080 Leu Gln Ile Val Pro Ile Leu Ala Leu Arg Gly Ser His Met Asn 1085 1090 1095 Trp Thr Arg Phe Asn Gln Met Thr Ser Val Met Gly Gly Gly Val 1100 1105 1110 Gly <210> 38 <211> 1056 <212> DNA <213> Bombyx mori <400> 38 ggccgcctcg tcaacgtgaa gaaattcaag caagcgcggg taaacggcgg gagtaactat 60 gactctctta agcgggagta actatgactc tcttaggggc gatacgcata attttaattt 120 ttcgattcaa atccagtcgt cttaatctgg tgaccagtgg cgcggtcacc agtatagtgc 180 acaggacgtg aatggctccg aggctggcgg agtcactcac tataagtgtg agagacgatg 240 tcctgtgcca agtatacgtc caaccctaac gggttaagtg aaattagttg ctcataacag 300 ggacggtgta cctgtttgct cgtggctggc tatcgaatgg acgggaccaa tacacccccc 360 tgttagtaat ggggtaagag agagcggtct gaaactatgg ccgagatcac gacgccccac 420 tcctacccat aacctgcacg tggtaccgcc gcacattgac cgatacggga ggaggggcag 480 cacttgaatc acgtagtctt ggtgtagcca ttgcgggact acagccctcg taagtgccgc 540 cttagaacgc aacggggcaa taggtgggcc ggggcgctag cgggggggag taatctcccc 600. tgttggcgtg caccgcactg ctccctctgg gggcagtgtc atccggaaac aggtgggccg 660 gggcgccacc aggggggagc aatccctcct gatgatggcg agcaccgcac tgtcccttat 720 gggacggtgt aacccggatg gctgtacacg tggtaacac gtgacagcag ccccgatgga 780 cggaccgcga ggaccgtcaa gcctagcagg taccttcggg tggggccttg cgatacctgc 840 gggcgaaccc tgtggtcggg tttgcagccc ggccacagtg ggttttttc ctgttgcaaa 900 aaagtcaaat aaagaaata gacctgaagc ctctggcctc ccgctggagt cagagaggac aggcgataac ccgactgtgc ggggttccgc cggcgcagat cctgtgggtc aggatgcgcc 1020 tggttggacc tgccagttct gcgaacgaac cttttc 1056 <210> 39 <211> 502 <212> DNA <213> Description (Bombyx mori) <400> 39 60. gagttaaggt ccataatcgg gcttcgggga ccgacactac aaatcgttcc gatactggcg ttgagaggtt cacacatgaa ctggaccagg ttcaatcaga tgacgtccgt catggggggc 120 ggcgtggtt gagccttgca cagtagtcca gcggtaaggg tgtagatcag gcccgtctgt 180 ttctcccccg gagctcgctc ccttggcttc ccttatatat tttaacatca gaaacagaca 240 ttaaacatct actgatccaa ttcgccggc gtacggccac gatcgggagg gtgggaatct 300 cgggggtctt ccgatcctaa tccatgatga ttacgacctg agtcactaaa gacgatggca 360 tgatgatccg gcgatgaaaa tagccaaatg cctcgtcatc taattagtga cgcgcatgaa 420 tggattaacg agattcccac tgtccctatc tactatctag cgaaaccaca gccaagggaa 480 cgggcttggg agaatcagcg gg 502

Claims

1. A gene delivery vector system, comprising: a) a first nucleic acid and a second nucleic acid, wherein: i) the first nucleic acid comprises a nucleotide sequence encoding an R2 retrotransposon R2 polypeptide; and ii) the second nucleic acid comprises a heterologous nucleotide sequence encoding one or more heterologous gene products, wherein the heterologous nucleotide sequence is flanked by an R2 retrotransposon 3' untranslated region (UTR) and an R2 retrotransposon 5' UTR, and wherein the heterologous nucleotide sequence has a length of at least 200 nucleotides; or b) polypeptides and nucleic acids, wherein: i) the polypeptide is an R2 retrotransposon R2 polypeptide; and ii) the nucleic acid comprises a heterologous nucleotide sequence encoding one or more heterologous gene products, wherein the heterologous nucleotide sequence is flanked by an R2 retrotransposon 3'UTR and an R2 retrotransposon 5'UTR, and wherein the heterologous nucleotide sequence has a length of at least 200 nucleotides.

2. The gene delivery vehicle system of claim 1, wherein the R2 polypeptide comprises an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO:

37.

3. The gene delivery vehicle system of claim 1 or claim 2, wherein the heterologous nucleotide sequence encodes a single heterologous gene product.

4. The gene delivery vehicle system of claim 3, wherein the single heterologous gene product is a polypeptide.

5. The gene delivery vehicle system of claim 3, wherein the single heterologous gene product is RNA.

6. The gene delivery vehicle system of claim 1 or claim 2, wherein the heterologous nucleotide sequence encodes at least a first heterologous gene product and a second heterologous gene product.

7. The gene delivery vehicle system of claim 6, wherein the first heterologous gene product is a polypeptide, and wherein the second heterologous gene product is RNA.

8. The gene delivery vehicle system of claim 4, wherein the polypeptide is a chimeric antigen receptor.

9. The gene delivery vehicle system of claim 6, wherein the first heterologous gene product is a first heterologous polypeptide, and wherein the second heterologous gene product is a second heterologous polypeptide.

10. The gene delivery vehicle system of claim 9, wherein the heterologous nucleotide sequence comprises, in 5' to 3' order: i) a nucleotide sequence encoding the first heterologous polypeptide; ii) an internal ribosome entry site or a nucleotide sequence encoding a self-cleaving polypeptide; and iii) a nucleotide sequence encoding the second heterologous polypeptide.

11. The gene delivery vehicle system of claim 9, wherein the first polypeptide and the second polypeptide together form a heterodimeric chimeric antigen receptor.

12. The gene delivery vehicle system of claim 7, wherein the polypeptide is an RNA-guided effector polypeptide, and wherein the RNA is a guide RNA that binds to the RNA-guided effector polypeptide.

13. The gene delivery vehicle system of claim 1 or claim 2, wherein the nucleotide sequence encoding the R2 polypeptide is codon optimized for expression in eukaryotic cells.

14. The gene delivery vehicle system of claim 1 or claim 2, wherein the heterologous nucleotide sequence encoding one or more heterologous gene products is operably linked to a transcriptional control element.

15. The gene delivery vehicle system of claim 14, wherein the transcriptional control element is a regulatable promoter.

16. The gene delivery vehicle system of claim 14, wherein the transcriptional control element is a constitutive promoter.

17. The gene delivery vehicle system of claim 1 or claim 2, wherein the heterologous nucleotide sequence has a length of at least 3 kb.

18. The gene delivery vehicle system of claim 1 or claim 2, wherein the heterologous nucleotide sequence has a length of 5 kb to 10 kb.

19. The gene delivery vehicle system of claim 1 or claim 2, wherein the heterologous nucleotide sequence has a length of 10 kb to 15 kb.

20. A medicine box, comprising: a) a first nucleic acid comprising a nucleotide sequence encoding an R2 retrotransposon R2 polypeptide; and b) a second nucleic acid comprising an insertion site for inserting a heterologous nucleotide sequence encoding one or more heterologous gene products, wherein the insertion site is flanked by an R2 retrotransposon 3' untranslated region (UTR) and an R2 retrotransposon 5' UTR, and wherein the heterologous nucleotide sequence has a length of at least 200 nucleotides.

21. The kit of claim 20, wherein the first nucleic acid and the second nucleic acid are in separate containers.

22. The kit of claim 20, wherein the nucleotide sequence encoding the R2 polypeptide is codon-optimized for expression in eukaryotic cells.

23. The kit of claim 20, wherein the second nucleic acid further comprises a transcriptional control element 5' to the insertion site.

24. A medicine kit, comprising: a) R2 retrotransposon R2 polypeptide; as well as b) a nucleic acid comprising an insertion site for inserting a heterologous nucleotide sequence encoding one or more heterologous gene products, wherein the insertion site is flanked by an R2 retrotransposon 3'UTR and an R2 retrotransposon 5'UTR, and wherein the heterologous nucleotide sequence has a length of at least 200 nucleotides.

25. The kit of claim 24, wherein the R2 polypeptide and the nucleic acid are in separate containers.

26. The kit of claim 24, wherein the nucleic acid further comprises a transcriptional control element 5' to the insertion site.

27. A method for delivering one or more target gene products to a eukaryotic cell in vitro or ex vivo, the method comprising contacting the cell with a gene delivery vehicle system as described in any one of claims 1-19, the method being a non-diagnostic and non-therapeutic method, and wherein the R2 polypeptide, the 5'UTR and the 3'UTR achieve insertion of a heterologous nucleic acid into the 28S region of the genome of the eukaryotic cell.

28. The method of claim 27, wherein the eukaryotic cell is a non-human animal cell.

29. The method of claim 27, wherein the eukaryotic cell is a human cell.

30. The method of claim 27, wherein the eukaryotic cell is a plant cell.

31. The method of claim 27, wherein the eukaryotic cell is an invertebrate cell.

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